Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains

The pathogenic strains of Vibrio cholerae that cause acute enteric infections in humans are derived from environmental nonpathogenic strains. To track the evolution of pathogenic V. cholerae and identify potential precursors of new pathogenic strains, we analyzed 324 environmental or clinical V. cholerae isolates for the presence of diverse genes involved in virulence or ecological fitness. Of 251 environmental non-O1, non-O139 strains tested, 10 (3.9%) carried the toxin coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, whereas another 10 isolates carried the TCP island alone, and were susceptible to transduction with CTX phage. Most V. cholerae O1 and O139 strains carried these two major virulence determinants, as well as the Vibrio seventh pandemic islands (VSP-1 and VSP-2), whereas 23 (9.1%) non-O1, non-O139 strains carried several VSP island genes, but none carried a complete VSP island. Conversely, 30 (11.9%) non-O1, non-O139 strains carried type III secretion system (TTSS) genes, but none of 63 V. cholerae O1 or O139 strains tested were positive for TTSS. Thus, the distribution of major virulence genes in the non-O1, non-O139 serogroups of V. cholerae is largely different from that of the O1 or O139 serogroups. However, the prevalence of putative accessory virulence genes (mshA, hlyA, and RTX) was similar in all strains, with the mshA being most prevalent (98.8%) followed by RTX genes (96.2%) and hlyA (94.6%), supporting more recent assumptions that these genes imparts increased environmental fitness. Since all pathogenic strains retain these genes, the epidemiological success of the strains presumably depends on their environmental persistence in addition to the ability to produce major virulence factors. Potential precursors of new pathogenic strains would thus require to assemble a combination of genes for both ecological fitness and virulence to attain epidemiological predominance.     

Distribution of "classic" virulence factors among Salmonella spp

Whether an infection with Salmonella spp. leads to a disease largely depends on the virulence of the strain and the constitution of the host. The virulence of the strain is determined by so-called virulence factors. Whereas a number of virulence factors of Salmonella have been identified only recently, others have been studied for decades. These latter virulence factors i.e., virulence-plasmids, toxins, fimbriae and flagella are therefore referred to as "classic" virulence factors. Here we present an overview on the distribution of (genes coding for) these virulence factors among Salmonella spp. The pathogenicity islands of Salmonella are also reviewed, all be it briefly, since they contain a major part of the virulence genes.     

The PathoChip,a functional gene array for assessing pathogenic properties of diverse microbial communities

Pathogens present in the environment pose a serious threat to human, plant and animal health as evidenced by recent outbreaks. As many pathogens can survive and proliferate in the environment, it is important to understand their population dynamics and pathogenic potential in the environment. To assess pathogenic potential in diverse habitats, we developed a functional gene array, the PathoChip, constructed with key virulence genes related to major virulence factors, such as adherence, colonization, motility, invasion, toxin, immune evasion and iron uptake. A total of 3715 best probes were selected from 13 virulence factors, covering 7417 coding sequences from 1397 microbial species (2336 strains). The specificity of the PathoChip was computationally verified, and approximately 98% of the probes provided specificity at or below the species level, proving its excellent capability for the detection of target sequences with high discrimination power. We applied this array to community samples from soil, seawater and human saliva to assess the occurrence of virulence genes in natural environments. Both the abundance and diversity of virulence genes increased in stressed conditions compared with their corresponding controls, indicating a possible increase in abundance of pathogenic bacteria under environmental perturbations such as warming or oil spills. Statistical analyses showed that microbial communities harboring virulence genes were responsive to environmental perturbations, which drove changes in abundance and distribution of virulence genes. The PathoChip provides a useful tool to identify virulence genes in microbial populations, examine the dynamics of virulence genes in response to environmental perturbations and determine the pathogenic potential of microbial communities.     

