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Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection
Monday, 2015/03/02 | 07:56:31

Kanna Nagamatsu, Thomas J. Hannan, Randi L. Guest, Maria Kostakioti, Maria Hadjifrangiskou, Jana Binkley, Karen Dodson, Tracy L. Raivio, and Scott J. Hultgren

Subject: MICROBIOLOGY

 

Significance

 

The majority of urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC). Upon UPEC infection, exfoliation of host bladder epithelial (urothelial) cells leads to sloughing of bacteria-laden cells into the urine for expulsion. However, it can also facilitate bacterial dissemination into deeper tissues. Thus, the balance and timing of exfoliation are important in determining disease outcomes. Here, we investigate host–pathogen dynamics in human urothelial cells in vitro and in murine model of acute cystitis. We discovered that the CpxR response regulator-CpxA sensor kinase two-component system regulates the expression of the pore-forming toxin α-hemolysin (HlyA) in response to environmental conditions. HlyA, in turn, is critical for fine-tuning the dynamics of host cell exfoliation and enhancing UPEC fitness during acute UTI.

 

Abstract

 

Urinary tract infections (UTIs) are among the most common bacterial infections, causing considerable morbidity in females. Infection is highly recurrent despite appropriate antibiotic treatment. Uropathogenic Escherichia coli (UPEC), the most common causative agent of UTIs, invades bladder epithelial cells (BECs) and develops into clonal intracellular bacterial communities (IBCs). Upon maturation, IBCs disperse, with bacteria spreading to neighboring BECs to repeat this cycle. This process allows UPEC to gain a foothold in the face of innate defense mechanisms, including micturition, epithelial exfoliation, and the influx of polymorphonuclear leukocytes. Here, we investigated the mechanism and dynamics of urothelial exfoliation in the early acute stages of infection. We show that UPEC α-hemolysin (HlyA) induces Caspase-1/Caspase-4–dependent inflammatory cell death in human urothelial cells, and we demonstrate that the response regulator (CpxR)-sensor kinase (CpxA) two-component system (CpxRA), which regulates virulence gene expression in response to environmental signals, is critical for fine-tuning HlyA cytotoxicity. Deletion of the cpxR transcriptional response regulator derepresses hlyA expression, leading to enhanced Caspase-1/Caspase-4– and NOD-like receptor family, pyrin domain containing 3-dependent inflammatory cell death in human urothelial cells. In vivo, overexpression of HlyA during acute bladder infection induces more rapid and extensive exfoliation and reduced bladder bacterial burdens. Bladder fitness is restored fully by inhibition of Caspase-1 and Caspase-11, the murine homolog of Caspase-4. Thus, we have discovered that fine-tuning of HlyA expression by the CpxRA system is critical for enhancing UPEC fitness in the urinary bladder. These results have significant implications for our understanding of how UPEC establishes persistent colonization.

 

See: http://www.pnas.org/content/112/8/E871.abstract.html?etoc

PNAS February 25, 2015; vol.112; no. 8 E871-E880

 

Fig. 6. ΔCpxR acute virulence is restored by inhibition of Caspase-1. (A) C3H/HeN mice (n = 4 for each group) were inoculated transurethrally with the indicated strains in the presence of DMSO or Ac-YVAD-CMK (Caspase-1/mCaspase-11 inhibitor). After 16 hpi, bladder specimens were homogenized and plated on LB plates. Colonies were counted to determine the number of bacteria per bladder. Data are representative of three independent experiments. All statistics shown are the results of a two-tailed Mann–Whitney t test. Horizontal bars indicate the geometric mean. (B) C3H/HeN mice (n = 4 for each group) were inoculated transurethrally with the indicated strains in the presence of DMSO or Ac-YVAD-CMK. After 16 hpi, urine sample were collected. Fixed urine samples were stained with WGA specific for the host cell surface (red) and TOPRO3 (blue). Arrowheads indicate bacteria. (Scale bar: 10 μm.) (C) Polymorphonuclear leukocytes and host nuclei scores of the urine sediments were determined at 16 hpi. Data are representative of three independent experiments. The statistics in C were determined by a two-tailed Mann–Whitney t test. The horizontal bars indicate the geometric mean. (D) Model for UPEC pathogenesis. UPEC HlyA triggers cell death and release of IL-1α and IL-1β. The dashed arrow indicates that it is not known how Caspase-4 synergizes with the assembled NLRP3 inflammasome to regulate Caspase-1 activation. *P < 0.05; ***P < 0.005.

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