Journal: Infection and Immunity

Article Title: Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages

doi: 10.1128/IAI.00695-19

Figure Lengend Snippet: ExsA directly regulates the transcription of impA. (A) Diagram of the impA promoter region and the lacZ transcriptional fusion. The region of the probe used for the EMSA and the LasR binding site are indicated. The conserved ExsA binding sequences of the reference strains PAK, PAO1, PA14, and LESB58 are aligned with the sequences of 90 sequenced P. aeruginosa genomes and 21 clinical isolates (underlined). Enlarged red letters in boldface type represent the exsA consensus binding sequence. Boxed blue and green letters represent the single-nucleotide polymorphism. The numbers at the right of the sequences represent the numbers of the sequenced strains that contain the same sequence. (B) PAK, the exsA::Ω mutant, and the exsA::Ω/exsA strain carrying either the PimpA-lacZ fusion or an empty vector (promoterless lacZ) were grown at 37°C with or without 5 mM EGTA for 3 h. The values (Miller units) shown are the means from three experiments. *, P < 0.05; **, P < 0.01; ns, not significant (by Student’s t test). (C) ExsA was incubated with a DNA probe containing the conserved exsA recognition sequence or mutated versions at 4°C for 25 min and then at 37°C for 5 min. Red and yellow letters represent the mutated nucleotides. Binding between ExsA and the probes was detected by an EMSA. (D) The ExsA binding sequence in PimpA-lacZ was mutated as described above for panel C, resulting in PimpA-lacZ Mut1 and Mut2. PAK carrying the original or mutated PimpA-lacZ was grown at 37°C with or without 5 mM EGTA for 3 h. The values (Miller units) shown are the means from three experiments. WT, wild type.

Article Snippet: Dynamic exclusion was employed, with a repeat duration of 12 s and an exclusion duration of 6 s. Mass spectra were searched against the P. aeruginosa protein database (strain ATCC 15692/DSM 22644/CIP 104116/JCM 14847/LMG 12228/1C/PRS 101/PAO1) by using Mascot (version 2.3.02).

Techniques: Binding Assay, Sequencing, Mutagenesis, Plasmid Preparation, Incubation

Journal: Infection and Immunity

Article Title: Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages

doi: 10.1128/IAI.00695-19

Figure Lengend Snippet: ImpA inhibits phagocytosis of P. aeruginosa by macrophages in vivo. Six-week-old female BALB/c mice were infected with PAK or the ΔimpA mutant at 1 × 107 CFU per mouse. At 3 h postinfection, the bacterial cells were collected from BALF, followed by RNA purification. (A and B) Relative mRNA levels of the T3SS genes exoS, pscF, popD, exsA (A), and impA (B) determined by quantitative real-time PCR. Data represent the means ± standard deviations from three samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by Student’s t test). (C) Six-week-old female BALB/c mice were infected by the GFP-expressing PAK or ΔimpA strain at 1 × 107 CFU per mouse. At 3 h postinfection, cells in the BALF were analyzed by flow cytometry. Macrophages were detected by using CD11b and F4/80. Data shown are representative of results for samples from 5 mice in each group. (D) Phagocytosis ratios representing the percentage of GFP-positive cells among the whole macrophage population. Data represent the means ± standard deviations.

Article Snippet: Dynamic exclusion was employed, with a repeat duration of 12 s and an exclusion duration of 6 s. Mass spectra were searched against the P. aeruginosa protein database (strain ATCC 15692/DSM 22644/CIP 104116/JCM 14847/LMG 12228/1C/PRS 101/PAO1) by using Mascot (version 2.3.02).

Techniques: In Vivo, Infection, Mutagenesis, Purification, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry

Journal: Infection and Immunity

Article Title: Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages

doi: 10.1128/IAI.00695-19

Figure Lengend Snippet: ImpA inhibits phagocytosis of P. aeruginosa by macrophages. (A) iBMDM cells were infected with the indicated strains at an MOI of 5 for 3 h, and bacterial cytotoxicity was determined by an LDH release assay. Error bars indicate standard deviations from triplicate assays. ns, not significant by Student’s t test. (B) Phagocytosis assay. iBMDM cells were infected with the indicated strains at an MOI of 5 for 30 min, followed by treatment with 250 μg/ml gentamicin for 10 min. The cells were then washed once with PBS and lysed in 1 ml cold sterile water. Bacterial CFU were determined by serial dilution and plating. *, P < 0.05; **, P < 0.01 (by Student’s t test). (C) GFP-expressing PAK and ΔimpA strains were incubated with iBMDM cells at an MOI of 50 for 30 min, followed by treatment with 250 μg/ml gentamicin for 10 min. The cells were then washed with PBS for 3 times. The bacterial cells that were taken up were observed under a fluorescence microscope. BF, bright field. Bars, 20 μm. (D) Ratios of uptake of bacterial cells by iBMDMs, calculated by counting 100 cells in at least three fields for each sample. (E) Representative flow cytometry analyses of iBMDMs infected with GFP-expressing PAK and ΔimpA strains. The bacterial cells were incubated with iBMDMs at an MOI of 50 for 30 min. Macrophage cells were identified with CD11b and F4/80. SSC, side scatter. (F) Phagocytosis ratios representing the percentages of GFP-positive cells among the whole population of examined cells. Data represent the means ± standard deviations from three samples.

Article Snippet: Dynamic exclusion was employed, with a repeat duration of 12 s and an exclusion duration of 6 s. Mass spectra were searched against the P. aeruginosa protein database (strain ATCC 15692/DSM 22644/CIP 104116/JCM 14847/LMG 12228/1C/PRS 101/PAO1) by using Mascot (version 2.3.02).

Techniques: Infection, Lactate Dehydrogenase Assay, Phagocytosis Assay, Sterility, Serial Dilution, Expressing, Incubation, Fluorescence, Microscopy, Flow Cytometry

Journal: Infection and Immunity

Article Title: Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages

doi: 10.1128/IAI.00695-19

Figure Lengend Snippet: Role of ImpA in intracellular survival of P. aeruginosa. iBMDMs were infected with the indicated strains as for the phagocytosis assay. Extracellular bacteria were eliminated by washing with PBS and treatment with 250 μg/ml gentamicin for 10 min. After that (indicated as time zero), the cells were incubated in DMEM with 10 μg/ml gentamicin for 4 h. The percentages of attached cells were quantified by crystal violet staining (A) and the intracellular bacteria were counted by plating (B) at the indicated time points. Error bars indicate standard deviations from triplicate assays. hpp, hours postphagocytosis. *, P < 0.05; ns, not significant (by Student’s t test).

Article Snippet: Dynamic exclusion was employed, with a repeat duration of 12 s and an exclusion duration of 6 s. Mass spectra were searched against the P. aeruginosa protein database (strain ATCC 15692/DSM 22644/CIP 104116/JCM 14847/LMG 12228/1C/PRS 101/PAO1) by using Mascot (version 2.3.02).

Techniques: Infection, Phagocytosis Assay, Bacteria, Incubation, Staining