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Autoinducer-2 (AI-2), a quorum-sensing signal molecule from the human pathogen Vibrio vulnificus , was assessed for its effect on the gut microbiome of mice. For this, we employed 16S rRNA sequencing to compare the gut microbiome of mice infected with either wild-type V. vulnificus or with the isotype Δ luxS that has a deletion in luxS which encodes the biosynthetic function of AI-2. The relative ratio of wild-type Vibrio species in the jejunum and ileum of mice infected with the wild type was significantly higher than that in mice infected with Δ luxS , suggesting that AI-2 plays an important role in the colonization of V. vulnificus in the small intestine. The bacterial composition in the gut of mice infected with Δ luxS comprises a higher proportion of Firmicutes , composed mainly of Lactobacillus , compared to the mice infected with wild-type cells. In the large intestine, Vibrio species were barely detected regardless of genetic background. Three Lactobacillus spp. isolated from fecal samples from mice infected with Δ luxS manifested significant antibacterial activities against V. vulnificus . Culture supernatants from these three species were dissolved by HPLC, and a substance in fractions showing inhibitory activity against V. vulnificus was determined to be lactic acid. Our results suggest that luxS in V. vulnificus affects not only the ability of the species to colonize the host gut but also its susceptibility to the growth-inhibiting activity of commensal bacteria including Lactobacillus . Key points • Gut microbiomes of ΔluxS-infected and WT Vibrio-infected mice differed greatly. • Difference was most prominent in the jejunum and ileum compared to the duodenum or large intestine. • In the small and large intestines of mice, the relative proportions of Vibrio and Lactobacillus species showed a negative relationship. • Effector molecules produced by Lactobacillus in mouse gut inhibit Vibrio growth.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V . parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella . Our results showed that two subpopulations (P1 and P2) of V . parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:Δ lldD ) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:Δ lldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Bile acids, the primary components of bile, are cholesterol-derived molecules synthesized in the liver and secreted to the small intestine. Besides their primary digestive roles, bile acids have antimicrobial properties and serve as an environmental cue for intestinal pathogens, modulating the expression of virulence factors, e.g., toxins and effector proteins. Whereas timely recognition and neutralization of pathogenic toxin effectors by the host is critical, our understanding of the effects of bile on their structure and function is limited. In this work, we found that bile effectively protected cultured IEC-18 enterocytes from the mixture of Aeromonas hydrophila secreted toxins, containing hemolysin, aerolysin, and RtxA (MARTX). To explore whether these effects have broad specificity, we employed biochemical and biophysical techniques to test the in vitro effects of bile and bile acids on several effector domains of MARTX and VgrG toxins from Vibrio cholerae and Aeromonas hydrophila, and catalytic domains of TcdA and TcdB toxins from Clostridioides difficile. Bile compromised the structural integrity of the tested effectors to various degrees in a protein charge-dependent manner. Bile and bile acids promoted exposure of hydrophobic residues and the unfolding of most, but not all, of the tested effectors, facilitating their precipitation and cleavage by chymotrypsin. Bile also inhibited specific activities of the tested effector enzymes, partially due to imposed oxidation of their catalytic residues. To summarize, this work validated bile as a non-proteinaceous factor of innate immunity, capable of compromising the structural integrity and function of the effector domains of various bacterial toxins.

In response to a major influx of freshwater to the Mississippi Sound following the opening of the Bonnet Carre Spillway, water samples were collected from three sites along the Mississippi shoreline to assess the impact of altered salinity on three pathogenic Vibrio species. Salinity readings across the affected area during the 2011 sample period ranged from 1.4 to 12.9 ppt (mean=7.0) and for the 2012 sample period from 14.1 to 23.6 ppt (mean=19.8). Analyses of the data collected in 2011 showed a reduction in densities of Vibrio parahaemolyticus and Vibrio vulnificus with a concurrent increase of Vibrio cholerae numbers, with V. cholerae becoming the only Vibrio detected once salinity readings dropped to 6 ppt. Follow-up samples taken in 2012 after recovery of the salinity in the sound showed that the relative densities of the three pathogenic vibrios had reverted back to normal levels. This study shows that although the spillway was open but a few weeks and the effects were therefore time limited, the Mississippi River water had a profound, if temporary, effect on Vibrio ecology in the Mississippi Sound.

