Etiological, epidemiological, and clinical features of acute diarrhea in China.

National-based prospective surveillance of all-age patients with acute diarrhea was conducted in China between 2009‒2018. Here we report the etiological, epidemiological, and clinical features of the 152,792 eligible patients enrolled in this analysis. Rotavirus A and norovirus are the two leading viral pathogens detected in the patients, followed by adenovirus and astrovirus. Diarrheagenic Escherichia coli and nontyphoidal Salmonella are the two leading bacterial pathogens, followed by Shigella and Vibrio parahaemolyticus. Patients aged <5 years had higher overall positive rate of viral pathogens, while bacterial pathogens were more common in patients aged 18‒45 years. A joinpoint analysis revealed the age-specific positivity rate and how this varied for individual pathogens. Our findings fill crucial gaps of how the distributions of enteropathogens change across China in patients with diarrhea. This allows enhanced identification of the predominant diarrheal pathogen candidates for diagnosis in clinical practice and more targeted application of prevention and control measures.

Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei.

The intestine is the primary target of pathogenic microbes during invasion. However, the interaction of Vibrio parahaemolyticus (V. parahaemolyticus) with intestinal epithelial cells and its effects on the intestinal function of Litopenaeus vannamei (L. vannamei) are poorly studied. Therefore, the aim of this study was to investigate the influence of V. parahaemolyticus infection on intestinal barrier function and nutrient absorption in L. vannamei. In the present study, a total of 90 shrimp were randomly divided into two groups including the control group and V. parahaemolyticus infection group (final concentration of 1 × 105 CFU/mL), with three replicates per group. The result showed that compared with the control group, V. parahaemolyticus infection increased (P < 0.05) serum diamine oxidase activity and endotoxin quantification, and down-regulated (P < 0.05) the mRNA levels of intestinal peroxinectin, integrin, midline fasciclin at 48 h and 72 h; V. parahaemolyticus infection decreased (P < 0.05) the mRNA expression of intestinal amino acid transporter (CAT1, EAAT3 and ASCT1) and glucose transporter (SGLT-1, GLUT) at 24 h, 48 h and 72 h, and increased (P < 0.05) serum glucose and amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His and Arg) concentration at 24 h. The results indicated that V. parahaemolyticus infection increased intestinal permeability, inhibited absorption of glucose and amino acid in L. vannamei.

Using NanoSIMS coupled with microfluidics to visualize the early stages of coral infection by Vibrio coralliilyticus.

BACKGROUND: Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral's capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature. RESULTS: Three coral fragments were inoculated with 15N-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. 15N-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative 15N-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of 15N-enriched hotspots, suggesting these tissues act as a "collection and digestion" site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these 15N-hotspots revealed these to be host phagosomal and secretory cells/vesicles. CONCLUSIONS: This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

Vibrio vulnificus infection induces the maturation and activation of dendritic cells with inflammatory Th17-polarizing ability.

Vibrio vulnificus (V. vulnificus) is a gram-negative bacterium, which causes life-threatening septicemia and gastroenteritis through the consumption of contaminated seafood or wound infection. In addition, V. vulnificus infection is known to stimulate the production of several pro-inflammatory cytokines, which are associated with inflammatory responses mediated predominantly by dendritic cells (DCs), functioning as antigen-presenting cells. The present study aimed to investigate whether V. vulnificus infection induced the maturation and activation of murine DCs, which have the ability to polarize T helper (Th) cells into Th17 cells. Dysregulated Th17 cell responses are known to cause tissue damage, promoting the penetration of pathogens; however, Th17 cells are also involved in host defense against infection. Infection with V. vulnificus significantly increased the expression of cell surface molecules, including CD40, CD80 and major histocompatibility complex class II, leading to the maturation and activation of DCs. In the present study, the analysis of the cytokine profiles of DCs upon infection with V. vulnificus revealed the preferential production of interleukin-1ß (IL-1ß) and IL-6, through which V. vulnificus-infected DCs induced the polarization of Th17 cells when naïve CD4+ T cells were co-incubated. The reduction of Th17 cell generation through the use of anti-IL-6 neutralizing antibodies indicated that the Th17-polarizing capacity of V. vulnificus was predominantly dependent on DC-derived IL-6. The in vivo administration of V. vulnificus-infected DCs consistently increased the Th17 cell population in the lymph nodes of mice. Finally, the oral administration of V. vulnificus in mice also increased Th17 cell responses in the lamina propria of the small intestine. These results collectively demonstrated that V. vulnificus induced inflammatory Th17 cell responses via DCs, which may be associated with the immunopathological effects caused by V. vulnificus infection.

