Vibrio cholerae embraces two major evolutionary traits as revealed by targeted gene sequencing (original) (raw)

Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1,269 bp) or luxR-hchA (LH) (~1,600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species. Vibrio cholerae is a gram-negative bacterial pathogen responsible for cholera, and several million cholera cases including 21,000-143,000 deaths occur worldwide each year 1. Serological grouping of V. cholerae has identified up to 206 O-serogroups 2. Epidemic/pandemic cholera is typically ascribed to serogroup O1; however, in 1992, a novel serogroup O139 V. cholerae caused outbreaks in Asian countries 3. V. cholerae carries several virulence-related genes to provoke pathogenic processes in the infected hosts. The key virulence factors of serogroups O1 and O139 include cholera toxin (CT), which is responsible for profuse watery diarrhea, and a pilus colonization factor known as toxin-coregulated pilus (TCP). Although most non-O1/non-O139 or environmental isolates of V. cholerae do not produce CT and lack the cholera toxin genes, some strains possess heat-stable enterotoxin (Stn) 4 , hemolysin (HlyA) 5,6 , repeat in toxin (RTX) 7 , Cholix toxin (ChxA) 8,9 , hemagglutinin protease (HAP) 10 , type 6 secretion system (T6SS) 11 , or type III secretion system (TTSS) 12. However, the pathogenic mechanisms of these isolates remain to be elucidated. High throughput sequencing facilitates the rapid and accurate identification of virulence factors of pathogenic bacteria, and can be used to identify the pathways of infectious disease transmission 13-15. Although genomic technologies are rapidly evolving, their widespread implementation in clinical microbiology laboratories and for monitoring public health is limited owing to the need for effective semi-automated pipelines, standardized quality control and data interpretation, bioinformatics expertise, and infrastructure 16. Relatedness and differences among V. cholerae isolates have been investigated by several molecular fingerprinting methods for a prolonged duration 17. Pulsed field gel electrophoresis (PFGE) has been used frequently for typing of the O1 and O139 serogroups of V. cholerae 18,19. Although PFGE is highly reproducible and its discriminatory power is sufficiently high, it is laborious, and is limited with regard to intra-and inter-laboratory comparison compared to sequence-based methods 17,20. Multilocus sequence typing (MLST) overcomes the poor portability of traditional and older molecular typing approaches 20. It is a technique whereby several internal control genes (loci) are sequenced, and