Comparative Genomic Analysis of Shrimp-Pathogenic Vibrio parahaemolyticus LC and Intraspecific Strains with Emphasis on Virulent Factors of Mobile Genetic Elements

Abstract

Vibrio parahaemolyticus exhibits severe pathogenicity in humans and animals worldwide. In this study, genome sequencing and comparative analyses were conducted for in-depth characterization of the virulence factor (VF) repertoire of V. parahaemolyticus strain LC, which presented significant virulence to shrimp Litopenaeus vannamei. Strain LC, harboring two circular chromosomes and three linear plasmids, demonstrated ≥98.14% average nucleotide identities with 31 publicly available V. parahaemolyticus genomes, including 13, 11, and 7 shrimp-, human-, and non-pathogenic strains, respectively. Phylogeny analysis based on dispensable genes of pan-genome clustered 11 out of 14 shrimp-pathogenic strains and 7 out of 11 clinical strains into two distinct clades, indicating the close association between host-specific pathogenicity and accessory genes. The VFDB database revealed that 150 VFs of LC were mainly associated with the secretion system, adherence, antiphagocytosis, chemotaxis, motility, and iron uptake, whereas no homologs of the typical pathogenic genes pirA, pirB, tdh, and trh were detected. Four genes, mshB, wbfT, wbfU, and wbtI, were identified in both types of pathogenic strains but were absent in non-pathogens. Notably, a unique cluster similar to Yen-Tc, which encodes an insecticidal toxin complex, and diverse toxin-antitoxin (TA) systems, were identified on the mobile genetic elements (MGEs) of LC. Conclusively, in addition to the common VFs, various unique MGE-borne VFs, including the Yen-Tc cluster, TA components, and multiple chromosome-encoded chitinase genes, may contribute to the full spectrum of LC virulence. Moreover, V. parahaemolyticus demonstrates host-specific virulence, which potentially drives the origin and spread of pathogenic factors.

Keywords: Vibrio parahaemolyticus; comparative genomics; mobile genetic element; shrimp; vibriosis; virulence factor.

Figure 1

The mortality rates of Litopenaeus vannamei injected with low and high dosages of V. parahaemolyticus LC (a), and the mortality rates of L. vannamei postlarvae subjected to immersion infection with LC at low and high dosage (b).

Figure 2

Average nucleotide identity analysis of genomes among V. parahaemolyticus LC with 31 intraspecific strains as well as two reference strains from V. alginolyticus and V. harveyi.

Figure 3

The phylogenomic trees were constructed based on the core genome of concatenated alignments of 3600 single-copy genes among 32 strains of Vibrio parahaemolyticus using the maximum likelihood method (a), and based on pangenome according to the presence/absence of 11,104 accessory genes using RAxML (b). Note: The scale bar indicates the number of substitutions per site (a) or the distance per 100 genes difference (b). Red circle: shrimp-pathogenic strains; blue circle: human-pathogenic strains; green circle: non-pathogenic strains. The abbreviations of CHN, USA, IND, MAL, KOR, JAP, and THA denote the strains originating from China, the United States, India, Malaysia, Korea, Japan, and Thailand, respectively.

Figure 4

The circular comparison images of V. parahaemolyticus LC (central reference) with six closely related strains (2210633, S01, 02-3, 12-1, 01-1 and 08-1), regarding the large chromosome (a) and the small chromosome (b).

Figure 5

Comparison of the sequence identity and gene contents of GI-5 from V. parahaemolyticus LC with the syntenic regions from strains 2210633, S01, 02-3, 12-1, 01-1, and 08-1. Note: Each gene is displayed by a horizontal arrow in the direction of its coding strand (to scale), and color-coded using the designated key of the six classes, as follows: virulence factor (red), integrase/transposase/recombinase (blue), acetyltransferase domain/family (orange), other enzyme/domain (yellow), general functional gene (green), and hypothetical protein (light gray).

Figure 6

Comparison of the sequence identity and gene contents of p96 from V. parahaemolyticus LC with plasmids from strains 01-1, 08-1, and 12-1. Note: Each gene is displayed by a horizontal arrow in the direction of its coding strand (to scale) and color-coded using the designated key of the seven classes, as follows: virulence factor (red); integrase/transposase/recombinase (blue), conjugative transfer component (light pink), type II/III/IV secretion system (orange), hypothetical protein (light grey), other enzyme/domain (yellow); general functional gene/protein (green).

Figure 7

Comparison of the sequence identity and gene contents of p71 from V. parahaemolyticus LC with plasmid from strain 01-1, and with the chromosome-borne Tc cluster from strains 02-3 and S01. Note: Each gene is displayed by a horizontal arrow in the direction of its coding strand (to scale) and color-coded using the designated key of six classes, as follows: toxin complex genes (red), integrase/transposase/recombinase (blue), conjugative transfer component (light pink), other enzyme/domain (yellow), general functional gene (green), hypothetical protein (light grey).