A Vibrio vulnificus VvpM Induces IL-1ß Production Coupled with Necrotic Macrophage Death via Distinct Spatial Targeting by ANXA2.

An inflammatory form of phagocyte death evoked by the Gram-negative bacterium Vibrio (V.) vulnificus (WT) is one of hallmarks to promote their colonization, but the virulence factor and infectious mechanism involved in this process remain largely unknown. Here, we identified extracellular metalloprotease VvpM as a new virulence factor and investigated the molecular mechanism of VvpM which acts during the regulation of the inflammatory form of macrophage death and bacterial colonization. Mutation of the vvpM gene appeared to play major role in the prevention of IL-1ß production due to V. vulnificus infection in macrophage. However, the recombinant protein (r) VvpM caused IL-1ß production coupled with necrotic cell death, which is highly susceptible to the knockdown of annexin A2 (ANXA2) located in both membrane lipid and non-lipid rafts. In lipid rafts, rVvpM recruited NOX enzymes coupled with ANXA2 to facilitate the production of ROS responsible for the epigenetic and transcriptional regulation of NF-κB in the IL-1ß promoter. rVvpM acting on non-lipid rafts increased LC3 puncta formation and autophagic flux, which are required for the mRNA expression of Atg5 involved in the autophagosome formation process. The autophagy activation caused by rVvpM induced NLRP3 inflammasome-dependent caspase-1 activation in the promoting of IL-1ß production. In mouse models of V. vulnificus infection, the VvpM mutant failed to elevate the level of pro-inflammatory responses closely related to IL-1ß production and prevented bacterial colonization. These findings delineate VvpM efficiently regulates two pathogenic pathways that stimulate NF-κB-dependent IL-1ß production and autophagy-mediated NLRP3 inflammasome via distinct spatial targeting by ANXA2.

Vibrio vulnificus MARTX cytotoxin causes inactivation of phagocytosis-related signaling molecules in macrophages.

BACKGROUND: Vibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans. We have previously shown that the multifunctional autoprocessing repeats in toxin (MARTXVv1) of a biotype 1 V. vulnificus strain promotes survival of this organism in the host by preventing it from engulfment by the phagocytes. The purpose of this study was to further explore how MARTXVv1 inhibits phagocytosis of this microorganism by the macrophage. METHODS: We compared between a wild-type V. vulnificus strain and its MARTXVv1-deficient mutant for a variety of phagocytosis-related responses, including morphological change and activation of signaling molecules, they induced in the macrophage. We also characterized a set of MARTXVv1 domain-deletion mutants to define the regions associated with antiphagocytosis activity. RESULTS: The RAW 264.7 cells and mouse peritoneal exudate macrophages underwent cell rounding accompanied by F-actin disorganization in the presence of MARTXVv1. In addition, phosphorylation of some F-actin rearrangement-associated signaling molecules, including Lyn, Fgr and Hck of the Src family kinases (SFKs), focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), phosphoinositide 3-kinase (PI3K) and Akt, but not p38, was decreased. By using specific inhibitors, we found that these kinases were all involved in the phagocytosis of MARTXVv1-deficient mutant in an order of SFKs-FAK/Pyk2-PI3K-Akt. Deletion of the effector domains in the central region of MARTXVv1 could lead to reduced cytotoxicity, depending on the region and size of deletion, but did not affect the antiphagocytosis activity and ability to cause rounding of macrophage. Reduced phosphorylation of Akt was closely associated with inhibition of phagocytosis by the wild-type strain and MARTXVv1 domain-deletion mutants, and expression of the constitutively active Akt, myr-Akt, enhanced the engulfment of these strains by macrophage. CONCLUSIONS: MARTXVv1 could inactivate the SFKs-FAK/Pyk2-PI3K-Akt signaling pathway in the macrophages. This might lead to impaired phagocytosis of the V. vulnificus-infected macrophage. The majority of the central region of MARTXVv1 is not associated with the antiphagocytosis activity.

In vitro and in vivo anti-Vibrio vulnificus activity of psammaplin A, a natural marine compound.

