Most microbiologists distinguish two groups of antimicrobial agents used in the treatment of infectious disease: antibiotics, which are natural substances produced by certain groups of microorganisms, and chemotherapeutic agents, which are chemically synthesized. A hybrid substance is a semisynthetic antibiotic, wherein a molecular version produced by the microbe is subsequently modified by the chemist to achieve desired properties. Furthermore, some antimicrobial compounds, originally discovered as products of microorganisms, can be synthesized entirely by chemical means. In the medical and parmaceutical worlds, all these antimicrobial agents used in the treatment of disease are referred to as antibiotics, interpreting the word literally.
The modern era of antimicrobial chemotherapy began in 1929, with Fleming’s discovery of the powerful bactericidal substance, penicillin, and Domagk’s discovery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity.
In the early 1940’s, spurred partially by the need for antibacterial agents in WW II, penicillin was isolated and purified and injected into experimental animals, where it was found not only to cure infections but also to possess incredibly low toxicity for the animals. This fact ushered into being the age of antibiotic chemotherapy, and an intense search for similar antimicrobial agents of low toxicity to animals that might prove useful in the treatment of infectious disease. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950’s, these and several other antibiotics were in clinical usage.
Microorganisms that Produce Antibiotics
The bacterial colonies at 10 o’clock, 2 o’clock and 8 o’clock on this agar plate are producing antibiotics that inhibit encroachment by the mold which is growing out from the center.
Most of the natural antibiotics that are being used in agriculture and medicine are produced by three unrelated groups of microbes, including eucaryotic molds and two types of spore-forming bacteria. However, many culturable, and some non culturable microbes, have been shown to produce various substances that inhibit other organisms that grow in their space. If we consider antibiotics as secondary metabolites of microbes, it narrows the field to the handful of microbes discussed below.
1. Penicillium and Cephalosporium molds produce beta-lactam antibiotics such as penicillin and cephalosporin and their relatives. They also produce the base molecule for development of semisynthetic beta-lactam antibiotics, such as amoxacillin and ampicillin. Beta-lactams are used to treat about one-third of outpatients with bacterial infections.
The natural habitat of molds is soil. And although sex is sometimes involved, they reproduce by spore formation. They are foremost in their abilities to degrade organic matter, and they play their most important role in natures in biodegradation and the carbon cycle. Most of us know that molds will grow on nearly anything that is organic and moist, so they are also responsible for a lot food spoilage as well as decomposition of our structural materials and textiles. “Nothing is forever”, with molds around.
Three colonies of a Penicillium mold growing on an agar medium. The green fuzzy appearance is the asexual spores of the fungus.
2. Actinomycetes, mainly Streptomyces species, produce tetracyclines, aminoglycosides (streptomycin and its relatives), macrolides (erythromycin and its relatives), chloramphenicol, ivermectin, rifamycins, and most other clinically-useful antibiotics that are not beta-lactams. Actinomycetes are the mainstay of the antibiotics industry.
Actinomycetes are a group of branched bacteria that reproduce by spore formation. They come from a phylum of Bacteria, Actinobacteria, and they are landed in Order Actinomycetales. Some of the representative family include such diverse bacteria as Actinomyces, Corynebacterium, Nocardia, Propionibacter, Streptomyces, Micromonospora and Frankia. Most actinomycetes are inhabitants of the soil. The characteristic odor of damp soil is due to the production of substances, called geosmins, by these bacteria
Two different actinomycetes were spotted in the center of the agar plate about two centimeters apart. This peculiar pattern of growth was observed after a 10-day incubation period. What could be going on? Courtesy of Jerry Ensign Department of Bacteriology. “Chance favors the prepared mind.”
3. Bacillus species, such as B. polymyxa and B. subtilis, produce polypeptide antibiotics (e.g. polymyxin and bacitracin), and B. cereus produces zwittermicin. Bacillus species have the relatively rare ability to form a type of resting cell called an endospore. Bacilli are Gram-positive, rod-shaped, aerobic bacteria that live in the soil. They play an important ecological role in aerobic decomposition, biodegradation and mineral recycling.
A swirl of Bacillus mycoides colonies growth amidst other bacteria and molds from the soil. The swirls are always counterclockwise, at least in the Northern Hemisphere where I have seen it.
These organisms all have in common that they live in soil and they form some sort of a spore or resting structure. It is not known why these microorganisms produce antibiotics, but the answer may be in the obvious – it affords them some nutritional or spatial advantage in their habitat by antagonizing the competition; or it may be in the subtle – it acts as some sort of hormone or signal molecule associated with sporulation or dormancy or germination. Antibiotics are secondary metabolites and they are produced at the same time that the cells begin their sporulation processes.
Antibiotics tend to be rather large, complicated organic molecules and may require as many as 30 separate enzymatic steps to synthesize. The maintenance of a substantial component of the bacterial genome devoted solely to the synthesis of an antibiotic leads one to conclude that the antibiotic is important, if not essential, to the survival of these organisms in their natural habitat.
Most of the microorganisms that produce antibiotics are resistant to the action of their own antibiotic, although the organisms are affected by other antibiotics, and their antibiotic may be effective against closely-related strains. In most cases, how or why bacteria are resistant to their own antibiotics is also unknown, but it may be worth pondering or studying if we are to understand the cellular and molecular basis of drug resistance in pathogens.
Antibiotics must have Selective Toxicity for the Microbe
Several hundreds of compounds with antibiotic activity have been isolated from microorganisms over the years, but only a few of them are clinically-useful. The reason for this is that only compounds with selective toxicity can be used clinically.
The selective toxicity of antibiotics means that they must be highly effective against the microbe but have minimal or no toxicity to humans. In practice, this is expressed by a drug’s therapeutic index (TI) – the ratio of the toxic dose (to the patient) to the therapeutic dose (to eliminate the infection). The larger the index, the safer is the drug (antibiotic) for human use.
The selective toxicity of antibiotics is brought about by finding vulnerable targets for the drug in the microbe that do not exist in the animal (eucaryote) that is given the drug. Most antibiotics in clinical usage are directed against bacterial cell wall synthesis, bacterial protein synthesis, or bacterial nucleic acid synthesis, which are unique in some ways to bacteria. For example, the beta lactam antibiotics (penicillin and its relatives) inhibit peptidoglycan synthesis in the cell wall. Humans have neither a cell wall nor peptidoglycan and so are unaffected by the action of the drug. Other antibiotics, including streptomycin and the tetracyclines, target bacterial protein synthesis because bacterial ribosomes (termed 70S ribosomes) are different from the ribosomes (80S) of humans and other eucaryotic organisms. Antibiotics such as the flouroqinolones (e.g. ciprofloxacin) inhibit procaryotic (not eucaryotic) DNA replication, and rifamycins inhibit bacterial (not eucaryotic) DNA transcription.
From a patient point of view, the most important property of an antimicrobial agent is its selective toxicity, i.e., that the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the patient.
Characteristics of Antibiotics
Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that is affected by a certain antibiotic is expressed as its spectrum of action. Antibiotics effective against procaryotes that kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum. If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum.
A clinically-useful antibiotic should have as many of these characteristics as possible.
-It should have a wide spectrum of activity with the ability to destroy or inhibit many different species of pathogenic organisms.
-It should be nontoxic to the host and without undesirable side effects.
-It should be nonallergenic to the host.
-It should not eliminate the normal flora of the host.
-It should be able to reach the part of the human body where the infection is occurring.
-It should be inexpensive and easy to produce.
-It should be chemically-stable (have a long shelf-life).
-Microbial resistance is uncommon and unlikely to develop.
Kinds of Antimicrobial Agents and their Primary Modes of Action
The table below is a summary of thetypes or classes of antibiotics and their properties including their biological source, spectrum and mode of action.
Classes of Antibiotics and their Properties
|Chemical class||Examples||Biological source||Spectrum (effective against)||Mode of action|
|Beta-lactams (penicillins and cephalosporins)||Penicillin G, Cephalothin||Penicillium notatum andCephalosporiumspecies||Gram-positive bacteria||Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly|
|Semisynthetic beta-lactams||Ampicillin, Amoxicillin||Gram-positive and Gram-negative bacteria||Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly|
|Clavulanic Acid||Augmentin is clavulanic acid plus Amoxicillin||Streptomyces clavuligerus||Gram-positive and Gram-negative bacteria||Inhibitor of bacterial beta-lactamases|
|Monobactams||Aztreonam||Chromobacterium violaceum||Gram-positive and Gram-negative bacteria||Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly|
|Carboxypenems||Imipenem||Streptomyces cattleya||Gram-positive and Gram-negative bacteria||Inhibits steps in cell wall (peptidoglycan) synthesis and murein assembly|
|Aminoglycosides||Streptomycin||Streptomyces griseus||Gram-positive and Gram-negative bacteria||Inhibits translation (protein synthesis)|
|Gentamicin||Micromonosporaspecies||Gram-positive and Gram-negative bacteria esp.Pseudomonas||Inhibits translation (protein synthesis)|
|Glycopeptides||Vancomycin||Amycolatopsis orientalisNocardia orientalis(formerly designated)||Gram-positive bacteria, esp.Staphylococcus aureus||Inhibits steps in murein (peptidoglycan) biosynthesis and assembly|
|Lincomycins||Clindamycin||Streptomyces lincolnensis||Gram-positive and Gram-negative bacteria esp. anaerobic Bacteroides||Inhibits translation (protein synthesis)|
|Macrolides||Erythromycin, Azithromycin||Streptomyces erythreus||Gram-positive bacteria, Gram-negative bacteria not enterics, Neisseria, Legionella, Mycoplasma||Inhibit translation (protein synthesis)|
|Polypeptides||Polymyxin||Bacillus polymyxa||Gram-negative bacteria||Damages cytoplasmic membranes|
|Bacitracin||Bacillus subtilis||Gram-positive bacteria||Inhibits steps in murein (peptidoglycan) biosynthesis and assembly|
|Polyenes||Amphotericin||Streptomyces nodosus||Fungi (Histoplasma)||Inactivate membranes containing sterols|
|Nystatin||Streptomyces noursei||Fungi (Candida)||Inactivate membranes containing sterols|
|Rifamycins||Rifampicin||Streptomyces mediterranei||Gram-positive and Gram-negative bacteria,Mycobacterium tuberculosis||Inhibits transcription (bacterial RNA polymerase)|
|Tetracyclines||Tetracycline||Streptomycesspecies||Gram-positive and Gram-negative bacteria, Rickettsias||Inhibit translation (protein synthesis)|
|Semisynthetic tetracycline||Doxycycline||Gram-positive and Gram-negative bacteria, RickettsiasEhrlichia, Borrelia||Inhibit translation (protein synthesis)|
|Chloramphenicol||Chloramphenicol||Streptomyces venezuelae||Gram-positive and Gram-negative bacteria||Inhibits translation (protein synthesis)|
|Quinolones||Nalidixic acid||synthetic||Mainly Gram-negative bacteria||Inhibits DNA
|Fluoroquinolones||Ciprofloxacin||synthetic||Gram-negative and some Gram-positive bacteria (Bacillus anthracis)||Inhibits DNA replication|
|Growth factor analogs||Sulfanilamide, Gantrisin, Trimethoprim||synthetic||Gram-positive and Gram-negative bacteria||Inhibits folic acid metabolism (anti-folate)|
|Isoniazid (INH)||synthetic||Mycobacterium tuberculosis||Inhibits mycolic acid synthesis; analog of pyridoxine (Vit B6)|
|para-aminosalicylic acid (PAS)||synthetic||Mycobacterium tuberculosis||Anti-folate|
Antimicrobial Agents Used in the Treatment of Infectious Disease
Examination of the foregoing table reveals that there are a handful of fundamental ways that antibacterial antibiotics work as therapeutic agents. Recall that the target of an antibiotic should be unique to the bacterium and not found, or not accessible to the antibiotic, in the patient. These are the most important targets in bacteria that have been exploited so far.
1. Attack bacterial cell wall synthesis. Bacteria have murein in their cell walls, not found in the host, and murein (peptidoglycan) is essential to the viability of the bacterium.
2. Interfere with protein synthesis. Attack is almost always ate the level of translation using 70S ribosomes in the translation machinery. 70S cytoplasmic ribosomes are absent in eucaryotes.
3. Interference with nucleic acid synthesis (RNA and DNA), which exploits differences between RNA polymerases and DNA replication strategies in bacteria and eucaryotes.
4. Inhibition of an essential metabolic pathway that exists in the bacterium but does not exist in the host. This is usually brought about through the use of competitive chemical analogs for bacterial enzymatic reactions.
5. Membrane inhibition or disruption doesn’t work too well because of the similarities between eucaryotic and bacterial membranes. However, the outer membrane of Gram-negative bacteria is a reasonable point of attack and some membrane inhibitors are included in the discussion below.
Cell wall synthesis inhibitors
Cell wall synthesis inhibitors generally inhibit some step in the synthesis of bacterial peptidoglycan. They exert their selective toxicity against bacteria because humans cells lack cell walls.
Beta lactam antibiotics. Chemically, these antibiotics contain a 4-membered beta lactam ring. They are the products of two genera of fungi, Penicillium and Cephalosporium, and are correspondingly represented by the penicillins and cephalosporins.
Chemical structures of some beta-lactam antibiotics. Clockwise: penicillin, cephalosporin, monobactam, carbapenem. Note the characteristic structure of the beta lactam ring.
The beta lactam antibiotics are stereochemically related to D-alanyl-D-alanine, which is a substrate for the last step in peptidoglycan synthesis, the final cross-linking between between peptide side chains. Penicillins bind to and inhibit the carboxypeptidase and transpeptidase enzymes that are required for this step in peptidoglycan biosynthesis. Beta lactam antibiotics are bactericidal and require that cells be actively growing in order to exert their toxicity.
Different beta lactams differ in their spectrum of activity and their effect on Gram-negative rods, as well as their toxicity, stability in the human body, rate of clearance from blood, whether they can be taken orally, ability to cross the blood-brain barrier, and susceptibility to bacterial beta-lactamases.
Natural penicillins, such as penicillin G or penicillin V (benzyl penicillin), are produced by fermentation of Penicillium chrysogenum. They are effective against streptococci, gonococci and staphylococci, except where resistance has developed. They are considered narrow spectrum since they are not effective against Gram-negative rods.
Penicillin G (Benzylpenicillin) is typically given by parenteral administration because it is unstable in the acid of the stomach. However, this achieves higher tissue concentrations than orally-administered penicillins and this increases its antibacterial potential. “PenG” may be used in treatment of bacterial endocarditis, gonorrhea, syphilis, meningitis, and pneumonia.
Semisynthetic penicillins first appeared in 1959. A mold produces the main part of the molecule (6-aminopenicillanic acid), which can be modified chemically by the addition of side chains. Many of these compounds have been developed to have distinct benefits or advantages over penicillin G, such as increased spectrum of activity (effectiveness against Gram-negative rods), resistance to penicillinase, effectiveness when administered orally, etc.; amoxicillin and ampicillin have broadened spectra against Gram-negative bacteria and are effective orally; methicillin is penicillinase-resistant.
The semisynthetic beta-lactam, amoxicillin. Amoxicillin is usually the drug of choice within the class because it is better absorbed following oral administration than other beta-lactam antibiotics. It is susceptible to degradation by bacterial beta-lactamase enzymes so it may be given with calvulanic acid (below) to decrease its susceptibility. It is used against a wide range of Gram-positive bacteria, including Streptococcus pyogenes, penicillin-sensitive Streptococcus pneumoniae, non beta-lactamase producing strains of Staphylococcus aureus and Enterococcus faecalis. Susceptible Gram-negative organisms include non beta-lactamase producing strains ofHaemophilus influenzae, Neisseria gonorrhoeae and N. meningitidis.
Clavulanic acid is a chemical sometimes added to a semisynthetic penicillin preparation. Thus, amoxicillin plus clavulanate is clavamox or augmentin. The clavulanate is not an antimicrobial agent. It inhibits beta lactamase enzymes and has given extended life to penicillinase-sensitive beta lactams.
The structure of calvulanic acid. Clavulanic acid is not an antibiotic. It is a beta-lactamase inhibitor sometimes combined with semisynthetic beta lactam antibiotics to overcome resistance in bacteria that produce beta-lactamase enzymes, which otherwise inactivate the antibiotic. Most commonly it is combined with amoxicillin (above) as Augmentin (trade name) or the veterinary preparation, clavamox.
Although nontoxic, penicillins occasionally cause death when administered to persons who are allergic to them. In the U.S. there are 300 – 500 deaths annually due to penicillin allergy. In allergic individuals the beta lactam molecule attaches to a serum protein and initiates an IgE-mediated inflammatory response.
