Figure 5

V-cGAPs regulate bacterial chemotaxis and intestinal colonization in vivo. (A) 293T cells were transfected with VCA0681 (WT or M2) and DncV. After 24 h, extracts from these cells were assayed for the presence of cGAMP activity, which was measured by detecting IRF3 phosphorylation after delivery to permeabilized THP-1 cells. (B) Quantitative RT-PCR assay (top) or immunoblot analysis (bottom) was performed to analyze the RNA or protein levels of V-cGAPs at the indicated times after arabinose (Ara) treatment in V. cholerae ΔdncV mutants expressing DncV from an arabinose-inducible plasmid. NS (nonspecific band) served as the loading control. (C, D) Examination of chemotactic behavior (top) of V. choleraeΔv-cGAPs mutants expressing control vector, arabinose-inducible DncV, and DncV together with IPTG-inducible V-cGAPs (WT or mutant) after induction by arabinose (left) or both arabinose and IPTG (right). Endogenous cGAMP activity assay (bottom) was performed as in A. VCA0210-HD/AA: H382A/D383A; VCA0931-HD/AA: H317A/D318A. (E) In vivo competition experiments measuring the ability of the indicated mutant strains to colonize the infant mouse intestine compared to the parental strain (C6706). Triple indicates the deletion of three V-cGAPs. Statistical significance was determined by comparing colonization ratio of mutants vs the parental strain WT C6706 (Student's t-test, ^***P < 0.001).