Vibrio vulnificus is known to induce severely fulminant and fatal septicemia in susceptible hosts. In the present study, the antimicrobial activity of natural marine product-derived compounds against V. vulnificus, were investigated in vitro and in vivo. Twelve pure compounds were isolated from natural marine products and their inhibitory effects on V. vulnificus-induced cytotoxicity were determined in INT­407 cells. Among the 12 pure compounds tested, treatment with psammaplin A significantly suppressed V. vulnificus­induced cytotoxicity in INT­407 cells. Notably, treatment with psammaplin A (5-50 µg) had improved survival rates compared with that in the untreated mice, when the mice were infected with V. vulnificus intraperitoneally. In addition, the bacterial load of V. vulnificus in several tissues (spleen, liver and small intestine) was significantly lower in psammaplin A­treated mice than in untreated mice. Furthermore, psammaplin A treatment significantly suppressed the growth of V. vulnificus. Taken together, these results indicate that psammaplin A may be a potential agent for the prevention and treatment of V. vulnificus infections.

Induction of mild enterocolitis in zebrafish Danio rerio via ingestion of Vibrio anguillarum serovar O1.

Vibrio anguillarum is the etiological agent of a fatal hemorrhagic disease known as vibriosis that affects a wide range of fish species, causing severe economic losses. Several investigations have been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. The aim of our study was to evaluate the most effective way to induce mild enteritis in a fish model, in order to allow further applications. The experiments were carried out using 2 methods of administration of V. anguillarum serotype O1 to adult zebrafish Danio rerio: via intraperitoneal injection and via ingestion of infected Artemia nauplii. The results showed that the intraperitoneal administration often caused massive fish death due to severe systemic involvement. In our experiments, the effect of intraperitoneal infection was evident 48 h post infection, with cumulative mortality within 7 d post infection with severe histopathological changes in kidney hematopoietic tissue and in the intestine. In contrast, oral infection via Artemia did not show systemic involvement and only a moderate degree of inflammatory influx of the mucosa, a partial recovery at 12 d post infection, and no mortality. For these reasons, oral infection with live food appears to be the most effective method to induce mild enteritis with a local inflammatory response.

Identification of Novel Inflammatory Cytokines and Contribution of Keratinocyte-Derived Chemokine to Inflammation in Response to Vibrio vulnificus Infection in Mice.

Currently, only tumor necrosis factor alpha (TNF-α) and interleukin family cytokines have been found to be elicited in Vibrio vulnificus (V. vulnificus)-infected animal models and humans. However, multiple other cytokines are also involved in the immune and inflammatory responses to foreign microorganism infection. Antibody array technology, unlike traditional enzyme-linked immunosorbent assay (ELISA), is able to detect multiple cytokines at one time. Therefore, in this study, we examined the proinflammatory cytokine profile in the serum and liver homogenate samples of bacterial-infected mice using antibody array technology. We identified nine novel cytokines in response to V. vulnificus infection in mice. We found that keratinocyte-derived chemokine (KC) was the most elevated cytokine and demonstrated that KC played a very important role in the V. vulnificus infection-elicited inflammatory response in mice, as evidenced by the fact that the blocking of KC by anti-KC antibody reduced hepatic injury in vivo and that KC induced by V. vulnificus infection in AML-12 cells chemoattracted neutrophils. Our findings implicate that KC may serve as a novel diagnostic biomarker and a possible therapeutic target for V. vulnificus infection.

Cyclo(Phe-Pro) produced by the human pathogen Vibrio vulnificus inhibits host innate immune responses through the NF-κB pathway.

Cyclo(Phe-Pro) (cFP) is a secondary metabolite produced by certain bacteria and fungi. Although recent studies highlight the role of cFP in cell-to-cell communication by bacteria, its role in the context of the host immune response is poorly understood. In this study, we investigated the role of cFP produced by the human pathogen Vibrio vulnificus in the modulation of innate immune responses toward the pathogen. cFP suppressed the production of proinflammatory cytokines, nitric oxide, and reactive oxygen species in a lipopolysaccharide (LPS)-stimulated monocyte/macrophage cell line and in bone marrow-derived macrophages. Specifically, cFP inhibited inhibitory κB (IκB) kinase (IKK) phosphorylation, IκBα degradation, and nuclear factor κB (NF-κB) translocation to the cell nucleus, indicating that cFP affects the NF-κB pathway. We searched for genes that are responsible for cFP production in V. vulnificus and identified VVMO6_03017 as a causative gene. A deletion of VVMO6_03017 diminished cFP production and decreased virulence in subcutaneously inoculated mice. In summary, cFP produced by V. vulnificus actively suppresses the innate immune responses of the host, thereby facilitating its survival and propagation in the host environment.

