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Tag words: pathogenic bacteria, bacterial pathogenicity, invasiveness, toxigenesis, colonization, specific adherence, adhesin, receptor, invasion, invasin, coagulase, leucocidin, hemolysin, streptokinase, phagocytosis, phagosome, lysosome, phagolysosome, immunological tolerance, antigenic disguise, immunosuppression, antigenic variation, protein toxins, botulinum toxin, diphtheria toxin, anthrax toxin, tetanus toxin, pertussis toxin, cholera enterotoxin, adenylate cyclase, staph enterotoxin, TSST, pyrogenic exotoxin, superantigen, shiga toxin, E. coli LT toxin, ST toxin, endotoxin, lipopolysaccharide, LPS, Lipid A, O antigen, O polysaccharide, toxoid, pathogenicity island.









Kenneth Todar currently teaches Microbiology 100 at the University of Wisconsin-Madison.  His main teaching interest include general microbiology, bacterial diversity, microbial ecology and pathogenic bacteriology.

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Mechanisms of Bacterial Pathogenicity (page 8)

(This chapter has 8 pages)

© Kenneth Todar, PhD

ENDOTOXINS

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 term endotoxin 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.
 
Table 5. CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND EXOTOXINS
PROPERTY ENDOTOXIN EXOTOXIN
CHEMICAL NATURE Lipopolysaccharide (mw = 10kDa) Protein (mw = 50-1000kDa)
RELATIONSHIP TO CELL Part of outer membrane Extracellular, diffusible
DENATURED BY BOILING No Usually
ANTIGENIC Yes Yes
FORM TOXOID No Yes
POTENCY Relatively low (>100ug) Relatively high (1 ug)
SPECIFICITY Low degree High degree
ENZYMATIC ACTIVITY No Usually
PYROGENICITY Yes Occasionally

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:

fever

changes in white blood cell counts

disseminated intravascular coagulation

tumor necrosis

hypotension

shock

lethality

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 PO4 groups, 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

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.




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