Urothelial Cultures Support Intracellular Bacterial Community Formation by Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) causes most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invades superficial bladder cells and proliferates rapidly, forming biofilm-like structures called intracellular bacterial communities (IBCs). Using a gentamicin protection assay and fluorescence microscopy, we developed an in vitro model for studying UPEC proliferation within immortalized human urothelial cells. By pharmacologic manipulation of urothelial cells with the cholesterol-sequestering drug filipin, numbers of intracellular UPEC CFU increased 8 h and 24 h postinfection relative to untreated cultures. Enhanced UPEC intracellular proliferation required that the urothelial cells, but not the bacteria, be filipin treated prior to infection. However, neither UPEC frequency of invasion nor early intracellular trafficking events to a Lamp1-positive compartment were modulated by filipin. Upon inspection by fluorescence microscopy, cultures with enhanced UPEC intracellular proliferation exhibited large, dense bacterial aggregates within cells that resembled IBCs but were contained with Lamp1-positive vacuoles. While an isogenic fimH mutant was capable of forming these IBC-like structures, the mutant formed significantly fewer than wild-type UPEC. Similar to IBCs, expression of E. coli iron acquisition systems was upregulated by intracellular UPEC. Expression of other putative virulence factors, including hlyA, cnf1, fliC, kpsD, and the biofilm adhesin yfaL also increased, while expression of fimA decreased and that of flu did not change. These results indicate that UPEC differentially regulates virulence factors in the intracellular environment. 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.Urinary tract infections (UTI) are the second most common infectious disease, in which uropathogenic Escherichia coli (UPEC) causes approximately 80% of community-acquired infections and 40% of nosocomial infections (51, 62, 63). UTI result in seven million clinic visits per year and cost $3.5 billion in treatment, representing a significant burden on the health care system (37). Half of all women will suffer a UTI during their lifetime, with a 25% recurrence rate within 6 months (10, 15). In 50% of these recurrent infections, the same UPEC strain causes both the primary and relapsing UTI (16, 25).Several host-pathogen interactions between urothelial cells and UPEC have been characterized, including UPEC induction of apoptosis, suppression of cytokine secretion, and invasion of urothelial cells (3, 4, 9, 11, 30, 31, 38, 40, 61). UPEC invasion can be mediated by the Dr adhesin, which binds type IV collagen and decay-accelerating factor, or by type 1 pili, which binds mannose residues by the FimH adhesin. Invasion by either mechanism can result in a persistent infection (18, 19, 41, 43, 44, 56, 57). When type 1 pilus-expressing UPEC is internalized, UPEC proliferates and differentiates into intracellular bacterial communities (IBCs), compact aggregates of intracellular bacteria with biofilm-like properties that have been characterized in a murine UTI model (1, 26, 71). IBCs are primarily identified visually by their morphology and location. IBCs are globular in shape, are tightly packed with coccoid bacteria, and typically occupy most of the host cell cytoplasm, causing urothelial cell distortion. Formation occurs 6 to 24 h postinfection, and IBCs express antigen 43 and type 1 pili and secrete a polysaccharide matrix (1, 26). Bacterial genes leuX, fimH, and surA are required for IBC formation, and several iron acquisition systems are upregulated in IBCs (22, 26, 48, 71). Similar to the murine model, IBC-like structures have been identified during human UTIs. Exfoliated cells exhibiting features resembling IBCs were found in patient urine samples, and UPEC isolates originating from asymptomic bacteriura, pyelonephritis, and cystitis patients formed IBCs in the murine UTI model (17, 52).Replicating the conditions required for IBC formation in vitro has proven to be difficult. One report described formation of IBC-like structures in a bladder carcinoma cell line after host cell permeabilization with a detergent (12). The absence of in vitro models is potentially due to the lack of cell lines that sufficiently recapitulate urothelial characteristics. Here we establish an in vitro model of IBC formation by pharmacologic manipulation of immortalized human urothelial cells. Our immortalized cell lines retain numerous characteristics of urothelial cells, including differentiation, inflammatory response, and apoptosis (3, 4, 30, 31, 39, 49, 64, 65). The IBC-like structures reported here are visually and morphologically similar to IBCs formed in the murine UTI model, occur with comparable kinetics, and similarly upregulate iron acquisition systems. Additionally, IBC formation is attenuated in a UPEC mutant lacking fimH. Thus, we have developed a model for studying UPEC IBC formation in vitro.