Complex regulatory networks of virulence factors in Vibrio vulnificus

Vibrio vulnificus produces various virulence factors that enhance its fitness in changing environments as well as host cell-damaging activities and inflammatory responses during infection.The spatiotemporal regulation of virulence factors is coordinated by numerous transcription factors that integrate diverse environmental signals such as nutrient availability, bacterial cell density, and antimicrobial agents.Small molecules that inhibit the activity of virulence-associated transcription factors can significantly attenuate the virulence phenotypes of V. vulnificus without affecting its in vitro growth.A comprehensive understanding of virulence regulation would allow us to determine effective control targets for the development of sustainable antivirulence therapies to combat Vibrio infection. The fulminating zoonotic pathogen Vibrio vulnificus is the causative agent of fatal septicemia in humans and fish, raising tremendous economic burdens in healthcare and the aquaculture industry. V. vulnificus exploits various virulence factors, including biofilm-related factors and exotoxins, for its persistence in nature and pathogenesis during infection. Substantial studies have found that the expression of virulence factors is coordinately regulated by numerous transcription factors that recognize the changing environments. Here, we summarize and discuss the recent discoveries of the physiological roles of virulence factors in V. vulnificus and their regulation by transcription factors in response to various environmental signals. This expanded understanding of molecular pathogenesis would provide novel clues to develop an effective antivirulence therapy against V. vulnificus infection. The fulminating zoonotic pathogen Vibrio vulnificus is the causative agent of fatal septicemia in humans and fish, raising tremendous economic burdens in healthcare and the aquaculture industry. V. vulnificus exploits various virulence factors, including biofilm-related factors and exotoxins, for its persistence in nature and pathogenesis during infection. Substantial studies have found that the expression of virulence factors is coordinately regulated by numerous transcription factors that recognize the changing environments. Here, we summarize and discuss the recent discoveries of the physiological roles of virulence factors in V. vulnificus and their regulation by transcription factors in response to various environmental signals. This expanded understanding of molecular pathogenesis would provide novel clues to develop an effective antivirulence therapy against V. vulnificus infection. V. vulnificus is a Gram-negative bacterium constituting the normal microbiota of marine and estuarine waters worldwide [1.Baker-Austin C. Oliver J.D. Vibrio vulnificus: new insights into a deadly opportunistic pathogen.Environ. Microbiol. 2018; 20: 423-430Crossref PubMed Scopus (129) Google Scholar,2.Baker-Austin C. et al.Vibrio spp. infections.Nat. Rev. Dis. Primers. 2018; 4: 8Crossref PubMed Scopus (297) Google Scholar]. This opportunistic human pathogen proliferates in warmer months and easily accumulates in molluscan shellfish that become a source of infection. In this regard, V. vulnificus infection occurs through the consumption of contaminated seafood, especially oysters, or the exposure of open wounds to contaminated water or seafood, resulting in fatal primary septicemia and wound infection, respectively. The mortality rate of V. vulnificus infection is the highest among those of foodborne pathogens, which accounts for 95% of seafood-related deaths in the USA [3.Jones M.K. Oliver J.D. Vibrio vulnificus: disease and pathogenesis.Infect. Immun. 2009; 77: 1723-1733Crossref PubMed Scopus (509) Google Scholar]. Accordingly, V. vulnificus has the highest per-case economic impact of all foodborne diseases [4.Hoffmann S. Ahn J.W. Updating Economic Burden of Foodborne Diseases Estimates for Inflation and Income Growth. U.S. Department of Agriculture, Economic Research Service, 2021https://ageconsearch.umn.edu/record/316343Google Scholar]. Furthermore, V. vulnificus can infect some fish that subsequently develop diseases with symptoms including hemorrhagic septicemia and death [5.Hernandez-Cabanyero C. Amaro C. Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus.Environ. Microbiol. 2020; 22: 4133-4148Crossref PubMed Scopus (15) Google Scholar]. Thus, V. vulnificus outbreaks in fish farms are responsible for substantial losses in the aquaculture industry. Recently, the geographic distribution of V. vulnificus and the incidence of infection have increased, which presumably results from global warming and significant rises in seawater temperature [1.Baker-Austin C. Oliver J.D. Vibrio vulnificus: new insights into a deadly opportunistic pathogen.Environ. Microbiol. 2018; 20: 423-430Crossref PubMed Scopus (129) Google Scholar,6.Baker-Austin C. et al.Non-cholera vibrios: the microbial barometer of climate change.Trends Microbiol. 2017; 25: 76-84Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar]. Therefore, there is a growing need for developing effective control strategies that inhibit the virulence of V. vulnificus. To this end, a comprehensive understanding of the molecular pathogenesis of the pathogen should be pursued. This review aims to highlight the recent advances in the study of the physiological roles of virulence factors in V. vulnificus and their spatiotemporal regulation by multiple transcription factors. V. vulnificus infection is notable for the rapid onset of symptoms and can progress to fatal systemic infection within a few days. The destructive nature of infection implies that the pathogenicity of the pathogen is a multifactorial and complex phenomenon involving numerous virulence factors. In this part of the review, we cover the newly identified virulence factors related to the biofilm (see Glossary) formation of V. vulnificus. Also, recent studies on the pathological function of its exotoxins, which focus on their modes of action and in vivo effects on host immune and inflammatory responses, are summarized together. Like many other pathogenic bacteria, V. vulnificus exists in a free-living planktonic state or a surface-attached biofilm state [7.Yildiz F.H. Visick K.L. Vibrio biofilms: so much the same yet so different.Trends Microbiol. 2009; 17: 109-118Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar,8.Ashrafudoulla M. et al.Current and future perspectives for controlling Vibrio biofilms in the seafood industry: a comprehensive review.Crit. Rev. Food Sci. Nutr. 2021; 61: 1827-1851Crossref PubMed Scopus (28) Google Scholar]. Bacteria in biofilms are more tolerant to environmental stresses such as nutrient limitation, antimicrobial agents, and host immune defenses than planktonic cells [9.Flemming H.C. et al.Biofilms: an emergent form of bacterial life.Nat. Rev. Microbiol. 2016; 14: 563-575Crossref PubMed Scopus (2785) Google Scholar]. In addition, biofilm formation largely contributes to colonization and persistence of V. vulnificus in oysters. As a result, the pathogen can accumulate in oysters, which may serve as reservoirs in nature and thus the primary source of oral infection [10.Park J.H. et al.Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies.Int. J. Food Microbiol. 2016; 236: 123-129Crossref PubMed Scopus (5) Google Scholar,11.Pu M. et al.A conserved tad pilus promotes Vibrio vulnificus oyster colonization.Environ. Microbiol. 2018; 20: 828-841Crossref PubMed Scopus (16) Google Scholar]. Given these characteristics of biofilms, the ability to form biofilms is closely linked to the transmission and pathogenesis of V. vulnificus. Developmental stages of biofilm formation consist of initial surface attachment, microcolony formation, maturation into a 3D matrix, and biofilm dispersal to colonize other niches [8.Ashrafudoulla M. et al.Current and future perspectives for controlling Vibrio biofilms in the seafood industry: a comprehensive review.Crit. Rev. Food Sci. Nutr. 2021; 61: 1827-1851Crossref PubMed Scopus (28) Google Scholar], which involves various virulence factors. Current studies have found that Type IV pili encoded by iam locus play an important role in the initial attachment stage [11.Pu M. et al.A conserved tad pilus promotes Vibrio vulnificus oyster colonization.Environ. Microbiol. 