Escherichia coli Uropathogenesis In Vitro : Invasion, Cellular Escape, and Secondary Infection Analyzed in a Human Bladder Cell Infection Model

Abstract

Uropathogenic Escherichia coli (UPEC) strains are capable of invading bladder epithelial cells (BECs) on the bladder luminal surface. Based primarily on studies in mouse models, invasion is proposed to trigger an intracellular uropathogenic cascade involving intracellular bacterial proliferation followed by escape of elongated, filamentous bacteria from colonized BECs. UPEC filaments on the mouse bladder epithelium are able to revert to rod-shaped bacteria, which are believed to invade neighboring cells to initiate new rounds of intracellular colonization. So far, however, these late-stage infection events have not been replicated in vitro. We have established an in vitro model of human bladder cell infection by the use of a flow chamber (FC)-based culture system, which allows investigation of steps subsequent to initial invasion. Short-term bacterial colonization on the FC-BEC layer led to intracellular colonization. Exposing invaded BECs to a flow of urine, i.e., establishing conditions similar to those faced by UPEC reemerging on the bladder luminal surface, led to outgrowth of filamentous bacteria similar to what has been reported to occur in mice. These filaments were capable of reverting to rods that could invade other BECs. Hence, under growth conditions established to resemble those present in vivo, the elements of the proposed uropathogenic cascade were inducible in a human BEC model system. Here, we describe the model and show how these characteristics are reproduced in vitro.

Quantification of filamentation by uropathogenic Escherichia coli during experimental bladder cell infection by using semi-automated image analysis

“…It has been suggested that UPEC filamentation is induced by intracellular stimuli from the invaded BEC itself or from recruited polymorphonuclear leukocytes (Horvath et al, 2011;Justice et al, 2006). However, recent results from our group showed that the physical and chemical growth conditions in the urinary tract can lead to strong and reversible filamentation of wild-type UPEC (Andersen et al, 2012). Specifically, we found that surface colonization in flow chambers in continuous flow of highly concentrated human urine leads to strong filamentation similar to what is observed in mouse UTI models (Justice et al, 2004) and urine samples from UTI patients (Rosen et al, 2007).…”

“…In this cyclic pathway UPEC invades the superficial bladder epithelial cell (BEC) layer, forms intracellular colonies and ultimately breaks out of the host cell in the form of highly filamentous bacteria (reviewed in Justice et al, 2008;Kau et al, 2005). By reverting back to its normal rod-shape through coordinated cell division each UPEC filament can give rise to multiple infectious single bacteria (Andersen et al, 2012;Justice et al, 2004), which can reinvade neighboring cells of the bladder epithelium. The intracellular uropathogenic cascade enables the bacterium to survive the host immune response as well as antibiotic treatment and is believed to account for the frequent recurrences of this type of infection.…”

“…This approach is based on the fact that filaments only form one colony on agar plates but contribute increasingly to the OD 600 -value or protein concentration with increasing size. The degree of filamentation may also be quantified using flow cytometry (Andersen et al, 2012;Horvath et al, 2011;Mattick et al, 2000), which relies on increased light scattering and passage time for filaments compared to rods. Both of these methods are fast and relatively straightforward to use but yield only relative estimates.…”

“…Data is obtained within minutes from several thousand bacteria detected in focus stacked images, yielding a strong statistical basis for estimation of mean bacterial cell lengths. We applied the method together with a recently established flow chamber (FC)-based bladder cell infection model which enables reproduction of the uropathogenic cascade in vitro (Andersen et al, 2012), to investigate the influence of BEC invasion and substratum surface, on the filamentous response by the prototypical UPEC strain UTI89. Our results show that filamentation is not strictly dependent on the uropathogenic cascade, as suggested by previous mouse studies (Justice et al, 2004(Justice et al, , 2006.…”

Strengths and Limitations of Model Systems for the Study of Urinary Tract Infections and Related Pathologies

“…The ease of use, low cost, and amenability to high-throughput assays, genetic manipulation, and biochemical analysis make cell culture-based systems attractive alternatives to many animal infection models. Recent advances with flow cell technology even make it possible to examine UPEC-host interactions using bladder cell monolayers in the presence of urine flow, mimicking conditions within the urinary tract during an actual UTI (181). Despite the proven utility of cell culture-based model systems, they cannot fully recapitulate the complexity of the host environment with its myriad cell types, complicated tissue architecture, variable nutrient levels, and teeming host defenses.…”