Molecular ecology of toxigenic Vibrio cholerae

Toxigenic Vibrio cholerae is the etiological agent of cholera, an acute dehydrating diarrhea that occurs in epidemic form in many developing countries. Although V. cholerae is a human pathogen, aquatic ecosystems are major habitats of Vibrio species, which includes both pathogenic and nonpathogenic strains that vary in their virulence gene content. V. cholerae belonging to the 01 and 0139 serogroups is commonly known to carry a set of virulence genes necessary for pathogenesis in humans. Recent studies have indicated that virulence genes or their homologues are also dispersed among environmental strains of V. cholerae belonging to diverse serogroups, which appear to constitute an environmental reservoir of virulence genes. Although the definitive roles of the virulence-associated factors in the environment, and the environmental selection pressures for V. cholerae-carrying virulence genes or their homologues is not clear, the potential for origination of new epidemic strains from environmental progenitors seems real. It is likely that the aquatic environment harbors different virulence-associated genes scattered among environmental vibrios, which possess a lower virulence potential than the epidemic strains. The ecosystem comprising the aquatic environment, V. cholerae, genetic elements mediating gene transfer, and the mammalian host appears to support the clustering of critical virulence genes in a proper combination leading to the origination of new V. cholerae strains with epidemic potential.     

Repression of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-associated proteins

The Hha/YmoA family of nucleoid-associated proteins is involved in gene regulation in enterobacteria. In Salmonella enterica serovar Typhimurium, virulence genes required for intracellular growth are induced following host cell invasion but the proteins responsible for repressing these genes prior to host cell entry have not been fully identified. We demonstrate here that Hha is the major repressor responsible for silencing virulence genes carried in Salmonella pathogenicity island 2 prior to bacteria sensing an intracellular environmental cue.     

Evolving ideas about genetics underlying insect virulence to plant resistance in rice-brown planthopper interactions

Many plant-parasite interactions that include major plant resistance genes have subsequently been shown to exhibit features of gene-for-gene interactions between plant Resistance genes and parasite Avirulence genes. The brown planthopper (BPH) Nilaparvata lugens is an important pest of rice (Oryza sativa). Historically, major Resistance genes have played an important role in agriculture. As is common in gene-for-gene interactions, evolution of BPH virulence compromises the effectiveness of singly-deployed resistance genes. It is therefore surprising that laboratory studies of BPH have supported the conclusion that virulence is conferred by changes in many genes rather than a change in a single gene, as is proposed by the gene-for-gene model. Here we review the behaviour, physiology and genetics of the BPH in the context of host plant resistance. A problem for genetic understanding has been the use of various insect populations that differ in frequencies of virulent genotypes. We show that the previously proposed polygenic inheritance of BPH virulence can be explained by the heterogeneity of parental populations. Genetic mapping of Avirulence genes indicates that virulence is a monogenic trait. These evolving concepts, which have brought the gene-for-gene model back into the picture, are accelerating our understanding of rice-BPH interactions at the molecular level.     

Derivation of a physical map of the chromosome of Bordetella pertussis Tohama I.

We have used pulsed-field gel electrophoresis to derive a restriction map of the chromosome of Bordetella pertussis for the enzymes XbaI, SpeI, PacI, and PmeI, which cleave 25, 16, 2, and 1 times, respectively. The apparent size of the genome is 3,750 kb. The positions of genes for major virulence determinants in the vir regulon and of some housekeeping genes were determined. Apart from the previously known linkage of the vir and fha loci, no significant linkage of virulence genes was demonstrated.     

Antimicrobials: targeting virulence genes necessary for intracellular multiplication

Intracellular bacteria constitute a major class of pathogens for humans and animals. Their pathogenicity is linked to their ability to multiply inside a host cell. A set of virulence genes (virulome) is required for this intracellular lifestyle. Recent studies have shown that blocking the enzymes encoded by these virulence genes impairs intracellular multiplication of the pathogen. These specific factors could constitute a new set of possible targets for antimicrobial drugs. The potential advantages, pitfalls and challenges of a strategy that targets these virulence factors are discussed.     

