mimicus strains, we compared the cytotoxicity of the wild-type an

mimicus strains, we compared the cytotoxicity of the wild-type and mutant strains for cultured cell lines. T3SS-deficient mutants were constructed by disruption of the homologue of the vscN2 gene, which encodes an ATPase of

T3SS2, in V. mimicus RIMD2218042 (α type) and RIMD2218067 (β type) strains. To confirm the deletion of the vscN2 gene, PCR amplification using oligonucleotide primer pairs was performed (see Additional file 1 and 8). The growth of the mutant strains MX69 in LB medium (1% NaCl) was indistinguishable from that of the parental strains (data not shown). Both V. mimicus RIMD2218042 and RIMD2218067 strains were cytotoxic for Caco-2 cells at 3 h post-infection. The cytotoxicity of both the T3SS2α- and T3SS2β-deficient mutant strains tended to decrease, but there were no significant differences between T3SS2α- and T3SS2β-deficient mutant strains and their parental strains (see Additional file 9). Discussion A recent study of ours demonstrated that two lines of distinct lineage of the T3SS2 gene cluster, T3SS2α and T3SS2β, are ARS-1620 purchase present in the KP-positive and trh-positive V. parahaemolyticus strains, respectively C59 purchase [20]. Although a previously reported study using dot blot

analysis could not detect the genes for T3SS2 in 16 Vibrio species, the probes and PCR primers used in previous studies were designed based on the sequence information of the T3SS2α genes in V. parahaemolyticus strain RIMD2210633 [7, 14]. Since the T3SS2β genes cannot be detected by either PCR amplifications or comparative genomic hybridization analysis targeting the T3SS2α genes [7, 15], we re-investigated the distribution of the T3SS2 genes, both T3SS2α and T3SS2β, in Vibrio species. To examine the distribution of the genes for T3SS2 in vibrios other than V. parahaemolyticus, we performed a PCR assay using PCR primer pairs targeting both the T3SS2α and T3SS2β genes. Of the 32 Vibrio species tested, the T3SS2-related genes were detected in three species, V. cholerae, which was previously reported, as well as V. hollisae and V. mimicus. In V. hollisae strains, only three genes for T3SS2α, Lepirudin vscN2, vscR2, and vscT2, were detected. Nevertheless,

the fact that the PCR reactions for these three genes were positive in all the five V. hollisae strains tested is intriguing. We speculate that the other genes for T3SS2α might be absent in these particular V. hollisae strains, or that the sequences of the other genes included variations that would make PCR amplification with the primer pairs used in this assay difficult. These possibilities should be examined in the future by more detailed genetic analyses, e.g. sequencing of the region flanking the T3SS2-related genes. A previous study showed that the T3SS2-related genes are present in V. mimicus strains [25]. In our study, the PCR assay also demonstrated the presence of the T3SS2 genes in V. mimicus strains. Of the 15 V.

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