Expression of RcrB confers resistance to hypochlorous acid in uropathogenic Escherichia coli

Abstract

To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acids side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections (UTIs), have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. The regulon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB . rcrB encodes the putative membrane protein RcrB, deletion of which substantially increases UPEC's susceptibility to HOCl. However, many questions regarding RcrB's role remain open including whether (i) the protein's mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. Here, we provide evidence that RcrB expression is sufficient to E. coli 's protection from HOCl and induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPEC's biofilm formation.

Importance: Bacterial infections pose an increasing threat to human health exacerbating the demand for alternative treatment options. UPEC, the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be well equipped with powerful defense systems to fend off the toxic effects of RCS. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPEC's most potent defense system towards HOCl-stress and phagocytosis. Thus, this novel HOCl-stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

FIG 1.. Recombinant expression of RcrB significantly improves HOCl resistance in different E. coli strains.

Growth phenotype analyses of the K-12 E. coli strain MG1655 and UPEC strains CFT073 and UTI89 carrying pET28a or rcrB-pET28a were performed in MOPSg media in the presence of the indicated HOCl concentrations. HOCl-mediated LPE was calculated for each strain (see Materials and Methods for a detailed protocol). Squares, MG1655; circles, CFT073; triangles, UTI89; orange filling, + RcrB. Expression of RcrB in (A) MG1655 and (B) UTI89 resulted in an increased HOCl resistance and is comparable to what we observed in the CFT073 strain, (n = 3–4, ± S.D.).

FIG 2.. The HOCl resistance of UPEC clinical isolates depends on the presence of RcrB.

(A) UPEC clinical isolates VUTI229, VUTI288, and VUTI 313 were grown to mid-log phase in MOPSg and changes in the expression of rcrA (black bars) and rcrB (white bars) upon 15 min exposure to 1 mM HOCl were determined by qRT-PCR. rcrA and rcrB mRNA level were elevated in all three strains in the presence of HOCl (n = 3, ± S.D.). (B-D) Growth phenotype analyses of the indicated UPEC clinical isolates causing (B) an asymptomatic infection, (C) cystitis, and (D) pyelonephritis were performed in MOPSg media in the presence of the indicated HOCl concentrations and compared to the K-12 E. coli strain MG1655 and UPEC strain CFT073. HOCl-mediated LPE was calculated for each strain (see Materials and Methods for a detailed protocol). The presence (+) and absence (–) of the rcrB gene in the genome of each strain is indicated in the legend. Diamonds, clinical isolates lacking rcrB; circles, UPEC carrying rcrB; squares, MG1655. The increased HOCl resistance of UPEC clinical isolates depends on the presence of rcrB, (n= 4–7, ± S.D.).

FIG 3.. RcrB plays an important role for UPEC’s HOCl resistance under various growth conditions.

(A, B) Growth phenotype analyses of UPEC strains CFT073 and ∆rcrB were performed in MOPSg media in the presence of the indicated HOCl concentrations. HOCl-mediated LPE was calculated for each strain (see Materials and Methods for a detailed protocol). (A) Exponential cultures (left): overnight cultures were diluted into fresh MOPSg to an OD₆₀₀ = 0.01 and grown until they reached an OD₆₀₀ = 0.1 before they were split up and cultivated in the presence of the indicated HOCl concentrations. (B) Late log cultures (right): overnight cultures were diluted 25-fold into fresh MOPSg and grown until they reached late log / early stationary phase (OD₆₀₀ ~2) before they were diluted again to OD₆₀₀ = 0.1 and cultivated in the presence of the indicated HOCl concentrations; (n = 3–4, ± S.D.). (C, D) Effects of HOCl on the survival of growing and non-growing UPEC strains CFT073 and ∆rcrB. CFT073 and ∆rcrB were grown in MOPSg media to (C) early exponential phase (OD₆₀₀ = 0.1) or (D) late log / early stationary phase (OD₆₀₀ ~2) before they were shifted to MOPS media in the presence or absence of glucose. The indicated concentrations of HOCl were added. After 145 min of incubation, excess HOCl was quenched by the addition of 5-fold excess thiosulfate, cells were serially diluted with PBS, spot-titered onto LB agar plates, and incubated overnight at 37°C. The experiments were repeated at least three independent times.

FIG 4.. RcrB does not protect from HOCl stress when UPEC is grown as biofilms.

Overnight cultures of CFT073 (black bars) and ∆rcrB (white bars) were diluted into LB without NaCl and cultivated under static conditions in 96-well plates in the absence and presence of the indicated HOCl concentrations. After 16 hours, OD₆₀₀ was determined, and planktonic cells removed. The biofilms were washed twice with PBS, stained with 0.1% crystal violet (CV) for 15 min, washed again twice with PBS to remove unbound CV dye. CV bound to biofilm cells was extracted with 30% (v/v) glacial acetic acid and OD₅₉₅ was determined. CV staining (OD₅₉₅) was normalized to the OD₆₀₀ of planktonic cells, (n = 2 [with 8 technical replicates each], ± S.D.).

FIG 5.. RcrB protects UPEC from HOCl stress when cultivated in artificial urine media (AUM).