Sialic acid mediated transcriptional modulation of a highly conserved sialometabolism gene cluster in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and its effect on virulence

Background  

Sialic acid has been shown to be a major virulence determinant in the pathogenesis of otitis media caused by the bacterium Haemophilus influenzae. This study aimed to characterise the expression of genes required for the metabolism of sialic acid and to investigate the role of these genes in virulence.     

Sequence analysis of the genes encoding for the major virulence factors of Bacillus anthracis vaccine strain 'Carbosap'

AIMS: This study was performed to analyse the molecular characteristics of genes encoding for the major virulence factors in Bacillus anthracis vaccine strain 'Carbosap' compared with the wild B. anthracis strain, to evaluate the basis of attenuation. METHODS AND RESULTS: The molecular characteristics of the B. anthracis 'Carbosap' vaccine strain, used as vaccine in Italy, were analysed in comparison with a B. anthracis virulent strain. Despite the presence of the two virulence plasmids pXO1 and pXO2, the 'Carbosap' strain proved to be protective for cattle. The presence of the regulatory genes atxA and pagR and the gerX operon, known to be involved in the virulence, was verified. In addition, all genes were sequenced. The results showed that no molecular differences between 'Carbosap' and the virulent strain were evident. CONCLUSIONS: The results of this study indicate that the attenuation of the 'Carbosap' vaccine strain is not due to the lack of virulence genes or to modifications occurring on the sequence of these genes. Therefore, other virulence factors, still unknown, could be involved in the pathogenic mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper adds new information regarding the molecular characteristics of the vaccine strain 'Carbosap' and highlights the need to better understand the virulence factors involved in the pathogenicity of B. anthracis strains.     

Prevalence of major virulence genes among various Vibrio cholerae El-TOR strains for evaluating their epidemiological significance

Specific oligonucleotide primers were chosen for identifying the fragments of the four major virulence genes of V. cholerae eltor (ctxA, tcpA, toxR, and hap) using the polymerase chain reaction (PCR). In order to estimate the efficiency of complex PCR testing of V. cholerae for evaluation of their epidemiological significance, a collection of 80 V. cholerae eltor strains with known virulence was selected, whose most important specific features had been studied previously. The hap was appropriate species-specific gene making it possible to detect V. cholerae strains regardless of their virulence. The most complete and objective data for evaluating the epidemic significance can be obtained by detecting the presence of three virulence genes (ctxA, tcpA, and toxR) in their chromosome. The prevalence of the above four genes in various V. cholerae strains isolated from the environment during epidemic and non-epidemic periods was studied.     

Virulence gene regulation by peptides in staphylococci and other Gram-positive bacteria

In staphylococci, autoinducing peptides activate agr. a global regulator of the expression of genes encoding virulence factors and other exoproteins. During the past year, there have been major advances in the structure-function analysis of these peptides and the regulation of a virulence factor by an autoinducing peptide in pneumococci has been demonstrated.     

