VicPred: A Vibrio cholerae Genotype Prediction Tool

Genomic information can be used to predict major pathogenic traits of pathogens without the need for laboratory experimentation. However, no Vibrio cholerae genome-based trait identification tools currently exist. The aim of this study was to develop a web-based prediction tool to identify Vibrio pathogenic traits using publicly available 796 whole-genome sequences of V. cholerae. Using this application, 68 structural O-antigen gene clusters belonging to 49 serogroups of V. cholerae were classified, and the composition of the genes within the O-antigen cluster of each serogroup was identified. The arrangement and location of the CTX prophage and related elements of the seventh cholera pandemic strains were also revealed. With the versatile tool, named VicPred, we analyzed the assemblage of various SXTs (sulfamethoxazole/trimethoprim resistance element) and major genomic islands (GIs) of V. cholerae, and the increasing trend in drug-resistance revealing high resistance of the V. cholerae strains to certain antibiotics. The pathogenic traits of newly sequenced V. cholerae strains could be analyzed based on these characteristics. The accumulation of further genome data will expedite the establishment of a more precise genome-based pathogenic traits analysis tool.

Molecular insights into the genome dynamics and interactions between core and acquired genomes ofVibrio cholerae

Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome ofVibrio choleraeharbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome ofV. choleraeand examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in theV. choleraechromosome. We have deleted more than 250 acquired genes from 6 different loci in theV. choleraechromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, includingEscherichia coli. In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypassV. choleraeimmunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome ofV. choleraeto remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes.

Prophage BPs Alters Mycobacterial Gene Expression and Antibiotic Resistance

Diseases caused by mycobacteria such as Mycobacterium tuberculosis are the leading cause of death worldwide. With the emergence of strains that are resistant to first-line anti-tuberculosis drugs and naturally drug-resistant pathogens such as M. abscessus, there is a need to increase our understanding of mycobacterial fitness and virulence and identify new targets for drugs. The majority of the pathogenic species of the bacterial genus Mycobacterium, including M. tuberculosis, carry integrated viral genomes (prophages) that are hypothesized to contribute to virulence. Though we know many of the ways in which phage genes directly contribute to pathogenesis, e.g., the CTX prophage encodes the toxin in Vibrio cholera, we know little about the impact of phages that encode no obvious toxin or virulence gene. Using an RNAseq approach, our lab recently showed for the first time that the presence of a prophage alters the expression of 7.4% of genes in the pathogenic mycobacterial species, M. chelonae. The presence of prophage BPs increased the expression of genes in the whiB7 regulon, including whiB7, eis2, and tap, and decreased the expression of a padR-family transcription factor. BP lysogens were more resistant to aminoglycosides (kanamycin and amikacin) and tetracycline than wild-type strains of M. chelonae. In order to determine how the BP prophage drives changes in bacterial gene expression and phenotype, we will test the effects of individual BP genes expressed during lysogeny, such as the immunity repressor, on bacterial gene expression and antibiotic resistance phenotypes.

Novel Cholera Toxin Variant and ToxT Regulon in EnvironmentalVibrio mimicusIsolates: Potential Resources for the Evolution ofVibrio choleraeHybrid Strains

ABSTRACTAtypical El Tor strains ofVibrio choleraeO1 harboring variantctxBgenes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics.Vibrio mimicusoccasionally produces CT, encoded byctxABon CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon inV. mimicusctx-positive (ctx+) strains (i.e.,V. mimicusstrains containingctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novelctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnvarray. Additionally, novel variants oftcpAandtoxT, respectively, showing phylogenetic lineage to a clade ofV. choleraenon-O1 and to a clade ofV. choleraenon-O139, were identified. TheV. mimicusstrains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lackedVibrioseventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats inctxABpromoter region similar to those seen with some classical strains ofV. choleraeO1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all thectx+V. mimicusstrains were clonally related. However, theirin vitroCT production andin vivotoxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmentalV. mimicusstrains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution ofV. choleraehybrid strains.IMPORTANCENatural diversification of CTXФ andctxABgenes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains ofV. cholerae. This report, showing the occurrence of CTXETcomprising a novel variant ofctxABinV. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains ofV. cholerae. Identification and cluster analysis of the newly discovered alleles oftcpAandtoxTsuggest their horizontal transfer from an uncommon clone ofV. cholerae. The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnvand of a CTXФETinV. mimicusprobably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybridV. choleraestrains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

