Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection - PubMed (original) (raw)

Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection

Kanna Nagamatsu et al. Proc Natl Acad Sci U S A. 2015.

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.

Keywords: microbial pathogenesis; persistent colonization; urinary tract infection; uropathogenic E. coli.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

CpxR regulates the expression of hemolysin. (A) Indicated strains were plated on blood agar plates and incubated overnight at 37 °C. The densitometry of hemolysis was performed using ImageJ. The densitometry of WT hemolysis is 1. Error bars represent ±SE. (B) In the absence or presence of WT CpxR, hly promoter-driven GFP expression in UTI89 WT and Δ_cpxR_ was observed by fluorescence microscopy. Total bacteria were randomly picked from the fluorescence micrographs, and the percentages of GFP-expressing bacteria were determined. Percentages were based on a count of 100 bacteria, and the values are means ± SD from three independent experiments. (C) HlyA expression in UTI89 WT, Δ_cpxR_, and Δ_hlyA_ was measured by qRT-PCR. The fold difference relative to the parent strain (UTI89 WT) was determined by the ΔΔCt method. (D) Mobility shift assays were performed with MBP-CpxR protein and a PCR product containing the hlyA, cpxP, or rpoD promoter region. Ac-P, acetyl phosphate. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Fig. 2.

Δ_cpxR_ exhibits a colonization defect during acute infection. (A) Graphs depict the colony-forming units determined for the bladders of C3H/HeN mice (n = 5 for each group) infected with 107 cfu of the indicated strains. Mice were killed at 16 hpi, and excised bladders were homogenized and plated on LB plates. Colonies were counted to determine the number of colonized bacteria per organ. (B) Graph depicts the colony-forming units determined for the bladders of C3H/HeN mice (n = 5 for each group) infected with the indicated strains. Mice were killed at 28 d, and excised bladders were homogenized and plated on LB plates. (C) Graph depicts intracellular bacterial titers in the bladders of C3H/HeN mice (n = 5 for each group) infected with the indicated strains. Mice were killed at 1, 3, and 16 hpi, and bladders were removed, bisected, and incubated in gentamicin to kill extracellular bacteria. Gentamicin-treated bladders were washed, homogenized, and plated on LB plates, and colonies were counted to determine the number of intracellular colony-forming units per bladder. Data are representative of two independent experiments. Horizontal bars indicate the geometric mean. (D) Graph depicts IBCs enumerated via LacZ staining in bladders of C3H/HeN mice (n = 5 for each group) infected with the indicated strains. Mice were killed at 6 hpi, and bladders were removed, bisected, and stained with LacZ to quantify IBCs. Data are from two independent experiments. (E) Fixed whole-mounted bladders were stained with DAPI and imaged by fluorescence microscopy. C3H/HeN mice (n = 3 for each group) were inoculated transurethrally with the indicated strains, and bladders removed at 6 hpi. (Scale bar: 50 μm.) The percentage of urothelial cells was based on a unit area (600 μm2). A value of 100 was set for uninfected bladders. Data are representative of three independent experiments. (F) C3H/HeN mice were infected with the indicated strains for 6 h. The homogenized bladder tissues were analyzed for the expression of UpIII by immunoblotting. The band intensity of UpIII was quantified and calculated in arbitrary units set to a value of 1 for uninfected bladder samples by the ImageJ application. *P < 0.05; **P < 0.01.

Fig. 3.

Fig. 3.

Δ_cpxR_ induces pyroptotic cell death in a hemolysin-dependent manner. (A) ATCC HTB-9 (5637) cells were treated for 1 h before and during infection with inhibitors specific for 10 μM Caspase-1/hCaspase-4 (Ac-YVAD-CHO) or Caspase-3 (Ac-DEVD-CHO). Cell death at an MOI of 10 for 4 hpi was determined by measuring LDH release in the presence of DMSO (vehicle control), Ac-YVAD-CHO, or Ac-DEVD-CHO. (B) 5637 cells were infected with the indicated strains in the presence of DMSO, Ac-YVAD-CHO, or Ac-DEVD-CHO. After 4 h, the amount of IL-1β in the culture medium was determined by ELISA. The depicted values are means ± SE from triplicate experiments. (C) 5637 cells were treated for 1 h before and during infection with inhibitors specific for Ac-YVAD-CHO or Ac-DEVD-CHO. After 4 h, the amount of IL-1α in the culture medium was determined by ELISA. The depicted values are means ± SE from triplicate experiments. (D) 5637 cells were transfected with siRNA against Caspase-1, Caspase-4, or NLRP3, or with scrambled siRNA. After 72 h, the cells were infected with UTI89-indicated strains for 4 h. Cell death was determined by measuring LDH release. The amount of IL-1β (E) and IL-1α (F) in the culture medium was determined by ELISA. *P < 0.05; **P < 0.01; ***P < 0.005.

Fig. 4.

Fig. 4.

HlyA triggers Caspase-1 activation and NLRP3 expression. (A) Immunofluorescence microscopy of 5637 cells infected for 4 h with the indicated UTI89 GFP-expressing strains. The 5637 cells were fixed and stained for Caspase-1 (red) and TOPRO3 (blue). Data are representative of three independent experiments. Arrowheads indicate Caspase-1 foci. (Scale bar: 10 μm.) (B) Percentage of cells with speckled, cytoplasmic FLICA staining. (C) 5637 cells were infected with UTI89-indicated strains for 4 h. The cells were analyzed for the expression of cleaved Caspase-1 in the supernatant (Sup) and procaspase-1, as well as for β-actin in the lysates (Lys) by immunoblotting. (D) Immunofluorescence microscopy of 5637 cells infected for 4 h with the indicated UTI89 GFP-expressing strains. The 5637 cells were fixed and stained for NLRP3 (red) and TOPRO3 (blue). Data are representative of three independent experiments. (Scale bar: 10 μm.) (E) 5637 cells infected with indicated strains were randomly picked from the immunofluorescence micrographs shown in D, and the percentages of cells showing NLRP3 staining were determined. Percentages were based on a count of 50 cells, and the values are means ± SD from three independent experiments. *P < 0.05; **P < 0.01.

Fig. 5.

Fig. 5.

Δ_CpxR_ increased pyroptotic cell death by detecting the effects of hemolysin. (A) C3H/HeN mice (n = 5 for each group) infected with the indicated strains were killed at 24 h after infection. The amount of IL-1β in the homogenized bladder specimens was determined by ELISA. All statistics in panels are the results of a two-tailed Mann–Whitney t test. (B) C3H/HeN mice (n = 2 for each group) infected with the indicated UTI89 strains were killed at 6 h after infection. Fixed whole bladders were stained with anti–cleaved Caspase-1 p10 (green), WGA (red), and TOPRO3 (blue). Data are representative of three independent experiments. (Scale bar: 20 μm.) (C) Percentages of bladder umbrella cells showing punctate staining of Caspase-1 were determined as shown in B. The values are means ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Fig. 6.

Δ_CpxR_ 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.

Comment in

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