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ABSTRACT Vibrio parahaemolyticus (Vpar) is a major cause of seafood-related gastroenteritis. A pandemic strain, RIMD2210633 (RIMD), has emerged from ancestors due to virulence mechanisms acquired through horizontal gene transfer (HGT). Mobile genetic elements play an essential role in RIMD pathogenicity, but they evolve rapidly and are challenging for sequence-based homology detection and functional inference. Guided by the Evolutionary Classification of protein Domains (ECOD), we utilized structure-assisted homology detection of high-quality AlphaFold models for RIMD proteins to augment their functional interpretation. This approach detected 7,107 domains from 3,970 RIMD proteins and classified them into the ECOD hierarchy, providing functional insights into 80% of the proteome. RIMD proteins originating from HGT were identified by comparison against 86 complete proteomes of other Vpar strains. The resulting RIMD mobilome is enriched with phage markers and bacterial defense domains. Evolutionary classification of known HGT proteins suggests they provide selective advantages against the human host, phage, and other stresses. Compared to a pre-pandemic strain, we found fast-evolving proteins with potential involvement in RIMD virulence and phage avoidance. These findings provide vital insights into bacterial evolution, pathogenic mechanisms, and adaptation strategies while offering testable hypotheses for experimental studies and an online resource for future investigation of the RIMD proteome.IMPORTANCEThe pandemic Vpar strain RIMD causes seafood-borne illness worldwide. Previous comparative genomic studies have revealed pathogenicity islands in RIMD that contribute to the success of the strain in infection. However, not all virulence determinants have been identified, and many of the proteins encoded in known pathogenicity islands are of unknown function. Based on the EOCD database, we used evolution-based classification of structure models for the RIMD proteome to improve our functional understanding of virulence determinants acquired by the pandemic strain. We further identify and classify previously unknown mobile protein domains as well as fast evolving residue positions in structure models that contribute to virulence and adaptation with respect to a pre-pandemic strain. Our work highlights key contributions of phage in mediating seafood born illness, suggesting this strain balances its avoidance of phage predators with its successful colonization of human hosts.

It has been proposed that bacterial membrane proteins may be produced via ‘transertion’, or concurrent transcription, translation and membrane insertion from membrane-associated genes. Here, Kaval et al. provide evidence supporting that Vibrio parahaemolyticus uses transertion to assemble a transmembrane complex (type III secretion system) used to inject virulence factors into host cells.

ABSTRACT Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase deamidating activity of VopC, a type 3 secretion system (T3SS) translocated effector, drives V. parahaemolyticus invasion. The intracellular pathogen uropathogenic Escherichia coli (UPEC) invades host cells by secreting a VopC homolog, the secreted toxin cytotoxic necrotizing factor 1 (CNF1). Because of the homology between VopC and CNF1, we hypothesized that topical application of CNF1 during V. parahaemolyticus infection could supplement VopC activity. Here, we demonstrate that CNF1 improves the efficiency of V. parahaemolyticus invasion, a bottleneck in V. parahaemolyticus infection, across a range of doses. CNF1 increases V. parahaemolyticus invasion independent of both VopC and the T3SS altogether but leaves a disproportionate fraction of intracellular bacteria unable to escape the endosome and complete their infection cycle. This phenomenon holds true in the presence or absence of VopC but is particularly pronounced in the absence of a T3SS. The native VopC, by contrast, promotes a far less efficient invasion but permits the majority of internalized bacteria to escape the endosome and complete their infection cycle. These studies highlight the significance of enzymatic specificity during infection, as virulence factors (VopC and CNF1 in this instance) with similarities in function (bacterial uptake), catalytic activity (deamidation), and substrates (Rho GTPases) are not sufficiently interchangeable for mediating a successful invasion for neighboring bacterial pathogens. IMPORTANCE Many species of intracellular bacterial pathogens target host small Rho GTPases to initiate invasion, including the human pathogens Vibrio parahaemolyticus and uropathogenic Escherichia coli (UPEC). The type three secretion system (T3SS) effector VopC of V. parahaemolyticus promotes invasion through the deamidation of Rac1 and CDC42 in the host, whereas the secreted toxin cytotoxic necrotizing factor 1 (CNF1) drives UPEC’s internalization through the deamidation of Rac1, CDC42, and RhoA. Despite these similarities in the catalytic activity of CNF1 and VopC, we observed that the two enzymes were not interchangeable. Although CNF1 increased V. parahaemolyticus endosomal invasion, most intracellular V. parahaemolyticus aborted their infection cycle and remained trapped in endosomes. Our findings illuminate how the precise biochemical fine-tuning of T3SS effectors is essential for efficacious pathogenesis. Moreover, they pave the way for future investigations into the biochemical mechanisms underpinning V. parahaemolyticus endosomal escape and, more broadly, the regulation of successful pathogenesis.