Characterization of Outer Membrane Vesicles Produced by Vibrio vulnificus.

Vibrio vulnificus (V. vulnificus) is a halophilic gram-negative bacterium that inhabits coastal warm water and induce severe diseases such as primary septicemia. To investigate the mechanisms of rapid bacterial translocation on intestinal infection, we focused on outer membrane vesicles (OMVs), which are extracellular vesicles produced by Gram-negative bacteria and deliver virulence factors. However, there are very few studies on the pathogenicity or contents of V. vulnificus OMVs (Vv-OMVs). In this study, we investigated the effects of Vv-OMVs on host cells. Epithelial cells INT407 were stimulated with purified OMVs and morphological alterations and levels of lactate dehydrogenase (LDH) release were observed. In cells treated with OMVs, cell detachment without LDH release was observed, which exhibited different characteristics from cytotoxic cell detachment observed in V. vulnificus infection. Interestingly, OMVs from a Vibrio Vulnificus Hemolysin (VVH) and Multifunctional-autoprocessing repeats-in -toxin (MARTX) double-deletion mutant strain also caused cell detachment without LDH release. Our results suggested that the proteolytic function of a serine protease contained in Vv-OMVs may contribute to pathogenicity of V. vulnificus by assisting bacterial translocation. This study reveals a new pathogenic mechanism during V. vulnificus infections. J. Med. Invest. 71 : 102-112, February, 2024.

Characteristic Metabolic Changes in Skeletal Muscle Due to Vibrio vulnificus Infection in a Wound Infection Model.

Vibrio vulnificus is a bacterium that inhabits warm seawater or brackish water environments and causes foodborne diseases and wound infections. In severe cases, V. vulnificus invades the skeletal muscle tissue, where bacterial proliferation leads to septicemia and necrotizing fasciitis with high mortality. Despite this characteristic, information on metabolic changes in tissue infected with V. vulnificus is not available. Here, we elucidated the metabolic changes in V. vulnificus-infected mouse skeletal muscle using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Metabolome analysis revealed changes in muscle catabolites and energy metabolites during V. vulnificus infection. In particular, succinic acid accumulated but fumaric acid decreased in the infected muscle. However, the virulence factor deletion mutant revealed that changes in metabolites and bacterial proliferation were abolished in skeletal muscle infected with a multifunctional-autoprocessing repeats-in-toxin (MARTX) mutant. On the other hand, mice that were immunosuppressed via cyclophosphamide (CPA) treatment exhibited a similar level of bacterial counts and metabolites between the wild type and MARTX mutant. Therefore, our data indicate that V. vulnificus induces metabolic changes in mouse skeletal muscle and proliferates by using the MARTX toxin to evade the host immune system. This study indicates a new correlation between V. vulnificus infections and metabolic changes that lead to severe reactions or damage to host skeletal muscle. IMPORTANCE V. vulnificus causes necrotizing skin and soft tissue infections (NSSTIs) in severe cases, with high mortality and sign of rapid deterioration. Despite the severity of the infection, the dysfunction of the host metabolism in skeletal muscle triggered by V. vulnificus is poorly understood. In this study, by using a mouse wound infection model, we revealed characteristic changes in muscle catabolism and energy metabolism in skeletal muscle associated with bacterial proliferation in the infected tissues. Understanding such metabolic changes in V. vulnificus-infected tissue may provide crucial information to identify the mechanism via which V. vulnificus induces severe infections. Moreover, our metabolite data may be useful for the recognition, identification, or detection of V. vulnificus infections in clinical studies.

Increased Vascular Permeability Due to Spread and Invasion of Vibrio vulnificus in the Wound Infection Exacerbates Potentially Fatal Necrotizing Disease.

Vibrio vulnificus is known to cause necrotizing soft tissue infections (NSTIs). However, the pathogenic mechanism causing cellulitis, necrotizing fasciitis, muscle necrosis, and rapidly developing septicemia in humans have not been fully elucidated. Here, we report a multilayer analysis of tissue damage after subcutaneous bacterial inoculation as a murine model of V. vulnificus NSTIs. Our histopathological examination showed the progression of cellulitis, necrotizing fasciitis, and muscle necrosis worsening as the infection penetrated deeper into the muscle tissue layers. The increase in vascular permeability was the primary cause of the swelling and congestion, which are acute signs of inflammation in soft tissue and characteristic of human NSTIs. Most importantly, our sequential analysis revealed for the first time that V. vulnificus not only spreads along the skin and subcutaneous tissues or fascia but also invades deeper muscle tissues beyond the fascia as the crucial process of its lethality. Also, increased vascular permeability enabled V. vulnificus to proliferate in muscle tissue and enter the systemic circulation, escalating the bacterium's lethality. Our finding may yield important clinical benefits to patients by helping physicians understand the impact of surgical debridement on the patient's quality of life. Furthermore, this study provides a promising system to accelerate studies of virulence factors and eventually help establish new therapies.