Multivalent adhesion molecule 7 clusters act as signaling platform for host cellular GTPase activation and facilitate epithelial barrier dysfunction.

Vibrio parahaemolyticus is an emerging bacterial pathogen which colonizes the gastrointestinal tract and can cause severe enteritis and bacteraemia. During infection, V. parahaemolyticus primarily attaches to the small intestine, where it causes extensive tissue damage and compromises epithelial barrier integrity. We have previously described that Multivalent Adhesion Molecule (MAM) 7 contributes to initial attachment of V. parahaemolyticus to epithelial cells. Here we show that the bacterial adhesin, through multivalent interactions between surface-induced adhesin clusters and phosphatidic acid lipids in the host cell membrane, induces activation of the small GTPase RhoA and actin rearrangements in host cells. In infection studies with V. parahaemolyticus we further demonstrate that adhesin-triggered activation of the ROCK/LIMK signaling axis is sufficient to redistribute tight junction proteins, leading to a loss of epithelial barrier function. Taken together, these findings show an unprecedented mechanism by which an adhesin acts as assembly platform for a host cellular signaling pathway, which ultimately facilitates breaching of the epithelial barrier by a bacterial pathogen.

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays.

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.

IscR is a global regulator essential for pathogenesis of Vibrio vulnificus and induced by host cells.

A mutant that exhibited less cytotoxic activity toward INT-407 human intestinal epithelial cells than the wild type was screened from a random transposon mutant library of Vibrio vulnificus, and an open reading frame encoding an Fe-S cluster regulator, IscR, was identified using a transposon-tagging method. A mutational analysis demonstrated that IscR contributes to mouse mortality as well as cytotoxicity toward the INT-407 cells, indicating that IscR is essential for the pathogenesis of V. vulnificus. A whole-genome microarray analysis revealed that IscR influenced the expression of 67 genes, of which 52 were upregulated and 15 were downregulated. Among these, 12 genes most likely involved in motility and adhesion to host cells, hemolytic activity, and survival under oxidative stress of the pathogen during infection were selected and experimentally verified to be upregulated by IscR. Accordingly, the disruption of iscR resulted in a significant reduction in motility and adhesion to INT-407 cells, in hemolytic activity, and in resistance to reactive oxygen species (ROS) such as H2O2 and tert-butyl hydroperoxide (t-BOOH). Furthermore, the present study demonstrated that iscR expression was induced by exposure of V. vulnificus to the INT-407 cells, and the induction appeared to be mediated by ROS generated by the host cells during infection. Consequently, the combined results indicated that IscR is a global regulator that contributes to the overall success in the pathogenesis of V. vulnificus by regulating the expression of various virulence and survival genes in addition to Fe-S cluster genes.

Characterization of a novel zinc transporter ZnuA acquired by Vibrio parahaemolyticus through horizontal gene transfer.

Vibrio parahaemolyticus is a clinically important foodborne pathogen that causes acute gastroenteritis worldwide. It has been shown that horizontal gene transfer (HGT) contributes significantly to virulence development of V. parahaemolyticus. In this study, we identified a novel znuA homolog (vpa1307) that belongs to a novel subfamily of ZnuA, a bacterial zinc transporter. The vpa1307 gene is located upstream of the V. parahaemolyticus pathogenicity island (Vp-PAIs) in both tdh-positive and trh-positive V. parahaemolyticus strains. Phylogenetic analysis revealed the exogenous origin of vpa1307 with 40% of V. parahaemolyticus clinical isolates possessing this gene. The expression of vpa1307 gene in V. parahaemolyticus clinical strain VP3218 is induced under zinc limitation condition. Gene deletion and complementation assays confirmed that vpa1307 contributes to the growth of VP3218 under zinc depletion condition and that conserved histidine residues of Vpa1307 contribute to its activity. Importantly, vpa1307 contributes to the cytotoxicity of VP3218 in HeLa cells and a certain degree of virulence in murine model. These results suggest that the horizontally acquired znuA subfamily gene, vpa1307, contributes to the fitness and virulence of Vibrio species.