Cephalosporins are beta lactam antibiotics with a similar mode of action to penicillins. They are produced by species of Cephalosporium molds. The have a low toxicity and a somewhat broader spectrum than natural penicillins. They are often used as penicillin substitutes against Gram-negative bacteria and in surgical prophylaxis. They are subject to degradation by some bacterial beta-lactamases, but they tend to be resistant to beta-lactamases from S. aureus.
The core structure of cephalosporin. Substituent groups added at position X on the six-membered ring generates variants of the antibiotic.
Two other classes of beta lactams are the carbapenems and monobactams. The latter are particularly useful for the treatment of allergic individuals. A person who becomes allergic to penicillin usually becomes allergic to the cephalosporins and the carbapenems as well. Such individuals can still be treated with the monobactams, which are structurally different so as not to induce allergy.
Aztreonam is a synthetic monocyclic beta lactam antibiotic (a monobactam) originally isolated from the bacterium Chromobacterium violaceum. It is not useful against Gram-positive bacteria but it has strong activity against a wide range of susceptible Gram-negative bacteria, includingPseudomonas aeruginosa, E. coli, Haemophilus and Klebsiella.
Bacitracin is a polypeptide antibiotic produced by Bacillus species. It prevents cell wall growth by inhibiting the release of the muropeptide subunits of peptidoglycan from the lipid carrier molecule that carries the subunit to the outside of the membrane. Teichoic acid synthesis, which requires the same carrier, is also inhibited. Bacitracin has a high toxicity which precludes its systemic use. It is present in many topical antibiotic preparations, and since it is not absorbed by the gut, it is given to “sterilize” the bowel prior to surgery.
Bacitracin is a polypeptide antibiotic produced by the licheniformis group of Bacillus subtilis var. Tracy. It is effective used topically, primarily against Gram-positive bacteria. It is used in ointment or cream form for topical treatment of a variety of localized skin and eye infections, as well as for the prevention of wound infections. A popular brand name Neosporin, contains bacitracin, neomycin and polymyxin B.
Cycloserine inhibits the early stages of murein synthesis where D-alanyl-D-alanine is added to the growing peptide side chain. The antibiotic resembles D-alanine in spatial structure, and it competitively inhibits the racemase reaction that converts L-alanine to D-alanine and the synthetase reaction that joins two D-alanine molecules. The affinity of cycloserine for these enzymes is about a hundred times greater than that of D-alanine. Cycloserine enters bacterial cells by means of an active transport system for glycine and can reach a relatively high intracellular concentration. This concentrating effect, along with its high affinity for susceptible enzymes, enables cycloserine to function as a very effective antimicrobial agent. However, it is fairly toxic and has limited use as a secondary drug for tuberculosis.
Cycloserine is an oral broad spectrum antibiotic effective against tuberculosis, by inhibiting cell wall synthesis of TB bacilli at the early stages of peptidoglycan synthesis. For the treatment against tuberculosis, it is classified as a second line drug.
Glycopeptides, such as the antibiotic vancomycin, inhibit both transglycosylation and transpeptidation reactions during peptidoglycan assembly. They bind to the muropeptide subunit as it is transferred out of the cell cytoplasm and inhibit subsequent polymerization reactions. Vancomycin is not effective against Gram-negative bacteria because it cannot penetrate their outer membrane. However, it has become important in clinical usage for treatment of infections by strains of Staphylococcus aureus that are resistant to virtually all other antibiotics (MRSA).
Vancomycin is a glycopeptide antibiotic used in the prophylaxis and treatment of infections caused by Gram-positive bacteria. It has traditionally been reserved as a drug of “last resort”, used only after treatment with other antibiotics had failed, although the emergence of vancomycin-resistant organisms means that it is increasingly being displaced from this role by linezolid and the carbapenems.
Cell membrane inhibitors
These antibiotics disorganize the structure or inhibit the function of bacterial membranes. The integrity of the cytoplasmic and outer membranes is vital to bacteria, and compounds that disorganize the membranes rapidly kill the cells. However, due to the similarities in phospholipids in eubacterial and eucaryotic membranes, this action is rarely specific enough to permit these compounds to be used systemically. The only antibacterial antibiotics of clinical importance that act by this mechanism are the polymyxins, produced by Bacillus polymyxa. Polymyxin is effective mainly against Gram-negative bacteria and is usually limited to topical usage. Polymyxins bind to membrane phospholipids and thereby interfere with membrane function. Polymyxin is occasionally given for urinary tract infections caused by Pseudomonas strains that are gentamicin, carbenicillin and tobramycin resistant. The balance between effectiveness and damage to the kidney and other organs is dangerously close, and the drug should only be given under close supervision in the hospital.
Polymyxin B. Polymyxins are cationic detergent antibiotics, with a general structure of a cyclic peptide with a long hydrophobic tail. They disrupt the structure of the bacterial cell membrane by interacting with its phospholipids. Polymyxins have a bactericidal effect on Gram-negative bacilli, especially on Pseudomonas and coliform bacteria. Polymyxin antibiotics are highly neurotoxic and nephrotoxic, and very poorly absorbed from the gastrointestinal tract. Polymyxins also have antifungal activity.
Protein synthesis inhibitors
Many therapeutically useful antibiotics owe their action to inhibition of some step in the complex process of protein synthesis. Their attack is always at one of the events occurring on the ribosome and never at the stage of amino acid activation or attachment to a particular tRNA. Most have an affinity or specificity for 70S (as opposed to 80S) ribosomes, and they achieve their selective toxicity in this manner. The most important antibiotics with this mode of action are the tetracyclines,chloramphenicol, the macrolides (e.g. erythromycin) and the aminoglycosides (e.g. streptomycin).
The aminoglycosides are products of Streptomyces species and are represented bystreptomycin, kanamycin, tobramycin and gentamicin. These antibiotics exert their activity by binding to bacterial ribosomes and preventing the initiation of protein synthesis.
Streptomycin binds to 30S subunit of the bacterial ribosome, specifically to the S12 protein which is involved in the initiation of protein synthesis. Experimentally, streptomycin has been shown to prevent the initiation of protein synthesis by blocking the binding of initiator N-formylmethionine tRNA to the ribosome. It also prevents the normal dissociation of ribosomes into their subunits, leaving them mainly in their 70S form and preventing the formation of polysomes. The overall effect of streptomycin seems to be one of distorting the ribosome so that it no longer can carry out its normal functions. This evidently accounts for its antibacterial activity but does not explain its bactericidal effects, which distinguishes streptomycin and other aminoglycosides from most other protein synthesis inhibitors.
Streptomycin is the first aminoglycoside antibiotic to be discovered, and was the first antibiotic to be used in treatment of tuberculosis. It was discovered in 1943, in the laboratory of Selman Waksman at Rutgers University. Waksman and his laboratory discovered several antibiotics, including actinomycin, streptomycin, and neomycin. Streptomycin is derived from the bacterium, Streptomyces griseus. Streptomycin stops bacterial growth by inhibiting protein synthesis. Specifically, it binds to the 16S rRNA of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This prevents initiation of protein synthesis.
Kanamycin and tobramycin have been reported to bind to the ribosomal 30S subunit and to prevent it from joining to the 50S subunit during protein synthesis. They may have a bactericidal effect because this leads to cytoplasmic accumulation of dissociated 30S subunits, which is apparently lethal to the cells.
Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria. Streptomycin has been used extensively as a primary drug in the treatment of tuberculosis. Gentamicin is active against many strains of Gram-positive and Gram-negative bacteria, including some strains of Pseudomonas aeruginosa. Kanamycin is active at low concentrations against many Gram-positive bacteria, including penicillin-resistant staphylococci. Gentamicin and Tobramycin are mainstays for treatment of Pseudomonasinfections. An unfortunate side effect of aminoglycosides has tended to restrict their usage: prolonged use is known to impair kidney function and cause damage to the auditory nerves leading to deafness.
Gentamicin is an aminoglycoside antibiotic, used mostly to treat Gram-negative infections. However, it is not used for Neisseria gonorrhoeae, Neisseria meningitidis or Legionella pneumophilainfections. It is synthesized by Micromonospora, a genus of Gram-positive bacteria widely distributed in water and soil. Like all aminoglycosides, when gentamicin is given orally, it is not systemically active because it is not absorbed to any appreciable extent from the small intestine. It is useful in treatment of infections caused by Pseudomonas aeruginosa.
The tetracyclines consist of eight related antibiotics which are all natural products of Streptomyces, although some can now be produced semisynthetically or synthetically. Tetracycline,chlortetracycline and doxycycline are the best known. The tetracyclines are broad-spectrum antibiotics with a wide range of activity against both Gram-positive and Gram-negative bacteria.Pseudomonas aeruginosa is less sensitive but is generally susceptible to tetracycline concentrations that are obtainable in the bladder. The tetracyclines act by blocking the binding of aminoacyl tRNA to the A site on the ribosome. Tetracyclines inhibit protein synthesis on isolated 70S or 80S (eucaryotic) ribosomes, and in both cases, their effect is on the small ribosomal subunit. However, most bacteria possess an active transport system for tetracycline that will allow intracellular accumulation of the antibiotic at concentrations 50 times as great as that in the medium. This greatly enhances its antibacterial effectiveness and accounts for its specificity of action, since an effective concentration cannot be accumulated in animal cells. Thus a blood level of tetracycline which is harmless to animal tissues can halt protein synthesis in invading bacteria.
The tetracyclines have a remarkably low toxicity and minimal side effects when taken by animals. The combination of their broad spectrum and low toxicity has led to their overuse and misuse by the medical community and the wide-spread development of resistance has reduced their effectiveness. Nonetheless, tetracyclines still have some important uses, such as the use of doxycycline in the treatment of Lyme disease.
Some newly discovered members of the tetracycline family (e.g. chelocardin) have been shown to act by inserting into the bacterial membrane, not by inhibiting protein synthesis.
The tetracycline core structure. The tetracyclines are a large family of antibiotics that were discovered as natural products of Streptomyces bacteria beginning in the late 1940s. Tetracycline sparked the development of many chemically altered antibiotics and in doing so has proved to be one of the most important discoveries made in the field of antibiotics. It is a classic “broad-spectrum antibiotic” used to treat infections caused by Gram-positive and Gram-negative bacteria and some protozoa.
Doxycycline is a semisynthetic tetracycline developed in the 1960s. It is frequently used to treat chronic prostatitis, sinusitis, syphilis, chlamydia, pelvic inflammatory disease, acne and rosacea. In addition, it is used in the treatment and prophylaxis of anthrax and in prophylaxis against malaria. It is also effective against Yersinia pestis (the infectious agent of bubonic plague) and is prescribed for the treatment of Lyme disease, ehrlichiosis and Rocky Mountain spotted fever. Because doxycycline is one of the few medications that is effective in treating Rocky Mountain spotted fever (with the next best alternative being chloramphenicol), it is indicated even for use in children for this illness.
Chloramphenicol is a protein synthesis inhibitor that has a broad spectrum of activity but it exerts a bacteriostatic effect. It is effective against intracellular parasites such as the rickettsiae. Unfortunately, aplastic anemia develops in a small proportion (1/50,000) of patients. Chloramphenicol was originally discovered and purified from the fermentation of a Streptomycesspecies, but currently it is produced entirely by chemical synthesis. Chloramphenicol inhibits the bacterial enzyme peptidyl transferase, thereby preventing the growth of the polypeptide chain during protein synthesis.
Chemical structure of chloramphenicol
Chloramphenicol is entirely selective for 70S ribosomes and does not affect 80S ribosomes. Its unfortunate toxicity towards the small proportion of patients who receive it is in no way related to its effect on bacterial protein synthesis. However, since mitochondria originated from procaryotic cells and have 70S ribosomes, they are subject to inhibition by some of the protein synthesis inhibitors including chloramphenicol. This likely explains the toxicity of chloramphenicol. The eucaryotic cells most likely to be inhibited by chloramphenicol are those undergoing rapid multiplication, thereby rapidly synthesizing mitochondria. Such cells include the blood forming cells of the bone marrow, the inhibition of which could present as aplastic anemia. Chloramphenicol was once a highly prescribed antibiotic and a number of deaths from anemia occurred before its use was curtailed. Now it is seldom used in human medicine except in life-threatening situations (e.g. typhoid fever).
The macrolide family of antibiotics is characterized by structures that contain large lactone rings linked through glycoside bonds with amino sugars. The most important members of the group areerythromycin and oleandomycin. Erythromycin is active against most Gram-positive bacteria,Neisseria, Legionella and Haemophilus, but not against the Enterobacteriaceae. Macrolides inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit. Binding inhibits elongation of the protein by peptidyl transferase or prevents translocation of the ribosome or both. Macrolides are bacteriostatic for most bacteria but are cidal for a few Gram-positive bacteria.
Chemical structure of a macrolide antibiotic, erythromycin.
Azithromycin, shown above, is a subclass of macrolide antibiotics. Azithromycin is one of the world’s best-selling antibiotics. It is s derived from erythromycin, but it differs chemically from erythromycin in that a methyl-substituted nitrogen atom is incorporated into the lactone ring, thus making the lactone ring 15-membered. Azithromycin is used to treat certain bacterial infections, most often bacteria causing middle ear infections, tonsillitis, throat infections, laryngitis, bronchitis, pneumonia and sinusitis. It is also effective against certain sexually transmitted diseases, such as non-gonococcal urethritis and cervicitis.
Lincomycin and clindamycin are a miscellaneous group of protein synthesis inhibitors with activity similar to the macrolides. Lincomycin has activity against Gram-positive bacteria and some Gram-negative bacteria (Neisseria, H. influenzae). Clindamycin is a derivative of lincomycin with the same range of antimicrobial activity, but it is considered more effective. It is frequently used as a penicillin substitute and is effective against Gram-negative anaerobes (e.g. Bacteroides).
Clindamycin is a lincosamide antibiotic. It is usually used to treat infections with anaerobic bacteria but can also be used to treat some protozoal diseases, such as malaria. It is a common topical treatment for acne, and can be useful against some methicillin-resistant Staphylococcus aureus (MRSA) infections. The most severe common adverse effect of clindamycin is Clostridium difficile-associated diarrhea (the most frequent cause of pseudomembranous colitis). Although this side-effect occurs with almost all antibiotics, including beta-lactam antibiotics, it is classically linked to clindamycin use.
Effects on Nucleic Acids
Some antibiotics and chemotherapeutic agents affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read. Either case, of course, can block the growth of cells. The majority of these drugs are unselective, however, and affect animal cells and bacterial cells alike and therefore have no therapeutic application. Two nucleic acid synthesis inhibitors which have selective activity against procaryotes and some medical utility are the quinolones andrifamycins.
Nalidixic acid is a synthetic chemotherapeutic agent that has activity mainly against Gram-negative bacteria. Nalidixic acid belongs to a group of compounds called quinolones. Nalidixic acid is a bactericidal agent that binds to the DNA gyrase enzyme (topoisomerase) which is essential for DNA replication and allows supercoils to be relaxed and reformed. Binding of the drug inhibits DNA gyrase activity.
Some quinolones penetrate macrophages and neutrophils better than most antibiotics and are thus useful in treatment of infections caused by intracellular parasites. However, the main use of nalidixic acid is in treatment of lower urinary tract infections (UTI). The compound is unusual in that it is effective against several types of Gram-negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and Proteus species which are common causes of UTIs. It is not usually effective against Pseudomonas aeruginosa, and Gram-positive bacteria may be resistant. Some quinolones have a broadened spectrum against Gram-positive bacteria. The fluoroquinolone, Cipro. (ciprofloxacin) was recently touted as the drug of choice for treatment and prophylaxis of anthrax, which is caused by a Gram-positive bacillus, Bacillus anthracis.
Ciprofloxacin (cipro), a fluoroquinolone is a broad-spectrum antimicrobial agent that is active against both Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a type II topoisomerase, which is an enzyme necessary to separate replicated DNA, and thereby inhibits cell division.
The rifamycins are a comparatively new group of antibiotics, also the products of Streptomycesspecies. Rifampicin is a semisynthetic derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis) and some Gram-negative bacteria. Rifampicinacts quite specifically on the bacterial RNA polymerase and is inactive towards DNA polymerase or RNA polymerase from animal cells. The antibiotic binds to the beta subunit of the polymerase and apparently blocks the entry of the first nucleotide which is necessary to activate the polymerase, thereby blocking mRNA synthesis. It has been found to have greater bactericidal effect against M. tuberculosis than other anti-tuberculosis drugs, and it has largely replaced isoniazid as one of the front-line drugs used to treat the disease, especially when isoniazid resistance is indicated. It is effective orally and penetrates the cerebrospinal fluid so it is useful for treatment of bacterial meningitis.