Characteristics of systemic infection and host responses in chickens experimentally infected with Salmonella enterica serovar Gallinarum biovar Gallinarum.

Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing systemic infection in poultry, which leads to significant economic losses due to high mortality. However, little is known about the dynamic process of systemic infection and pathogenic characteristics of S. Gallinarum in chickens. In the present study, we developed an oral infection model that reproduces the pathology of S. Gallinarum and clarified the host immune response of the infected chickens. Chickens at 20 days of age orally inoculated at a dose of 108 colony forming unit (CFU) showed typical clinical signs of fowl typhoid and died between 6 and 10 days post infection. The inoculated S. Gallinarum rapidly disseminated to multple organs and the bacterial counts increased in the liver and spleen at 3 days post infection. Pathological changes associated wirh inflammation in the liver and spleen became apparent at 4 days post infection, and increased expression of interferon (IFN)-γ and interleuikin (IL)-12 in the liver and spleen did not observed until 3 days post infection. These results indicate that S. Gallinarum rapidly spread to entire body through intestine, and the low-level of inflammatory responses in the liver during the early stage of infection may contribute to rapid, systemic dissemination of the bacteria. Our infection model and findings will contribute to the better understanding of the pathogenic mechanism of S. Gallinarum, and provide new insights into the prevention and control of fowl typhoid.

MukB Is a Gene Necessary for Rapid Proliferation of Vibrio vulnificus in the Systemic Circulation but Not at the Local Infection Site in the Mouse Wound Infection Model.

Vibrio vulnificus causes rapid septicemia in susceptible individuals who have ingested contaminated foods or have open wounds exposed to seawater contaminated with the bacteria. Despite antibiotic therapy and aggressive debridement, mortality from septicemia is high. In this study, we showed that MukB mutation (mukB::Tn) affected the proliferation of V. vulnificus in the systemic circulation but not at the inoculation site in the wound infection model. A comparison of mukB::Tn with WT and a mukB complement strain (mukB::Tn/pmukB) on the bacterial burden in the muscle at the infection site showed that spreading and proliferation of the mukB::Tn strain was similar to those of the other strains. However, the bacterial burden of mukB::Tn in the spleen was reduced compared to that of the WT strain in the wound infection model. In a competition experiment, we found a lower bacterial burden of mukB::Tn in the spleen than that of the WT strain infecting the systemic circulation. Here, we report on a gene required for the rapid proliferation of V. vulnificus only in the systemic circulation and potentially required for its survival. Our finding may provide a novel therapeutic target for V. vulnificus septicemia.

New inducible mast cell-deficient mouse model (Mcpt5/Cma1DTR).

Mast cell-deficient mice are helpful for understanding the roles of mast cells in vivo. To date, a dozen mouse models for mast cell deficiency have been reported. However, mice with a specific depletion of all populations of mast cells have not been reported. We generated knock-in mice, termed Mcpt5/Cma1DTR mice, expressing human diphtheria toxin A (DT) receptor under the endogenous promoter of Mcpt5 (also known as Cma1), which encodes mouse mast cell protease-5. Flow cytometry and histological analysis showed that intraperitoneal injection of DT induced almost complete depletion of mast cells in heterozygote Mcpt5/Cma1DTR/+ mice. The deletion rates of mast cells in peritoneal cavity, mesentery, abdominal skin, ear skin, and glandular stomach were 99.9%, 100%, 98.7%, 97.7%, and 100%, respectively. Passive cutaneous anaphylaxis reaction also revealed mast cell deficiency in ear skin after DT treatment. Other than mast cells, a small percentage of marginal zone B cells in Mcpt5/Cma1DTR/+ mice were killed by DT treatment. In conclusion, the Mcpt5/Cma1DTR/+ mouse model is valuable for achieving conditional depletion of all populations of mast cells without inducing a marked reduction in other cells.

Transcriptional inhibition of feline immunodeficiency virus by alpha-amanitin.

