Abstract
Uropathogenic Escherichia coli (UPEC) is the causative bacterium in most urinary tract infections (UTIs). UPEC cells adhere to and invade bladder epithelial cells (BECs) and cause uropathogenicity. Invading UPEC cells may encounter one of several fates, including degradation in the lysosome, expulsion to the extracellular milieu for clearance, or survival as an intracellular bacterial community and quiescent intracellular reservoir that can cause later infections. Here we considered the possibility that UPEC cells secrete factors that activate specific host cell signaling networks to facilitate the UPEC invasion of BECs. Using GFP-based reporters and Western blot analysis, we found that the representative human cystitis isolate E. coli UTI89 and its derivative UTI89ΔFimH, which does not bind to BECs, equally activate phosphatidylinositol 4,5-bisphosphate 3-OH kinase (PI3K), Akt kinase, and mTOR complex (mTORC) 1 and 2 in BECs. We also found that conditioned medium taken from UTI89 and UTI89ΔFimH cultures similarly activates epidermal growth factor receptor (EGFR), PI3K, Akt, and mTORC and that inhibition of EGFR and mTORC2, but not mTORC1, abrogates UTI89 invasion in vitro and in animal models of UTI. Our results reveal a key molecular mechanism of UPEC invasion and the host cells it targets, insights that may have therapeutic utility for managing the ever-increasing number of persistent and chronic UTIs.
Highlights
Uropathogenic Escherichia coli (UPEC) is the causative bacterium in most urinary tract infections (UTIs)
Our results suggest that PI3K, Akt, and mTOR complex 2 (mTORC2) facilitate bacterial invasion, but how these intracellular enzymes become activated in response to extracellularly found UPEC (UTI89⌬FimH) and bacterial conditioned medium (CM) remains unclear
It is well-accepted that UPEC invade host bladder epithelial cells, which may be accomplished through phagocytosis, via induction of actin polymerization, and/or pinocytosis of caveolae and clathrin-coated pits assembled on the plasma membrane
Summary
Our work has implicated dynamin in vesicle trafficking and UPEC invasion [16, 17], but the exact mechanisms involved remain incomplete. UTI89⌬FimH bacteria are deficient in binding to BECs (Fig. S1A) and, like UTI89, are fully capable of activating PI3K and Akt (Fig. 1), suggesting possible involvement of bacterial secreted factor(s). The Western blotting results showed a time-dependent increase of p-Akt by UTI89 (and UTI89⌬FimH) and their respective CM (Fig. S2C), supporting the conclusion that UPEC CM–found factor(s) act upon host BECs to activate specific signaling networks involved in bacterial invasion. Neither knockdown of Raptor nor Rictor had any effect on UTI89 attachment to BECs (Fig. 2I) These results demonstrate a requirement for mTORC2, but not mTORC1, in UTI89 entry but not attachment to BECs. Our results suggest that PI3K, Akt, and mTORC2 facilitate bacterial invasion, but how these intracellular enzymes become activated in response to extracellularly found UPEC (UTI89⌬FimH) and bacterial CM remains unclear.
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