Takashige Kashimoto

Vibrio vulnificus induces macrophage apoptosis in vitro and in vivo.

ABSTRACT In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of V. vulnificus.

Matrix metalloproteinase-2 stimulates collagen-I expression through phosphorylation of focal adhesion kinase in rat cardiac fibroblasts

Collagen-I is thought to be the main component of the extracellular matrix in cardiac fibrosis, the accumulation of which occurs with excessive activation of matrix metalloproteinase-2 (MMP-2). MMP-2 degrades the extracellular matrix; however, the relative importance of MMP-2 to collagen-I synthesis in cardiac fibroblasts remains unclear. We investigated whether extracellular activation of MMP-2 regulates collagen-I synthesis and phosphorylation of focal adhesion kinase (FAK) in rat cardiac fibroblasts. Primary cultures of rat cardiac fibroblasts were incubated with purified active MMP-2 to determine whether extracellular MMP-2 affects collagen-I synthesis and FAK phosphorylation in cardiac fibroblasts. Exogenous MMP-2 significantly stimulated FAK (Tyr397) phosphorylation and induced collagen-I expression in a time-dependent manner. Simultaneous treatment with the FAK inhibitor PF573228 abolished exogenous MMP-2-enhanced FAK (Tyr397) phosphorylation and collagen-I expression. Cells were then stimulated with norepinephrine (NE) to investigate whether endogenous MMP-2 could also induce collagen-I expression through FAK (Tyr397) phosphorylation. NE-stimulated endogenous MMP-2 activation in conditioned medium was significantly attenuated by simultaneous treatment with the MMP inhibitor PD166793. Similarly, NE-induced FAK (Tyr397) phosphorylation and collagen-I expression were significantly inhibited by simultaneous treatment with PD166793 or PF573228. Furthermore, MMP-2 knockdown induced by small interfering RNA (siRNA) significantly abolished endogenous MMP-2 expression and activation. MMP-2 siRNA significantly abolished NE-induced FAK (Tyr397) phosphorylation and collagen-I expression. These findings suggest that the extracellular activation of MMP-2 accelerated collagen-I synthesis in rat cardiac fibroblasts and that FAK phosphorylation (Tyr397) plays a pivotal role in MMP-2-stimulated collagen-I synthesis.

Dominant-negative Rho, Rac, and Cdc42 facilitate the invasion process of Vibrio parahaemolyticus into Caco-2 cells.

ABSTRACT To clarify the invasive process of Vibrio parahaemolyticus, an invasion assay was performed using cells expressing dominant negative small GTPases of the Rho family. This assay showed that the dominant negative host phenotype facilitates bacterial invasion, suggesting that the mechanism of V. parahaemolyticus invasion differs from that reported for other invasive bacteria.

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.

Identification of a Receptor-Binding Domain of Bordetella Dermonecrotic Toxin

ABSTRACT Bordetella dermonecrotic toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating or polyaminating Gln63 of the small GTPase Rho. DNT is an A-B toxin which is composed of an N-terminal receptor-binding (B) domain and a C-terminal enzymatically active (A) domain. In this study, to analyze the functional and structural organization of DNT, we prepared 10 clones of hybridoma producing anti-DNT monoclonal antibodies. One of these antibodies, 2B3, neutralized the effects of DNT on target cells when mixed with the toxin. When microinjected into cells, however, 2B3 did not inhibit the intoxication by DNT. Western blot analysis revealed that 2B3 recognized the N-terminal region of DNT. To delineate the DNT-binding domain, we examined a series of truncated DNT mutants for the ability to competitively inhibit the intoxication of cells by the full-length DNT and found that a fragment consisting of the N-terminal 54 amino acids (DNT1-54) was the smallest inhibitory fragment. The radioiodinated DNT1-54 actually bound to target cells, which was inhibited by 2B3. These results suggest that the N-terminal 54 amino acids of DNT are responsible for the binding to target cells. DNT1-54 bound to none of the DNT-resistant cells, implying the presence of a cell surface receptor specific to DNT-sensitive cells.

A novel mechanism of autolysis in Helicobacter pylori : Possible involvement of peptidergic substances

Background.  Helicobacter pylori survival in a hostile acidic environment is known to be caused by its production of urease, which is not released by known secretion pathways. It has been proposed that H. pylori cells undergo spontaneous autolysis during cultivation and that urease becomes surface‐associated only concomitant with bacterial autolysis. The aim of this study was to elucidate mechanisms by which H. pylori cells undergo autolysis during cultivation.

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO3) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO3 in the kidneys. A significant increase in 8-hydroxy-2′-deoxyguanosine in nuclei of the kidney was detected only in the KBrO3 treated mice, while the comet assay showed that the Fe-NTA and KBrO3 treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.

The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization of Vibrio vulnificus Hemolysin

Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.

Efficacy of Soluble Recombinant FliC Protein from Salmonella enterica Serovar Enteritidis as a Potential Vaccine Candidate Against Homologous Challenge in Chickens

Summary. FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 109 colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-&ggr; secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed. Resumen. Eficacia de la proteína recombinante soluble FliC de Salmonella enterica serovar Enteritidis como un candidato potencial para una vacuna contra el desafío homólogo en pollos. El antígeno de flagelina FliC de Salmonella Enteritidis, se puso a prueba como un candidato para vacuna en su efecto protector frente a un desafío homólogo en pollos. Después de la inmunización con FliC recombinante (rFliC) o la administración de solución amoriguada de fosfatos (PBS) a los 56 días de edad, los pollos fueron desafiados con 109 unidades formadoras de colonias de Salmonella Enteritidis a los 76 días de edad. Las aves vacunadas mostraron una disminución significativa de los conteos bacterianos en el hígado y en los contenidos cecales en comparación con las aves a las que se les administró PBS a los siete días después del desafío, pero la protección fue parcial. El experimento de replicación también mostró un resultado similar. En ambos experimentos, la vacunación indujo un aumento de IgG anti-rFliC en el suero, que era también reactiva a los flagelos nativos. El nivel de IgA intestinal fue ligeramente mayor en las aves vacunadas en comparación con las aves del grupo control. Sin embargo, ni la respuesta proliferativa, ni la secreción de &ggr;-interferón por las células del bazo en respuesta a la estimulación con rFliC fue inducida. Por lo tanto, el efecto de la proteína rFliC como una vacuna es limitada, y se requiere de un perfeccionamiento posterior.

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.