2018; 20: 828-841Crossref PubMed Scopus (16) Google Scholar,12.Pu M. Rowe-Magnus D.A. A Tad pilus promotes the establishment and resistance of Vibrio vulnificus biofilms to mechanical clearance.NPJ Biofilms Microbiomes. 2018; 4: 10Crossref PubMed Scopus (26) Google Scholar]. Therefore, Iam pili facilitate biofilm formation and autoaggregation, thereby contributing to oyster colonization. The extracellular polymeric matrix of biofilms is composed of exopolysaccharides (EPSs), proteins, nucleic acids, and lipids [13.Flemming H.C. Wingender J. The biofilm matrix.Nat. Rev. Microbiol. 2010; 8: 623-633Crossref PubMed Scopus (6177) Google Scholar]. As EPSs are the most predominant component of a biofilm matrix, V. vulnificus possesses three distinct loci involved in the assembly and export of EPSs: the rbd locus, the brp locus, and the EPS-III locus [14.Kim H.S. et al.Role of NtrC-regulated exopolysaccharides in the biofilm formation and pathogenic interaction of Vibrio vulnificus.Mol. Microbiol. 2009; 74: 436-453Crossref PubMed Scopus (46) Google Scholar, 15.Guo Y. Rowe-Magnus D.A. Identification of a c-di-GMP-regulated polysaccharide locus governing stress resistance and biofilm and rugose colony formation in Vibrio vulnificus.Infect. Immun. 2010; 78: 1390-1402Crossref PubMed Scopus (35) Google Scholar, 16.Guo Y. Rowe-Magnus D.A. Overlapping and unique contributions of two conserved polysaccharide loci in governing distinct survival phenotypes in Vibrio vulnificus.Environ. Microbiol. 2011; 13: 2888-2990Crossref PubMed Scopus (18) Google Scholar]. In addition, the products of the brpLG operon have been newly identified to promote EPS production, which is essential for robust biofilm and rugose colony formation [17.Hwang S.H. et al.A master regulator BrpR coordinates the expression of multiple loci for robust biofilm and rugose colony development in Vibrio vulnificus.Front. Microbiol. 2021; 12679854Crossref Scopus (4) Google Scholar]. Meanwhile, flagellin-homologous proteins FlaE and FlaF encoded by flaEF genes and a calcium-binding protein CabA encoded by a cabABC operon are secretory matrix proteins that function as other crucial components of the biofilm matrix [10.Park J.H. et al.Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies.Int. J. Food Microbiol. 2016; 236: 123-129Crossref PubMed Scopus (5) Google Scholar,18.Park J.H. et al.The cabABC operon essential for biofilm and rugose colony development in Vibrio vulnificus.PLoS Pathog. 2015; 11e1005192Google Scholar,19.Jung Y.C. et al.Role of flagellin-homologous proteins in biofilm formation by pathogenic Vibrio species.mBio. 2019; 10e01793-19Crossref Scopus (13) Google Scholar]. They are secreted to the extracellular matrix and strengthen the biofilm matrix in association with EPSs. Last, the production of an elastolytic protease VvpE and a capsular polysaccharide (CPS) facilitates the dispersal of biofilm to colonize new niches [20.Kim S.M. et al.LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment, and its expression is induced by host cells.Infect. Immun. 2013; 81: 3721-3730Crossref PubMed Scopus (51) Google Scholar,21.Lee K.J. et al.Role of capsular polysaccharide (CPS) in biofilm formation and regulation of CPS production by quorum-sensing in Vibrio vulnificus.Mol. Microbiol. 2013; 90: 841-857Crossref PubMed Scopus (61) Google Scholar]. Upon entering the host, V. vulnificus expresses several exotoxins required for its severe invasiveness and tissue-damaging ability toward host cells. The multifunctional autoprocessing repeats‐in‐toxin (MARTX) toxin encoded by the rtxA gene is a key exotoxin with cytotoxic/cytopathic activities, which is a single polypeptide protein composed of repeat-containing regions at the N- and C-termini and central effector domains (Box 1) [22.Kim B.S. et al.Distinct roles of the repeat-containing regions and effector domains of the Vibrio vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin.mBio. 2015; 6e00324-15Crossref Scopus (42) Google Scholar,23.Kim B.S. The modes of action of MARTX toxin effector domains.Toxins (Basel). 