(A) Growth phenotype analyses of the K-12 E. coli strain MG1655 and UPEC strains CFT073 and ∆rcrB were performed in AUM media in the presence of 380 μM HOCl. HOCl-mediated LPE was calculated for each strain (see Materials and Methods for a detailed protocol). HOCl exposure in MG1655 and ∆rcrB caused ~6 hours higher LPE compared to CFT073, (n = 5, ± S.D.). (B) Survival of MG1655, CFT073, and ∆rcrB upon HOCl exposure was examined in a time-killing assay. Overnight strains grown in LB were diluted ~25-fold into AUM (OD₆₀₀ = 0.1) and exposed to 668 μM HOCl. After 60 min of incubation, cells were serially diluted in PBS, spot-titered onto LB agar, and incubated overnight for CFU counts. Survival of MG1655 and ∆rcrB were significantly reduced compared to CFT073, (n= 6, ± S.D.). (C) UPEC clinical isolates VUTI308 (possesses chromosomal rcrB) and VUTI207 (chromosomal rcrB absent) were grown in AUM in the presence of the indicated HOCl concentrations. After 16 hours of growth at 37°C, OD₆₀₀ was measured and plotted against the HOCl concentrations tested. VUTI308 showed growth between 300–400 μM HOCl exposure while VUTI207 did not, (n= 4, ± S.D.).

FIG 6.. Transcription of the RcrR regulon is induced over a longer time following exposure to HOCl.

UPEC strain CFT073 was grown in MOPSg to mid-log phase (OD₆₀₀ ~0.5–0.55) before it was incubated with the indicated HOCl concentrations for the indicated time. Transcription was stopped by the addition of ice-cold methanol. RNA was extracted, residual DNA removed, and mRNA reverse transcribed into cDNA. Transcript levels of the indicated genes were determined by qRT-PCR. Gene expression was normalized to the housekeeping gene rrsD and calculated as fold changes based on expression levels in the untreated control. (A) Fold-change in transcript level of select members of the RcrR regulon following treatment with increasing concentrations of HOCl for 15 min, (n=3, ± S.D.). (B-D) Fold-changes in transcript level of select members of the (B) RcrR, (C) NemR, and (D) RclR regulons following treatment with 1 mM HOCl for the indicated time points, (n=3, ± S.D.).

FIG 7.. Transcription of the RcrR regulon is induced in the presence of HOCl and its byproducts but not H₂O₂ or HOSCN.

UPEC strain CFT073 was grown in MOPSg to mid-log phase (OD₆₀₀ ~0.5–0.55) prior to incubation with sublethal concentrations of the indicated stressors for 15 min (HOCl, 1 mM; NCT, 0.3 mM; NH₂Cl, 0.3 mM; HOSCN, 0.35 mM; HOBr, 0.5 mM; Ca(OCl)₂, 0.2 mM; Gly-Cl, 0.5 mM; H₂O₂, 10 mM). Transcription was stopped by the addition of ice-cold methanol. RNA was extracted, residual DNA removed, and mRNA reverse transcribed into cDNA. The induction of transcript levels of the indicated genes was determined by qRT-PCR. Gene expression was normalized to the housekeeping gene rrsD and calculated as fold changes based on expression levels in the untreated control. rcrB and rcrR transcript level were increased following treatment with HOCl, NCT, HOBr, NH₂Cl, Ca(OCl)₂, and Gly-Cl, respectively. No change in transcript level was seen in the presence of H₂O₂, while exposure to HOSCN resulted in downregulation of rcrB/rcrR (n=3, ± S.D.).

FIG 8.. The RcrR regulon protects from HOCl stress and its byproducts but not from H₂O₂ and HOSCN.

Growth phenotype analyses of the K-12 E. coli strain MG1655 carrying pET28a or rcrB-pET28a, respectively, as well as UPEC strains CFT073, ∆rcrB, and ∆rcrR were performed in MOPSg media in the presence of the indicated stressor concentrations. LPE were calculated for each strain (see Materials and Methods for a detailed protocol). Endogenous and recombinant expression of RcrB in CFT073 and MG1655, respectively, resulted in an increase in resistance to all stressors tested except to HOSCN and H₂O₂.

FIG 9.. RcrB and RclC do not complement each other during HOCl exposure.

Complementation analyses of the UPEC strains CFT073, ∆rcrB, ∆rclC, and ∆rcrB∆rclC in the presence of rcrB-pET28a, rclC-pET28a, and the empty vector control pET28a were performed in MOPSg media in the presence of the indicated HOCl concentrations. HOCl-mediated LPE was calculated for each strain (see Materials and Methods for a detailed protocol). The absence of CFT073 rclC does not cause increased HOCl sensitivity nor does recombinant RclC expression complement the ∆rcrB strain, (n = 3, ± S.D.).

FIG 10.. Summary of the findings of this study.

The RcrR regulon plays an important role for UPEC’s survival during RCS stress, including their exposure to HOCl, HOBr, and the byproducts of HOCl, such as NCT, Gly-Cl, NH₂Cl, and Ca(OCl)₂, respectively. The protective effect of the RcrR regulon is exclusively mediated by RcrB given that expression of plasmid encoded RcrB is sufficient for an increased HOCl resistance in E. coli strains that lack the RcrR regulon. In contrast, RcrR neither responds to HOSCN nor to H₂O₂. RCS induces RcrB expression in exponentially growing and stationary phase UPEC in minimal and artificial urine media. No such protective effect was observed in biofilm-forming UPEC cells. In contrast to the NemR and RclR regulons which play significant roles for protection of E. coli K12 strains from HOCl, expression of the RcrR regulon appears to be the main RCS defense system in UPEC given its prolonged expression during RCS exposure.