MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 10. INFECTION AND THE INFECTIOUS PROCESS

GENETIC CONTROL OF PATHOGENICITY AND VIRULENCE

The pathogenicity factors we have examined are genetically encoded traits. Genes encoding pathogenicity factors are typically located in the bacterial genome as clusters known as pathogenicity islands (PAIs). METABOLISM/2.html">THE CONCEPT OF PAIs was first proposed in 1980 by J. Hacker and colleagues while investigating the GENETIC BASIS OF virulence in enteropathogenic E. coli (EPEC) strain 536. The researchers demonstrated that a deletion of PAIs in these Bacteria turns EPEC into non-pathogenic E. coli. Later, PAIs were discovered in other species of microorganisms. It is precisely the presence of PAIs that distinguishes pathogenic microorganisms from non-pathogenic ones. These islands encode the synthesis of various pathogenicity factors: adhesins, toxins, exoenzymes, aggressins, as well as corresponding secretion systems. Pathogenicity islands may have specific designations depending on the bacterial type they belong to; for instance, Vibrio pathogenicity islands (VPI) are known in cholera vibrio, the locus of enterocyte effacement (LEE) in enteropathogenic Escherichia, and Salmonella pathogenicity islands (SPI) in salmonellae. It is believed that bacteria acquire these Gene clusters during evolution via Horizontal Gene Transfer. PAIs possess A number of specific characteristics. First, PAIs are large genomic regions (up to 200 kb) containing many pathogenicity genes. Second, it has been found that the percentage of G-C nucleotide pairs in PAIs differs from that of the rest of the bacterial Cell's chromosomal material. Thus, the G-C nucleotide content in non-pathogenic bacteria ranges from 25 to 75 %, whereas in pathogenic microorganisms it is within 40-60 %, specifically due to the enrichment with these NUCLEOTIDES in pathogenicity islands. The reasons for this difference remain unknown today. However, the conservation of The nucleotide sequence composition of PAIs is specific to certain genera and species of microorganisms. Third, PAIs are typically associated with Mobile Genetic Elements, being flanked, for example, by direct repeats (DRs). Direct repeats are DNA sequences (16-20 bp) consisting of repeated nucleotide pairs that serve as integration sites for Bacteriophages. In addition, DRs serve as recognition sites for Enzymes that excise mobile genetic elements, which ultimately leads to the instability of PAIs flanked by DRs. Some PAIs contain bacteriophage integrase or transposase genes. Integrases originate from lysogenic bacteriophages and ensure the Integration of the phage genome into The Genome

of the host Organism, as well as the reverse process (excision of the phage genome) during the lytic phase of infection. These genes function in some PAIs, and the enzymes encoded by them can mediate the excision and loss of PAIs. Other PAIs contain genes similar to transposon integrase and resolvase genes. These mobile genetic elements can change their localization within the chromosome, as well as between the chromosome and Plasmids. Insertion sequences (ISs) are also detected within PAIs. Fourth, PAIs are unstable Regions of the genome. The functioning of PAIs is disrupted at a frequency much higher than the mutation frequency of ordinary genes. Genetic analysis of this phenomenon has proven that the disruption of PAI functioning does not occur through Mutations of individual pathogenicity genes, but is mediated by the loss of large PAI regions or the entire "pathogenicity island" completely. Such PAI disruptions are frequently observed during the cultivation of pathogenic microorganisms in vitro or during prolonged persistent infection. This indicates that PAIs have an inherent propensity for genetic instability. Fifth, PAIs are represented mosaically in the genome, unlike the remaining homogeneous segments of DNA. Some PAIs represent the insertion of a single genetic element. Other PAIs have a complex Structure because during evolution, bacterial Cells acquired various genetic elements independently, at different times, and from different sources.

The fact that important pathogenicity factors are similar across different species of microorganisms can only be explained by horizontal gene transfer. The process of horizontal genetic Information Transfer in bacteria can occur via mechanisms of transformation, conjugation, and Transduction. Certain types of bacteria are capable of natural transformation. During certain growth phases, cells express transport systems capable of taking up free DNA from their microenvironment. Although most of this absorbed foreign DNA will be degraded, some fragments may nevertheless become "useful" genes that integrate into the recipient cell's genome. It is believed that this mechanism enables the uptake of DNA from distant microbial species and the acquisition of new properties by the recipient cell.