2018; 10: 507Crossref Scopus (14) Google Scholar]. Once secreted, the MARTX toxin binds to host plasma membranes with the repeat regions, forms a porelike structure, and translocates its central effector domains into the host cytosol. Interestingly, the effectors are autoprocessed and liberated to the host subcellular compartments in response to host-specific molecules [24.Egerer M. Satchell K.J. Inositol hexakisphosphate-induced autoprocessing of large bacterial protein toxins.PLoS Pathog. 2010; 6e1000942Crossref PubMed Scopus (54) Google Scholar, 25.Gavin H.E. Satchell K.J. MARTX toxins as effector delivery platforms.Pathog. Dis. 2015; 73ftv092PubMed Google Scholar, 26.Woida P.J. Satchell K.J.F. Coordinated delivery and function of bacterial MARTX toxin effectors.Mol. Microbiol. 2018; 107: 133-141Crossref PubMed Scopus (17) Google Scholar]. Each of the released effectors exhibits diverse cytopathic/cytotoxic activities, resulting in cytoskeleton and Golgi disruption, dysregulation of host cell signaling, and apoptotic cell death [23.Kim B.S. The modes of action of MARTX toxin effector domains.Toxins (Basel). 2018; 10: 507Crossref Scopus (14) Google Scholar,27.Zhou Y. et al.Nε-fatty acylation of Rho GTPases by a MARTX toxin effector.Science. 2017; 358: 528-531Crossref PubMed Scopus (33) Google Scholar, 28.Biancucci M. et al.The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypical mechanism to disrupt Ras-ERK signaling.Sci. Signal. 2018; 11eaat8335Crossref PubMed Scopus (27) Google Scholar, 29.Jang S.Y. et al.Structural basis of inactivation of Ras and Rap1 small GTPases by Ras/Rap1-specific endopeptidase from the sepsis-causing pathogen Vibrio vulnificus.J. Biol. Chem. 2018; 293: 18110-18122Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 30.Lee Y. et al.Makes caterpillars floppy-like effector-containing MARTX toxins require host ADP-ribosylation factor (ARF) proteins for systemic pathogenicity.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 18031-18040Crossref PubMed Scopus (12) Google Scholar, 31.Herrera A. et al.N-terminal autoprocessing and acetylation of multifunctional-autoprocessing repeats-in-toxins (MARTX) Makes Caterpillars Floppy-like effector is stimulated by adenosine diphosphate (ADP)-ribosylation factor 1 in advance of Golgi fragmentation.Cell. Microbiol. 2020; 22e13133Crossref PubMed Scopus (6) Google Scholar]. Based on its mode of action, the MARTX toxin is essential for V. vulnificus to improve antiphagocytosis, colonization, and dissemination to the bloodstream and other organs, and thereby lethality in mice [32.Jeong H.G. Satchell K.J. Additive function of Vibrio vulnificus MARTXVv and VvhA cytolysins promotes rapid growth and epithelial tissue necrosis during intestinal infection.PLoS Pathog. 2012; 8e1002581Crossref PubMed Scopus (105) Google Scholar,33.Gavin H.E. et al.The effector domain region of the Vibrio vulnificus MARTX toxin confers biphasic epithelial barrier disruption and is essential for systemic spread from the intestine.PLoS Pathog. 2017; 13e1006119Crossref PubMed Scopus (28) Google Scholar]. Also, recent studies further revealed that the expression of the MARTX toxin moderates immune responses of gut epithelial cells but stimulates inflammatory signaling of immune cells [34.Murciano C. et al.MARTX toxin in the zoonotic serovar of Vibrio vulnificus triggers an early cytokine storm in mice.Front. Cell. Infect. Microbiol. 2017; 7: 332Crossref PubMed Scopus (20) Google Scholar, 35.Lee A. et al.Vibrio vulnificus RtxA is a major factor driving inflammatory T helper type 17 cell responses in vitro and in vivo.Front. Immunol. 2018; 9: 2095Crossref PubMed Scopus (5) Google Scholar, 36.Kim B.S. et al.MARTX toxin-stimulated interplay between human cells and Vibrio vulnificus.mSphere. 2020; 5e00659-20Crossref Google Scholar].Box 1The mode of action of MARTX toxinsAs a large single polypeptide protein, MARTX toxins consist of variable effector domains between conserved N- and C-terminal repeat regions (Figure IA) [23.Kim B.S. The modes of action of MARTX toxin effector domains.Toxins (Basel). 2018; 10: 507Crossref Scopus (14) Google Scholar]. Even different strains in the same species can possess different types of effectors, resulting in their varied pathological effects. For example, the MARTX toxin of V. vulnificus R99 strain induces a strong early cytokine storm in mice, which is not triggered by that of V. vulnificus YJ016 strain [34.Murciano C. et al.MARTX toxin in the zoonotic serovar of Vibrio vulnificus triggers an early cytokine storm in mice.Front. Cell. Infect. Microbiol. 