Rifampicin (or rifampin) is a bactericidal antibiotic from the rifamycin group. It is a semisynthetic compound derived from Amycolatopsis rifamycinica (formerly known as Amycolatopsis mediterranei and Streptomyces mediterranei). Rifampicin is typically used to treat Mycobacteriuminfections, including tuberculosis and leprosy; and also has a role in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) in combination with fusidic acid. It is used in prophylactic therapy against Neisseria meningitidis (meningococcal) infection. It is also used to treat infection by Listeria monocytogenes, Neisseria gonorrhoeae, Haemophilus influenzae andLegionella pneumophila.
Many of the synthetic chemotherapeutic agents (synthetic antibiotics) are competitive inhibitorsof essential metabolites or growth factors that are needed in bacterial metabolism. Hence, these types of antimicrobial agents are sometimes referred to as anti-metabolites or growth factor analogs, since they are designed to specifically inhibit an essential metabolic pathway in the bacterial pathogen. At a chemical level, competitive inhibitors are structurally similar to a bacterial growth factor or metabolite, but they do not fulfill their metabolic function in the cell. Some are bacteriostatic and some are bactericidal. Their selective toxicity is based on the premise that the bacterial pathway does not occur in the host.
Sulfonamides were introduced as chemotherapeutic agents by Domagk in 1935, who showed that one of these compounds (prontosil) had the effect of curing mice with infections caused by beta-hemolytic streptococci. Chemical modifications of the compound sulfanilamide gave rise to compounds with even higher and broader antibacterial activity. The resulting sulfonamides have broadly similar antibacterial activity, but differ widely in their pharmacological actions. Bacteria which are almost always sensitive to the sulfonamides include Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli. The sulfonamides have been extremely useful in the treatment of uncomplicated UTI caused by E. coli, and in the treatment of meningococcal meningitis (because they cross the blood-brain barrier).
The sulfonamides (e.g. Gantrisin and Trimethoprim) are inhibitors of the bacterial enzymes required for the synthesis of tetrahydofolic acid (THF), the vitamin form of folic acid essential for 1-carbon transfer reactions. Sulfonamides are structurally similar to para aminobenzoic acid (PABA), the substrate for the first enzyme in the THF pathway, and they competitively inhibit that step. Trimethoprim is structurally similar to dihydrofolate (DHF) and competitively inhibits the second step in THF synthesis mediated by the DHF reductase. Animal cells do not synthesize their own folic acid but obtain it in a preformed fashion as a vitamin. Since animals do not make folic acid, they are not affected by these drugs, which achieve their selective toxicity for bacteria on this basis.
The chemical structures of sulfanilamide and para-aminobenzoic acid (PABA). In bacteria, sulfanilamide acts as a competitive inhibitor of the enzyme dihydropteroate synthetase, DHPS, which catalyses the conversion of PABA to dihydropteroate, a key step in folate synthesis. Folate is necessary for the cell to synthesize nucleic acids (DNA and RNA), and in its absence, cells will be unable to divide. Hence, sulfanilamide and other sulfonamides exhibit a bacteriostatic rather than bactericidal effect.
Three additional synthetic chemotherapeutic agents have been used in the treatment of tuberculosis: isoniazid (INH), para-aminosalicylic acid (PAS), and ethambutol. The usual strategy in the treatment of tuberculosis has been to administer a single antibiotic (historically streptomycin, but now, most commonly, rifampicin is given) in conjunction with INH and ethambutol. Since the tubercle bacillus rapidly develops resistance to the antibiotic, ethambutol and INH are given to prevent outgrowth of a resistant strain. It must also be pointed out that the tubercle bacillus rapidly develops resistance to ethambutol and INH if either drug is used alone. Ethambutol inhibits incorporation of mycolic acids into the mycobacterial cell wall. Isoniazid has been reported to inhibit mycolic acid synthesis in mycobacteria and since it is an analog of pyridoxine (Vitamin B6) it may inhibit pyridoxine-catalyzed reactions as well. Isoniazid is activated by a mycobacterial peroxidase enzyme and destroys several targets in the cell. PAS is an anti-folate, similar in activity to the sulfonamides. PAS was once a primary anti-tuberculosis drug, but now it is a secondary agent, having been largely replaced by ethambutol.
Isoniazid is also called isonicotinyl hydrazine or INH. Isoniazid is a first-line anti-tuberculosis medication used in the prevention and treatment of tuberculosis. Isoniazid is never used on its own to treat active tuberculosis because resistance quickly develops.
A pathogen is a microorganism that is able to cause disease in a plant, animal or insect.Pathogenicity is the ability to produce disease in a host organism. Microbes express their pathogenicity by means of their virulence, a term which refers to the degree of pathogenicity of the microbe. Hence, the determinants of virulence of a pathogen are any of its genetic or biochemical or structural features that enable it to produce disease in a host.
The relationship between a host and a pathogen is dynamic, since each modifies the activities and functions of the other. The outcome of such a relationship depends on the virulence of the pathogen and the relative degree of resistance or susceptibility of the host, due mainly to the effectiveness of the host defense mechanisms.
Staphylococcus aureus, arguably the most prevalent pathogen of humans, may cause up to one third of all bacterial diseases ranging from boils and pimples to food poisoning, to septicemia and toxic shock. Electron micrograph from Visuals Unlimited, with permission.
The Underlying Mechanisms of Bacterial Pathogenicity
Two broad qualities of pathogenic bacteria underlie the means by which they cause disease:
1. Invasiveness is the ability to invade tissues. It encompasses mechanisms for colonization(adherence and initial multiplication), production of extracellular substances which facilitate invasion (invasins) and ability to bypass or overcome host defense mechanisms.
2. Toxigenesis is the ability to produce toxins. Bacteria may produce two types of toxins calledexotoxins and endotoxins. Exotoxins are released from bacterial cells and may act at tissue sites removed from the site of bacterial growth. Endotoxins are cell-associated substance. (In a classic sense, the term endotoxin refers to the lipopolysaccharide component of the outer membrane of Gram-negative bacteria). However, endotoxins may be released from growing bacterial cells and cells that are lysed as a result of effective host defense (e.g. lysozyme) or the activities of certain antibiotics (e.g. penicillins and cephalosporins). Hence, bacterial toxins, both soluble and cell-associated, may be transported by blood and lymph and cause cytotoxic effects at tissue sites remote from the original point of invasion or growth. Some bacterial toxins may also act at the site of colonization and play a role in invasion.
Acid-fast stain of Mycobacterium tuberculosis, the agent of tuberculosis (TB). The bacteria are the small pink-staining rods. More than one-third of the world population is infected. The organism has caused more human deaths than any other bacterium in the history of mankind. Although its ability to produce disease is multifactorial, it is not completely understood. American Society of Microbiology, with permission.
The first stage of microbial infection is colonization: the establishment of the pathogen at the appropriate portal of entry. Pathogens usually colonize host tissues that are in contact with the external environment. Sites of entry in human hosts include the urogenital tract, the digestive tract, the respiratory tract and the conjunctiva. Organisms that infect these regions have usually developed tissue adherence mechanisms and some ability to overcome or withstand the constant pressure of the host defenses at the surface.
Bacterial Adherence to Mucosal Surfaces. In its simplest form, bacterial adherence or attachment to a eucaryotic cell or tissue surface requires the participation of two factors: a receptorand an ligand. The receptors so far defined are usually specific carbohydrate or peptide residues on the eucaryotic cell surface. The bacterial ligand, called an adhesin, is typically a macromolecular component of the bacterial cell surface which interacts with the host cell receptor. Adhesins and receptors usually interact in a complementary and specific fashion. Table 1 is a list of terms that are used in medical microbiology to refer to microbial adherence to surfaces or tissues.
|Adhesin||A surface structure or macromolecule that binds a bacterium to a specific surface|
|Receptor||A complementary macromolecular binding site on a (eucaryotic) surface that binds specific adhesins or ligands|
|Lectin||Any protein that binds to a carbohydrate|
|Ligand||A surface molecule that exhibits specific binding to a receptor molecule on another surface|
|Mucous||The mucopolysaccharide layer of glucosaminoglycans covering animal cell mucosal surfaces|
|Fimbriae||Filamentous proteins on the surface of bacterial cells that may behave as adhesins for specific adherence|
|Common pili||Same as fimbriae|
|Sex pilus||A specialized pilus that binds mating procaryotes together for the purpose of DNA transfer|
|Type 1 fimbriae||Fimbriae in Enterobacteriaceae which bind specifically to mannose terminated glycoproteins on eucaryotic cell surfaces|
|Type 4 pili||Pili in certain Gram-positive and Gram-negative bacteria. In Pseudomonas, thought to play a role in adherence and biofilm formation|
|S-layer||Proteins that form the outermost cell envelope component of a broad spectrum of bacteria, enabling them to adhere to host cell membranes and environmental surfaces in order to colonize.|
|Glycocalyx||A layer of exopolysaccharide fibers on the surface of bacterial cells which may be involved in adherence to a surface. Sometimes a general term for a capsule.|
|Capsule||A detectable layer of polysaccharide (rarely polypeptide) on the surface of a bacterial cell which may mediate specific or nonspecific attachment|
|Lipopolysaccharide (LPS)||A distinct cell wall component of the outer membrane of Gram-negative bacteria with the potential structural diversity to mediate specific adherence. Probably functions as an adhesin|
|Teichoic acids and lipoteichoic acids (LTA)||Cell wall components of Gram-positive bacteria that may be involved in nonspecific or specific adherence|
Specific Adherence of Bacteria to Cell and Tissue Surfaces
Several types of observations provide indirect evidence for specificity of adherence of bacteria to host cells or tissues:
1. Tissue tropism: particular bacteria are known to have an apparent preference for certain tissues over others, e.g. S. mutans is abundant in dental plaque but does not occur on epithelial surfaces of the tongue; the reverse is true for S. salivarius which is attached in high numbers to epithelial cells of the tongue but is absent in dental plaque.
2. Species specificity: certain pathogenic bacteria infect only certain species of animals, e.g. N. gonorrhoeae infections are limited to humans; Enteropathogenic E. coli K-88 infections are limited to pigs; E. coli CFA I and CFA II infect humans; E. coli K-99 strain infects calves.; Group A streptococcal infections occur only in humans.
3. Genetic specificity within a species: certain strains or races within a species are genetically immune to a pathogen , e.g. Certain pigs are not susceptible to E. coli K-88 infections; Susceptibility to Plasmodium vivax infection (malaria) is dependent on the presence of the Duffy antigens on the host’s redblood cells.
Although other explanations are possible, the above observations might be explained by the existence of specific interactions between microorganisms and eucaryotic tissue surfaces which allow microorganisms to become established on the surface.
Mechanisms of Adherence to Cell or Tissue Surfaces
The mechanisms for adherence may involve two steps:
1. nonspecific adherence: reversible attachment of the bacterium to the eucaryotic surface (sometimes called “docking”)
2. specific adherence: reversible permanent attachment of the microorganism to the surface (sometimes called “anchoring”).
The usual situation is that reversible attachment precedes irreversible attachment but in some cases, the opposite situation occurs or specific adherence may never occur.
Nonspecific adherence involves nonspecific attractive forces which allow approach of the bacterium to the eucaryotic cell surface. Possible interactions and forces involved are:
1. hydrophobic interactions
2. electrostatic attractions
3. atomic and molecular vibrations resulting from fluctuating dipoles of similar frequencies
4. Brownian movement
5. recruitment and trapping by biofilm polymers interacting with the bacterial glycocalyx (capsule)
Specific adherence involves permanent formation of many specific lock-and-key bonds between complementary molecules on each cell surface. Complementary receptor and adhesin molecules must be accessible and arranged in such a way that many bonds form over the area of contact between the two cells. Once the bonds are formed, attachment under physiological conditions becomes virtually irreversible.
Specific adherence involves complementary chemical interactions between the host cell or tissue surface and the bacterial surface. In the language of medical microbiologist, a bacterial “adhesin” attaches covalently to a host “receptor” so that the bacterium “docks” itself on the host surface. The adhesins of bacterial cells are chemical components of capsules, cell walls, pili or fimbriae. The host receptors are usually glycoproteins located on the cell membrane or tissue surface.
Several types of experiments provide direct evidence that receptor and/or adhesin molecules mediate specificity of adherence of bacteria to host cells or tissues. These include:
1. The bacteria will bind isolated receptors or receptor analogs.
2. The isolated adhesins or adhesin analogs will bind to the eucaryotic cell surface.
3. Adhesion (of the bacterium to the eucaryotic cell surface) is inhibited by:a. isolated adhesin or receptor molecules
b. adhesin or receptor analogs
c. enzymes and chemicals that specifically destroy adhesins or receptors
d. antibodies specific to surface components (i.e., adhesins or receptors.
Some Specific Bacterial Adhesins and their Receptors
The adhesins of E. coli are their common pili or fimbriae. A single strain of E. coli is known to be able to express several distinct types of fimbriae encoded by distinct regions of the chromosome or plasmids. This genetic diversity permits an organism to adapt to its changing environment and exploit new opportunities presented by different host surfaces. Many of the adhesive fimbriae of E. coli have probably evolved from fimbrial ancestors resembling Type-I and Type IV fimbriae.
Type-I fimbriae enable E. coli to bind to D-mannose residues on eucaryotic cell surfaces. Type-I fimbriae are said to be “mannose-sensitive” since exogenous mannose blocks binding to receptors on red blood cells. Although the primary 17kDa fimbrial subunit is the major protein component of Type-1 fimbriae, the mannose-binding site is not located here, but resides in a minor protein (28-31kDa) located at the tips or inserted along the length of the fimbriae. By genetically varying the minor “tip protein” adhesin, the organisms can gain ability to adhere to different receptors. For example, tip proteins on pyelonephritis-associated (pap) pili recognize a galactose-galactose disaccharide, while tip proteins on S-fimbriae recognize sialic acid.
Pseudomonas, Vibrio and Neisseria possess Type IV pili that contain protein subunit with a methylated amino acid, often phenylalanine, at or near its amino terminus. These “N-methylphenylalanine pili” have been established as virulence determinants in pathogenesis ofPseudomonas aeruginosa lung infection in cystic fibrosis patients. These type of fimbriae occur inNeisseria gonorrhoeae and their receptor is thought to be an oligosaccharide. Type IV pili are the tcp (toxin coregulated pili) fimbriae used in attachment of Vibrio cholerae to the gastrointestinal epithelium.
Gram stain of Neisseria gonorrhoeae, the agent of the STD gonorrhea. The bacteria are seen as pairs of cocci (diplococci) in association with host pmn’s (polymorphonuclear leukocytes). Gonorrhea is the second most prevalent STD in the U.S. behind chlamydia. The bacterium has multiple determinants of virulence including the ability to attach to and enter host cells, resist phagocytic killing and produce endotoxins which eventually lead to an intense inflammatory response. CDC.
The adhesins of Streptococcus pyogenes are controversial. In 1972, Gibbons and his colleagues demonstrated that attachment of streptococci to the oral mucosa of mice is dependent on M protein. Olfek and Beachey argued that lipoteichoic acid (LTA), rather than M protein, was responsible for streptococcal adherence to buccal epithelial cells. In 1996, Hasty and Courtney proposed a two-step model of attachment that involved both M protein and teichoic acids. They suggested that LTA loosely tethers streptococci to epithelial cells, and then M protein secures a firmer, irreversible association. In 1992, protein F was discovered and found to be a fibronectin binding protein. More recently, in 1998, M proteins M1 and M3 were also found to bind to fibronectin. Apparently, S. pyogenes produces multiple adhesins with varied specificities.
Electron micrograph of Streptococcus pyogenes (Group A strep) by Maria Fazio and Vincent A. Fischetti, Ph.D. with permission. The Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University. The cell surface fibrils, that consist primarily of M protein, are clearly evident. The M protein has several possible roles in virulence: it is involved in adherence, resistance to phagocytosis, and in antigenic variation of the pathogen.
Staphylococcus aureus also binds to the amino terminus of fibronectin by means of a fibronectin-binding protein which occurs on the bacterial surface. Apparently S. aureus and Group A streptococci use different mechanisms but adhere to the same receptor on epithelial surfaces.
