When the mutant was complemented with pBAD24-tatABC, CT production of the N16961-dtatABC-cp strain increased compared to that of the mutant strains, N169-dtatABC and N169-dtatABC(pUC18) (P < 0.05 for the N16961-dtatABC-cp/N16961 comparison, and P < 0.05 for the N169-dtatABC-cp/N169-dtatABC comparison, One-Way ANOVA: Post Hoc Multiple Comparisons method, Fig. 6), indicating that the decrease in CT production in the

supernatant of the mutant may result from a defect in the Tat system. Figure 6 CT production in the supernatant of strains N16961, N169-dtatABC, and N169-dtatABC-cp. The strains were cultured find more using the AKI method. Data were obtained in independent triplicate cultures for each strain. We also measured the amount of CT in the cytoplasm. The CT concentration

in the cytoplasm of both N16961 and N169-dtatABC cells was much lower (< 5 ng/ml/OD600) than that in the culture supernatant (14–19 μg/ml/OD600), indicating that most of the CT was exported. The percentages of toxin secreted in the wild type strain and the tatABC mutant were nearly identical (99.97% and Crenigacestat molecular weight 99.93%, respectively). Although CT was still exported in the mutant, its production was markedly decreased compared to that of the wild type strain. We then examined CT gene transcription in the tat mutant and wild type strain with quantitative RT-PCR. We determined that, for the ctxB gene, the difference ΔΔCt of N169-dtatABC/N16961 was 1.523 with thyA as the internal reference and 1.506 with the 16S rDNA gene as the internal reference. Based on 2-ΔΔCt method, the ctxB gene transcription level of N169-dtatABC was 0.348 times compared to N16961 when using thyA as reference, and 0.352 times when using 16s-rDNA gene as reference, showing that cholera toxin gene was downregulated in the Tat mutant when compared to the wild type strain. In vivo colonization and

in vitro cell attachment experiments Colonization in the host intestine is required for the pathogenicity of V. cholerae. To analyze the colonization buy BMS-907351 ability of the tat mutant strain, science a suckling mouse intestine model was used in competitive experiments. We found that the colonization ability of the mutant was less than that of the wild type strain, as the colonization competitive ratio of the wild type strain N16961 to the mutant strain N169-dtatABC was 84:1 (from 40 to 120). Additionally, in the cell culture model, attachment to HT-29 was lower for the mutant than for the wild type strain (Fig. 7A to 7D). The attachment competitive ratio for the wild type strain N16961 to the mutant strain N169-dtatABC was 39: 1 (from 16 to 49). When the mutant strain was complemented with pTatABC-N16961, the attachment ability was restored (Fig. 7D). Figure 7 Colonization and attachment attenuation of the tatABC mutant N169-dtatABC. A.