2017; 7: 332Crossref PubMed Scopus (20) Google Scholar].After the effector domains are translocated into the host cytoplasm, a conserved cysteine protease domain (CPD) becomes activated by binding to the eukaryotic cell‐specific molecule, inositol hexakisphosphate (InsP6) [24.Egerer M. Satchell K.J. Inositol hexakisphosphate-induced autoprocessing of large bacterial protein toxins.PLoS Pathog. 2010; 6e1000942Crossref PubMed Scopus (54) Google Scholar]. Then, the activated form of CPD processes the effector domain regions, resulting in the release of effectors into the host cytoplasm (Figure IB) [25.Gavin H.E. Satchell K.J. MARTX toxins as effector delivery platforms.Pathog. Dis. 2015; 73ftv092PubMed Google Scholar,26.Woida P.J. Satchell K.J.F. Coordinated delivery and function of bacterial MARTX toxin effectors.Mol. Microbiol. 2018; 107: 133-141Crossref PubMed Scopus (17) Google Scholar]. The intermediate effector modules are further processed and fully activated by a makes caterpillars floppy-like domain (MCF) that interacts with ADP-ribosylation factors (ARFs) in the host cells [30.Lee Y. et al.Makes caterpillars floppy-like effector-containing MARTX toxins require host ADP-ribosylation factor (ARF) proteins for systemic pathogenicity.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 18031-18040Crossref PubMed Scopus (12) Google Scholar,31.Herrera A. et al.N-terminal autoprocessing and acetylation of multifunctional-autoprocessing repeats-in-toxins (MARTX) Makes Caterpillars Floppy-like effector is stimulated by adenosine diphosphate (ADP)-ribosylation factor 1 in advance of Golgi fragmentation.Cell. Microbiol. 2020; 22e13133Crossref PubMed Scopus (6) Google Scholar].Each liberated effector exhibits various cytopathic activities [23.Kim B.S. The modes of action of MARTX toxin effector domains.Toxins (Basel). 2018; 10: 507Crossref Scopus (14) Google Scholar]. The MARTX toxin of clinical isolate V. vulnificus MO6-24/O strain contains four effector domains: the domain of unknown function in the first position (DUF1), Rho GTPase-inactivation domain (RID), alpha/beta hydrolase domain (ABH), and MCF. DUF1 interacts with prohibitin 1 in the host membrane which is predicted to be an initial receptor for the early binding of MARTX toxins. RID inactivates Rho-family GTPases that regulate cell cytoskeleton, leading to host cell rounding. ABH is a phosphatidylinositol 3-phosphate-specific phospholipase A1 inhibiting the autophagic pathway and endosomal trafficking of host cells. MCF results in apoptotic cell death and Golgi disruption. In addition to these four effectors, other clinical isolates CMCP6 and YJ016 strains have Ras/Rap1-specific endopeptidase (RRSP) that dysregulates host cell signaling. On the other hand, another biotype 3 clinical isolate BAA87 strain does not contain MCF and RRSP but possesses ExoY-like adenylate cyclase domain (ExoY) and domain X (DmX) causing intracellular accumulation of cAMP and Golgi disruption, respectively. As a large single polypeptide protein, MARTX toxins consist of variable effector domains between conserved N- and C-terminal repeat regions (Figure IA) [23.Kim B.S. The modes of action of MARTX toxin effector domains.Toxins (Basel). 2018; 10: 507Crossref Scopus (14) Google Scholar]. Even different strains in the same species can possess different types of effectors, resulting in their varied pathological effects. For example, the MARTX toxin of V. vulnificus R99 strain induces a strong early cytokine storm in mice, which is not triggered by that of V. vulnificus YJ016 strain [34.Murciano C. et al.MARTX toxin in the zoonotic serovar of Vibrio vulnificus triggers an early cytokine storm in mice.Front. Cell. Infect. Microbiol. 2017; 7: 332Crossref PubMed Scopus (20) Google Scholar]. After the effector domains are translocated into the host cytoplasm, a conserved cysteine protease domain (CPD) becomes activated by binding to the eukaryotic cell‐specific molecule, inositol hexakisphosphate (InsP6) [24.Egerer M. Satchell K.J. Inositol hexakisphosphate-induced autoprocessing of large bacterial protein toxins.PLoS Pathog. 