Treponema pallidum has three related surface adhesins (P1, P2 and P3) which bind to a four-amino acid sequence (Arg-Gly-Asp-Ser) of the cell-binding domain of fibronectin. It is not clear if T. pallidum uses fibronectin to attach to host surfaces or coats itself with fibronectin to avoid host defenses (phagocytes and immune responses).
Treponema pallidum, the spirochete that causes syphilis. Silver stain. CDC.
TABLE 2. EXAMPLES OF SPECIFIC ATTACHMENTS OF BACTERIA TO HOST CELL OR TISSUE SURFACES
|Streptococcus pyogenes||Protein F||Amino terminus of fibronectin||Pharyngeal epithelium||Sore throat|
|Streptococcus mutans||Glycosyl transferase||Salivary glycoprotein||Pellicle of tooth||Dental caries|
|Streptococcus salivarius||Lipoteichoic acid||Unknown||Buccal epithelium of tongue||None|
|Streptococcus pneumoniae||Cell-bound protein||N-acetylhexos-
|Staphylococcus aureus||Cell-bound protein||Amino terminus of fibronectin||Mucosal epithelium||Various|
|Neisseria gonorrhoeae||Type IV pili (N-methylphenyl- alanine pili)||Glucosamine-
|Enterotoxigenic E. coli||Type-I fimbriae||Species-specific carbohydrate(s)||Intestinal epithelium||Diarrhea|
|UropathogenicE. coli||Type I fimbriae||Complex carbohydrate||Urethral epithelium||Urethritis|
|UropathogenicE. coli||P-pili (pap)||Globobiose linked to ceramide lipid||Upper urinary tract||Pyelonephritis|
|Bordetella pertussis||Fimbriae (“filamentous hemagglutinin”)||Galactose on sulfated glycolipids||Respiratory epithelium||Whooping cough|
|Fucose and mannose carbohydrate||Intestinal epithelium||Cholera|
|Treponema pallidum||Peptide in outer membrane||Surface protein (fibronectin)||Mucosal epithelium||Syphilis|
|Mycoplasma||Membrane protein||Sialic acid||Respiratory epithelium||Pneumonia|
|Chlamydia||Unknown||Sialic acid||Conjunctival or urethral epithelium||Conjunctivitis or urethritis|
The invasion of a host by a pathogen may be aided by the production of bacterial extracellular substances which act against the host by breaking down primary or secondary defenses of the body. Medical microbiologists have long referred to these substances as invasins. Most invasins are proteins (enzymes) that act locally to damage host cells and/or have the immediate effect of facilitating the growth and spread of the pathogen. The damage to the host as a result of this invasive activity may become part of the pathology of an infectious disease.
The extracellular proteins produced by bacteria which promote their invasion are not clearly distinguished from some extracellular protein toxins (“exotoxins”) which also damage the host. Invasins usually act at a short range (in the immediate vicinity of bacterial growth) and may not actually kill cells as part of their range of activity; exotoxins are often cytotoxic and may act at remote sites (removed from the site of bacterial growth). Also, exotoxins typically are more specific and more potent in their activity than invasins. Even so, some classic exotoxins (e.g. diphtheria toxin, anthrax toxin) may play some role in colonization or invasion in the early stages of an infection, and some invasins (e.g. staphylococcal leukocidin) have a relatively specific cytopathic effect.
A Survey of Bacterial Invasins
“Spreading Factors” is a descriptive term for a family of bacterial enzymes that affect the physical properties of tissue matrices and intercellular spaces, thereby promoting the spread of the pathogen.
Hyaluronidase. is the original spreading factor. It is produced by streptococci. staphylococci, and clostridia. The enzyme attacks the interstitial cement (“ground substance”) of connective tissue by depolymerizing hyaluronic acid.
Collagenase is produced by Clostridium histolyticum and Clostridium perfringens. It breaks down collagen, the framework of muscles, which facilitates gas gangrene due to these organisms.
Neuraminidase is produced by intestinal pathogens such as Vibrio cholerae and Shigella dysenteriae. It degrades neuraminic acid (also called sialic acid), an intercellular cement of the epithelial cells of the intestinal mucosa.
Streptokinase and staphylokinase are produced by streptococci and staphylococci, respectively. Kinase enzymes convert inactive plasminogen to plasmin which digests fibrin and prevents clotting of the blood. The relative absence of fibrin in spreading bacterial lesions allows more rapid diffusion of the infectious bacteria.
Enzymes that Cause Hemolysis and/or Leucolysis
These enzymes usually act on the animal cell membrane by insertion into the membrane (forming a pore that results in cell lysis), or by enzymatic attack on phospholipids, which destabilizes the membrane. They may be referred to as lecithinases or phospholipases, and if they lyse red blood cells they are sometimes called hemolysins. Leukocidins, produced by staphylococci andstreptolysin produced by streptococci specifically lyse phagocytes and their granules. These latter two enzymes are also considered to be bacterial exotoxins.
Phospholipases, produced by Clostridium perfringens (i.e., alpha toxin), hydrolyze phospholipids in cell membranes by removal of polar head groups.
Lecithinases, also produced by Clostridium perfringens, destroy lecithin (phosphatidylcholine) in cell membranes.
Hemolysins, notably produced by staphylococci (i.e., alpha toxin), streptococci (i.e., streptolysin) and various clostridia, may be channel-forming proteins or phospholipases or lecithinases that destroy red blood cells and other cells (i.e., phagocytes) by lysis.
Beta-hemolytic Streptococcus. This is the characteristic appearance of a blood agar plate culture of the bacterium. Note the translucency around the bacterial colonies, representing hemolysis of the red cells in the culture medium due to production of a diffusible hemolysin (streptolysin).
Coagulase, formed by Staphylococcus aureus, is a cell-associated and diffusible enzyme that converts fibrinogen to fibrin which causes clotting. Coagulase activity is almost always associated with pathogenic S. aureus and almost never associated with nonpathogenic S. epidermidis, which has led to much speculation as to its role as a determinant of virulence. Possibly, cell bound coagulase could provide an antigenic disguise if it clotted fibrin on the cell surface. Or a staphylococcal lesion encased in fibrin (e.g. a boil or pimple) could make the bacterial cells resistant to phagocytes or tissue bactericides or even drugs which might be unable to diffuse to their bacterial target.
Extracellular Digestive Enzymes
Heterotrophic bacteria, in general, produce a wide variety of extracellular enzymes includingproteases, lipases, glycohydrolases, nucleases, etc., which are not clearly shown to have a direct role in invasion or pathogenesis. These enzymes presumably have other functions related to bacterial nutrition or metabolism, but may aid in invasion either directly or indirectly.
Toxins With Short-Range Effects Related to Invasion
Bacterial protein toxins which have adenylate cyclase activity, are thought to have immediate effects on host cells that promote bacterial invasion. One component of the anthrax toxin (EF or Edema Factor) is an adenylate cyclase that acts on nearby cells to cause increased levels of cyclic AMP and disruption of cell permeability. One of the toxins of Bordetella pertussis, the agent of whooping cough, has a similar effect. These toxins may contribute to invasion through their effects on macrophages or lymphocytes in the vicinity which are playing an essential role to contain the infection. For example, since they use ATP as a substrate, they may deplete phagocyte reserves of energy needed for ingestion. Edema is seen as a pathology because the increase in cAMP in affected cells disrupts equilibrium.
Gelatinous edema seen in a cutaneous anthrax lesion. CDC.
The following table summarizes the activities of many bacterial proteins that are noted for their contribution to bacterial invasion of tissues.
|Hyaluronidase||Streptococci, staphylococci and clostridia||Degrades hyaluronic of connective tissue|
|Collagenase||Clostridiumspecies||Dissolves collagen framework of muscles|
|Neuraminidase||Vibrio choleraeand Shigella dysenteriae||Degrades neuraminic acid of intestinal mucosa|
|Coagulase||Staphylococcus aureus||Converts fibrinogen to fibrin which causes clotting|
|Kinases||Staphylococci and streptococci||Converts plasminogen to plasmin which digests fibrin|
|Leukocidin||Staphylococcus aureus||Disrupts neutrophil membranes and causes discharge of lysosomal granules|
|Streptolysin||Streptococcus pyogenes||Repels phagocytes and disrupts phagocyte membrane and causes discharge of lysosomal granules|
|Hemolysins||Streptococci, staphylococci and clostridia||Phospholipases or lecithinases that destroy red blood cells (and other cells) by lysis|
|Lecithinases||Clostridium perfringens||Destroy lecithin in cell membranes|
|Phospholipases||Clostridium perfringens||Destroy phospholipids in cell membrane|
|Anthrax EF||Bacillus anthracis||One component (EF) is an adenylate cyclase which causes increased levels of intracellular cyclic AMP|
|Pertussis AC||Bordetella pertussis||One toxin component is an adenylate cyclase that acts locally producing an increase in intracellular cyclic AMP|
EVASION OF HOST DEFENSES
Some pathogenic bacteria are inherently able to resist the bactericidal components of host tissues. For example, the poly-D-glutamate capsule of Bacillus anthracis protects the organisms against cell lysis by cationic proteins in sera or in phagocytes. The outer membrane of Gram-negative bacteria is a formidable permeability barrier that is not easily penetrated by hydrophobic compounds such as bile salts which are harmful to the bacteria. Pathogenic mycobacteria have a waxy cell wall that resists attack or digestion by most tissue bactericides. And intact lipopolysaccharides (LPS) of Gram-negative pathogens may protect the cells from complement-mediated lysis or the action of lysozyme.
Most successful pathogens, however, possess additional structural or biochemical features which allow them to resist the main lines of host internal defense against them, i.e., the phagocytic and immune responses of the host.
Overcoming Host Phagocytic Defenses
Microorganisms invading tissues are first and foremost exposed to phagocytes. Bacteria that readily attract phagocytes, and that are easily ingested and killed, are generally unsuccessful as parasites. In contrast, most bacteria that are successful as parasites interfere to some extent with the activities of phagocytes or in some way avoid their attention.
Microbial strategies to avoid phagocytic killing are numerous and diverse, but are usually aimed at blocking one or of more steps in the phagocytic process. Recall the steps in phagocytosis:
1. Contact between phagocyte and microbial cell
3. Phagosome formation
4. Phagosome-lysosome fusion
5. Killing and digestion
Avoiding Contact with Phagocytes
Bacteria can avoid the attention of phagocytes in a number of ways.
1. Invade or remain confined in regions inaccessible to phagocytes. Certain internal tissues (e.g. the lumen of glands) and surface tissues (e.g. the skin) are not patrolled by phagocytes.
2. Avoid provoking an overwhelming inflammatory response. Some pathogens induce minimal or no inflammation required to focus the phagocytic defenses.
3. Inhibit phagocyte chemotaxis. e.g. Streptococcal streptolysin (which also kills phagocytes) suppresses neutrophil chemotaxis, even in very low concentrations. Fractions of Mycobacterium tuberculosis are known to inhibit leukocyte migration. Clostridium ø toxin inhibits neutrophil chemotaxis.
4. Hide the antigenic surface of the bacterial cell. Some pathogens can cover the surface of the bacterial cell with a component which is seen as “self” by the host phagocytes and immune system. Phagocytes cannot recognize bacteria upon contact and the possibility of opsonization by antibodies to enhance phagocytosis is minimized. For example, pathogenic Staphylococcus aureus produces cell-bound coagulase which clots fibrin on the bacterial surface. Treponema pallidum binds fibronectin to its surface. Group A streptococci are able to synthesize a capsule composed of hyaluronic acid.
Inhibition of Phagocytic Engulfment
Some bacteria employ strategies to avoid engulfment (ingestion) if phagocytes do make contact with them. Many important pathogenic bacteria bear on their surfaces substances that inhibit phagocytic adsorption or engulfment. Clearly it is the bacterial surface that matters. Resistance to phagocytic ingestion is usually due to a component of the bacterial cell wall, or fimbriae, or a capsule enclosing the bacterial wall. Classical examples of antiphagocytic substances on the bacterial surface include:
Polysaccharide capsules of S. pneumoniae, Haemophilus influenzae, Treponema pallidum andKlebsiella pneumoniae
M protein and fimbriae of Group A streptococci
Surface slime (polysaccharide) produced by Pseudomonas aeruginosa
O antigen associated with LPS of E. coli
K antigen of E. coli or the analogous Vi antigen of Salmonella typhi
Cell-bound or soluble Protein A produced by Staphylococcus aureus
Streptococcus pneumoniae, FA stain showing its antphagocytic capsule (CDC). S. pneumoniaecells that possess a capsule are virulent; nonencapsulated strains are avirulent. Although S. pneumoniae strains possess a variety of determinants of virulence, this illustrates the essential role of their capsule in ability to resist phagocytosis by alveolar macrophages in order to initiate disease.
Survival Inside of Phagocytes
Some bacteria survive inside of phagocytic cells, in either neutrophils or macrophages. Bacteria that can resist killing and survive or multiply inside of phagocytes are considered intracellular parasites. The environment of the phagocyte may be a protective one, protecting the bacteria during the early stages of infection or until they develop a full complement of virulence factors. The intracellular environment guards the bacteria against the activities of extracellular bactericides, antibodies, drugs, etc.
Most intracellular parasites have special (genetically-encoded) mechanisms to get themselves into their host cell as well as special mechanisms to survive once they are inside. Intracellular parasites usually survive by virtue of mechanisms which interfere with the bactericidal activities of the host cell. Some of these bacterial mechanisms include:
1. Inhibition of phagosome-lysosome fusion. The bacteria survive inside of phagosomes because they prevent the discharge of lysosomal contents into the phagosome environment. Specifically phagolysosome formation is inhibited in the phagocyte. This is the strategy employed bySalmonella, M. tuberculosis, Legionella and the Chlamydiae.
Intracellular Mycobacterium tuberculosis in lung. Ziehl-Neelsen acid fast stain (CDC).
2. Survival inside the phagolysosome. With some intracellular parasites, phagosome-lysosome fusion occurs but the bacteria are resistant to inhibition and killing by the lysosomal constituents. Also, some extracellular pathogens can resist killing in phagocytes utilizing similar resistance mechanisms. Little is known of how bacteria can resist phagocytic killing within the phagocytic vacuole, but it may be due to the surface components of the bacteria or due to extracellular substances that they produce which interfere with the mechanisms of phagocytic killing. Bacillus anthracis, Mycobacterium tuberculosis and Staphylococcus aureus all possess mechanisms to survive intracellular killing in macrophages.
3. Escape from the phagosome. Early escape from the phagosome vacuole is essential for growth and virulence of some intracellular pathogens. This is a very clever strategy employed by the Rickettsias which produce a phospholipase enzyme that lyses the phagosome membrane within thirty seconds of after ingestion.
Products of Bacteria that Kill or Damage Phagocytes
One obvious strategy in defense against phagocytosis is direct attack by the bacteria upon the professional phagocytes. Any of the substances that pathogens produce that cause damage to phagocytes have been referred to as “aggressins”. Most of these are actually extracellular enzymes or toxins that kill phagocytes. Phagocytes may be killed by a pathogen before or after ingestion.
Killing phagocytes before ingestion. Many Gram-positive pathogens, particularly the pyogenic cocci, secrete extracellular enzymes which kill phagocytes. Many of these enzymes are called “hemolysins” because their activity in the presence of red blood cells results in the lysis of the rbcs.
Pathogenic streptococci produce streptolysin. Streptolysin O binds to cholesterol in membranes. The effect on neutrophils is to cause lysosomal granules to explode, releasing their contents into the cell cytoplasm.
Pathogenic staphylococci produce leukocidin, which also acts on the neutrophil membrane and causes discharge of lysosomal granules.
Other examples of bacterial extracellular proteins that inhibit phagocytosis include the Exotoxin A ofPseudomonas aeruginosa which kills macrophages, and the bacterial exotoxins that are adenylate cyclases (e.g. anthrax toxin EF and pertussis AC) which decrease phagocytic activity.
Gram stain of a pustular exudate from a mixed bacterial infection. Pus is the usual outcome of the battle between phagocytes and bacterial strategies to kill them.
Killing phagocytes after ingestion. Some bacteria exert their toxic action on the phagocyte after ingestion has taken place. They may grow in the phagosome and release substances which can pass through the phagosome membrane and cause discharge of lysosomal granules, or they may grow in the phagolysosome and release toxic substances which pass through the phagolysosome membrane to other target sites in the cell. Many bacteria which are the intracellular parasites of macrophages (e.g. Mycobacteria, Brucella, Listeria) usually destroy macrophages in the end, but the mechanisms are not understood.
