Abstract
ABSTRACTUropathogenic Escherichia coli (UPEC) is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs). Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs) of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies.
Highlights
Uropathogenic Escherichia coli (UPEC) accounts for over 85% of reported community-acquired urinary tract infections (UTI) [1]
Mouse model studies have shown that the ability of UPEC strains to form intracellular bacterial communities (IBCs) allows UPEC to persist in the face of a stringent population bottleneck during acute cystitis, leading to a range of infection outcomes such as the formation of quiescent intracellular reservoirs (QIRs) or the development of chronic cystitis, which is characterized by persistent high-titer bacteriuria (Ͼ104 CFU/ ml) and high-titer bacterial bladder burdens (Ͼ104 CFU) 2 or more weeks after inoculation, accompanied by chronic inflammation [7, 15]
Our analysis of the IBC transcriptome identified several similar transcriptional profiles found in studies that have probed the UPEC transcriptome of bacteria collected from the urine of patients diagnosed with a UTI; those studies did not screen for the presence of IBCs [26,27,28], which can be sloughed into the urine, depending on the timing of the onset of IBC formation and collection of the urine [12]
Summary
Uropathogenic Escherichia coli (UPEC) accounts for over 85% of reported community-acquired urinary tract infections (UTI) [1]. These factors include micturition, exfoliation, expulsion, and the innate immune response [9, 10, 16] Such bottlenecks prevent the use of large-scale in vivo transposon screens to identify factors involved in mediating IBC expansion and UTI progression and have impeded substantial progress in this area of UPEC pathogenesis research. Several genes associated with alternative sugar metabolism processes, including lacZ and srlA, were upregulated, suggesting the absence of glucose and the presence of nonglucose sugars for utilization These data corroborated previous circumstantial observations of lacZ upregulation in IBCs, based on strong X-Gal (5bromo-4-chloro-3-indolyl--D-galactopyranoside) staining of IBCs indicative of beta galactosidase activity within the intracellular bacterial community [17, 18, 25]. Our findings further our insights into conditions within the unique intracellular niche of IBCs and provide direction for future studies focused on the development of therapeutics targeting UPEC UTIs (both acute and chronic/recurrent)
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