Seasonality, clinical types and prognostic factors of Vibrio vulnificus infection.

INTRODUCTION: Vibrio vulnificus infection, an uncommon but life-threatening illness, manifests as two main types, primary septicemia and primary wound infections.  Little information regarding the seasonality of V. vulnificus infections in tropical areas and prognostic factors of primary V. vulnificus wound infections is available. METHODOLOGY: This retrospective study was conducted to include 159 V. vulnificus-infected admissions at our institution in southern Taiwan, 63 with primary septicemia (Group 1) and 96 with primary wound infections (Group 2), from 1999 to 2008, for analysis. RESULTS: The case-fatality rate was 24%. Eighty-eight percent of these cases occurred during April to November. During December to March, patients in Group 2 were less likely to have acquired the infection compared with those in Group 1. Group 1 was more likely to have comorbidities and a higher case-fatality rate compared to Group 2. In multivariate analysis, hemorrhagic bullous skin lesions/necrotizing fasciitis (P=0.024), lesions involving two or more limbs (P=0.043), and shock on admission (P=0.015) were related to an increased mortality risk, while surgery < 24 hours after admission (P=0.001) was related to a decreased mortality risk in Group 1; however, hemorrhagic bullous skin lesions/necrotizing fasciitis (P=0.045) was the only prognostic factor in Group 2. CONCLUSION: The presence of hemorrhagic bullous lesion/necrotizing fasciitis is the main prognostic factor for primary septicemia or primary wound infections caused by V. vulnificus. Persons with an underlying immunocompromised status should avoid consuming raw/undercooked seafood or exposing wounds to seawater and should wear clothing during handling of seafood/fishing, especially in warmer months.

The Laboratory Risk Indicator for Necrotizing Fasciitis score for discernment of necrotizing fasciitis originated from Vibrio vulnificus infections.

BACKGROUND: The Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score has been verified as a useful diagnostic tool for detecting necrotizing fasciitis (NF). Its application, however, is mainly for NF types I and II. The practical relevance of the LRINEC score for Vibro vulnificus-related skin and soft tissue infection (SSTI) was hardly ever investigated. The aim of this study was to assess the applicability of the LRINEC scoring system and to identify NF-predicting factors in patients with V. vulnificus-caused SSTI. METHODS: A retrospective study was conducted, enrolling 125 consecutive patients diagnosed with V. vulnificus-related SSTI who were admitted to a teaching hospital between January 2003 and December 2011. Demographics, laboratory data, comorbidities, treatment, and outcomes were collected for each patient and extracted for analysis. Logistic regression and receiver operating characteristic curve analyses were performed. RESULTS: The mean (SD) age of the 125 patients was 63.0 (10.9) years; 58% of the patients were male. The mean (SD) LRINEC score at admission was 2.4 (1.9) points. Of the 125 patents, 72 (58%) had NF. Multivariate analysis revealed that the presence of hemorrhagic bullous lesions (p = 0.002) and higher LRINEC scores at admission (p < 0.0001) were significantly associated with the presence of NF. In addition, the area under the receiver operating characteristic curve for the LRINEC scoring model for detecting NF was 0.783 (p < 0.0001). An optimal cutoff LRINEC score of 2 or greater had a sensitivity of 71%, a specificity of 83%, and a positive predictive value of 85%, with an 11.9-fold increased risk for the presence of NF (p < 0.0001). CONCLUSION: We have demonstrated that the LRINEC score and hemorrhagic bullous/blistering lesions are significant predictors of NF in patients with V. vulnificus-related SSTI. V. vulnificus-infected patients having hemorrhagic bullous/blistering lesions or with an LRINEC score of 2 or greater should be thoughtfully evaluated for the presence of NF. LEVEL OF EVIDENCE: Diagnostic test study, level II.

Vibrio parahaemolyticus ExsE is requisite for initial adhesion and subsequent type III secretion system 1-dependent autophagy in HeLa cells.