Antibodies that are bound to bacterial surfaces will activate complement by the classical pathway and bacterial polysaccharides activate complement by the alternative pathway. Bacteria in serum and other tissues, especially Gram-negative bacteria, need protection from the antimicrobial effects of complement before and during an immunological response.
One role of capsules in bacterial virulence is to protect the bacteria from complement activation and the ensuing inflammatory response. Polysaccharide capsules can hide bacterial components such as LPS or peptidoglycan which can induce the alternate complement pathway. Some bacterial capsules are able to inhibit formation of the C3b complex on their surfaces, thus avoiding C3b opsonization and subsequent formation of C5b and the membrane attack complex (MAC) on the bacterial cell surface. Capsules that contain sialic acid (a common component of host cell glycoproteins), such as found in Neisseria meningitidis, have this effect.
One of the principal targets of complement on Gram-negative bacteria is LPS. It serves as the attachment site for C3b and triggers the alternative pathway of activation. It also binds C5b.
LPS can be modified by pathogens in two ways that affects its interaction with complement. First, by attachment of sialic acid residues to the LPS O antigen, a bacterium can prevent the formation of C3 convertase just as capsules that contain sialic acid can do so. Both Neisseria meningitidis andHaemophilus influenzae, which cause bacterial meningitis, are able to covalently attach sialic acid residues to their O antigens resulting in resistance to MAC. Second, LPS with long, intact O antigen side-chains can prevent effective MAC killing. Apparently the MAC complex is held too far from the vulnerable outer membrane to be effective.
Bacteria that are not killed and lysed in serum by the complement MAC are said to be serum resistant. As might be expected many of the Gram-negative bacteria that cause systemic infections, (bacteremia or septicemia) are serum resistant. Gram-positive bacteria are naturally serum-resistant since their cells are not enclosed in an outer membrane.
Other ways that pathogens are able to inhibit the activity of complement is to destroy one or more of the components of complement. Pseudomonas aeruginosa produces an extracellular elastaseenzyme that inactivates components of complement.
Avoiding Host Immunological Responses
On epithelial surfaces the main antibacterial immune defense of the host is the protection afforded by secretory antibody (IgA). Once the epithelial surfaces have been penetrated, however, the major host defenses of inflammation, complement, phagocytosis, Antibody-mediated Immunity (AMI), and Cell-mediated Immunity (CMI) are encountered. If there is a way for a pathogen to successfully bypass or overcome these host defenses, then some bacterial pathogen has probably discovered it. Bacteria evolve very rapidly in relation to their host, so that most of the feasible anti-host strategies are likely to have been tried out and exploited. Ability to defeat the immune defenses may play a major role in the virulence of a bacterium and in the pathology of disease. Several strategic bacterial defenses are described below.
Immunological Tolerance to a Bacterial Antigen
Tolerance is a property of the host in which there is an immunologically-specific reduction in the immune response to a given Ag. Tolerance to a bacterial Ag does not involve a general failure in the immune response but a particular deficiency in relation to the specific antigen(s) of a given bacterium. If there is a depressed immune response to relevant antigens of a parasite, the process of infection is facilitated. Tolerance can involve either AMI or CMI or both arms of the immunological response.
Tolerance to an Ag can arise in a number of ways, but three are possibly relevant to bacterial infections.
1. Fetal exposure to Ag
2. High persistent doses of circulating Ag
3. Molecular mimicry. If a bacterial Ag is very similar to normal host “antigens”, the immune responses to this Ag may be weak giving a degree of tolerance. Resemblance between bacterial Ag and host Ag is referred to as molecular mimicry. In this case the antigenic determinants of the bacterium are so closely related chemically to host “self” components that the immunological cells cannot distinguish between the two and an immune response cannot be raised. Some bacterial capsules are composed of polysaccharides (hyaluronic acid, sialic acid) so similar to host tissue polysaccharides that they are not immunogenic.
Bacteria may be able to coat themselves with host proteins (fibrin, fibronectin, antibody molecules) or with host polysaccharides (sialic acid, hyaluronic acid) so that they are able to hide their own antigenic surface components from the immunological system.
Some pathogens (mainly viruses and protozoa, rarely bacteria) cause immunosuppression in the infected host. This means that the host shows depressed immune responses to antigens in general, including those of the infecting pathogen. Suppressed immune responses are occasionally observed during chronic bacterial infections such as leprosy and tuberculosis.
Persistence of a Pathogen at Bodily Sites Inaccessible to the Immune Response
Some pathogens can avoid exposing themselves to immune forces.
Intracellular pathogens can evade host immune responses as long as they stay inside of infected cells and they do not allow microbial Ag to form on the cell surface. Macrophages support the growth of the bacteria and at the same time give them protection from immune responses.
Some pathogens persist on the luminal surfaces of the GI tract, oral cavity and the urinary tract, or the lumen of the salivary gland, mammary gland or the kidney tubule.
Induction of Ineffective Antibody
Many types of antibody are formed against a given Ag, and some bacterial components may display various antigenic determinants. Antibodies tend to range in their capacity to react with Ag (the ability of specific Ab to bind to an Ag is called avidity). If Abs formed against a bacterial Ag are of low avidity, or if they are directed against unimportant antigenic determinants, they may have only weak antibacterial action. Such “ineffective” (non-neutralizing) Abs might even aid a pathogen by combining with a surface Ag and blocking the attachment of any functional Abs that might be present.
Antibodies Absorbed by Soluble Bacterial Antigens
Some bacteria can liberate antigenic surface components in a soluble form into the tissue fluids. These soluble antigens are able to combine with and “neutralize” antibodies before they reach the bacterial cells. For example, small amounts of endotoxin (LPS) may be released into surrounding fluids by Gram-negative bacteria.
One way bacteria can avoid forces of the immune response is by periodically changing antigens, i.e., undergoing antigenic variation. Some bacteria avoid the host antibody response by changing from one type of fimbriae to another, by switching fimbrial tips. This makes the original AMI response obsolete by using new fimbriae that do not bind the previous antibodies. Pathogenic bacteria can vary (change) other surface proteins that are the targets of antibodies. Antigenic variation is prevalent among pathogenic viruses as well.
Changing antigens during the course of an infection
Antigens may vary or change within the host during the course of an infection, or alternatively antigens may vary among multiple strains (antigenic types) of a parasite in the population. Antigenic variation is an important mechanism used by pathogenic microorganisms for escaping the neutralizing activities of antibodies. Antigenic variation usually results from site-specific inversions or gene conversions or gene rearrangements in the DNA of the microorganisms.
Changing antigens between infections
Many pathogenic bacteria exist in nature as multiple antigenic types or serotypes, meaning that they are variant strains of the same pathogenic species. For example, there are multiple serotypes ofSalmonella typhimurium based on differences in cell wall (O) antigens or flagellar (H) antigens. There are 80 different antigenic types of Streptococcus pyogenes based on M-proteins on the cell surface. There are over one hundred strains of Streptococcus pneumoniae depending on their capsular polysaccharide antigens. Based on minor differences in surface structure chemistry there are multiple serotypes of Vibrio cholerae, Staphylococcus aureus, Escherichia coli, Neisseria gonorrhoeae and an assortment of other bacterial pathogens.
Two types of bacterial toxins
At a chemical level there are two types of bacterial toxins:
lipopolysaccharides, which are associated with the cell walls of Gram-negative bacteria.
proteins, which may be released into the extracellular environment of pathogenic bacteria.
The lipopolysaccharide (LPS) component of the Gram-negative bacterial outer membrane bears the name endotoxin because of its association with the cell wall of bacteria.
Most of the protein toxins are thought of as exotoxins, since they are “released” from the bacteria and act on host cells at a distance.
BACTERIAL PROTEIN TOXINS
The protein toxins are typically soluble proteins secreted by living bacteria during exponential growth. The production of protein toxins is generally specific to a particular bacterial species (e.g. only Clostridium tetani produces tetanus toxin; only Corynebacterium diphtheriae produces the diphtheria toxin). Usually, virulent strains of the bacterium produce the toxin (or range of toxins) while nonvirulent strains do not, such that the toxin is the major determinant of virulence. Both Gram-positive and Gram-negative bacteria produce soluble protein toxins. Bacterial protein toxins are the most potent poisons known and may show activity at very high dilutions.
The protein toxins resemble enzymes in a number of ways. Like enzymes, bacterial exotoxins:
are denatured by heat, acid, proteolytic enzymes
have a high biological activity (most act catalytically)
exhibit specificity of action
As enzymes attack specific substrates, so bacterial protein toxins are highly specific in the substrate utilized and in their mode of action. The substrate (in the host) may be a component of tissue cells, organs, or body fluid. Usually the site of damage caused by the toxin indicates the location of the substrate for that toxin. Terms such as “enterotoxin”, “neurotoxin”, “leukocidin” or “hemolysin” are sometimes used to indicate the target site of some well-defined protein toxins.
Certain protein toxins have very specific cytotoxic activity (i.e., they attack specific cells, for example, tetanus or botulinum toxins), but some (as produced by staphylococci, streptococci, clostridia, etc.) have fairly broad cytotoxic activity and cause nonspecific death of tissues (necrosis). Toxins that are phospholipases may be relatively nonspecific in their cytotoxicity because they cleave phospholipids which are components of host cell membranes resulting in the death of the cell by leakage of cellular contents. This is also true of pore-forming “hemolysins” and “leukocidins”.
A few protein toxins obviously bring about the death of the host and are known as “lethal toxins”, and even though the tissues affected and the target sites may be known, the precise mechanism by which death occurs is not understood (e.g. anthrax toxin).
As “foreign” substances to the host, most of the protein toxins are strongly antigenic. In vivo,specific antibody (antitoxin) neutralizes the toxicity of these bacterial proteins. However, in vitro, specific antitoxin may not fully inhibit their enzymatic activity. This suggests that the antigenic determinant of the toxin is distinct from the active (enzymatic) portion of the protein molecule. The degree of neutralization of the enzymatic site may depend on the distance from the antigenic site on the molecule. However, since the toxin is fully neutralized in vivo, this suggests that other (host) factors must play a role.
Protein toxins are inherently unstable: in time they lose their toxic properties but retain their antigenic ones. This was first discovered by Ehrlich and he coined the term toxoid for this product.Toxoids are detoxified toxins which retain their antigenicity and their immunizing capacity. The formation of toxoids can be accelerated by treating toxins with a variety of reagents including formalin, iodine, pepsin, ascorbic acid, ketones, etc. The mixture is maintained at 37o at pH range 6 to 9 for several weeks. The resulting toxoids can be use for artificial immunization against diseases caused by pathogens where the primary determinant of bacterial virulence is toxin production. Toxoids are the immunizing agents against diphtheria and tetanus that are part of the DPT vaccine.
A + B Subunit Arrangement of Protein Toxins
Many protein toxins, notably those that act intracellularly (with regard to host cells), consist of two components: one component (subunit A) is responsible for the enzymatic activity of the toxin; the other component (subunit B) is concerned with binding to a specific receptor on the host cell membrane and transferring the enzyme across the membrane. The enzymatic component is not active until it is released from the native toxin. Isolated A subunits are enzymatically active and but lack binding and cell entry capability. Isolated B subunits may bind to target cells (and even block the binding of the native A+B toxin), but they are nontoxic. There are a variety of ways that toxin subunits may be synthesized and arranged: A-B or A-5B indicates that subunits synthesized separately and associated by noncovalent bonds; A/B denotes subunit domains of a single protein that may be separated by proteolytic cleavage; A + B indicates separate protein subunits that interact at the target cell surface; 5B indicates that the binding domain is composed of 5 identical subunits.
Tertiary structure of the pertussis toxin produced by Bordetella pertussis. Pertussis toxin is a member of the A-B bacterial toxin superfamily. It is a hexameric protein comprising five distinct subunits, designated S1-S5. S2, S3, S4 and S5 comprise the B oligomer, responsible for binding the toxin to the cell surface. Each subunit is translated separately with an amino-terminal signal sequence which is cleaved during transport to the periplasm. S2 and S3 function as adhesins, S2 binds specifically to a glycolipid called lactosylceramide, which is found primarily on the ciliated epithelial cells. S3 binds to a glycoprotein found mainly on phagocytic cells.
Attachment and Entry of Toxins
There are at least two mechanisms of toxin entry into target cells. In one mechanism called direct entry, the B subunit of the native toxin (A+B) binds to a specific receptor on the target cell and induces the formation of a pore in the membrane through which the A subunit is transferred into the cell cytoplasm. In an alternative mechanism, the native toxin binds to the target cell and the A+B structure is taken into the cell by the process of receptor-mediated endocytosis (RME). The toxin is internalized in the cell in a membrane-enclosed vesicle called an endosome. H+ ions enter the endosome lowering the internal pH which causes the A+B subunits to separate. Somehow, the B subunit affects the release of the A subunit from the endosome so that it will reach its target in the cell cytoplasm. The B subunit remains in the endosome and is recycled to the cell surface. In both cases, a large protein molecule must insert into and cross a membrane lipid bilayer. This activity is reflected in the ability of most A/B native toxins, or their B components, to insert into artificial lipid bilayers, creating ion permeable pathways.
In keeping with the observation that genetic information for functions not involved in viability of bacteria is frequently located extrachromosomally, the genes encoding toxin production are generally located on plasmids or in lysogenic bacteriophages. Thus the processes of genetic exchange in bacteria, notably conjugation and transduction, can mobilize these genetic elements between strains of bacteria, and therefore may play a role in determining the pathogenic potential of a bacterium.
Why certain bacteria produce such potent toxins is mysterious and is analogous to asking why an organism should produce an antibiotic. The production of a toxin may play a role in adapting a bacterium to a particular niche, but it is not essential to the viability of the organism. Many toxigenic bacteria are free-living in Nature and in associations with humans in a form which is phenotypically identical to the toxigenic strain but lacking the ability to produce the toxin.
There is conclusive evidence for the pathogenic role of diphtheria, tetanus and botulinum toxins, various enterotoxins, staphylococcal toxic shock syndrome toxin, and streptococcal erythrogenic toxin. And there is clear evidence for the pathological involvement of pertussis toxin, anthrax toxin, shiga toxin and the necrotizing toxins of clostridia in host-parasite relationships.
|NAME OF TOXIN||BACTERIUM INVOLVED||ACTIVITY|
|Anthrax toxin (EF)||Bacillus anthracis||Edema Factor (EF) is an adenylate cyclase that causes increased levels in intracellular cyclic AMP in phagocytes and formation of ion-permeable pores in membranes (hemolysis)|
|Adenylate cyclase toxin||Bordetella pertussis||Acts locally to increase levels of cyclic AMP in phagocytes and formation of ion-permeable pores in membranes (hemolysis)|
|Cholera enterotoxin||Vibrio cholerae||ADP ribosylation of G proteins stimulates adenylate cyclase and increases cAMP in cells of the GI tract, causing secretion of water and electrolytes|
|E. coli LT toxin||Escherichia coli||Similar to cholera toxin|
|Shiga toxin||Shigella dysenteriae||Enzymatically cleaves rRNA resulting in inhibition of protein synthesis in susceptible cells|
|Botulinum toxin||Clostridium botulinum||Zn++ dependent protease that inhibits neurotransmission at neuromuscular synapses resulting in flaccid paralysis|
|Tetanus toxin||Clostridium tetani||Zn++ dependent protease that inhibits neurotransmission at inhibitory synapses resulting in spastic paralysis|
|Diphtheria toxin||Corynebacterium diphtheriae||ADP ribosylation of elongation factor 2 leads to inhibition of protein synthesis in target cells|
|Pertussis toxin||Bordetella pertussis||ADP ribosylation of G proteins blocks inhibition of adenylate cyclase in susceptible cells|
|Staphylococcus enterotoxins*||Staphylococcus aureus||Massive activation of the immune system, including lymphocytes and macrophages, leads to emesis (vomiting)|
|Toxic shock syndrome toxin (TSST-1)*||Staphylococcus aureus||Acts on the vascular system causing inflammation, fever and shock|
|Pyrogenic exotoxins (SPE) e.g. Erythrogenic toxin (scarlet fever toxin)*||Streptococcus pyogenes||Causes localized erythematous reactions|
* The “pyrogenic exotoxins” produced by Staphylococcus aureus and Streptococcus pyogeneshave been designated as superantigens. They represent a family of molecules with the ability to elicit massive activation of the immune system. These proteins share the ability to stimulate T cell proliferation by interaction with Class II MHC molecules on APCs and specific V beta chains of the T cell receptor. The important feature of this interaction is the resultant production of IL-1, TNF, and other lymphokines which appear to be the principal mediators of disease processes associated with these toxins.