Monitoring virulence in Asian rice gall midge populations in India

The Asian rice gall midge, Orseolia oryzae (Wood‐Mason) (Diptera: Cecidomyiidae), is a major pest of rice [Oryza sativa L. (Poaceae)] in India. Breeding resistant varieties and their cultivation has been the main approach to manage this pest. However, the breakdown of resistance conferred by the major genes, deployed one at a time, through evolution of virulent biotypes has become a major setback to this approach. Development of polymerase chain reaction‐based molecular markers for eight of the 10 resistance genes and their possible use in marker‐assisted selection has enabled breeders to pyramid resistance genes for achieving durable resistance. However, the choice of resistance genes needs to be made with a better understanding of the virulence composition of the pest populations in the target area and the genetics of plant resistance and insect virulence, as the rice–gall midge interaction is a gene‐for‐gene one. We adopted a single‐female test and coupled it with a modified F₂ screen test to note the virulence composition of gall midge populations and estimated the frequency of virulence alleles for adaptation at three pest endemic locations in India, namely, Warangal, Ragolu, and Raipur. Results on biotype composition showed heterogeneous pest populations in all the tests and at all the locations. Tests at Warangal repeated after 8 years showed a rapid increase in frequency of the virulence allele conferring adaptation to the plant resistance gene Gm2 as compared to that of the allele for adaptation to the resistance gene Gm1. This is probably the first direct measurement of a durability parameter of plant genes conferring insect resistance. Results supported earlier observations that sex‐linked virulence against Gm2 makes it less durable. The sex ratio did not deviate from the expected 1:1 ratio at Warangal, but at Ragolu females outnumbered males.     

Variations in the distribution of genes encoding virulence and extracellular proteins in group A streptococcus are largely restricted to 11 genomic loci

Group A streptococcus (GAS) is a human pathogen associated with a wide range of human diseases that vary in symptoms and clinical severity. In this report we describe the use of a targeted low density array representing genes encoding classical virulence factors, purported virulence factors and other extracellular proteins to examine differences in the genetic profiles of 68 clinical GAS isolates. Of the 226 genes on the array (encoding 217 virulence factors or putative extracellular proteins and nine positive control house-keeping proteins) 62 had distributions that were statistically associated with specific GAS M-types. While 32 of these genes were bacteriophage related, the remaining 30 have not previously been described as bacteriophage associated. We show that these 'non-bacteriophage related' genes are found in 11 loci located in five greater chromosomal regions, often near classical GAS virulence factors, and often accompanied by genes associated with mobile genetic elements (MGEs). Many of these loci also demonstrated genetic variation within strains of the same M-type, suggesting these regions to be recombinatorial and mutational hotspots. Evidence for acquisition of genes from other species is also apparent in these loci. Our data suggests that imprecise recombination events involving MGEs not only result in acquisition of new genes, but can also result in deletion of flanking chromosomal genes. Thus MGE related events would appear to be the major contributor to variation of discrete virulence loci, which could account for the disease causing propensity of individual strains. We believe that profiling of the 11 loci could be a meaningful tool in epidemiological GAS typing studies.     

Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.     

Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC

Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC’s survival in the host’s low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.     

Quantitative trait locus (QTL) mapping reveals a role for unstudied genes in Aspergillus virulence

Infections caused by the fungus Aspergillus are a major cause of morbidity and mortality in immunocompromised populations. To identify genes required for virulence that could be used as targets for novel treatments, we mapped quantitative trait loci (QTL) affecting virulence in the progeny of a cross between two strains of A. nidulans (FGSC strains A4 and A91). We genotyped 61 progeny at 739 single nucleotide polymorphisms (SNP) spread throughout the genome, and constructed a linkage map that was largely consistent with the genomic sequence, with the exception of one potential inversion of ～527 kb on Chromosome V. The estimated genome size was 3705 cM and the average intermarker spacing was 5.0 cM. The average ratio of physical distance to genetic distance was 8.1 kb/cM, which is similar to previous estimates, and variation in recombination rate was significantly positively correlated with GC content, a pattern seen in other taxa. To map QTL affecting virulence, we measured the ability of each progeny strain to kill model hosts, larvae of the wax moth Galleria mellonella. We detected three QTL affecting in vivo virulence that were distinct from QTL affecting in vitro growth, and mapped the virulence QTL to regions containing 7-24 genes, excluding genes with no sequence variation between the parental strains and genes with only synonymous SNPs. None of the genes in our QTL target regions have been previously associated with virulence in Aspergillus, and almost half of these genes are currently annotated as "hypothetical". This study is the first to map QTL affecting the virulence of a fungal pathogen in an animal host, and our results illustrate the power of this approach to identify a short list of unknown genes for further investigation.