Overall structure of CprR obtained from Alphafold.

(A) Ribbon representation of the CprR. The overall structure of CprR comprises 8 α-helixes and 10 β-sheets. (B) Part of sequence alignment on CprR and other homologous proteins with known structures, including Thermotoga maritima PhoB, Mycobacterium tuberculosis H37Rv Regx3 and PhoP. The potential residues involved in DNA interaction are labeled with solid asterisk. (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

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

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

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

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

CprRS is conserved in <i>P</i>. <i>aeruginosa</i> and contributes to its full virulence.

(A) Schematic representation of CprRS system genes and proteins. (B) Conservation analysis of cprRS genes in 872 P. aeruginosa strains. Dots represent outliers from the respective groups. (C) The top sequence Blast hits of CprS on other pathogen genomes by NCBI. The identities to CprS are also marked. (D) Growth curves of WT and mutants on LB medium. (E) Measurement virulence of the WT and mutant strains in a Galleria mellonella infection model. Each G. mellonella was injected with 10 μL of P. aeruginosa dilution (5 × 103 CFU/mL), and the PBS-injected larvae were the negative control. The larvae were monitored for 24 h after the infection (Mantel-Cox test for statistics, *P cprS strains. (G) Volcano plot displaying the proteomic profiles of WT and ΔcprS strains. The significantly up- and down-regulated proteins are labeled with red and cyan, respectively. The significant expressed proteins are also categorized by functional category in (H). Student’s t-test was used to assess the significance of differential expression of proteins (DEPs) between two groups. Proteins that have significance level of P 1.5 or < –1.5 were considered as DEPs.</p

Bacteria strains and plasmids.

Pseudomonas aeruginosa is a highly pathogenic bacterium known for its ability to sense and coordinate the production of virulence factors in response to host immune responses. However, the regulatory mechanisms underlying this process have remained largely elusive. In this study, we investigate the two-component system CprRS in P. aeruginosa and unveil the crucial role of the sensor protein CprS in sensing the human host defense peptide LL-37, thereby modulating bacterial virulence. We demonstrate that CprS acts as a phosphatase in the presence of LL-37, leading to the phosphorylation and activation of the response regulator CprR. The results prove that CprR directly recognizes a specific sequence within the promoter region of the HigBA toxin-antitoxin system, resulting in enhanced expression of the toxin HigB. Importantly, LL-37-induced HigB expression promotes the production of type III secretion system effectors, leading to reduced expression of proinflammatory cytokines and increased cytotoxicity towards macrophages. Moreover, mutations in cprS or cprR significantly impair bacterial survival in both macrophage and insect infection models. This study uncovers the regulatory mechanism of the CprRS system, enabling P. aeruginosa to detect and respond to human innate immune responses while maintaining a balanced virulence gene expression profile. Additionally, this study provides new evidence and insights into the complex regulatory system of T3SS in P. aeruginosa within the host environment, contributing to a better understanding of host-microbe communication and the development of novel strategies to combat bacterial infections.</div

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