Exploring the Genomic Traits of Non-toxigenic Vibrio parahaemolyticus Strains Isolated in Southern Chile

Abstract

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism.

Keywords: RTX; Vibrio parahaemolyticus; Zot; accessory genome; genomic island; non-toxigenic; prophage; virulence.

FIGURE 1

Pan-genome and phylogenetic tree of V. parahaemolyticus strains. (A) Dynamics of pan-, core, and accessory genes found by progressive addition of V. parahaemolyticus genomes. (B) Maximum-likelihood tree obtained from a concatenated nucleotide sequence alignment of the orthologous core genes (3,943 genes) for the V. parahaemolyticus strains. The VpKX strain was included in the tree. Bootstrap values < 80% are not shown. The horizontal bar at the base of the figure represents 0.001 substitutions per nucleotide site.

FIGURE 2

Comparative analysis of the genomic context of the T3SS1 gene cluster in V. parahaemolyticus strains. DNA-based comparison of the T3SS1 gene cluster of strains VpKX, PMA2.15, PMA1.15, PMA14.14, PMC54.13, and PMC53.7. The region encoding the putative EmrAB efflux pump and EmrR regulator is highlighted in a box. BlastN analysis was performed using WebACT and displayed with the ACT software.

FIGURE 3

Comparative analysis of the T3SS2 gene clusters identified in strain PMA1.15 of V. parahaemolyticus. Translated DNA-based comparison of the T3SS2 gene cluster of PMA1.15 and the T3SS2-α and T3SS2-β of the VpKX and TH3996 strains of V. parahaemolyticus, respectively. TBlastX analysis was performed using WebACT and displayed with the ACT software.

FIGURE 4

Schematic representation of accessory elements carrying virulence or fitness factors in the V. parahaemolyticus strains. (A) Genomic islands in V. parahaemolyticus strains PMC 53.7, PMC 54.13, PMA 1.15, PMA 2.15, and PMA 3.15. Position of GIs and prophage-like element are shown in Tables 4, 5, respectively. The colors were assigned according to the possible role of each ORF as shown in the figure. (B) Prophage-related elements in V. parahaemolyticus strains PMC 53.7, PMA 2.15, PMA 3.15, and PMA 1.15 that encode a gene related to zonula occludens toxin (zot) or RTX toxin.

FIGURE 5

Phylogeny of Zot toxin sequences in Vibrio species. Phylogenetic tree based on Zot protein sequences found in diverse Vibrio species. Maximum-likelihood method, 100 bootstrap values.

FIGURE 6

Diversity of Zot toxin sequences in Vibrio species. Alignment of Zot amino acid sequences found in V. parahaemolyticus strains and V. cholerae as outgroup.

FIGURE 7

The cytotoxic effect of V. parahaemolyticus strains in Caco-2 cells. The release of lactate dehydrogenase (LDH) into the medium was measured by optical density. Different letters indicate as follows: a, medium cytotoxicity; b, medium-high cytotoxicity; c, high cytotoxicity; and d, very high cytotoxicity. ANOVA post hoc Bonferroni (p < 0.05).