| Uropathogenic E. coli (UPEC) cause most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invade superficial bladder cells and proliferate rapidly, forming biofilm-like structures called intracellular bacterial communities (IBC). Using a gentamicin protection assay and fluorescence microscopy, I have developed an in vitro model for studying UPEC invasion and proliferation within immortalized human urothelial cells. By pharmacologic manipulation and RNA interference, a number of host proteins were identified that are involved in E. coli invasion of urothelial cells. Using isogenic mutants, bacterial components FimH and lipopolysaccharide (LPS) were demonstrated to play a role in E. coli invasion. These studies showed that E. coli invades urothelial cells by multiple mechanisms, and that UPEC and K-12 E. coli invade by different mechanisms. UPEC, but not K-12 E. coli, engaged in beta1 integrin-dependent adherence and invasion. Additionally, UPEC strains with rough-LPS were more invasive than strains with smooth LPS in a TLR4-dependent manner. UPEC proliferated within urothelial cells, while K-12 strains could not. Intracellular proliferation was dependent on the expression of full length LPS, and proliferating UPEC were eradicated by exposure to lysosomotropic drugs chloroquine or propranolol. This in vitro model is a valuable tool for identifying host and bacterial components required for urothelial invasion and intracellular proliferation by UPEC, and for facilitating the identification of potential treatments for UTI.;Pre-incubation of urothelial cells with the cholesterol-sequestering drug filipin resulted in increased numbers of intracellular UPEC relative to untreated cultures. Upon inspection by fluorescence microscopy, filipin-treated cultures exhibited large, dense bacterial aggregates within cells that resembled IBC. In vitro IBC required that the urothelial cells, but not the bacteria, be filipin-treated prior to infection. IBC-like structures were morphologically indistinguishable from, and shared many characteristics with IBC in the murine model of UTI. These characteristics include the kinetics of formation, the expression of antigen 43, a requirement for fimH , and the up-regulation of iron acquisition systems. Thus, immortalized urothelial cultures that recapitulate IBC formation in vitro represent a novel system for the molecular and biochemical characterization of the UPEC intracellular life cycle. |
