The use of molecular biology in the experimental study of infectious disease agent's pathogenicity brought us closer to understanding the biological nature of the phenomenon, based on the interaction of prokaryotic and eukaryotic organisms. Nonetheless, there is still no definitive answer to which category of biological events the bacterial pathogenicity phenomenon should belong to. Based on the definition of ecology* as a science, an infectious disease can be considered a special model of an ecosystem, in which a living organism is a pathogen, and its natural environment is multi-cellular tissues of humans, animals or plants. In this habitat the microbe is able to perform its vital functions and interact with the tissue cells of its host's body. Death of the host's body as an outcome of infectious disease is a type of the ecological-like disaster, causing collapse of this agent's natural environment, followed by death of the majority of pathogen population.
In the very distant past, our planet was inhabited by microbial society that consisted of saprophytes (non-infectious microorganisms) whose natural environment was water and soil. The development of pathogenic germs that were able to induce infectious diseases was connected with appearance of multi-cellular organisms. For example, the potential ecological space represented by human and animal tissues provided pathogens with definite advantages in comparison to natural reservoirs of water and soil. The warm-blooded environment protected microbes from volatile characteristics of the wild, such as changes in temperature. Under stable temperatures, organisms could reproduce and increase the number of individuals that possessed newly developed biological capabilities. Heredity of these characteristics was regulated through natural selection that preserved the genetic structures of the best adapted individuals, and eventually resulted in origination of new species. It leads to appearance of a new group of paratrophic microorganisms capable of inhabiting various plants and animals.
The appearance of saprophyte individuals that could interact not only with hard inert surfaces but also with living cells of multi-cellular tissues can be viewed as the first step in the establishment of new a ecological space. In these bacteria, specific macromolecules appeared and morphological structures composed of them emerged as a result of expansion of the cell walls. Eventually, they transformed into functional active organelles with an adhesive role. These new morphological structures are called fimbriae or common pili (1). Saprophyte bacteria can use adhesive organelles to attach to surrounding hard substrates and coalesce into colonies. This property of saprophytes was particularly useful for the bacteria that are involved in decomposition of organic remains in soil and water. For example, the process of putrefaction induced by putrefactive bacteria is more intensive when performed by a microbial colony as opposed to a collection of individual organisms.
The adhesive property of bacteria turned out to be very important as the organisms took possession of the new ecological niche. Among the adhesive macromolecules variations that had resulted from various gene mutations, some showed high affinity to the process of physic-chemical binding with eukaryotic cells. In contrast to the simple electrostatic contact with hard surfaces that was typical for many saprophytes, the ligand-receptor interaction of bacterial adhesions and the cell surface demonstrated a high degree of specificity. After adherence to a cell surface, microorganisms begin to reproduce intensely and increase their population while colonizing parts of the host tissue. The capability to extend microbial population in a more complex multi-cellular environment (as opposed to soil and water) meant that it was a new property allowing new germ species to broaden their habitat.
Microbial cells that possessed such adhesive properties in relation to animal or plant tissues became commensals and potential pathogens. Natural selection completed formation of the new species and contributed to the promotion of the specificity of adhesive function. For example, 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.
Another morphological structure of bacterial cell, the capsule, also played an important role in helping microbes to conquer multi-cellular environment. The capsule is located on the surface of a bacterial cell and has a gelatinous consistence usually reinforced by chains or threads of linear polymers. When separated from a cell, a capsule can easily transform into hydrophilic gel.
In saprophyte organisms that inhabited water and soil, the capsule was primarily a protective device against mechanical damage of the bacterial cell and a barrier to poisons that could penetrate it. The role of the capsule for bacteria that colonized tissues in humans, animals, and plants was amplified since in the new environment it acquired a new function protecting the pathogen from destructive biological mechanisms of the host organism. The mammals were armed with an immune system and phagocytosis as the first powerful line of defense. To resist compliment activation and absorption by phagocytes the capsule of microbial cells needed certain chemical traits.
A good example of the new biological properties of the capsule can be found in some pathogenic bacteria of the Bacillus genus. One of these species, Bacillus anthracis, is the agent that causes anthrax in human and animals. The critical development in its evolution was the mutation of a gene coding the glutamic acid biosynthesis and the assembly of its linear polymers in its capsule. The bacterial cells that had a capsule consisting of D-isomers glutamic acid became invulnerable to host phagocytes. Indeed, it is known that peptides that consist of D-amino acids isomers are biologically resistant to the action of proteolytic degradation. The saprophyte members of this family such as B. subtilis and B. megatherium also have a capsule but their capsule polypeptides are built of L- and D-glutamic acids isomers. In the process of colonization of the new ecological niche, a few individuals with the D-isomer mutant gene were selected from an array of saprophytes bacilli through natural selection. These genetic changes contributed to growth of microbial population residing in multi-cellular media and, finally, helped to form a new species named Bacillus anthracis. However, in order to become a real pathogen, the new microorganism had to acquire an offensive weapon - the toxin
Many 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. These bacterial substances had different molecular and chemical structure. This group of bio-molecules consisted of peptides and proteins (for example, staphylococcal protein A and streptococcal protein M, lipopolysacharides (LPS) produced by gram-negative bacteria, glycoproteins and other mixed polymers. Some pathogens such as Streptococcus pyogenes, Staphylococcus aureus and Treponema pallidum use fibronectin-binding proteins to provide an antigenic disguise if they clotted fibrin on the cell surface and to avoid host defenses. 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.