Endotoxins are part of the outer cell wall of bacteria. Endotoxins are invariably associated with Gram-negative bacteria as constituents of the outer membrane of the cell wall. Although the termendotoxin is occasionally used to refer to any “cell-associated” bacterial toxin, it should be reserved for the lipopolysaccharide complex associated with the outer envelope of Gram-negative bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, and other leading pathogens. Lipopolysaccharide (LPS) participates in a number of outer membrane functions that are essential for bacterial growth and survival, especially within the context of a host-parasite interaction.
The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity (antigenicity) is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. LPS activates complement by the alternative (properdin) pathway and may be a part of the pathology of most Gram-negative bacterial infections.
For the most part, endotoxins remain associated with the cell wall until disintegration of the bacteria. In vivo, this results from autolysis, external lysis, and phagocytic digestion of bacterial cells. It is known, however, that small amounts of endotoxin may be released in a soluble form, especially by young cultures.
Compared to the classic exotoxins of bacteria, endotoxins are less potent and less specific in their action, since they do not act enzymatically. Endotoxins are heat stable (boiling for 30 minutes does not destabilize endotoxin), but certain powerful oxidizing agents such as , superoxide, peroxide and hypochlorite degrade them. Endotoxins, although strongly antigenic, cannot be converted to toxoids. A comparison of the properties of bacterial endotoxins compared to classic exotoxins is shown in Table 5.
|CHEMICAL NATURE||Lipopolysaccharide (mw = 10kDa)||Protein (mw = 50-1000kDa)|
|RELATIONSHIP TO CELL||Part of outer membrane||Extracellular, diffusible|
|DENATURED BY BOILING||No||Usually|
|POTENCY||Relatively low (>100ug)||Relatively high (1 ug)|
|SPECIFICITY||Low degree||High degree|
Lipopolysaccharides are complex amphiphilic molecules with a mw of about 10kDa, that vary widely in chemical composition both between and among bacterial species. In a basic ground plan common to all endotoxins, LPS consists of three components or regions:
(1) Lipid A—- (2) Core polysaccharide—- (3) O polysaccharide
Lipid A is the lipid component of LPS. It contains the hydrophobic, membrane-anchoring region of LPS. Lipid A consists of a phosphorylated N-acetylglucosamine (NAG) dimer with 6 or 7 fatty acids (FA) attached. Usually 6 FA are found. All FA in Lipid A are saturated. Some FA are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present. The structure of Lipid A is highly conserved among Gram-negative bacteria. Among Enterobacteriaceae Lipid A is virtually constant.
The Core (R) polysaccharide is attached to the 6 position of one NAG. The R antigen consists of a short chain of sugars. For example: KDO – Hep – Hep – Glu – Gal – Glu – GluNAc.
Two unusual sugars are usually present, heptose and 2-keto-3-deoxyoctonoic acid (KDO), in the core polysaccharide. KDO is unique and invariably present in LPS and so has been an indicator in assays for LPS (endotoxin).
With minor variations, the core polysaccharide is common to all members of a bacterial genus (e.g.Salmonella), but it is structurally distinct in other genera of Gram-negative bacteria. Salmonella,Shigella and Escherichia have similar but not identical cores.
The O polysaccharide (also referred to as the O antigen or O side chain) is attached to the core polysaccharide. It consists of repeating oligosaccharide subunits made up of 3-5 sugars. The individual chains vary in length ranging up to 40 repeat units. The O polysaccharide is much longer than the core polysaccharide and it maintains the hydrophilic domain of the LPS molecule. Often, a unique group of sugars, called dideoxyhexoses, occurs in the O polysaccharide.
A major antigenic determinant (antibody-combining site) of the Gram-negative cell wall resides in the O polysaccharide. Great variation occurs in the composition of the sugars in the O side chain between species and even strains of Gram-negative bacteria.
LPS and virulence of Gram-negative bacteria
Endotoxins are toxic to most mammals. They are strong antigens but they seldom elicit immune responses which give full protection to the animal against secondary challenge with the endotoxin. They cannot be toxoided. Endotoxins released from multiplying or disintegrating bacteria significantly contribute to the symptoms of Gram-negative bacteremia and septicemia, and therefore represent important pathogenic factors in Gram-negative infections. Regardless of the bacterial source, all endotoxins produce the same range of biological effects in the animal host. The injection of living or killed Gram-negative cells, or purified LPS, into experimental animals causes a wide spectrum of nonspecific pathophysiological reactions related to inflammation such as:
changes in white blood cell counts
disseminated intravascular coagulation
The sequence of events follows a regular pattern: 1. latent period; 2. physiological distress (fever, diarrhea, prostration, shock); 3. death. How soon death occurs varies on the dose of the endotoxin, route of administration, and species of animal. Animals vary in their susceptibility to endotoxin.
The role of Lipid A
The physiological activities of endotoxins are mediated mainly by the Lipid A component of LPS. Lipid A is the toxic component of LPS, as evidence by the fact that injection of purified Lipid A into an experimental animal will elicit the same response as intact LPS. The primary structure of Lipid A has been elucidated, and Lipid A has been chemically synthesized. Its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide, the PO4groups, the acyl chains, and also the KDO-containing inner core. Lipid A is known to react at the surfaces of macrophages causing them to release cytokines that mediate the pathophysiological response to endotoxin.
The role of the O polysaccharide
Although nontoxic, the polysaccharide side chain (O antigen) of LPS may act as a determinant of virulence in Gram-negative bacteria. The O polysaccharide is responsible for the property of “smoothness” of bacterial cells, which may contribute to their resistance to phagocytic engulfment. The O polysaccharide is hydrophilic and may allow diffusion or delivery of the toxic lipid in the hydrophilic (in vivo) environment. The long side chains of LPS afforded by the O polysaccharide may prevent host complement from depositing on the bacterial cell surface which would bring about bacterial cell lysis. The O polysaccharide may supply a bacterium with its specific ligands (adhesins) for colonization which is essential for expression of virulence. Lastly, the O-polysaccharide is antigenic, and the usual basis for antigenic variation in Gram-negative bacteria rests in differences in their O polysaccharides.
Pathogenicity Islands (PAI) are a distinct class of genomic islands which are acquired by horizontal gene transfer. They are incorporated in the genome of pathogenic bacteria but are usually absent from non-pathogenic organisms of the same or closely related species. They usually occupy relatively large genomic regions ranging from 10-200 kb and encode genes which contribute to virulence of the pathogen. Typical examples are adhesins, toxins, iron uptake systems, invasins, etc.
One species of bacteria may have more than one pathogenicity island. For example, in Salmonella, five pathogenicity islands have been identified. They are found mainly in Gram-negative bacteria, but have been shown in a few Gram-positives. They are found in pathogens that undergo gene transfer by plasmid, phage, or a conjugative transposon and are typically transferred through mechanisms of horizontal gene transfer (HGT).
Pathogenicity islands may be located on the bacterial chromosome or may be a part of a plasmid. They are high in Guanine + Cytosine content. They are flanked by direct repeats i.e., the sequence of bases at two ends are the same. They are associated with tRNA genes, which target sites for the integration of DNA. They have characteristics of transposons in that they carry functional genes, e.g. integrase, transposase, or part of insertion sequences, and may move from one tRNA locus to another on the chromosome or plasmid.
Pathogenicity islands play a pivotal role in the virulence of bacterial pathogens of humans and are also essential for virulence in pathogens of animals and plants. The availability of a large number of genome sequences of pathogenic bacteria and their nonpathogenic relatives has allowed the identification of novel pathogen-specific genomic islands. PAI apparently have been acquired during the speciation of pathogens from their nonpathogenic or environmental ancestors. The acquisition of PAI not only is an ancient evolutionary event that led to the appearance of bacterial pathogens on a timescale of millions of years but also may represent a mechanism that contributes to the appearance of new pathogens within a human life span. The acquisition of knowledge about PAI, their structure, their mobility, and the pathogenicity factors they encode not only is helpful in gaining a better understanding of bacterial evolution and interactions of pathogens with eucaryotic host cells but also may have important practical implications such as providing delivery systems for vaccination and tools for the development of new strategies for therapy of bacterial infections.
PAIs represent distinct genetic elements encoding virulence factors of pathogenic bacteria, but they belong to a more general class of genomic islands, which are common genetic elements sharing a set of unifying features. Genomic islands have been acquired by horizontal gene transfer. In recent years many different genomic islands have been discovered in a variety of pathogenic as well as non-pathogenic bacteria. Because they promote genetic variability, genomic islands play an important role in microbial evolution.
A diverse microbial flora is associated with the skin and mucous membranes of every human being from shortly after birth until death. The human body, which contains about 1013 cells, routinely harbors about 1014 bacteria (Fig. 6-1). This bacterial population constitutes the normal microbial flora . The normal microbial flora is relatively stable, with specific genera populating various body regions during particular periods in an individual’s life. Microorganisms of the normal flora may aid the host (by competing for microenvironments more effectively than such pathogens as Salmonella spp or by producing nutrients the host can use), may harm the host (by causing dental caries, abscesses, or other infectious diseases), or may exist as commensals (inhabiting the host for long periods without causing detectable harm or benefit). Even though most elements of the normal microbial flora inhabiting the human skin, nails, eyes, oropharynx, genitalia, and gastrointestinal tract are harmless in healthy individuals, these organisms frequently cause disease in compromised hosts. Viruses and parasites are not considered members of the normal microbial flora by most investigators because they are not commensals and do not aid the host.
Numbers of bacteria that colonize different parts of the body. Numbers represent the number of organisms per gram of homogenized tissue or fluid or per square centimeter of skin surface.
Significance of the Normal Flora
The fact that the normal flora substantially influences the well-being of the host was not well understood until germ-free animals became available. Germ-free animals were obtained by cesarean section and maintained in special isolators; this allowed the investigator to raise them in an environment free from detectable viruses, bacteria, and other organisms. Two interesting observations were made about animals raised under germ-free conditions. First, the germ-free animals lived almost twice as long as their conventionally maintained counterparts, and second, the major causes of death were different in the two groups. Infection often caused death in conventional animals, but intestinal atonia frequently killed germ-free animals. Other investigations showed that germ-free animals have anatomic, physiologic, and immunologic features not shared with conventional animals. For example, in germ-free animals, the alimentary lamina propria is underdeveloped, little or no immunoglobulin is present in sera or secretions, intestinal motility is reduced, and the intestinal epithelial cell renewal rate is approximately one-half that of normal animals (4 rather than 2 days).
Although the foregoing indicates that bacterial flora may be undesirable, studies with antibiotic treated animals suggest that the flora protects individuals from pathogens. Investigators have used streptomycin to reduce the normal flora and have then infected animals with streptomycin-resistant Salmonella. Normally, about 106 organisms are needed to establish a gastrointestinal infection, but in streptomycin-treated animals whose flora is altered, fewer than 10 organisms were needed to cause infectious disease. Further studies suggested that fermentation products (acetic and butyric acids) produced by the normal flora inhibitedSalmonella growth in the gastrointestinal tract. Figure 6-2 shows some of the factors that are important in the competition between the normal flora and bacterial pathogens.
Mechanisms by which the normal flora competes with invading pathogens. Compare this schematic with Figure 6-3.
The normal flora in humans usually develops in an orderly sequence, or succession, after birth, leading to the stable populations of bacteria that make up the normal adult flora. The main factor determining the composition of the normal flora in a body region is the nature of the local environment, which is determined by pH, temperature, redox potential, and oxygen, water, and nutrient levels. Other factors such as peristalsis, saliva, lysozyme secretion, and secretion of immunoglobulins also play roles in flora control. The local environment is like a concerto in which one principal instrument usually dominates. For example, an infant begins to contact organisms as it moves through the birth canal. A Gram-positive population (bifidobacteria arid lactobacilli) predominates in the gastrointestinal tract early in life if the infant is breast-fed. This bacterial population is reduced and displaced somewhat by a Gram-negative flora (Enterobacteriaceae) when the baby is bottle-fed. The type of liquid diet provided to the infant is the principal instrument of this flora control; immunoglobulins and, perhaps, other elements in breast milk may also be important.
What, then, is the significance of the normal flora? Animal and some human studies suggest that the flora influences human anatomy, physiology, lifespan, and, ultimately, cause of death. Although the causal relationship of flora to death and disease in humans is accepted, of her roles of the human microflora need further study.
Normal Flora of Skin
Skin provides good examples of various microenvironments. Skin regions have been compared to geographic regions of Earth: the desert of the forearm, the cool woods of the scalp, and the tropical forest of the armpit. The composition of the dermal microflora varies from site to site according to the character of the microenvironment. A different bacterial flora characterizes each of three regions of skin: (1) axilla, perineum, and toe webs; (2) hand, face and trunk; and (3) upper arms and legs. Skin sites with partial occlusion (axilla, perineum, and toe webs) harbor more microorganisms than do less occluded areas (legs, arms, and trunk). These quantitative differences may relate to increased amount of moisture, higher body temperature, and greater concentrations of skin surface lipids. The axilla, perineum, and toe webs are more frequently colonized by Gram-negative bacilli than are drier areas of the skin.
The number of bacteria on an individual’s skin remains relatively constant; bacterial survival and the extent of colonization probably depend partly on the exposure of skin to a particular environment and partly on the innate and species-specific bactericidal activity in skin. Also, a high degree of specificity is involved in the adherence of bacteria to epithelial surfaces. Not all bacteria attach to skin; staphylococci, which are the major element of the nasal flora, possess a distinct advantage over viridans streptococci in colonizing the nasal mucosa. Conversely, viridans streptococci are not seen in large numbers on the skin or in the nose but dominate the oral flora.
The microbiology literature is inconsistent about the density of bacteria on the skin; one reason for this is the variety of methods used to collect skin bacteria. The scrub method yields the highest and most accurate counts for a given skin area. Most microorganisms live in the superficial layers of the stratum corneum and in the upper parts of the hair follicles. Some bacteria, however, reside in the deeper areas of the hair follicles and are beyond the reach of ordinary disinfection procedures. These bacteria are a reservoir for recolonization after the surface bacteria are removed.
S. epidermidis is a major inhabitant of the skin, and in some areas it makes up more than 90 percent of the resident aerobic flora.
The nose and perineum are the most common sites for S. aureus colonization, which is present in 10 percent to more than 40 percent of normal adults. S. aureus is prevalent (67 percent) on vulvar skin. Its occurrence in the nasal passages varies with age, being greater in the newborn, less in adults. S. aureus is extremely common (80 to 100 percent) on the skin of patients with certain dermatologic diseases such as atopic dermatitis, but the reason for this finding is unclear.
Micrococci are not as common as staphylococci and diphtheroids; however, they are frequently present on normal skin.Micrococcus luteus, the predominant species, usually accounts for 20 to 80 percent of the micrococci isolated from the skin.
The term diphtheroid denotes a wide range of bacteria belonging to the genus Corynebacterium. Classification of diphtheroids remains unsatisfactory; for convenience, cutaneous diphtheroids have been categorized into the following four groups: lipophilic or nonlipophilic diphtheroids; anaerobic diphtheroids; diphtheroids producing porphyrins (coral red fluorescence when viewed under ultraviolet light); and those that possess some keratinolytic enzymes and are associated with trichomycosis axillaris (infection of axillary hair). Lipophilic diphtheroids are extremely common in the axilla, whereas nonlipophilic strains are found more commonly on glabrous skin.
Anaerobic diphtheroids are most common in areas rich in sebaceous glands. Although the name Corynebacterium acnes was originally used to describe skin anaerobic diphtheroids, these are now classified as Propionibacterium acnes and as P. granulosum.P. acnes is seen eight times more frequently than P. granulosum in acne lesions and is probably involved in acne pathogenesis. Children younger than 10 years are rarely colonized with P. acnes. The appearance of this organism on the skin is probably related to the onset of secretion of sebum (a semi-fluid substance composed of fatty acids and epithelial debris secreted from sebaceous glands) at puberty. P. avidum, the third species of cutaneous anaerobic diphtheroids, is rare in acne lesions and is more often isolated from the axilla.
Streptococci, especially β-hemolytic streptococci, are rarely seen on normal skin. The paucity of β-hemolytic streptococci on the skin is attributed at least in part to the presence of lipids on the skin, as these lipids are lethal to streptococci. Other groups of streptococci, such as α-hemolytic streptococci, exist primarily in the mouth, from where they may, in rare instances, spread to the skin.
