CprS contributes to virulence and Polymyxin B resistance in <i>P</i>. <i>aeruginosa</i>.

(A) Hierarchical clustering of the z-scored extracted ion chromatogram (left panel) was used to evaluate the reproducibility of the proteome quantification in WT and ΔcprS strains under LL-37 treatment, the significant expressed proteins are categorized by functional category (right panel). (B) Hierarchical clustering of the zscored extracted ion chromatogram (left panel) was used to evaluate the reproducibility of the proteome quantification in ΔcprS strains before and after LL-37 treatment, the significant expressed proteins are categorized by functional category (right panel). (C) Polymyxin B MICs of WT and mutants in LB medium. (TIF)</p

Significant downregulated proteins in Δ<i>cprS</i>Δ<i>higB</i> compared with WT after LL-37 treatment.

Significant downregulated proteins in ΔcprSΔhigB compared with WT after LL-37 treatment.</p

CprRS controls the T3SS expression through HigBA.

(A) Volcano plot displaying the proteomic profiles of ΔcprRΔhigB and (C) ΔcprSΔhigB compared to WT after LL-37 treatment. (B) and (D) The significant expressed proteins in Fig 5A and 5C are categorized by functional category, respectively. (E) The expression changes of T3SS genes in WT and mutant strains were measured with or without LL-37 treatment, respectively. (F) Raw264.7 cell were infected by different strains at an MOI of 10. At indicated time points, the relative cytotoxicity was determined by the LDH release assay. Error bars indicate the means ± SD of three independent experiments. *P P P < 0.001.</p

HigBA system is activated in the presence of LL-37.

(A) β-galactosidase reporter system to determine the transcription activity of higB promoter in P. aeruginosa. (B) The mRNA levels of higA in mutants compared WT. Error bars indicate the means ± SD of three independent experiments. *P P P higB and higA in P. aeruginosa under LL-37 treatment. higA mRNA could be expressed separately from a promoter inside higB (marked as P2). (D) Degradation of HigA under LL-37 treatment. PAO1 expressing HigA-His6 were treated with LL-37 as (C) and analyzed by Western blotting, and the anti-RNA polymerase beta (RNAP) antibody was used as a negative control to determine the level of HigA. (TIF)</p

CprR directly activates the expression of type II TA system HigBA.

(A) The potential CprR recognition motif and binding site in higB promoter. (B) EMSAs showing that CprR binds the promoter region of higB. Each reaction mixture contains PCR products of higB (1 μM) and the protein concentrations were indicated above the lane. (C) EMSAs of CprR with mutant higB promoter. (D) Construction of β-galactosidase reporter system to determine the transcription regulation ability of CprR. The higB promoter were cloned ahead of a promoterless lacZ gene in pRG970km to construct lacZ fusions and then co-transformed into E. coli BL21 (DE3) with pET22b-cprR. The bacteria carrying the vectors were grown in LB medium as OD600 reached to 0.6 and supplied with 0.1 mM IPTG for 4 h at 37°C. Then the cells were collected and β-Galactosidase activities were described in methods. (E) AcP treatment enhanced the DNA-binding ability of CprR. (F) The DNA-binding ability of CprRD53A was not affected by AcP.</p

CprS specifically senses and responds to LL-37 signal.

(A) The expression levels of cprS and cprR treated with LL-37 and Polymyxin B were measured by qRT-PCR, respectively. The oprL gene was used as a normalizer. (B) The binding affinities of CprSFL and CprSSD toward different cationic peptides were measured with MST. The final protein concentration was 100 nM and cationic peptides have 16 doubling dilutions started from 500 μM. The experiment was repeated three times.</p

Significant downregulated proteins in <i>cprS</i>-mutant compared with WT.

Significant downregulated proteins in cprS-mutant compared with WT.</p

Significant downregulated proteins in Δ<i>cprR</i>Δ<i>higB</i> compared with WT after LL-37 treatment.

Significant downregulated proteins in ΔcprRΔhigB compared with WT after LL-37 treatment.</p

CprR directly interacts with <i>higB</i> promoter.

(A) The binding affinity of CprR toward higB promoter was measured with MST. The CprR was pretreated with AcP and then labeled with Monolith His-Tag Labelling Kit RED-tris-NTA 2nd Generation Kit. The final protein concentration was 100 nM and DNA fragments have 16 doubling dilutions started from 10 μM (B) EMSAs showing that native CprR rather than mutant CprR could bind to the promoter region of bkdA1. Each reaction mixture contains PCR products (1 μM) and the CprR protein concentrations were indicated above the lane. (TIF)</p

MICs to different antibiotics of the WT, the <i>cprR</i> and <i>cprS</i> mutants.

MICs to different antibiotics of the WT, the cprR and cprS mutants.</p