Despite of different mechanisms of resistance to phagocytosis, these substances played the same functional role - they protected microbial germs from the host phagocyte and immune system.
Another property that turned out to be helpful in conquering multi-cellular media was the ability to penetrate intercellular space or invasive capacity. Only a small number of pathogens acquired this ability. To that effect, bacteria employed the enzymes that saprophytes used to degrade of organic remains in water and soil. Using enzymes such as hyaluronidase, lecithinase, proteases, some glycopeptidases etc. pathogen that colonized tissue surface was able to split intercellular concreted compounds and invade the tissue. These enzymes have other functions related to bacterial nutrition or metabolism, but may aid in invasion either directly or indirectly. The invasive function is another property of the pathogenicity complex, in addition to selective adhesion to host eukaryotic cells and anti-phagocyte capacity.
We can assume that as a result of the first step of evolution all three functions played a very important role in the process of colonization of the new ecological niche by microbial pathogens, and guaranteed its necessary life in the host organism. Their biological action was devoid of aggressive features and did not induce any damage to human or animal multi-cellular systems. Thus, during this first stage of bacterial colonization of the new environment, the interaction between the pathogen and the tissue cells of the host organism can be characterized as a kind of a symbiotic balance. Typical examples are the oral microflora, the intestinal flora, the urogenital flora etc. in the human host organism.
Formation of the pathogenic complex was further complicated after the damaging function developed in the bacterial cells, which possessed the above mentioned properties. The aggressive activity was directed to induce various types of dysfunction in the host tissue cells. Development of damaging function was realized in two ways: bio-synthesis of endo- and exo-toxins.
Endotoxons are constituents of the outer membrane of the bacterial cell wall. The biological activity of endotoxin is associated with lipopolysaccaride (LPS). LPS participate in a number of outer membrane functions that are essential for bacterial growth and survival in host-pathogen interaction. The above mentioned feature is a basic argument for hypothesis that the damaging factor had been developed in Gram-negative bacteria on the early steps of evolutionary process of pathogenic function.
Toxicity of the cell unbound endotoxins is associated with the lipid component (Lipid A) and antigenic activity is associated with the polysaccharide components. LPS activates complement by the alternative pathway and may be a part of pathology of Gram-negative bacterial infections induced by E. coli, Salmonella, Pseudomonas, Neisseria, Haemophilus and other pathogens. Regardless of the bacterial source, all endtoxins produce the same range of biological effects in the host organism. They cause a wide spectrum of non-specific pathophysiological reactions and are responsible for fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock and lethality. Lipid A is known to react at the surfaces of macrophages causing them to release tumor necrosis factor (TNF-a) and probably other cytokines. The polysaccharide part of LPS may supply a bacterium with its specific ligands for adhesion which is essential for colonization as the first stage of any infection. In contrast to the protein exotoxins, endotoxins do not act enzymatically and they are less potent and less specific in their action. Blood and lymphoid cells as well as immune system cells and compliment system, all are targets that undergo endotoxin action.
Another family of aggressive factors is represented by protein exotoxins that were able to damage or to destroy the vitally important physiological systems in the organ tissue cells. Production of protein toxins is specific to a particular pathogenic bacteria species although it consists of both populations of virulent and non-virulent strains. Virulent strains of pathogen are able to produce the toxin while non-virulent are not. Both Gram-positive and Gram-negative bacteria produce soluble protein toxins. The most part of exotoxins possess an enzymatic activity that can be realized in contact with the host tissue target cells. Bacterial protein exotoxins are different in their molecular structure: some of them are represented by simple polypeptide molecules and others have a complicated subunit structure.
The bacterial cell produces toxin molecules that are not toxic for the prokaryotic organism, but their damaging actions are aimed at eukaryotic cells. There is no direct connection between production of these toxin macromolecules and viability of pathogenic bacteria. Facts show that the capacity of exotoxin synthesis is caused by a biological stimulus to an increasing number of the microbial population.
We cannot rule out the possibility that the development of the major determinant of pathogenicity resulted from functional mimicry that pathogen borrowed from the host cell environment. Close contacts between two types of cells, prokaryotic (microorganism) and eukaryotic, lead to communicative events at the molecular level and had dramatic consequences for both the pathogen and the multi-cellular host environment. Apparently, under the action of the outside genetic information, new elements of non-chromosome heredity appeared in the bacterial genome. These elements usually were not integrated in microbial chromosome but they contained some genes that regulated production and assembly of original protein macromolecules. In spite of limited information it was shown that the genes responsible for biosyntheses of some bacterial exo-toxins are located on plasmids or in lysogenic bacteriophages (B. anthracis toxin, Ps. aeruginosa toxin, Cl. botulinum toxin, Cl. tetani toxin, diphtheria toxin etc.).
