Fig 4.
YbcLUTI is secreted. (A and B) The level of PMN migration elicited by various strains of E. coli (A) or conditioned medium (B) was evaluated using the transepithelial PMN migration model. YbcL variants tethered to either the inner or outer bacterial membrane (YbcLIM or YbcLOM, respectively) were unable to suppress PMN migration when expressed episomally in UTI89 ΔybcL. The trends in PMN migration observed with conditioned medium mimicked those observed when the inoculum included live bacteria. Asterisks in panels A and B indicate statistically significant (P < 0.05) increases in PMN migration compared to that with wild-type UTI89. (C) Localization of YbcL variants was assessed by Western blotting. After 1 h of infection of 5637 cells or PMN with the indicated strains of E. coli, the supernatant (S) and eukaryotic cell lysate (L) fractions were filter sterilized, TCA precipitated and resolved by SDS-PAGE. During infection of either cell type, YbcLUTI and YbcLOM were clearly detected in the supernatant fractions, while YbcLIM was minimally detected in those fractions. All three variants were detected in the PMN lysate; however, only YbcLUTI was detected in the 5637 cell lysate. An equivalent volume of each bacterial inoculum (I) is shown for comparison across strains.