Bacteria face tough competition in polymicrobial communities. To persist in a specific niche, many species produce toxic extracellular effectors to interfere with the growth of nearby microbes. These effectors include the recently reported non-canonical D-amino acids (NCDAAs). In Vibrio cholerae , the causative agent of cholera, NCDAAs control cell wall integrity in stationary phase. Here, an analysis of the composition of the extracellular medium of V. cholerae revealed the unprecedented presence of D-Arg. Compared with other D-amino acids, D-Arg displayed higher potency and broader toxicity in terms of the number of bacterial species affected. Tolerance to D-Arg was associated with mutations in the phosphate transport and chaperone systems, whereas D-Met lethality was suppressed by mutations in cell wall determinants. These observations suggest that NCDAAs target different cellular processes. Finally, even though virtually all Vibrio species are tolerant to D-Arg, only a few can produce this D-amino acid. Indeed, we demonstrate that D-Arg may function as part of a cooperative strategy in vibrio communities to protect non-producing members from competing bacteria. Because NCDAA production is widespread in bacteria, we anticipate that D-Arg is a relevant modulator of microbial subpopulations in diverse ecosystems.

Vibrio species, which include several pathogens, are autochthonous to estuarine and warm coastal marine environments, where biofilm formation bolsters their ecological persistence and transmission. Here, we identify a bicistronic operon, rcbAB , whose products synergistically inhibit motility and promote biofilm maturation post-attachment by modulating intracellular c-di-GMP levels in the human and animal pathogen V. vulnificus . RcbA contains an N-terminal tetratricopeptide repeat (TPR) domain and a structured C-terminal region of unknown function, while RcbB possesses an N-terminal TPR domain and a C-terminal GGDEF domain characteristic of diguanylate cyclases. The TPR domain of RcbB represses its diguanylate cyclase activity, while RcbA’s TPR domain and C-terminal region co-operatively de-repress it. Localization of both proteins to the flagellar pole is TPR-dependent but not co-dependent, although RcbA anchors RcbB to the pole in the absence of polar landmarks such as HubP and flagella. The conservation of rcbAB across diverse bacterial taxa substantiates its fundamental importance in bacterial biology. This work demonstrates how spatio-genetically coordinated TPR domain-containing proteins modulate c-di-GMP signaling, contributing to our understanding of biofilm formation in Vibrio species and potentially other bacteria. It also reveals the first evidence of inter-protein interaction via the TPR domains of both partners, challenging the conventional paradigm in which only one bears the domain.

Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells.

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria ( Vibrio parahaemolyticus , Vp; V. campbellii , Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.

Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear. We therefore examined the distribution of the genes for T3SS2 in vibrios other than V. parahaemolyticus by using a PCR assay targeting both T3SS2α and T3SS2β genes. Among the 32 Vibrio species tested in our study, several T3SS2-related genes were detected in three species, V. cholerae, V. mimicus and V. hollisae, and most of the essential genes for type III secretion were present in T3SS2-positive V. cholerae and V. mimicus strains. Moreover, both V. mimicus strains possessing T3SS2α and T3SS2β were identified. The gene organization of the T3SS2 gene clusters in V. mimicus strains was fundamentally similar to that of V. parahaemolyticus and V. cholerae in both T3SS2α- and T3SS2β-possessing strains. This study is the first reported evidence of the presence of T3SS2 gene clusters in V. mimicus strains. This finding thus provides a new insight into the pathogenicity of the V. mimicus species.

The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress.