g AvrA Tight Junction A Control pCMV AvrA C186A Occludin-1 ZO-1 Claudin-1 Villin AvrA B Relative density 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 C pCMV AvrA C186A Occludin P<0.05, P<0.01 ZO1 Claudin1 the report by Kohler et al.. In Fig. 1B, the immunoblot intensity analysis demonstrated that occludin and ZO-1 expression was significantly increased by the presence of PhoPc with AvrA protein expression, whereas the AvrA-deficient strain and wild-type Salmonella 14028s with insufficient AvrA protein induced a significantly less in ZO-1 and occludin expression. AvrA expression also stabilized TJ proteins in HT-29CL19A monolayers. AvrA overexpression in epithelial cells increases TJ protein expression To determine whether AvrA expression directly regulates TJ protein, we transfected human colonic epithelial HT29CL19A cells with a pCMV-myc-AvrA wild-type gene construct, a pCMVmyc-AvrAC186A AvrA mutant construct, or a control pCMV-myc plasmid. The AvrA mutant C186A is a single amino acid residue transition which is mutated at the key cysteine required for AvrA activity as previously described. As shown in Fig. 2, AvrA overexpression in colonic epithelial cells increased ZO-1, claudin1, and occludin-1 expression significantly, whereas the AvrA mutant C186A was able to reverse the effect and decrease the TJ protein expression to the levels comparable to those in the cells transfected with empty pCMV-myc vector. These data indicate that AvrA expression directly increases 10336422 TJ protein expression. The cysteine site required for the AvrA activity is involved in AvrA regulation of TJ protein expression. AvrA expression alters tight junction protein distribution in vitro We further examined tight junction protein distribution. Epithelial cells colonized with AvrA-sufficient or -deficient strains were analyzed for the location of claudin-1 and ZO-1. ZO-1: ZO-1 is a cytoplasmic plaque tight junction protein. In control monolayers without any treatment, ZO-1 was restricted to cellular borders and distributed in a smooth arc-like pattern. In AvrA Tight Junction Control PhoPc AvrA- ZO-1 Claudin-1 ZO-1 Claudin-1 DAPI Zsection PhoPc treated cells, the distribution of ZO-1 was very similar to that in the control cells. The appearance of ZO-1 in the PhoPc group was similar to the control group when cells were viewed in cross-Z-section. However, in cells treated with Salmonella derivative AvrA- mutant, the normally smooth arc-like ZO-1 profiles were transformed into a complex series of irregular undulations. Further, ZO-1 staining became thinner and more sinuous. The Z-section panel in Fig. 3 shows the weak staining of ZO-1 in AvrA2. Claudin-1: Claudin-1 is highly enriched at sites of cell-cell contact, co-localizing with the TJ marker, ZO-1. AvrA absence induced a disorganization of transmembrane protein claudin-1, and the protein was expanded intracellularly. Interestingly, PhoPC treatment also LY2109761 slightly changed the distribution of claudin-1. Intracellular AvrA Tight Junction claudin-1 was detectable in the cytosol 
of the cells colonized with PhoPc. This indicated that additional bacterial proteins may be involved in 18347139 regulating TJs. Overall, our immunofluorescence data suggest that AvrA modulates junctional localization of ZO-1 and claudin-1 proteins. sion was able to significantly decrease cell permeability. Our data demonstrated that AvrA-sufficient bacteria significantly decrease the intestinal permeability compared to AvrA-deficient bacteria. It indicates the