2010; 6e1000942Crossref PubMed Scopus (54) Google Scholar]. Then, the activated form of CPD processes the effector domain regions, resulting in the release of effectors into the host cytoplasm (Figure IB) [25.Gavin H.E. Satchell K.J. MARTX toxins as effector delivery platforms.Pathog. Dis. 2015; 73ftv092PubMed Google Scholar,26.Woida P.J. Satchell K.J.F. Coordinated delivery and function of bacterial MARTX toxin effectors.Mol. Microbiol. 2018; 107: 133-141Crossref PubMed Scopus (17) Google Scholar]. The intermediate effector modules are further processed and fully activated by a makes caterpillars floppy-like domain (MCF) that interacts with ADP-ribosylation factors (ARFs) in the host cells [30.Lee Y. et al.Makes caterpillars floppy-like effector-containing MARTX toxins require host ADP-ribosylation factor (ARF) proteins for systemic pathogenicity.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 18031-18040Crossref PubMed Scopus (12) Google Scholar,31.Herrera A. et al.N-terminal autoprocessing and acetylation of multifunctional-autoprocessing repeats-in-toxins (MARTX) Makes Caterpillars Floppy-like effector is stimulated by adenosine diphosphate (ADP)-ribosylation factor 1 in advance of Golgi fragmentation.Cell. Microbiol. 2020; 22e13133Crossref PubMed Scopus (6) Google Scholar]. Each liberated effector exhibits various cytopathic activities [23.Kim B.S. The modes of action of MARTX toxin effector domains.Toxins (Basel). 2018; 10: 507Crossref Scopus (14) Google Scholar]. The MARTX toxin of clinical isolate V. vulnificus MO6-24/O strain contains four effector domains: the domain of unknown function in the first position (DUF1), Rho GTPase-inactivation domain (RID), alpha/beta hydrolase domain (ABH), and MCF. DUF1 interacts with prohibitin 1 in the host membrane which is predicted to be an initial receptor for the early binding of MARTX toxins. RID inactivates Rho-family GTPases that regulate cell cytoskeleton, leading to host cell rounding. ABH is a phosphatidylinositol 3-phosphate-specific phospholipase A1 inhibiting the autophagic pathway and endosomal trafficking of host cells. MCF results in apoptotic cell death and Golgi disruption. In addition to these four effectors, other clinical isolates CMCP6 and YJ016 strains have Ras/Rap1-specific endopeptidase (RRSP) that dysregulates host cell signaling. On the other hand, another biotype 3 clinical isolate BAA87 strain does not contain MCF and RRSP but possesses ExoY-like adenylate cyclase domain (ExoY) and domain X (DmX) causing intracellular accumulation of cAMP and Golgi disruption, respectively. Phospholipase A2 PlpA is the most recently discovered exotoxin that causes necrotic cell death of epithelial cells and lyses human erythrocytes of which membranes contain sufficient phosphatidylcholine [37.Jang K.K. et al.Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.J. Biol. Chem. 2017; 292: 17129-17143Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar]. Notably, PlpA cooperates sequentially with the MARTX toxin to facilitate necrotic cell death by disrupting host cell blebs induced by the MARTX toxin [38.Cho C. et al.Vibrio vulnificus PlpA facilitates necrotic host cell death induced by the pore forming MARTX toxin.J. Microbiol. 2022; 60: 224-233Crossref PubMed Scopus (2) Google Scholar]. Consistent with this, PlpA is crucial for systemic infection, liver damage, and thus lethality in mice [37.Jang K.K. et al.Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.J. Biol. Chem. 2017; 292: 17129-17143Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar]. Still, further investigations are required to elucidate the detailed mode of action of PlpA during infection. Cytolysin/hemolysin VvhA is a pore-forming toxin conferring powerful hemolytic activity to V. vulnificus [39.Kaus K. et al.Glycan specificity of the Vibrio vulnificus hemolysin lectin outlines evolutionary history of membrane targeting by a toxin family.J. Mol. Biol. 2014; 426: 2800-2812Crossref PubMed Scopus (28) Google Scholar,40.Yuan Y. et al.Vibrio vulnificus hemolysin: biological activity, regulation of vvhA expression, and role in pathogenesis.Front. Immunol. 