These so-called "chimera" proteins (4) had both hormone-like as well as enzyme-like properties and played an important role in the development of the pathogenic complex. Their precursors were molecules secreted by germ cells into the liquid media. Under the exposure of limited proteolysis, the precursor polypeptide chain was cleaving and eventually developed into a bi-functional molecular structure. The hormone-like component of the macromolecule was able to recognize specific membrane receptor on the sensitive tissue cell and bind to the cell surface. After the ligand-receptor binding, the enzymatic active components of the bi-functional structure were subjected to endocytosis or pore-forming mechanism and targeted one of the vital systems of the host cell. Microbial protein toxins with this type of "chimera" structure were highly damaging to eukaryotic cells.
Molecular model of the "chimera"toxin complex is often represented by the formula A + B = (?) where the subunit B is the peptide (or peptides) responsible for the membrane receptor binding and the subunit "A" is the peptide that is able to penetrate the cell and damage the intracellular target. Membrane receptors for many bacterial toxins are the same lipid-containing components (for example, gangliosides) that are used by hormone molecules.
Certain protein toxins have broad cytotoxic activity and cause both very specific as well as nonspecific damages of tissue cells. Pore-forming mechanism of action underlies cytotoxicity of some exotoxins (hemolysins and leukocidins). A family of staphylococcal and streptococcal exotoxins such as enterotoxins, TSST, pyrogenic toxins and others was named as superantigens. These simple proteins possess the distinct domain structure and are able to elicit massive activation of the immune system and to inhibit antibody response simultaneously. They stimulate T cell proliferation by interaction with Class II MHC molecules on APCs and specific Vß chains of the T cell receptor. Production of IL-1, TNF, and other lymphokines is a very important result of this interaction.
The study of the genetic bases of bacterial pathogenicity showed that the development of pathogenic potency had been synchronized with appearance of the distinct genetic elements into the bacterial genome of pathogenic organism. These genomic islands are acquired by horizontal gene transfer, and they encode genes which contribute to production of pathogenic determinants. Pathogenicity islands (PAI) are usually absent from non-pathogenic organisms of the same species. PAI are found in pathogens that undergo gene transfer by plasmid, phage or conjugative transposon. They may be incorporated in the bacterial chromosome or may be a part of a plasmid.
PAI apparently have been acquired during the specification of pathogens from their non-pathogenic or environmental ancestors (6). The acquisition of PAI can be considered as an ancient evolutionary event that led to the appearance of phenomenon based on the interaction of prokaryotic and eukaryotic organisms on a timescale of millions of years. We can assume that the feature of the multi-cellular environment surrounded a bacterial cell could result to development of new mechanisms of horizontal gene transfer.
Given the current understanding of bio-molecules produced by various causative microorganisms, it can be concluded that the development of the pathogenicity in some non-pathogenic forms and the colonization of a new ecological niche developed in parallel. The laws of Nature dictate that the supreme goal of all biological species, including pathogens, is unrestrained growth of the population. This means in turn that microbial mass in a tissue colonized by a pathogen can grow logarithmically with a corresponding growth in the amount of the tissue-damaging substance. The damaging action frequently results in degradation and eventual death of the multi-cellular host organism, also killing the majority of the colonizing bacteria. Interaction between pathogen and its human or animal environment ends in ecological-like catastrophe.
Bacterial species are saved from disappearance by a few individual cells that escape the host and infect other human or animal subjects. Some germs are also able to survive in air, water, or soil using the capabilities inherited from their saprophyte precursors.
Is it possible to prevent catastrophic destruction of an organism invaded by pathogens? Modern civilization has acquired an arsenal of epidemiological and medical tools for prevention and treatment of infectious diseases. However, as we are constantly reminded by sporadic outbreaks, the causative microorganisms can not be eradicated for as long as there are carriers they can inhabit.
*Ecology is the study of the interaction of organisms with their physical environment and each with other.
1. Beachey E.H. Bacterial Adherence: adhesin receptor interaction mediating the attachment of bacteria to mucosal surface. J. Infect. Dis. 1981, 143:325-345
2. Ezepchuk Yu.V. Biomolecular Bases of Bacterial Pathogenicity. Nauka, Moscow 1977, 215 pp
3. Ezepchuk Yu. V. Pathogenicity as a Function of Biomolecules. Medicine, Moscow 1984, 238 p
4. Jaljaszevicz J. and T. Wadstrom Bacterial Toxins and Cell Membranes, Academic Press, NewYork, 1978
5, Leung D.Y.M., B.T. Huber, P.M. Schlivert Superantigens Marcel Dekker,Inc., NewYork, 1997, 607 p
6. Schmidt H. and Hensel M. Pathogenicity Islands in Bacterial Pathogenesis. Clin. Microbiol.Rev. 2006 January, 19(1) 257
7. Todar Kenneth. The Mechanism of Bacterial Pathogenicity, The Microbial World, 2009, 1-26.
Yurii V. Ezepchuk, Ph.D.
Professor of Biochemistry
Denver, Colorado, U.S.A.