Novel cholera toxin variant and ToxT regulon in environmentalVibrio mimicusstrains: potential resources for the evolution ofVibrio choleraehybrid strains

ABSTRACTAtypical El Tor strains ofVibrio choleraeO1 harboring variantctxBgenes of cholera toxin (CT) are gradually becoming a major cause of recent cholera epidemics.Vibrio mimicusoccasionally contains virulence factors associated with cholera, e.g., CT, encoded byctxABon CTXФ genome; and TCP, the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon inctx+vestrains ofV. mimicusisolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence related-genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novelctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized in an array of RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv. Additionally, a novel variant oftcpAandtoxTrespectively, showing clonal lineage to a phylogenetic clade ofV. choleraenon-O1/O139, was identified. TheV. mimicusstrains lacked the RTX and TLC elements, andVibrioseventh pandemic islands of the El Tor strains, but contained five heptamer (TTTTGAT) repeats inctxABpromoter region like some classical strains ofV. choleraeO1. PFGE analysis showed all thectx+veV. mimicusstrains were clonally related. However, theirin vitroCT production andin vivotoxigenecity were variable, which could be explained by differential transcription of virulence genes along with ToxR regulon. Taken together, our findings strongly suggest that environmentalV. mimicusstrains act as potential reservoir of atypical virulence factors, including variant CT and ToxT regulon, and may contribute to the evolution ofV. choleraehybrid strains.IMPORTANCENatural diversification of CTXФ andctxABgenes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains ofV. cholerae. This study showing the occurrence of CTXETcomprising a novel variant ofctxABinV. mimicuspoints out a previously unnoticed evolutionary event, independent to that of the El Tor strains ofV. cholerae. Identification and cluster analysis of the newly-discovered alleles oftcpAandtoxTindicates their horizontal transfer from an uncommon clone ofV. cholerae. The genomic content of ToxT regulon, and tandemly arranged multiple pre-CTXФEnvand a CTXФETinV. mimicusprobably act as salient raw materials inducing natural recombination among the hallmark virulence genes of hybridV. choleraestrains. This study will facilitate deeper understanding of the evolution of new variant CT and ToxT regulon, influencing cholera epidemiology.

RS1 Satellite Phage Promotes Diversity of Toxigenic Vibrio cholerae by Driving CTX Prophage Loss and Elimination of Lysogenic Immunity

ABSTRACTIn El Tor biotype strains of toxigenicVibrio cholerae, the CTXϕ prophage often resides adjacent to a chromosomally integrated satellite phage genome, RS1, which produces RS1ϕ particles by using CTX prophage-encoded morphogenesis proteins. RS1 encodes RstC, an antirepressor against the CTXϕ repressor RstR, which cooperates with the host-encoded LexA protein to maintain CTXϕ lysogeny. We found that superinfection of toxigenic El Tor strains with RS1ϕ, followed by inoculation of the transductants into the adult rabbit intestine, caused elimination of the resident CTX prophage-producing nontoxigenic derivatives at a high frequency. Further studies usingrecAdeletion mutants and a clonedrstCgene showed that the excision event wasrecAdependent and that introduction of additional copies of the clonedrstCgene instead of infection with RS1ϕ was sufficient to enhance CTXϕ elimination. Our data suggest that once it is excised from the chromosome, the elimination of CTX prophage from host cells is driven by the inability to reestablish CTXϕ lysogeny while RstC is overexpressed. However, with eventual loss of the additional copies ofrstC, the nontoxigenic derivatives can act as precursors of new toxigenic strains by acquiring the CTX prophage either through reinfection with CTXϕ or by chitin-induced transformation. These results provide new insights into the role of RS1ϕ inV. choleraeevolution and the emergence of highly pathogenic clones, such as the variant strains associated with recent devastating epidemics of cholera in Asia, sub-Saharan Africa, and Haiti.