Gram-negative bacteria make up a small proportion of the skin flora. In view of their extraordinary numbers in the gut and in the natural environment, their scarcity on skin is striking. They are seen in moist intertriginous areas, such as the toe webs and axilla, and not on dry skin. Desiccation is the major factor preventing the multiplication of Gram-negative bacteria on intact skin.Enterobacter, Klebsiella, Escherichia coli, and Proteus spp. are the predominant Gram-negative organisms found on the skin.Acinetobacter spp also occurs on the skin of normal individuals and, like other Gram-negative bacteria, is more common in the moist intertriginous areas.
The microbiology of a normal nail is generally similar to that of the skin. Dust particles and other extraneous materials may get trapped under the nail, depending on what the nail contacts. In addition to resident skin flora, these dust particles may carry fungi and bacilli. Aspergillus, Penicillium, Cladosporium, and Mucor are the major types of fungi found under the nails.
Oral and Upper Respiratory Tract Flora
The oral flora is involved in dental caries and periodontal disease, which affect about 80 percent. of the population in the Western world. The oral flora, its interactions with the host, and its response to environmental factors are thoroughly discussed in another Chapter. Anaerobes in the oral flora are responsible for many of the brain, face, and lung infections that are frequently manifested by abscess formation.
The pharynx and trachea contain primarily those bacterial genera found in the normal oral cavity (for example, α-and β-hemolytic streptococci); however, anaerobes, staphylococci, neisseriae, diphtheroids, and others are also present. Potentially pathogenic organisms such as Haemophilus, mycoplasmas, and pneumococci may also be found in the pharynx. Anaerobic organisms also are reported frequently. The upper respiratory tract is so often the site of initial colonization by pathogens (Neisseria meningitides,C. diphtheriae, Bordetella pertussis, and many others) and could be considered the first region of attack for such organisms. In contrast, the lower respiratory tract (small bronchi and alveoli) is usually sterile, because particles the size of bacteria do not readily reach it. If bacteria do reach these regions, they encounter host defense mechanisms, such as alveolar macrophages, that are not present in the pharynx.
Gastrointestinal Tract Flora
The stomach is a relatively hostile environment for bacteria. It contains bacteria swallowed with the food and those dislodged from the mouth. Acidity lowers the bacterial count, which is highest (approximately 103 to 106 organisms/g of contents) after meals and lowest (frequently undetectable) after digestion. Some Helicobacter species can colonize the stomach and are associated with type B gastritis and peptic ulcer disease. Aspirates of duodenal or jejunal fluid contain approximately 103 organisms/ml in most individuals. Most of the bacteria cultured (streptococci, lactobacilli, Bacteroides) are thought to be transients. Levels of 105 to about 107 bacteria/ml in such aspirates usually indicate an abnormality in the digestive system (for example, achlorhydria or malabsorption syndrome). Rapid peristalsis and the presence of bile may explain in part the paucity of organisms in the upper gastrointestinal tract. Further along the jejunum and into the ileum, bacterial populations begin to increase, and at the ileocecal junction they reach levels of 106 to 108 organisms/ml, with streptococci, lactobacilli, Bacteroides, and bifidobacteria predominating.
Concentrations of 109 to 1011 bacteria/g of contents are frequently found in human colon and feces. This flora includes a bewildering array of bacteria (more than 400 species have been identified); nonetheless, 95 to 99 percent belong to anaerobic genera such as Bacteroides, Bifidobacterium, Eubacterium, Peptostreptococcus, and Clostridium. In this highly anaerobic region of the intestine, these genera proliferate, occupy most available niches, and produce metabolic waste products such as acetic, butyric, and lactic acids. The strict anaerobic conditions, physical exclusion (as is shown in many animal studies), and bacterial waste products are factors that inhibit the growth of other bacteria in the large bowel.
Although the normal flora can inhibit pathogens, many of its members can produce disease in humans. Anaerobes in the intestinal tract are the primary agents of intra-abdominal abscesses and peritonitis. Bowel perforations produced by appendicitis, cancer, infarction, surgery, or gunshot wounds almost always seed the peritoneal cavity and adjacent organs with the normal flora. Anaerobes can also cause problems within the gastrointestinal lumen. Treatment with antibiotics may allow certain anaerobic species to become predominant and cause disease. For example, Clostridium difficile, which can remain viable in a patient undergoing antimicrobial therapy, may produce pseudomembranous colitis. Other intestinal pathologic conditions or surgery can cause bacterial overgrowth in the upper small intestine. Anaerobic bacteria can then deconjugate bile acids in this region and bind available vitamin B12 so that the vitamin and fats are malabsorbed. In these situations, the patient usually has been compromised in some way; therefore, the infection caused by the normal intestinal flora is secondary to another problem.
More information is available on the animal than the human microflora. Research on animals has revealed that unusual filamentous microorganisms attach to ileal epithelial cells and modify host membranes with few or no harmful effects. Microorganisms have been observed in thick layers on gastrointestinal surfaces (Fig. 6-3) and in the crypts of Lieberkuhn. Other studies indicate that the immune response can be modulated by the intestinal flora. Studies of the role of the intestinal flora in biosynthesis of vitamin K and other host-utilizable products, conversion of bile acids (perhaps to cocarcinogens), and ammonia production (which can play a role in hepatic coma) show the dual role of the microbial flora in influencing the health of the host. More basic studies of the human bowel flora are necessary to define their effect on humans.
(A) Scanning electron micrograph of a cross-section of rat colonic mucosa. The bar indicates the thick layer of bacteria between the mucosal surface and the lumen (L) (X 262,) (B) Higher
The type of bacterial flora found in the vagina depends on the age, pH, and hormonal levels of the host. Lactobacillus spp. predominate in female infants (vaginal pH, approximately 5) during the first month of life. Glycogen secretion seems to cease from about I month of age to puberty. During this time, diphtheroids, S. epidermidis, streptococci, and E. coli predominate at a higher pH (approximately pH 7). At puberty, glycogen secretion resumes, the pH drops, and women acquire an adult flora in which L. acidophilus, corynebacteria, peptostreptococci, staphylococci, streptococci, and Bacteroides predominate. After menopause, pH again rises, less glycogen is secreted, and the flora returns to that found in prepubescent females. Yeasts (Torulopsis andCandida) are occasionally found in the vagina (10 to 30 percent of women); these sometimes increase and cause vaginitis.
In the anterior urethra of humans, S. epidermidis, enterococci, and diphtheroids are found frequently; E. coli, Proteus, and Neisseria(nonpathogenic species) are reported occasionally (10 to 30 percent). Because of the normal flora residing in the urethra, care must be taken in clinically interpreting urine cultures; urine samples may contain these organisms at a level of 104/ml if a midstream (clean-catch) specimen is not obtained.
The conjunctival flora is sparse. Approximately 17 to 49 percent of culture samples are negative. Lysozyme, secreted in tears, may play a role in controlling the bacteria by interfering with their cell wall formation. When positive samples show bacteria, corynebacteria, Neisseriae, and Moraxellae are cultured. Staphylococci and streptococci are also present, and recent reports indicate that Haemophilus parainfluenzae is present in 25 percent of conjunctival samples.
Host Infection by Elements of the Normal Flora
This chapter has briefly described the normal human flora; however, the pathogenic mechanisms of various genera or the clinical syndromes in which they are involved was not discussed. Although such material is presented in other chapters, note that a breach in mucosal surfaces often results in infection of the host by members of the normal flora. Caries, periodontal disease, abscesses, foul-smelling discharges, and endocarditis are hallmarks of infections with members of the normal human flora (Fig. 6-4). In addition, impairment of the host (for example, those with heart failure or leukemia) or host defenses (due to immunosuppression, chemotherapy, or irradiation) may result in failure of the normal flora to suppress transient pathogens or may cause members of the normal flora to invade the host themselves. In either situation, the host may die.
Clinical conditions that may be caused by members of the normal flora.
|Primary Defects||Acquired Defects|
|Lymphoid Stem Cells|
|HypoGG||Pre B-cells||Thymus||DiGeorge Syndrome|
- Type I: Immediate Hypersensitivity
- Type II: Cytotoxic Hypersensitivity
- Type III: Immune Complex Hypersensitivity
- Type IV: Delayed Hypersensitivity
This page will describe the four types of hypersensitivity, giving examples of diseases that may result.
- First, a guinea pig is injected intravenously with an antigen. For this example, bovine serum albumin (BSA, a protein) will be used. After two weeks, the same antigen will be reinjected into the same animal. Within a few minutes, the animal begins to suffocate and dies by a process called anaphylactic shock.
- Instead of reinjecting the immunized guinea pig, serum is transferred from this pig to a “naive” (unimmunized) pig. When this second guinea pig is now injected with BSA, it also dies of anaphylactic shock. However, if the second pig is injected with a different antigen (e.g. egg white albumin), the pig shows no reaction.
- If immune cells (T-cells and macrophages instead of serum) are transfered from the immunized pig to a second pig, the result is very different; injection of the second pig with BSA has no effect.
- The reaction elicited by antigen occurs very rapidly (hence the name “immediate hypersensitivity”).
- The hypersensitivity is mediated via serum-derived components (i.e. antibody).
- The hypersensitivity is antigen-specific (as one might expect for an antibody-mediated reaction).
The details of this reaction can be summarized as follows (click the image to animate):
- Initial introduction of antigen produces an antibody response. More specifically, the type of antigen and the way in which it is administered induce the synthesis of IgE antibody in particular.
- Immunoglobulin IgE binds very specifically to receptors on the surface of mast cells, which remain circulating.
- Reintroduced antigen interacts with IgE on mast cells causing the cells to degranulate and release large amounts of histamine, lipid mediators and chemotactic factors that cause smooth muscle contraction, vasodilation, increased vascular permeability, broncoconstriction and edema. These reactions occur very suddenly, causing death.
Examples of Type I hypersensitivities include allergies to penicillin, insect bites, molds, etc. A person’s sensitivity to these allergens can be tested by a cutaneous reaction. If the specific antigen in question is injected intradermally and the patient is sensitive, a specific reaction known as wheal and flare can be observed within 15 minutes. Individuals who are hypersensitive to such allergens must avoid contact with large inocula to prevent anaphylactic shock.
There are many examples of Type II hypersensitivity. These include:
- Pemphigus: IgG antibodies that react with the intracellular substance found between epidermal cells.
- Autoimmune hemolytic anemia (AHA): This disease is generally inspired by a drug such as penicillin that becomes attached to the surface of red blood cells (RBC) and acts as hapten for the production of antibody which then binds the RBC surface leading to lysis of RBCs.
- Goodpasture’s syndrome: Generally manifested as a glomerulonephritis, IgG antibodies that react against glomerular basement membrane surfaces can lead to kidney destruction.
One example of a Type III hypersensitivity is serum sickness, a condition that may develop when a patient is injected with a large amount of e.g. antitoxin that was produced in an animal. After about 10 days, anti-antitoxin antibodies react with the antitoxin forming immune complexes that deposit in tissues. Type III hypersensitivities can be ascertained by intradermal injection of the antigen, followed by the observance of an “Arthus” reaction (swelling and redness at site of injection) after a few hours.
- First, a guinea pig is injected with a sub-lethal dose of Mycobacterium tuberculosis (MT). Following recovery of the animal, injection of a lethal dose of MT under the skin produces only erythema (redness) and induration (hard spot) at the site of injection 1-2 days later.
- Instead of reinjecting the immunized guinea pig, serum is transfered from this pig to a “naive” (unimmunized) pig. When this second guinea pig is now injected with MT, it dies of the infection.
- If immune cells (T-cells and macrophages instead of serum) are transfered from the immunized pig to a second pig, the result is very different; injection of the second pig with MT causes only erythema and induration at the site of injection 1-2 days later.
- In a separate experiment, if the immunized guinea pig is injected with a lethal dose of Listeria monocytogenes (LM) instead of MT, it dies of the infection. However, if the pig is simultaneously injected with both LM and MT, it survives.
- The reaction elicited by antigen occurs relatively slowly (hence the name “delayed hypersensitivity”).
- The hypersensitivity is mediated via T-cells and macrophages.
- The hypersensitivity illustrates both antigen-specific (T-cell) and antigen non-specific (macrophage) characteristics.
The details of this reaction can be summarized as follows (click the image to animate):
- Initial introduction of antigen produces a cell-mediated response. Mycobacterium tuberculosis is an intracellular pathogen and recovery requires induction of specific T-cell clones with subsequent activation of macrophages.
- Memory T-cells respond upon secondary injection of the specific (i.e. MT) antigen, but not the non-specific (i.e. LM) antigen.
- Induction of the memory T-cells causes activation of macrophages and destruction of both specific (MT) and non-specific (LM) microorganisms.
- making bacteria more susceptible to phagocytosis
- directly lysing some bacteria and foreign cells
- producing chemotactic substances
- increasing vascular permeability
- causing smooth muscle contraction
- promoting mast cell degranulation
The complement system can be activated via two distinct pathways; the classical pathway and the alternate pathway. Once initiated, a cascade of events (the “complement cascade”) ensues, providing the functions listed above.Most of the complement components are numbered (e.g. C1, C2, C3, etc.) but some are simply refered to as “Factors”. Some of the components must be enzymatically cleaved to activate their function; others simply combine to form complexes that are active. The following table lists these components and their functions.
|Components of the Classical Pathway|
|Native component||Active component(s)||Function(s)|
|C1(q,r,s)||C1q||Binds to antibody that has bound antigen, activates C1r.|
|C1r||Cleaves C1s to activate protease function.|
|C1s||Cleaves C2 and C4.|
|C2b||Active enzyme of classical pathway; cleaves C3 and C5.|
|C3||C3a||Mediates inflammation; anaphylatoxin.|
|C3b||Binds C5 for cleavage by C2b.
Binds cell surfaces for opsonization and activation of alternate pathway.
|C4b||Binds C2 for cleavage by C1s. Binds cell surfaces for opsonization.|
|Components of the Alternate Pathway|
|Native component||Active component(s)||Function(s)|
|C3||C3a||Mediates inflammation; anaphylatoxin.|
|C3b||Binds cell surfaces for opsonization and activation of alternate pathway.|
|Factor B||B||Binds membrane bound C3b. Cleaved by Factor D.|
|Bb||Cleaved form stabilized by P produces C3 convertase.|
|Factor D||D||Cleaves Factor B when bound to C3b.|
|Properdin||P||Binds and stabilizes membrane bound C3bBb.|
|Components of the Membrane-Attack Complex|
|Native component||Active component(s)||Function(s)|
|C5||C5a||Mediates inflammation; anaphylatoxin, chemotaxin.|
|C5b||Initiates assembly of the membrane-attack complex (MAC).|
|C6||C6||Binds C5b, forms acceptor for C7.|
|C7||C7||Binds C5b6, inserts into membrane, forms acceptor for C8.|
|C8||C8||Binds C5b67, initiates C9 polymerization.|
|C9||C9n||Polymerizes around C5b678 to form channel that causes cell lysis.|
The classical pathway starts with C1; C1 binds to immunoglobulin Fc (primarily IgM and IgG); C1 is recognition complex composed of 22 polypeptide chains in 3 subunits; C1q, C1r, C1s. C1q is the actual recognition portion, a glycoprotein containing hydroxyproline and hydroxylysine that looks like a tulip flower. Upon binding via C1q, C1r is activated to become a protease that cleaves C1s to a form that activates (cleaves) both C2 and C4 to C2a/b and C4a/b. C2b and C4b combine to produce C3 convertase (C3 activating enzyme). C4a has anaphylactic activity (inflammatory response).
C3 is central to both the classical and alternative pathways. In classical, C4b2b convertase cleaves C3 into C3a/b. C3a is a potent anaphylatoxin. C3b combines with C4b2b to form C4b2b3b complex that is a C5 convertase. C3b can also bind directly to cells making them susceptible to phagocytosis.
C5 is converted by C5 convertase (i.e. C4b2b3b) to C5a/b. C5a has potent anaphylatoxic and chemotaxic activities. C5b functions as an anchor on the target cell surface to which the lytic membrane-attack complex (MAC) forms. MAC includes C5b, C6, C7, C8 and C9. Once C9 polymerizes to form a hole in the cell wall, lysis ensues.
The alternate pathway may be initiated by immunologic (e.g. IgA or IgE) or non-immunologic (e.g. LPS) means. The cascade begins with C3. A small amount of C3b is always found in circulation as a result of spontaneous cleavage of C3 but the concentrations are generally kept very low (see below). However, when C3b binds covalently to sugars on a cell surface, it can become protected. Then Factor B binds to C3b. In the presence of Factor D, bound Factor B is cleaved to Ba and Bb; Bb contains the active site for a C3 convertase. Next. properdin binds to C3bBb to stabilize the C3bBb convertase on cell surface leading to cleavage of C3. Finally, a C3bBb3b complex forms and this is a C5 convertase, cleaving C5 to C5a/b. Once formed, C5b initiates formation of the membrane attack complex as described above.Generally, only Gram-negative cells can be directly lysed by antibody plus complement; Gram-positive cells are mostly resistant. However, phagocytosis is greatly enhanced by C3b binding (phagocytes have C3b receptors on their surface) and antibody is not always required. In addition, complement can neutralize virus particles either by direct lysis or by preventing viral penetration of host cells.