Conjugation allows plasmid transfer between bacteria. Such plasmids can subsequently replicate autonomously from the bacterial chromosome, although under certain conditions they can integrate into the chromosome.

Certain pathogenicity factors can be encoded by the bacteriophage genome. Bacteriophages are isolated from any bacterial species. The accidental transfer of pathogenicity genes by phages allows the recipient bacterium to acquire new properties and gain an advantage over similar microorganisms in colonizing a new eco-niche. Many PAIs have

excessively large sizes, sometimes even equivalent to the sizes of the phages themselves. In such cases, transfer can occur via The Mechanism of generalized transduction. As a rule, during phage Replication, copies of the genome are packaged into the phage "HEAD". During this replication, Bacterial DNA is fragmented, and a portion of such DNA can be accidentally packaged into the phage "head". Such errors occur with a frequency of 1 : 1000. Defective phages still retain The ability to infect new bacterial cells. In this way, bacterial DNA that may encode pathogenicity factors is transmitted.

What exactly determines the localization of PAIs in the bacterial cell genome is not yet known. It has been found that similar virulence gene clusters can be contained in both the bacterial chromosome and plasmids. For example, the mxi and spa genes encoding the type III secretion system necessary for Shigella spp. invasion are localized in a giant plasmid of the bacterium, whereas a similar SPI-1 gene cluster in Salmonella enterica is localized in the chromosome.

The formation of common-type pili involved in adhesion is generally controlled by chromosomal genes. At the same time, certain Escherichia adhesins (CFA/I, CFA/II, CFA/III) are encoded by plasmid genes.

The production of BIOLOGICALLY ACTIVE SUBSTANCES involved in penetration can be encoded by both plasmid (e.g., in Shigella sonnei) and chromosomal genes.

The synthesis of antiphagocytic and anticomplementary substances, such as protein A of S. aureus, M-protein of S. pyogenes, and the capsular polysaccharide of S. pneumoniae, is predominantly controlled by chromosomal genes.

The Genetic control of toxin production is quite diverse and can be carried out with the participation of chromosomal genes, various plasmids (F, R, Col) containing tox-Transposons, as well as converting bacteriophages. Chromosomal tox genes control the production of choleragen, exfoliatin, C. perfringens enterotoxin, etc. Tox genes controlling the production of diphtheria histotoxin, S. pyogenes erythrogenic toxin, and botulinum neurotoxin have been found within the Chromosomes of lysogenic cultures carrying a prophage. Sometimes the genetic control of toxin production can be complex. For example, many tox-plasmids control the production not of the toxins themselves, but of protoxins that require an additional enzyme for their activation. The production of the protease enzyme itself is under the control of chromosomal genes. Proteases are involved in the activation of many protoxins, such as diphtheria histotoxin, botulinum protoxin, etc. Consequently, in the bacterial cell, joint control

by plasmid and chromosomal genes is exercised over the production of functionally active toxins.

Thus, PAIs are very widespread in the bacterial world precisely due to horizontal gene transfer, plasmid transfer, phage activity, and bacterial competence for free DNA uptake.

All pathogenicity factors and the degree of their manifestation are subject to phenotypic and genotypic changes. The causes of such changes are the action of various physiological and Chemical factors. Since the time of L. Pasteur, the artificial reduction of virulence—attenuation (Latin attenuo meaning to weaken)—has been the basis for the production of certain Vaccines.

A genotypic reduction in virulence is possible through mutations, recombinations, and the loss of extrachromosomal hereditary factors (plasmids, transposons).

A phenotypic reduction in virulence can occur when the pathogen encounters unfavorable conditions. In vitro, this happens As a result of unfavorable cultivation regimens and nutrient medium composition, and the action of selective adverse factors. A decrease in virulence in vivo develops during prolonged persistence of the pathogen in the host organism. Those bacterial cells that survive acquire resistance to the action of immune factors and antimicrobial agents, but lose their pathogenic properties (for example, due to the loss of plasmid or chromosomal pathogenicity genes). Methods for reducing the virulence of pathogenic microorganisms are of great practical importance for obtaining vaccine strains.

An increase in virulence is achieved by cultivating bacteria in vitro under optimal conditions or through repeated passages of a low-virulence culture through the body of a sensitive laboratory animal. In this case, Selection of virulent individuals present in a heterogeneous bacterial population also takes place, which can ultimately lead to an increase in its virulence.