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OUTER MEMBRANE PROTEINS AS VIRULENCE DETERMINANTS OF ESCHERICHIA COLI
URINARY TRACT INFECTION
T F Nicholson, K M Watts, J A Loughman, P Gilmore, R R Townsend, and D A Hunstad
Washington University School of Medicine, St. Louis, MO
Background: Urinary tract infections (UTIs) in women and children are most often caused by
uropathogenic E. coli (UPEC). Recent studies reveal that bladder epithelial binding by UPEC is followed
by an extensive intracellular phase of pathogenesis, including the development of biofilm-like intracellular
bacterial communities (IBCs) within bladder epithelial cells. The genetic determinants of IBC formation
and intraepithelial persistence, which may seed clinical recurrences of UTI, are largely unknown. Our
previous work demonstrated that intracellular pathogenesis of UPEC depends on SurA, a conserved
periplasmic chaperone thought to support the maturation of outer membrane proteins (OMPs).
Hypothesis: SurA-dependent OMPs are critical determinants of the intracellular phase of UTI caused by
UPEC. Design/Method: To identify SurA-dependent proteins in the E. coli outer membrane (OM), a
comparative membrane proteomics approach was used. Targeted mutation in the UPEC outer membrane
protein A (OmpA) gene was subsequently achieved, and the phenotypes of this mutant were examined in in
vitro and in vivo models of UTI. Results: Proteins whose steady-state levels in the OM were reduced in
the UPEC surA mutant were uniformly beta-barrel porins; the OMP demonstrating the largest fold
reduction in the surA mutant OM was OmpA. The UPEC ompA mutant bound cultured bladder epithelial
cells normally and achieved normal epithelial invasion in the murine bladder. However, the ompA mutant
failed to replicate to wild-type levels in vivo and formed 3-fold fewer IBCs (p < 0.02) that appeared small
and stunted. Further, chronic persistence within the bladder was reduced in the ompA mutant (3 logs fewer
colony-forming units per bladder, p < 0.001); this phenotype was even more evident during competitive coinfections with wild-type UPEC. Persistence was rescued in mice functionally deficient in Toll-like
receptor 4, the primary instigator of the inflammatory response during UTI, suggesting that OmpA may
interact with host neutrophil responses. The UPEC ompA mutant was unable to suppress transepithelial
neutrophil migration in vitro, in contrast to wild-type UPEC (p < 0.001). Conclusions: Comparative
membrane proteomics was used successfully to identify E. coli OM proteins whose maturation depends on
the chaperone SurA. We have identified OmpA as a SurA-dependent OMP important for intracellular
pathogenesis of UTI. Further studies aim to establish the mechanism and molecular basis of these effects.