- C1 Inhibitor inhibits the production of C3b by combining with and inactivating C1r and C1s. This prevents formation of the C3 convertase, C4b2b.
- Protein H inhibits the production of C3b by inhibiting the binding of Factor B to membrane-bound C3b, thereby preventing cleavage of B to Bb and production of the C3 convertase, C3bBb.
- Factor I inhibits the production of C3b by cleaving C3b into C3c and C3d, which are inactive. Factor I only works on cell membrane bound C3b, mostly on red blood cells (i.e. non-activator surfaces).
- Phagocytosis and killing of intracellular pathogens
- Direct cell killing by cytotoxic T cells
- Direct cell killing by NK and K cells
These responses are especially important for destroying intracellular bacteria, eliminating viral infections and destroying tumor cells. This page will discuss the cell-mediated immune response, focusing on the mechanisms involved.
|Non-encapsulated microorganisms are easily phagocytosed and killed within macrophages.|
|Encapsulated microorganisms require the production of antibody in order to be effectively phagocytosed. Once engulfed, however, they are easily killed.|
|Intracellular microorganisms elicit the production of antibody, which allows effective phagocytosis. Once engulfed, however, they survive within the phagocyte and eventually kill it.|
|Intracellular microorganisms also activate specific T-cells, which then release lymphokines (e.g. IFN, TNF) that cause macrophage activation. Activated (“killer”) macrophages are then very effective at destroying the intracellular pathogens.|
- Inoculation of an unimmunized guinea pig with a lethal dose of the intracellular pathogen Mycobacterium tuberculosis (MT) results in death of the animal. Inoculation with a sub-lethal dose induces immunity.
- Inoculation of an MT-immunized guinea pig with a lethal dose of MT causes a local reaction (“delayed hypersensitivity”) one to two days later.
- Inoculation of an MT-immunized guinea pig with a lethal dose of a different intracellular pathogen, Listeria monocytogenes (LM) again results in death of the animal.
- Inoculation of an MT-immunized guinea pig with a lethal dose of LM and MT causes a delayed hypersensitivity reaction.
These results demonstrate the specific (T-cell mediated) and non-specific (macrophage mediated) aspects of this type of cell mediated immunity.
CTLs, like other T-cells are both antigen and MHC-restricted. That is, CTLs require i) recognition of a specific antigenic determinant and ii) recognition of “self” MHC (Click here to review these requirements). Briefly, CTLs recognize antigen via their T-cell receptor. This receptor makes specific contacts with the antigenic determinant and the target cell’s class I MHC molecule. CTLs also express CD8, which may assist the antigen recognition process. Once recognition is successful, the CTL “programs” the target cell for self-destruction. This process is thought to occur in one of several possible ways. First, CTLs may release a substance known as perforin in the space between the CTL and its target. In the presence of calcium ions, the perforin polymerizes, forming channels in the target cell’s membrane. These channels may cause the target cell to lyse. Second, the CTL may also release various enzymes that pass through the polyperforin channels, causing target cell damage. Third, the CTL may release lymphokines and/or cytokines that interact with specific receptors on the target cell surface, causing internal responses that lead to destruction of the target cell. CTLs principally act to eliminate endogenous antigens. Click here for more information.
NK cells are part of a group know as the “large granular lymphocytes”. These cells are generally non-specific, MHC-unrestricted cells involved primarily in the elimination of neoplastic or tumor cells. The precise mechanism by which they recognize their target cells is not clear. Probably, there is some type of NK-determinant expressed by the target cells that is recognized by an NK-receptor on the NK cell surface. Once the target cell is recognized, killing occurs in a manner similar to that produced by the CTL.
K-cells are probably not a separate cell type but rather a separate function of the NK group. K-cells contain immunoglobulin Fc receptors on their surface and are involved in a process known as Antibody-dependent Cell-mediated Cytotoxicity (ADCC). ADCC occurs as a consequence of antibody being bound to a target cell surface via specific antigenic determinants expressed by the target cell. Once bound, the Fc portion of the immunoglobulin can be recognized by the K-cell. Killing then ensues by a mechanism similar to that employed by CTLs. This type of CMIR can also result in Type II hypersensitivities.
Interactions between antigen and antibody involve non-covalent binding of an antigenic determinant (epitope) to the variable region (complementarity determining region, CDR) of both the heavy and light immunoglobulin chains. These interactions are analogous to those observed in enzyme-substrate interactions and they can be defined similarly. To describe the strength of the antigen-antibody interaction, one can define the affinity constant (K) as shown:
|Affinity K =||[Ab – Ag]
[Ab] × [Ag]
|= 104 to 1012 L/mol|
|Affinity K =||1
1 × 1
|= 100 L/mol|
|Amount of precipitate|
|Amount of Ag
|If one then measures the amount of antigen and antibody remaining in the supernatant, one sees the following:|
Click the button to illustrate the process
- Antibody excess might occur when a person is exposed to a virus from which they have recently recovered. Hence, their body would contain a relatively large concentration of antiviral antibodies. These antibodies could quickly act to block cell receptors on the viral surface and prevent adsorption to host cells, thereby preventing disease.
- Antigen excess might occur early in the first infection by a microorganism. A person would have relatively few antibodies and these would form complexes but they would be very small. Such small complexes probably would not be phagocytosed or removed by the kidneys and could become lodged near tissue surfaces. Later, when antibody becomes available, the size of the complexes can increase leading to effective elimination by phagocytes or tissue damage where the smaller complexes had become lodged. Click here for more information.
- Equivalence would occur when a person is exposed to an agent to which they have circulating antibodies. The correct ratio of antigen to antibody would produce extensive lattice formation, leading to enhanced phagocytosis, opsonization or agglutination, effectively eliminating the foreign agent.
Crossreactivity also forms the basis for several diagnostic tests. For example, infection with Treponema pallidum (syphilis) causes the production of antibodies that cross-react with a substance found in cardiac muscle, cardiolipin. Since it is much easier to obtain pure cardiolipin than pure Treponemal antigens, this cross-reaction is used to test for syphilis (Wassermann test). Likewise, antibodies produced against certain Rickettsia cross-react with antigens from Proteus. Since the latter are much easier to obtain, they can be used to test for the former.
Expansion and Differentiation phase: Induced lymphocyte clones proliferate and mature to a functional stage (i.e. Ag receptor cells mature to Ag effector cells)
Effector phase: Abs or T cells exert biological effects either:
- Independently or
- Through the action of macrophages, complement, other non-specific agents
This page will discuss induction, differentiation and regulation of the humoral immune response, focusing on the production of Abs.
Induction of the humoral immune response begins with the recognition of antigen. Through a process of clonal selection, specific B-cells are stimulated to proliferate and differentiate. However, this process requires the intervention of specific T-cells that are themselves stimulated to produce lymphokines that are responsible for activation of the antigen-induced B-cells. In other words, B cells recognize antigen via immunoglobulin receptors on their surface but are unable to proliferate and differentiate unless prompted by the action of T-cell lymphokines. In order for the T-cells to become stimulated to release lymphokines, they must also recognize specific antigen. However, while T-cells recognize antigen via their T-cell receptors, they can only do so in the context of the MHC molecules. This “antigen-presentation” is the responsibility of the antigen-presenting cells (APCs).
Several types of cells may serve the APC function. Perhaps the best APC is, in fact, the B-cell itself. When B-cells bind antigen, the antigen becomes internalized, processed and expressed on the surface of the B-cell. Expression occurs within the class II MHC molecule, which can then be recognized by T-helper cells (CD4+). Click the image to animate.
Other types of antigen-presenting cells include the macrophage and dendritic cells. These cells either actively phagocytose or pinocytose foreign antigens. The antigens are then processed in a manner similar to that observed for the B-cells. Next, specific antigen epitopes are expressed on the macrophage or dendritic cell surface. Again, this expression occurs within the class II MHC molecule, where T-cell recognition occurs. The stimulated T-cells then release lymphokines that act upon “primed” B-cells (B-cells that have already encountered antigen), inducing B-cell proliferation and differentiation. Click the image to animate.
B-cells begin their lives in the bone marrow as multipotential stem cells. These completely undifferentiated cells serve as the source for all of the cellular components of the blood and lymphoid system. The initial differentiation step that ultimately leads to the mature B-cell involves DNA rearrangements joining the D and J segments of the immunoglobulin heavy chain genes (click here for more information). Next, DNA rearrangements joining the variable (V) region to the DJ segments of the immunoglobulin heavy chain, as well as similar rearrangements within the light chain genes gives rise to the pre-B-cell. Establishment of the B-cell specificity and consequent expression of surface immunoglobulin gives rise to the “virgin”, fully functional B-cell. Each of these steps is entirely independent of antigen.
The antigen-dependent stages of B-lymphocyte differentiation occur in the spleen, lymph nodes and other peripheral tissue. These stages are, of course, initiated upon encounter with antigen and activation by T-cell lymphokines. The activated B-cell first develops into a B-lymphoblast, becoming much larger and shedding all surface immunoglobulin. The B-lymphoblast then develops into a plasma cell, which is, in essence, an antibody factory. This terminal differentiation stage is responsible for production of primarily IgM antibody during the “primary response”. Some B-cells, however, do not differentiate into plasma cells. Instead, these cells undergo secondary DNA rearrangements that place the constant region of the IgG, IgA or IgE genes in conjunction with the VDJ genes. This “class switch” establishes the phenotype of these newly differentiated B-cells; these cells remain as long-lived “memory cells”. Upon subsequent encounter with antigen, these cells respond very quickly to produce large amounts of IgG, IgA or IgE antibody, generating the “secondary response”.
|Regulation of the immune response is possibly mediated in several ways. First, a specific group of T-cells, suppressor T-cells, are thought to be involved in turning down the immune response. Like helper T-cells, suppressor T-cells are stimulated by antigen but instead of releasing lymphokines that activate B-cells (and other cells), suppressor T-cells release factors that suppress the B-cell response. While immunosuppression is not completely understood, it appears to be more complicated than the activation pathway, possibly involving additional cells in the overall pathway.|
|Other means of regulation involve interactions between antibody and B-cells. One mechanism, “antigen blocking”, occurs when high doses of antibody interact with all of the antigen’s epitopes, thereby inhibiting interactions with B-cell receptors. A second mechanism, “receptor cross linking”, results when antibody, bound to a B-cell via its Fc receptor, and the B-cell receptor both combine with antigen. This “cross-linking” inhibits the B-cell from producing further antibody.|
|Another means of regulation that has been proposed is the idiotypic network hypothesis. This theory suggests that the idiotypic determinants of antibody molecules are so unique that they appear foreign to the immune system and are, therefore, antigenic. Thus, production of antibody in response to antigen leads to the production of anti-antibody in response, and anti-anti-antibody and so on. Eventually, however, the level of [anti]n-antibody is not sufficient to induce another round and the cascade ends.|
The Major Histocompatibility Complex (MHC) is a set of molecules displayed on cell surfaces that are responsible for lymphocyte recognition and “antigen presentation”. The MHC molecules control the immune response through recognition of “self” and “non-self” and, consequently, serve as targets in transplantation rejection. The Class I and Class II MHC molecules belong to a group of molecules known as the Immunoglobulin Supergene Family, which includes immunoglobulins, T-cell receptors, CD4, CD8, and others. This page will describe the MHC molecules and the process of antigen presentation.
The major histocompatibility complex is encoded by several genes located on human chromosome 6. Class I molecules are encoded by the BCA region while class II molecules are encoded by the D region. A region between these two on chromosome 6 encodes class III molecules, including some complement components.
Class I molecules are composed of two polypeptide chains; one encoded by the BCA region and another (ß2-microglobulin) that is encoded elsewhere. The MHC-encoded polypeptide is about 350 amino acids long and glycosylated, giving a total molecular weight of about 45 kDa. This polypeptide folds into three separate domains called alpha-1, alpha-2 and alpha-3. ß2-microglobulin is a 12 kDa polypeptide that is non-covalently associated with the alpha-3 domain. Between the alpha-1 and alpha-2 domains lies a region bounded by a beta-pleated sheet on the bottom and two alpha helices on the sides. This region is capable of binding (via non-covalent interactions) a small peptide of about 10 amino acids. This small peptide is “presented” to a T-cell and defines the antigen “epitope” that the T-cell recognizes (see below). The following images illustrate the structure of the class I MHC as seen schematically, and three dimensionally from the side and from the top (T-cell perspective). The MHC-encoded polypeptide is shown in blue, the ß2-microglobulin is green and the peptide antigen is red.
|Class I MHC||Side view||Top view|
Class II molecules are composed of two polypeptide chains, both encoded by the D region. These polypeptides (alpha and beta) are about 230 and 240 amino acids long, respectively, and are glycosylated, giving molecular weights of about 33 kDa and 28 kDa. These polypeptides fold into two separate domains; alpha-1 and alpha-2 for the alpha polypeptide, and beta-1 and beta-2 for the beta polypeptide. Between the alpha-1 and beta-1 domains lies a region very similar to that seen on the class I molecule. This region, bounded by a beta-pleated sheet on the bottom and two alpha helices on the sides, is capable of binding (via non-covalent interactions) a small peptide of about 10 amino acids. This small peptide is “presented” to a T-cell and defines the antigen “epitope” that the T-cell recognizes (see below). The following images illustrate the structure of the class II MHC as seen schematically, and three dimensionally from the side and from the top (T-cell perspective). The MHC-encoded polypeptides are shown in yellow and green, while the peptide antigen is shown in red.
|Class II MHC||Side view||Top view|
While class I and class II molecules appear somewhat structurally similar and both present antigen to T-cells, their functions are really quite distinct. First, class I molecules are found on virtually every cell in the human body. Class II molecules, in contrast, are only found on B-cells, macrophages and other “antigen-presenting cells” (APCs). Second, class I molecules present antigen to cytotoxic T-cells (CTLs) while class II molecules present antigen to helper T-cells (TH-cells). This specificity reflects the third difference, the type of antigen presented. Class I molecules present “endogenous” antigen while class II molecules present “exogenous” antigens. An endogenous antigen might be fragments of viral proteins or tumor proteins. Presentation of such antigens would indicate internal cellular alterations that if not contained could spread throughout the body. Hence, destruction of these cells by CTLs is advantageous to the body as a whole. Exogenous antigens, in contrast, might be fragments of bacterial cells or viruses that are engulfed and processed by e.g. a macrophage and then presented to helper T-cells. The TH-cells, in turn, could activate B-cells to produce antibody that would lead to the destruction of the pathogen.
The T-cell receptor molecule (TCR) is structurally and functionally similar to the B-cell immunoglobulin receptor. TCR is composed of two, disulfide-linked polypeptide chains, alpha and beta, each having separate constant and variable domains much like immunoglobulins. The variable domain contains three hypervariable regions that are responsible for antigen recognition. Genetic diversity is ensured in a manner analogous to that for immunoglobulins (click here for more information). Thus, just like the B-cell surface immunoglobulin provides antigen specificity to its B-cell, the TCR allows T-cells to recognize their particular antigenic moiety. However, T-cells cannot recognize antigen without help; the antigenic determinant must be presented by an appropriate (i.e. self) MHC molecule. Upon recognition of a specific antigen, the signal is passed to the CD3 molecule and then into the T-cell, prompting T-cell activation and the release of lymphokines. The following images illustrate the structure of the TCR as seen schematically, and three dimensionally from the side.
The TCR provides the specificity for an individual T-cell to recognize its particular antigen. However, this recognition is “MHC-restricted” because the TCR also requires interactions with MHC. Also, interactions between the CD4 molecule (found on helper T-cells) and class II MHC or the CD8 molecule (found on cytotoxic T-cells) and class I MHC stabilize and consummate the antigen recognition process, allowing helper T-cells to respond to “exogenous” antigens (leading to B-cell activation and the production of antibody) or cytotoxic T-cells to respond to “endogenous” antigens (leading to target cell destruction). The following images illustrate these processes schematically, and three dimensionally.
|TCR – APC (class II)||TCR – Target cell (class I)|
|Antigen Presentation by MHC-II to TCR|