Vibrio parahaemolyticus pandemic serotype O3 : K6 causes acute gastroenteritis, wound infections and septicaemia in humans. This organism encodes two type III secretion systems (T3SS1 and T3SS2); host-cell cytotoxicity has been attributed to T3SS1. Synthesis and secretion of T3SS1 proteins is positively regulated by ExsA, which is presumptively regulated by the ExsCDE pathway, similar to Pseudomonas aeruginosa. Herein we deleted the putative exsE from V. parahaemolyticus and found constitutive expression of the T3SS1 in broth culture as expected. More importantly, however, in a cell culture model, the ΔexsE strain was unable to induce cytotoxicity, as measured by release of lactate dehydrogenase (LDH), or autophagy, as measured by LC3 conversion. This is markedly different from P. aeruginosa, where deletion of exsE has no effect on host-cell cytolysis. Swarming and cytoadhesion were reduced for the deletion mutant and could be recovered along with T3SS1-induced HeLa cell cytotoxicity by in cis expression of exsE in the ΔexsE strain. Loss of adhesion and swarming motility was associated with the loss of flagella biogenesis in the exsE-deficient strain. Mouse mortality was unaffected by the deletion of exsE compared with a wild-type control, suggesting that additional adhesins are important for intoxication in vivo. Based on these data, we conclude that ExsE contributes to the negative regulation of T3SS1 and, in addition, contributes to regulation of an adherence phenotype that is requisite for translocation of effector proteins into HeLa cells.

RTX toxin enhances the survival of Vibrio vulnificus during infection by protecting the organism from phagocytosis.

Vibrio vulnificus is a marine bacterium causing serious septicemia and wound infection in humans. It produces an RTX toxin that can lyse a variety of cells and is important for virulence in mice. In this study, we explored the role of RTX in pathogenesis by characterizing an RTX-deficient mutant. This mutant showed an ∼2-log reduction in virulence for mice infected by various routes. Survival of the mutant at the infection site and subsequent spread into the bloodstream were impaired. In mice pretreated with cyclophosphamide to deplete the neutrophils, both the virulence and survival at the infection site of this mutant were enhanced. This mutant was further shown to be more readily cleared from the macrophage-rich mouse peritoneal cavity and phagocytosed by murine macrophages. These findings suggest that the RTX of V. vulnificus is required for bacterial survival during infection by protecting the organism from phagocytosis.

Histopathological studies on Vibrio harveyi- infected tiger puffer, Takifugu rubripes (Temminck et Schlegel), cultured in Japan.

Vibrio harveyi infection occurred with a moderate mortality in tiger puffer, Takifugu rubripes (Temminck et Schlegel), in autumn 2007, at a mariculture farm in western Japan. The diseased fish showed nodular lesions in the branchial chamber and the inner surface of the operculum. Histopathologically, the lesions comprised granulation tissue containing many suppurative foci allowing propagation of the bacteria and granuloma encapsulating abscesses with a decrease in bacteria. The bacteria were disseminated in visceral organs including the spleen, kidney, liver, and myocardium, resulting in the formation of granulomatous lesions. Two groups of tiger puffer juveniles were artificially infected by an intramuscular injection with an isolate (1.0_10(8) CFU/fish). During the experimental period, 20% mortality occurred within 4-6 days post-infection (d.p.i). The fish sampled on 4 d.p.i showed abscesses in the lateral musculature at the injection site. The fish sampled 5 d.p.i. displayed the production of granulation tissue containing many suppurative foci, which replaced the necrotic dermis and lateral musculature. Surviving fish (15 d.p.i.) had granulomatous lesions in the lateral musculature at the injection site. Pyogranulomatosis is pathognomonic in V. harveyi infection of tiger puffer.

The primitive immune system of amphioxus provides insights into the ancestral structure of the vertebrate immune system.

Amphioxus is considered to be the basal chordate. However, the structural and anatomical features of the amphioxus immune system are still elusive. Here we report a profile of structural studies of the amphioxus gill and gut, the first line of defending against microbes, through optical and electron microscopy. The amphioxus gut and gill are characterized by the following morphological criteria compared with vertebrates: primary and secondary lymphoid-like tissue clustered in the gill, a thicker basement membrane with a large villus channel and lack of muscular layer in the gut, along with blood vessels that fill with phagocytes following microbial challenge. The phenomena of tissue repair after microbial invasion was observed, though no phagocytes were observed in the region of tissue necrosis. The epithelium cells of amphioxus gut showed active phagocytosis after the microbial challenge. A small number of free and fixed macrophage-like cells were also found in the amphioxus gut. The current results described the structure of the immune system and cellular defense against infection in a protochordate, which may help us in understanding the structural origin of the vertebrate immune system.