Alpha-amanitin, one of the amatoxins in egg amanita, has a cyclic peptide structure, and was reported as having antiviral activity against several viruses. We investigated whether α-amanitin has antiviral activity against feline immunodeficiency virus (FIV). FL-4 cells persistently infected with FIV Petaluma were cultured with α-amanitin. Reverse transcriptase (RT) activity in the supernatant of FL-4 cells was significantly inhibited by α-amanitin. In addition, the production of FIV core protein in FL-4 cells was inhibited by α-amanitin when analyzed by western blotting. Furthermore, α-amanitin inhibited the transcription of FIV in real-time RT-PCR. These data suggested that α-amanitin showed anti-FIV activity by inhibiting the RNA transcription level.

Expansion of Necrosis Depending on Hybrid Motor-Driven Motility of Aeromonas hydrophila in a Murine Wound Infection Model.

The gram-negative bacterium Aeromonas hydrophila is a cause of fulminant and lethal necrotizing soft tissue infections (NSTIs). Suppressing the rapid proliferation of the pathogen and expansion of the necrosis caused in the host is an important issue in clinical practice, but the pathogenic mechanism for the rapid aggravation has not been clarified. In this study, we characterized the function of two types of motor stators in A. hydrophila and explored the role of motility during wound infection. In vitro analysis showed that the motility was reliably maintained while being complemented by the stators. We created a non-motile strain that lacked genes encoding two types of motor stators and analyzed the role of motility in a murine wound infection model. Examination of the bacterial burden in the local infection site and systemic circulation revealed that motility was not essential for the proliferation of A. hydrophila in the host. However, the extent of necrosis at the lesions was lower, and survival times were prolonged in mice infected with the non-motile strain compared with mice infected with the parent strain. These results provide evidence that the rapid expansion of necrosis and the progression to death within a short time period is dependent on the motility of A. hydrophila.

Chemotactic invasion in deep soft tissue by Vibrio vulnificus is essential for the progression of necrotic lesions.

Necrotizing soft tissue infections (NSTI) progress to severe necrosis and result in fatal sepsis within a short time. Vibrio vulnificus is a causative agent and can spread from the initial infection site through soft tissue finally to the systemic circulation of the host. The motility and chemotaxis of this bacterium are essential for proliferation and lethality in a murine model of the infection, but their role in pathogenicity has not been characterized. In this study, we revealed the roles of motility and chemotaxis during the process of V. vulnificus infection. We compared a nonmotile mutant and two nonchemotactic mutants with their parent strain (WT) with regard to bacterial spread using an in vivo imaging system (IVIS) and invasion by detection of bacteria from the muscle and spleen of a murine infection model. WT rapidly spread throughout the infected thigh and invaded deep muscle causing severe tissue damage. The detection rate in the systemic circulation and the lethality were high. On the other hand, the nonmotile mutant stayed at the inoculation site, and the nonchemotactic mutants spread only slowly through the soft tissue of the infected thigh. Detection in the systemic circulation, the degree of tissue damage, and the lethality of nonchemotactic mutants were significantly reduced in mice compared with WT. This study demonstrated that chemotaxis is essential for invasion from the infection site to the deep and distant tissues and the main pathogenic factor for the rapid progression leading to sepsis in V. vulnificus NSTI.

Vibiro vulnificus hemolysin associates with gangliosides.

BACKGROUND: Vibrio vulnificus hemolysin (VVH) is a pore-forming toxin secreted by Vibrio vulnificus. Cellular cholesterol was believed to be the receptor for VVH, because cholesterol could bind to VVH and preincubation with cholesterol inhibited cytotoxicity. It has been reported that specific glycans such as N-acetyl-D-galactosamine and N-acetyl-D-lactosamine bind to VVH, however, it has not been known whether these glycans could inhibit the cytotoxicity of VVH without oligomer formation. Thus, to date, binding mechanisms of VVH to cellular membrane, including specific receptors have not been elucidated. RESULTS: We show here that VVH associates with ganglioside GM1a, Fucosyl-GM1, GD1a, GT1c, and GD1b by glycan array. Among them, GM1a could pulldown VVH. Moreover, the GD1a inhibited the cytotoxicity of VVH without the formation of oligomers. CONCLUSION: This is the first report of a molecule able to inhibit the binding of VVH to target cells without oligomerization of VVH.

Identification of in vivo Essential Genes of Vibrio vulnificus for Establishment of Wound Infection by Signature-Tagged Mutagenesis.