2020; 11599439Crossref Scopus (14) Google Scholar]. VvhA triggers apoptosis, necrotic cell death, and autophagy by dysregulation of host cell signaling, leading to host tissue damage [41.Lee S.J. et al.Vibrio vulnificus VvhA induces NF-κB-dependent mitochondrial cell death via lipid raft-mediated ROS production in intestinal epithelial cells.Cell Death Dis. 2015; 6: 1655Crossref PubMed Scopus (39) Google Scholar,42.Song E.J. et al.Vibrio vulnificus VvhA induces autophagy-related cell death through the lipid raft-dependent c-Src/NOX signaling pathway.Sci. Rep. 2016; 627080Google Scholar]. Also, injection of purified VvhA induces proinflammatory cytokine and inflammatory chemokines in a mouse model [43.Qin K. et al.Vibrio vulnificus cytolysin induces inflammatory responses in RAW264.7 macrophages through calcium signaling and causes inflammation in vivo.Microb. Pathog. 2019; 137103789Crossref PubMed Scopus (10) Google Scholar], which presumably results in septic shock. Elastolytic protease VvpE is an extracellular zinc metalloprotease with diverse proteolytic activities leading to host tissue damage and inflammation [44.Kothary M.H. Kreger A.S. Purification and characterization of an elastolytic protease of Vibrio vulnificus.J. Gen. Microbiol. 1987; 133: 1783-1791PubMed Google Scholar,45.Miyoshi S. Vibrio vulnificus infection and metalloprotease.J. Dermatol. 2006; 33: 589-595Crossref PubMed Scopus (48) Google Scholar]. It has been reported that VvpE induces necrotic cell death via interleukin (IL)-1β production in host intestinal epithelial cells [46.Lee S.J. et al.Vibrio vulnificus VvpE stimulates IL-1β production by the hypomethylation of the IL-1β promoter and NF-κB activation via lipid raft-dependent ANXA2 recruitment and reactive oxygen species signaling in intestinal epithelial cells.J. Immunol. 2015; 195: 2282-2293Crossref PubMed Scopus (20) Google Scholar]. Furthermore, VvpE alters intestinal barrier function by inhibiting the expression of mucin 2, one of the major protein components of the mucus layer, and disrupting tight junctions, which contributes to the intestinal colonization of V. vulnificus [47.Lee S.J. et al.Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells.Cell Death Dis. 2015; 6e1787Google Scholar,48.Lee S.J. et al.VvpE mediates the intestinal colonization of Vibrio vulnificus by the disruption of tight junctions.Int. J. Med. Microbiol. 2016; 306: 10-19Crossref PubMed Scopus (20) Google Scholar]. Considering the significant effects of purified VvhA and VvpE, it is quite surprising that mutation of vvhA and vvpE cannot attenuate the virulence of V. vulnificus in mice [49.Fan J.J. et al.Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin.Infect. Immun. 2001; 69: 5943-5948Crossref PubMed Scopus (75) Google Scholar], unlike that of rtxA and plpA. This might be attributable to the overwhelming potency of the MARTX toxin. Nonetheless, several pieces of evidence suggested the additive function of the MARTX toxin and VvhA for in vivo growth, intestinal tissue necrosis, inflammation, and dissemination of infection [32.Jeong H.G. Satchell K.J. Additive function of Vibrio vulnificus MARTXVv and VvhA cytolysins promotes rapid growth and epithelial tissue necrosis during intestinal infection.PLoS Pathog. 2012; 8e1002581Crossref PubMed Scopus (105) Google Scholar]. Thus, the cooperative or synergistic actions between exotoxins should be further examined at molecular levels to grasp their actual roles during infection. It would also be helpful to investigate at which stage of infection each exotoxin is substantially produced within the host. To coordinate the expression of virulence factors, V. vulnificus takes into account a wide range of environmental signals. Thus, diverse transcription factors are exploited to integrate all the signals and produce each virulence factor at adequate levels. These complex regulatory networks enable the pathogen to effectively achieve maximum fitness in nature and within the host. Recent studies have elucidated the regulatory mechanisms of several transcription factors regarding their cognate signals and regulons. B