Comparative analysis of Vibrio cholerae isolates from Ghana reveals variations in genome architecture and adaptation of outbreak and environmental strains

Abstract

Recurrent epidemics of cholera denote robust adaptive mechanisms of Vibrio cholerae for ecological shifting and persistence despite variable stress conditions. Tracking the evolution of pathobiological traits requires comparative genomic studies of isolates from endemic areas. Here, we investigated the genetic differentiation among V. cholerae clinical and environmental isolates by highlighting the genomic divergence associated with gene decay, genome plasticity, and the acquisition of virulence and adaptive traits. The clinical isolates showed high phylogenetic relatedness due to a higher frequency of shared orthologs and fewer gene variants in contrast to the evolutionarily divergent environmental strains. Divergence of the environmental isolates is linked to extensive genomic rearrangements in regions containing mobile genetic elements resulting in numerous breakpoints, relocations, and insertions coupled with the loss of virulence determinants acf, zot, tcp, and ctx in the genomic islands. Also, four isolates possessed the CRISPR-Cas systems with spacers specific for Vibrio phages and plasmids. Genome synteny and homology analysis of the CRISPR-Cas systems suggest horizontal acquisition. The marked differences in the distribution of other phage and plasmid defense systems such as Zorya, DdmABC, DdmDE, and type-I Restriction Modification systems among the isolates indicated a higher propensity for plasmid or phage disseminated traits in the environmental isolates. Our results reveal that V. cholerae strains undergo extensive genomic rearrangements coupled with gene acquisition, reflecting their adaptation during ecological shifts and pathogenicity.

Keywords: CRISPR-Cas system; Vibrio cholerae; genetic recombination; genome plasticity; phage and plasmid defense; pseudogenes.

FIGURE 1

Ortholog and SNP-based phylogeny of the V. cholerae isolates. (A) Combined whole genome phylogenetic tree and Ortholog-based matrix showing the relatedness and divergence of the isolates based on the ortholog assignment of genes. (B) SNP-based phylogenetic tree and matrix showing the relationship between the clinical and environmental isolates. Included in the analysis are the V. cholerae strains N16961, VC1374, O395, GXFL-4, VC-hf7, SA3G, and O77 RIMD genomes obtained from the NCBI datasets.

FIGURE 2

Virulence genes in V. cholerae isolates. (A) Schematic gene organization and alignments of the region containing the acf and tcp gene clusters in the N16961, SA3G, and the environmental isolates E30 and E32. The E30 and E32 share high similarities (between 93 and 100%) in gene organization around the location containing the virulence gene clusters in the reference strain. (B) Gene organization and alignment of the zot, ctxAB region in N16961, SA3G and the environmental isolates E30 and E32. (C) The gene order and alignment of the zot and ctx gene clusters in E4, E19, S35, S1, N16961, and O77 RIMD. The absence of the zot and ctx genes and disruption in synteny around the genomic regions is observed in the E30 and E32 isolates. The decay in the gene order in the zot and ctx region compared to the reference strain is shown by discontinuous regions of genomic fragments aligned above and below the N16961 gene cluster. The grayscale represents nucleotide homology.

FIGURE 3

Virulence genes in V. cholerae isolates. (A) The gene organization and alignment of the acf and tcp region in the N16961, O77 RIMD and the environmental isolates S1 and E4. (B) Genetic organization of the tcp and acf gene cluster region in N16961, VC-hf7 and the isolates E19 and S35. The grayscale bars show the nucleotide homology in the aligned regions. Insertion elements, DNA metabolism genes, the phage sequences are in green.

FIGURE 4

Homologous recombination events detected in V. cholerae strains. The phylogenetic tree is generated from the number of SNPs. The homologous recombination events are shown by blocks, which are colored blue for unique events in strains and red for events occurring in multiple strains. The recombination hotspots are marked at the top of the plot.

FIGURE 5

The CRISPR-Cas system in V. cholerae isolates. (A) The CRISPR-Cas system and alignment of the region in the clinical isolate C6, V. cholerae 0395, and the N16961. The C6 harbors a type I-E CRISPR-cas system homologous to the one in V. cholerae 0395 with similar GC% patterns of the cas genes. The region flanking the CRISPR-Cas system in the C6 is conserved in the N16961. (B) Genetic organization of the CRISPR-Cas loci in the environmental isolate E4 and E19 and their alignment with V. cholerae RFB05 and Shewanella sp SNU WT4. The type I-F CRISPR-Cas system in E4 is homologous to the one in V. cholerae RFB05. The type I-F CRISPR-Cas system in the E19 is homologous to the one in Shewanella sp. SNU WT4 and shares almost similar GC%, which is anomalously low and probably acquired by lateral gene transfer (%GC of Cas genes in E19 is 41% compared to the overall GC% of 47.7% in E19. The grayscale bars show the nucleotide homology in the aligned regions.

FIGURE 6

(A) Genetic organization of the CRISPR-Cas system identified in the V. cholerae isolate S1. The Tn7- like transposon regions with the associated cargo carrying the mini type I-F CRISPR-Cas system in both V. cholerae isolate S1 and the Vibrio sp 2521_89 were aligned and compared using BLAST and EasyFig. The Tn7 element is inserted into the site downstream of the signal recognition particle RNA (srp_RNA). (B) The distribution of phage defense mechanisms in the clinical and environmental vibrio isolates. (C) Schematic of the gene cluster containing the phage and plasmid defense components commonly found in VP_II. The gene order is representative of the clinical isolates from this study and the N16961. (D) Schematic of the ddmABC gene cluster in the V. cholerae clinical isolates.