PrtR homeostasis contributes to Pseudomonas aeruginosa pathogenesis and resistance against ciprofloxacin

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced by P. aeruginosa that are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component of P. aeruginosa. In this study, we demonstrate that mutation in prtR results in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by a tac promoter repressed the endogenous prtR promoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

FIG 1

Role of PrtR in a mouse acute pneumonia model. (A) Role of PrtR in the colonization of P. aeruginosa. Female BALB/c mice (6 to 8 weeks old) were intranasally inoculated with 5 × 10⁸ bacterial cells of wild-type PAK or its isogenic ΔprtN::Gm, ΔprtNprtR::Gm, or ΔprtNprtR::Gm mutant containing exsA driven by a tac promoter (ΔprtNR::Gm/Ptac-exsA). Mice were sacrificed 16 h postinfection (hpi). Lungs were isolated and homogenized. Bacterial loads were determined by serial dilution and plating. Bars represent medians, and error bars represent standard errors of the means (SEM). Significance by the Mann-Whitney test: *, P < 0.05; **, P < 0.01. (B) In vivo and in vitro competitive index (CI) between the prtR::Tn mutant and wild-type PAK. The two strains were mixed 1:1 and used for infection in a mouse acute pneumonia model (eight mice) or coculture in vitro. The competitive index was calculated as follows: CI = (mutant output/WT output)/(mutant input/WT input). **, P < 0.01, by Student's t test. (C) Cellular and secreted ExoS in PAK or ΔprtN::Gm, ΔprtNprtR::Gm, and ΔprtNR::Gm/Ptac-ExsA mutant strains. Overnight bacterial cultures were diluted to 2% in LB or 5% in LB plus 5 mM EGTA and grown at 37°C for 3 h. Supernatants and pellets from equivalent bacterial cell numbers were loaded, separated by SDS-PAGE gels, and immunoblotted with anti-ExoS antibody. ExoS is indicated by arrows. ΔprtNR::Gm represents ΔprtNprtR::Gm.

FIG 2

Expression of PrtR during mouse lung infection. The BALFs from three wild-type-PAK-infected mice were collected at the indicated time points. The BALF at 0 hpi was collected immediately after bacterial inoculation. Total RNA was isolated from the BALF, and the expression levels of prtR were determined by real-time PCR. The 30S ribosomal protein gene rpsL was used as an internal standard, and all reported changes are normalized to the levels of RpsL RNA. **, P < 0.01 compared to the other time points by Student's t test.

FIG 3

Signals that induce PrtR expression. (A) Numbers of host cells (means ± SEM) in the BALFs at indicated time points. **, P < 0.01 by the Mann-Whitney test. (B) Cells in the BALFs stained with Wright's stain. The neutrophil percentage is 76.6% ± 9.91% (average ± standard deviation), as counted based on cell morphology of samples from 3 infected mice. (C) A549 cells, Beas-2B cells, HL-60 cells, and differentiated HL-60 (dHL-60) cells were infected with PAK (MOI = 30). After 1 h of incubation, total RNA was extracted. The mRNA levels of PrtR were determined by real-time PCR. All reported changes are normalized to the levels of RpsL RNA. White bars (0 h) indicate RNA that was extracted immediately after bacteria were mixed with cells. (D) Luminometry of ROS production by A549, Beas-2B, HL-60, differentiated HL-60 cells infected with PAK (MOI = 30), and differentiated HL-60 cells treated with PMA. RU, relative units.

FIG 4

Autoregulation of PrtR. (A) Expression of prtR-1-lacZ in PAK and in ΔprtNprtR::Gm and ΔprtN::Gm mutants containing empty vector or PrtR-overexpressing plasmid. **, P < 0.01, compared to PAK or ΔprtN::Gm mutant by one-way analysis of variance (ANOVA) test. (B) Fragments of the prtR promoter region fused with promoterless lacZ gene or prtR-His fusion. The sequence shown is the putative PrtR binding site. Italic nucleotides represent conserved sequences in the putative PrtR-binding sites in prtR and prtN promoters. (C) Expression of prtR-1-lacZ, prtR-2-lacZ, and prtR-3-lacZ in PAK containing empty vector or prtR overexpression plasmid. *, P < 0.05 compared with prtR-1-lacZ or prtR-2-lacZ in PAK containing empty vector, by one-way ANOVA test; **, P < 0.01, compared with prtR-1-lacZ or prtR-2-lacZ in PAK containing prtR overexpression plasmid, by one-way ANOVA test. (D) Expression of PrtR-His with different lengths of promoter in PAK containing empty vector or prtR overexpression plasmid. (E) EMSA displaying binding of PrtR with the potential PrtR binding site in the promoter of prtR. A 0.5-pmol volume of double-stranded DNA fragment (containing the consensus motif or an altered conserved sequence) was electrophoresed alone (lanes 1 and 3) or after incubation with 1 pmol of purified PrtR protein (lanes 2 and 4).

FIG 5

Protein levels of PrtR-His under ciprofloxacin treatment. Wild-type PAK cells containing PrtR-His driven by the prtR promoter with the PrtR binding site (PprtR-1) (A) or without the binding site (PprtR-3) (B) were treated with indicated concentrations of ciprofloxacin for 1 h. Samples from equal numbers of bacteria were loaded onto SDS-PAGE gels, and levels of PrtR-His were detected with anti-His antibody. Note that due to the low expression level of prtR-His driven by PprtR-1, the exposure time in panel A was much longer than that in panel B.

FIG 6

Effect of PrtR overexpression on T3SS genes and ptrB expression during infection. Mice were infected with PAK containing empty vector or PrtR overexpression plasmid. BALFs were isolated at 6 hpi, and RNAs were purified. mRNA levels of T3SS genes and ptrB were quantified by real-time PCR. All reported changes are normalized to the levels of RpsL RNA. **, P < 0.01 compared to PAK containing empty vector by Student's t test.

FIG 7

Role of PrtR in biofilm resistance against ciprofloxacin. PAK cells containing empty vector or PrtR overexpression plasmid were inoculated in wells of a 96-well plate. (A) Crystal violet staining of biofilms. Upper panel, biofilms without ciprofloxacin treatment; lower panel, biofilms treated with ciprofloxacin at a concentration of 4× MIC (0.625 μg/ml) for 60 min. (B) The biofilm-associated dye was quantified by optical density measurement. (C) The biofilms were dissociated from the wells by gentle sonication, and bacterial numbers were quantified by plating. The bacterial survival rates after ciprofloxacin treatment were calculated based on live bacterial numbers in biofilms with or without ciprofloxacin treatment. **, P < 0.01, *, P < 0.05, compared to the values of wild-type PAK containing empty vector by one-way ANOVA test. (D) mRNA levels of indicated genes in biofilms formed by PAK containing empty vector or PrtR overexpression plasmid after ciprofloxacin treatment. All reported changes are normalized to the levels of RpsL RNA. **, P < 0.01 compared to PAK containing empty vector by Student's t test. (E) Survival rates of the biofilms formed by PAK and by ΔprtNprtR::Gm and ΔprtN::Gm mutants after ciprofloxacin treatment. *, P < 0.05 compared to PAK by one-way ANOVA test. CIP, ciprofloxacin.