Forced resurgence and targeting of intracellular uropathogenic Escherichia coli reservoirs.

Intracellular quiescent reservoirs of uropathogenic Escherichia coli (UPEC), which can seed the bladder mucosa during the acute phase of a urinary tract infection (UTI), are protected from antibiotic treatments and are extremely difficult to eliminate. These reservoirs are a potential source for recurrent UTIs that affect millions annually. Here, using murine infection models and the bladder cell exfoliant chitosan, we demonstrate that intracellular UPEC populations shift within the stratified layers of the urothelium during the course of a UTI. Following invasion of the terminally differentiated superficial layer of epithelial cells that line the bladder lumen, UPEC can multiply and disseminate, eventually establishing reservoirs within underlying immature host cells. If given access, UPEC can invade the superficial and immature bladder cells equally well. As infected immature host cells differentiate and migrate towards the apical surface of the bladder, UPEC can reinitiate growth and discharge into the bladder lumen. By inducing the exfoliation of the superficial layers of the urothelium, chitosan stimulates rapid regenerative processes and the reactivation and efflux of quiescent intracellular UPEC reservoirs. When combined with antibiotics, chitosan treatment significantly reduces bacterial loads within the bladder and may therefore be of therapeutic value to individuals with chronic, recurrent UTIs.

The Cpx stress response system potentiates the fitness and virulence of uropathogenic Escherichia coli.

Strains of uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections, representing one of the most widespread and successful groups of pathogens on the planet. To colonize and persist within the urinary tract, UPEC must be able to sense and respond appropriately to environmental stresses, many of which can compromise the bacterial envelope. The Cpx two-component envelope stress response system is comprised of the inner membrane histidine kinase CpxA, the cytosolic response regulator CpxR, and the periplasmic auxiliary factor CpxP. Here, by using deletion mutants along with mouse and zebrafish infection models, we show that the Cpx system is critical to the fitness and virulence of two reference UPEC strains, the cystitis isolate UTI89 and the urosepsis isolate CFT073. Specifically, deletion of the cpxRA operon impaired the ability of UTI89 to colonize the murine bladder and greatly reduced the virulence of CFT073 during both systemic and localized infections within zebrafish embryos. These defects coincided with diminished host cell invasion by UTI89 and increased sensitivity of both strains to complement-mediated killing and the aminoglycoside antibiotic amikacin. Results obtained with the cpxP deletion mutants were more complicated, indicating variable strain-dependent and niche-specific requirements for this well-conserved auxiliary factor.

Regulation of membrane protein degradation by starvation-response pathways.

The multivesicular body (MVB) pathway delivers membrane proteins to the lumen of the vacuole/lysosome for degradation. The resulting amino acids are transported to the cytoplasm for reuse in protein synthesis. Our study shows that this amino acid recycling system plays an essential role in the adaptation of cells to starvation conditions. Cells respond to amino acid starvation by upregulating both endocytosis and the MVB pathway, thereby providing amino acids through increased protein turnover. Our data suggest that increased Rsp5-dependent ubiquitination of membrane proteins and a drop in Ist1 levels, a negative regulator of endosomal sorting complex required for transport (ESCRT) activity, cause this response. Furthermore, we found that target of rapamycin complex 1 (TORC1) and a second, unknown nutrient-sensing system are responsible for the starvation-induced protein turnover. Together, the data indicate that protein synthesis and turnover are linked by a common regulatory system that ensures adaptation and survival under nutrient-stress conditions.