Vibrio vulnificus can cause severe necrotic lesions within a short time. Recently, it has been reported that the numbers of wound infection cases in healthy hosts are increasing, for which surgical procedures are essential in many instances to eliminate the pathogen owing to its rapid proliferation. However, the mechanisms by which V. vulnificus can achieve wound infection in healthy hosts have not been elucidated. Here, we advance a systematic understanding of V. vulnificus wound infection through genome-wide identification of the relevant genes. Signature-tagged mutagenesis (STM) has been developed to identify functions required for the establishment of infection including colonization, rapid proliferation, and pathogenicity. Previously, STM had been regarded to be unsuitable for negative selection to detect the virulence genes of V. vulnificus owing to the low colonization and proliferation ability of this pathogen in the intestinal tract and systemic circulation. Alternatively, we successfully identified the virulence genes by applying STM to a murine model of wound infection. We examined a total of 5418 independent transposon insertion mutants by signature-tagged transposon mutagenesis and detected 71 clones as attenuated mutants consequent to disruption of genes by the insertion of a transposon. This is the first report demonstrating that the pathogenicity of V. vulnificus during wound infection is highly dependent on its characteristics: flagellar-based motility, siderophore-mediated iron acquisition system, capsular polysaccharide, lipopolysaccharide, and rapid chromosome partitioning. In particular, these functions during the wound infection process and are indispensable for proliferation in healthy hosts. Our results may thus allow the potential development of new strategies and reagents to control the proliferation of V. vulnificus and prevent human infections.

Accurate prediction of anti-phagocytic activity of Vibrio vulnificus by measurement of bacterial adherence to hydrocarbonsPrediction of Anti-Phagocytic Activity.

Vibrio vulnificus can cause necrotizing soft tissue infection via exposure through an open wound, and the incubation period in cases of wound infection is only about 16 h. These facts strongly suggest that mechanisms to evade innate immune cell phagocytosis are essential for its pathogenicity. Hydrophobic interaction is one of the binding mechanisms between bacteria and phagocytes. Several factors that maintain cell surface hydrophobicity (CSH) can contribute to anti-phagocytic activity. In this study, we tried to identify V. vulnificus genes involved in maintaining the CSH, in order to elucidate mechanisms of anti-phagocytic activity. We obtained 143 mutants that had lost their ability to proliferate in the host, using signature-tagged transposon basis mutagenesis (STM). The CSH of these mutants was measured by the bacterial adherence to hydrocarbons (BATH) assay. The CSH of only four mutants differed significantly from that of wild type (WT). Of these four mutants, degS mutant (degS::Tn) showed lesser anti-phagocytic activity than WT in the opsonophagocytosis assay, even though degS::Tn showed opaque-type colonies. Furthermore, survival times of mice subcutaneously inoculated with degS::Tn were prolonged. These facts indicated that the BATH assay is a more suitable method of analyzing the anti-phagocytic activity of V. vulnificus than the comparison of colony morphology.

Immunogenicity and protective efficacy of Vibrio vulnificus flagellin protein FlaB in a wound infection model.

Vibrio vulnificus is known as an opportunistic bacterial pathogen that causes primary septicemia and wound infection in humans. Recently, the incidence of wound infection by V. vulnificus is increasing in warm countries. In this study, we examined a vaccine antigen against V. vulnificus in mice. FlaB, a component protein of the V. vulnificus flagellum, was expressed as a recombinant protein, named rFlaB. After immunization of mice with rFlaB, the mice were challenged by subcutaneous inoculation with V. vulnificus. Bacterial burdens in muscular tissue at the infection site in rFlaB-immunized mice were significantly decreased compared with those of control mice. We found that rFlaB immunization can partially suppress proliferation of V. vulnificus at the local infection site.

Both polarity and aromatic ring in the side chain of tryptophan 246 are involved in binding activity of Vibrio vulnificus hemolysin to target cells.

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis against a variety of mammalian cells by forming pores on the cellular membrane. VVH is known to bind to the cellular membrane as a monomer, and then convert to a pore-forming oligomer. However, the structural basis for binding of this toxin to target cells remains unknown. We show here that the polarity and indole ring on the side chain of Trp 246 (W246) of VVH, which sits on a bottom loop, participates in binding to cellular membrane. To clarify the binding mechanisms of VVH, we generated a series of W246 point mutants that were substituted with Arg (W246R), Ala (W246A), or Tyr (W246Y), and tested their binding and cytotoxicity on Chinese hamster ovary (CHO) cells. At a final concentration of 1 µg/ml of VVH, wild type (Wt), W246A and W246Y could bind and induce cytotoxicity to CHO cells, whereas W246R could not. The cytotoxic activity of W246A was significantly lower than that of Wt. These findings indicate that both the polarity and indole ring on the side chain of W246 were involved in the binding of this toxin to the target cellular membrane. The indole ring plays a particularly important role in toxin binding.

