HigB of Pseudomonas aeruginosa Enhances Killing of Phagocytes by Up-Regulating the Type III Secretion System in Ciprofloxacin Induced Persister Cells

Abstract

Bacterial persister cells are dormant and highly tolerant to lethal antibiotics, which are believed to be the major cause of recurring and chronic infections. Activation of toxins of bacterial toxin-antitoxin systems inhibits bacterial growth and plays an important role in persister formation. However, little is known about the overall gene expression profile upon toxin activation. More importantly, how the dormant bacterial persisters evade host immune clearance remains poorly understood. Here we demonstrate that a Pseudomonas aeruginosa toxin-antitoxin system HigB-HigA is required for the ciprofloxacin induced persister formation. Transcriptome analysis of a higA::Tn mutant revealed up regulation of type III secretion systems (T3SS) genes. Overexpression of HigB increased the expression of T3SS genes as well as bacterial cytotoxicity. We further demonstrate that wild type bacteria that survived ciprofloxacin treatment contain higher levels of T3SS proteins and display increased cytotoxicity to macrophage compared to vegetative bacterial cells. These results suggest that P. aeruginosa accumulates T3SS proteins during persister formation, which can protect the persister cells from host clearance by efficiently killing host immune cells.

Keywords: Pseudomonas aeruginosa; gene regulation; persistence; toxin/antitoxin; type III secretion system.

Figure 1

HigA negatively regulates the higB-higA operon. (A) Sketch map of the higB-higA operon. Arrows indicate the directions and locations of the primers for RT-PCR. (B) Total RNA was isolated from PA14 and the higA::Tn mutant. cDNA was synthesized and used as templates in PCR. RNAs were used in RT-PCR as negative controls. (C,D) The relative mRNA levels of higB and higA genes in PA14 and the higA::Tn mutant. Total RNA was isolated and the mRNA levels were determined by quantitative RT-PCR with the 16S rRNA as the internal control. Data represents the mean ± standard deviation from three independent experiments performed in triplicate. ^*p < 0.05; ^**p < 0.01 compared to wild type PA14 by Student's t-test. (E) Promoter region of the higB-higA operon. The predicted −10 and −35 elements of the promoter are boxed. The transcriptional start site is indicated by a black arrow, and the start codon of higB is underlined. The palindromic sequences of hypothetical HigA binding sites are indicated by gray arrows. (F) EMSA displaying binding of HigA to the higB-higA promoter. Purified HigA-His protein was incubated with the 38-bp DNA fragment indicated by the box with dashed lines in (E) or altered sequence. The mixtures were electrophoresed and observed by ethidium bromide staining. (G) Cleavage of HigA by the Lon protease. Wild type PA14, the clpP::Tn and lon::Tn mutants carrying pMMB67EH-higA-His were cultured in the presence of 1 mM IPTG for 1 h. Then 50 μg/ml spectinomycin was added to the medium. At indicated time points, the HigA-His levels were determined by Western blot analysis with an anti-His antibody. The relative density of each band was determined with Image J.

Figure 2

Role of HigA-HigB in persister formation. (A) Wild type PA14, a higA::Tn mutant and a complemented strain were treated with 0.25 μg/ml ciprofloxacin. At indicated time points the survival rates were determined by plating assay. (B) Wild type PA14, the ΔhigA and ΔhigAΔhigB mutants were cultured in the presence or absence of 0.025 μg/ml ciprofloxacin for 2 h and then treated with 0.25 μg/ml ciprofloxacin. The survival rates were determined by plating assay. (C,D) Wild type PA14 was treated with 0.025 μg/ml ciprofloxacin for 2 h and the mRNA levels of higB or higA were determined by quantitative RT-PCR. Error bars represent the standard error. ^*p < 0.05, by Student's t-test. (E) Wild type PA14 carrying a P_tac driven higB gene or the empty vector were cultured with 1 mM IPTG for 2 h, followed by treatment with 0.25 μg/ml ciprofloxacin. The survival rates were determined by plating. Error bars represent the standard errors. The graphs are representatives of three independent experiments.

Figure 3

Expression levels of T3SS genes in wild type PA14, the higA::Tn mutant and complemented strain. (A) Relative mRNA levels of exsC, exsA, pcrV, and exoU in indicated strains at exponential growth phase (OD₆₀₀ = 0.8~1.0). Error bars represent the standard errors. ^*p < 0.05, ^**p < 0.01, ^***p < 0.005 compared to wild type PA14 by Student's t-test. (B) Bacteria carrying an exoU-His driven by its native promoter (P_exoU-exoU-His) were grown in LB at 37°C. At the OD₆₀₀ of 1.0, bacteria were collected. Samples from equivalent bacterial cells were loaded into SDS-PAGE gels and stained with Coomassie blue or probed with an anti-His antibody. (C) Raw264.7 cell were infected by indicated strains at an MOI of 10. At indicated time points, the relative cytotoxicity was determined by the LDH release assay.

Figure 4

HigB promotes T3SS mediated cytotoxicity. Wild type PA14 and an exsA::Tn mutant containing pMMB67EH-higB-His or pMMB67EH was grown in the presence of indicated concentrations of IPTG for 3 h. Quantitative RT-PCR was used to determine the relative mRNA levels of exsC (A), exsA (B), pcrV (C), and exoU (D). (E) PA14 or exsA::Tn containing exoU-His driven by its native promoter (P_exoU-exoU-His) and pMMB67EH-higB or pMMB67EH was grown in the presence of indicated concentrations of IPTG for 3 h. Samples from equivalent bacterial cells were loaded into SDS-PAGE gels and stained with Coomassie blue or or probed with an anti-His antibody. The relative density of each band was determined by Image J. (F) Raw264.7 cells were infected with the bacteria at an MOI of 10 for 3.5 h, followed by LDH release assay. ND, not detectable.

Figure 5

Levels of HigB, PcrV and cytotoxicity of bacterial cells that survived ciprofloxacin treatment. PA14/P_higB-gfp was cultured in the presence of 0.025 μg/ml ciprofloxacin for 2 h and then treated with 0.125 μg/ml ciprofloxacin for 30 min. The bacteria were washed with PBS twice, stained with PI (A) or fixed and permeabilized and then stained with rabbit anti-PcrV, followed by Alex Fluor 594–labeled goat anti–rabbit immunoglobulin. Bar = 10 μm (B). Quantification of fluorescence positive cells was based on analysis of about 100 cells from three different samples. (C) Wild type PA14 was cultured in the presence of 0.025 μg/ml ciprofloxacin for 2 h and then treated with 0.125 μg/ml ciprofloxacin for 30 min. Raw264.7 cells were infected with the surviving bacteria or bacteria grown in LB at an MOI of 10 for 3.5 h. The anti-PcrV antibody was added to the medium at indicated dilutions. The relative cytotoxicity was determined by the LDH release assay. Error bars represent the standard errors. (D) Wild type PA14, the ΔhigA or ΔhigAΔhigB mutant was cultured in the presence of 0.025 μg/ml ciprofloxacin for 2 h and then treated with 0.125 μg/ml ciprofloxacin for 30 min. Raw264.7 cells were infected with the survived bacteria or bacteria grown in LB at an MOI of 10 for 3.5 h. The relative cytotoxicity was determined by the LDH release assay. Error bars represent the standard errors. Each graph represents the results of three independent experiments. ^*p < 0.05, ^***p < 0.005 by Student's t-test.