Importance of fumarate and nitrate reduction regulatory protein for intestinal proliferation of Vibrio vulnificus.

The sepsis caused by Vibrio vulnificus is characterized by an average incubation period of 26 h and a high mortality rate exceeding 50%. The fast growth and dissemination of V. vulnificus in vivo lead to poor clinical outcomes in patients. Therefore, elucidation of the proliferation mechanisms of this organism in vivo may lead to the development of an effective therapeutic strategy. In this study, we focused on the low oxygen concentration in the intestinal milieu because of its drastic difference from that in air. Fumarate and nitrate reduction regulatory protein (FNR) is known to be a global transcriptional regulator for adaptation to anaerobic conditions in various bacteria. We generated a strain of V. vulnificus in which the fnr gene was replaced with an erythromycin resistance gene (fnr::erm mutant). When the fnr::erm mutant was tested in a growth competition assay against the wild-type (WT) in vivo, the competitive index of fnr::erm mutant to WT in the intestinal loop and liver was 0.378 ± 0.192 (mean ± SD) and 0.243 ± 0.123, respectively. These data suggested that FNR is important for the proliferation of V. vulnificus in the intestine to achieve a critical mass to be able to invade the systemic circulation.

A silkworm infection model to investigate Vibrio vulnificus virulence genes.

The halophilic marine bacterium, Vibrio vulnificus, occasionally causes fatal septicemia in immunocompromised patients. Mice are commonly used as experimental animals to investigate the virulence of V. vulnificus, however, a large number of mice are generally required for bioassays. The present study examined whether the invertebrate species, silkworms, can be used instead of mice to investigate V. vulnificus virulence. When the silkworms were inoculated with 1.2x107 colony forming units of V. vulnificus OPU1­Rf, a virulent strain of V. vulnificus, all injected silkworms died within 48 h, however, those injected with culture filtrate or diluent did not. This silkworm infection model was then used to isolate attenuated V. vulnificus mutants from 1,016 transposon­inserted mutants. Consequently, a harmless mutant, SW998, was isolated. In this strain, the transposon was inserted into the rtxA gene, which is a known V. vulnificus virulence gene. In conclusion, the present study demonstrated that silkworms are useful animals for investigating the virulence of V. vulnificus.

Vibrio vulnificus detected in the spleen leads to fatal outcome in a mouse oral infection model.

Vibrio vulnificus causes rapid disseminating septicemia by oral infection in infected individuals who have an underlying disease, especially chronic liver diseases. Although the elucidation of specific risk factors for V. vulnificus infection in patients with liver diseases is of urgent importance, no appropriate experimental animal model that mimics the liver diseases in this bacterial infection has been available so far. To discover these risk factors, we generated a liver disordered mouse by performing bile duct ligation (BDL). Hepatitis developed in the BDL mice; however, this did not affect mortality in mice after orogastric administration of V. vulnificus, suggesting that the liver disorders caused by the BDL were not risk factors for V. vulnificus septicemia. When the dead and surviving mice were compared, V. vulnificus could be detected from the spleen only in the dead group. Furthermore, significantly higher numbers of V. vulnificus were detected from the intestines in the dead group than in the surviving group ( P < 0.001). These findings suggested that proliferation of the challenge inoculum in the intestine was needed for the oral infection with V. vulnificus, and that the elimination of V. vulnificus in the liver and/or spleen plays a critical role in survival of the host.

Signature-tagged mutagenesis of Vibrio vulnificus.

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.

The Vibrio parahaemolyticus effector VopC mediates Cdc42-dependent invasion of cultured cells but is not required for pathogenicity in an animal model of infection.

Vibrio parahaemolyticus is a Gram-negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homologue of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by non-phagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results alsosuggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibres in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC-mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhoea and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.

Inhibition of binding of Vibrio vulnificus hemolysin (VVH) by MßCD.

Vibrio vulnificus secrets a pore-forming toxin called Vibrio vulnificus hemolysin (VVH). In this study, we showed that methyl-beta-cyclodextrin (MßCD), an oligosaccharide, decreased binding of VVH to Chinese hamster ovary (CHO) cells, resulting in inhibition of its cytotoxicity. When the VVH was incubated with MßCD, cytotoxicity of the toxin was inhibited from 100.3 ± 7.2% to 19.6 ± 5.3%. Binding analysis showed that the amount of VVH on the cells was decreased from 101.4 ± 9.2% to 18.1 ± 8.0% only when MßCD was present in the culture media. Our results indicate that the inhibition of cytotoxicity of VVH by MßCD was due to a decrease in the amount of toxin binding to CHO cells.