Acetylation of CspC Controls the Las Quorum-Sensing System through Translational Regulation of rsaL in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a ubiquitous pathogenic bacterium that can adapt to a variety environments. The ability to effectively sense and respond to host local nutrients is critical for the infection of P. aeruginosa. However, the mechanisms employed by the bacterium to respond to nutrients remain to be explored. CspA family proteins are RNA binding proteins that are involved in gene regulation. We previously demonstrated that the P. aeruginosa CspA family protein CspC regulates the type III secretion system in response to temperature shift. In this study, we found that CspC regulates the quorum-sensing (QS) systems by repressing the translation of a QS negative regulatory gene, rsaL. Through RNA immunoprecipitation coupled with real-time quantitative reverse transcription-PCR (RIP-qRT-PCR) and electrophoretic mobility shift assays (EMSAs), we found that CspC binds to the 5' untranslated region of the rsaL mRNA. Unlike glucose, itaconate (a metabolite generated by macrophages during infection) reduces the acetylation of CspC, which increases the affinity between CspC and the rsaL mRNA, leading to upregulation of the QS systems. Our results revealed a novel regulatory mechanism of the QS systems in response to a host-generated metabolite. IMPORTANCE Bacterial infectious diseases impose a severe threat to human health. The ability to orchestrate virulence determinant in response to the host environment is critical for the pathogenesis of bacterial pathogens. Pseudomonas aeruginosa is a leading pathogen that causes various infections in humans. In P. aeruginosa, the quorum-sensing (QS) systems play an important role in regulating the production of virulence factors. In this study, we find that a small RNA binding protein, CspC, regulates the QS systems by repressing the expression of a QS negative regulator. We further demonstrate that CspC is acetylated in response to a host-derived metabolite, itaconate, which alters the function of CspC in regulating the QS system. The importance of this work is in elucidation of a novel regulatory pathway that regulates virulence determinants in P. aeruginosa in response to a host signal.

Keywords: CspC; Pseudomonas aeruginosa; acetylation; itaconate; quorum sensing.

FIG 1

CspC is required for pyocyanin production. Indicated strains were grown overnight in LB at 37°C. (a) Pyocyanin levels in the supernatants of the overnight bacterial cultures. (b) mRNA levels of the pyocyanin synthesis genes, including overall phzA (phzA1 and phzA2) and phzM, were determined by qRT-PCR. Data represent the means from three independent experiments, and error bars indicate standard deviations. **, P < 0.01; *, P < 0.05 by Student's t test.

FIG 2

CspC controls the production of QS signal molecules. (a) The relative levels of 3-oxo-C12-HSL and C4-HSL of indicated strains. The indicated strains were grown overnight. The bacterial supernatants were mixed with the reporter strains at a volume ratio of 1:1. The GFP fluorescence and OD₆₀₀ were measured every 30 min at 37°C for 12 h. The data were calculated as GFP fluorescence/OD₆₀₀. (b) 3-Oxo-C12-HSL and C4-HSL contents of indicated strains measured with E. coli reporter strains in LB. Miller units are used for the mean results from at least three independent experiments. ***, P < 0.001; **, P < 0.01 by Student's t test. (c) Relative mRNA levels of lasI, lasR, rhlI, and rhlR. Total RNA of indicated strains was isolated from bacteria grown overnight, and mRNA levels of lasI, lasR, rhlI, and rhlR were determined by qRT-PCR. Data represent the means from three independent experiments, and error bars indicate standard deviations. **, P < 0.01; *, P < 0.05 by Student's t test. (d) Bacteria carrying P_lasI-lacZ and P_rhlI-lacZ were grown in LB to an OD₆₀₀ of 0.3. Miller units are used for mean results from three independent experiments. **, P < 0.01 by Student's t test.

FIG 3

CspC binds to the 5′UTR of the rsaL mRNA. (a) Fold enrichment of the indicated fragments tested by an RIP-coupled qRT-PCR assay. Locations of the primers used in the qRT-PCR are indicated by arrows. The exsA gene was used as a positive control, and the rpsL gene was used as an internal control. Data represent the means from three independent experiments, and error bars indicate standard deviations. ***, P < 0.001 compared to the other samples by Student's t test. (b) Schematic diagram of the rsaL-GST fusions driven by a tac promoter with indicated length of upstream regions. (c) Bacteria carrying the rsaL-GST with indicated upstream segments (P_tac-74/37/-rsaL-GST) were grown in LB containing 150 μg/mL carbenicillin. The RsaL-GST and RpoA levels were determined by Western blotting. (d) The purified CspC-GST was incubated with a 37-nt ssDNA that represents the 37-nt 5′-UTR of the rsaL mRNA and a complementary ssDNA for 30 min at 25°C. The samples were subjected to electrophoresis in a native gel, followed by staining with SYBR Gold nucleic acid gel stain.

FIG 4

CspC controls the QS systems through rsaL. The bacteria were grown overnight in LB at 37°C. (a) The relative mRNA levels of lasI and lasR were determined by qRT-PCR. Data represent the mean from three independent experiments, and error bars indicate standard deviations. (b) The relative 3-oxo-C12-HSL levels in the supernatant of the bacterial cultures were measured with the E. coli reporter strain. Miller units are the mean results from three independent experiments. (c) The pyocyanin levels in the supernatant of the bacterial cultures. Data represent the means from three independent experiments, and error bars indicate standard deviations. ***, P < 0.001; **, P < 0.01 by Student's t test.

FIG 5

K41 residue of CspC is involved in the translational regulation of rsaL. (a) Equal amounts of CspC(WT)-GST, CspC(K41Q)-GST, and CspC(K41R)-GST were incubated with the 37-nt ssDNA for 30 min at 25°C. The samples were subjected to electrophoresis in a native gel, followed by staining with SYBR Gold nucleic acid gel stain. (b) Indicated strains carrying P_tac-37-rsaL-GST were grown to an OD₆₀₀ of 1.0 in LB containing 150 μg/mL carbenicillin. The RsaL-GST and RpoA levels were determined by Western blotting. (c and d) Indicated strains were grown overnight in LB containing 150 μg/mL carbenicillin and 1 mM IPTG. (c) The mRNA levels of overall phzA, lasI, and lasR were determined by qRT-PCR. (d) The pyocyanin levels in the supernatants. Data represent the means from three independent experiments, and error bars indicate standard deviations. ***, P < 0.001; **, P < 0.01 by Student's t test.

FIG 6

Acetylation of CspC modulates the expression of rsaL and the Las QS system in response to itaconate. (a and b) Wild-type PA14 carrying a cspC-GST driven by its own promoter was grown at 37°C overnight in M9 medium with glucose or itaconate as the sole carbon source. (a) The amounts of CspC-GST and RpoA were determined by Western blotting. (b) Acetylation and the total amounts of the purified CspC-GST were determined by Western blotting. (c and d) Wild-type PA14 was grown in M9 medium with glucose or itaconate as the sole carbon source at 37°C. (c) The mRNA levels of lasI, lasR, and overall phzA were determined by qRT-PCR. Data represent the means from three independent experiments, and error bars indicate standard deviations. ***, P < 0.001; **, P < 0.01; *, P < 0.05 by Student's t test. (d) Pyocyanin levels in the supernatants of the bacterial cultures. (e) Indicated strains carrying P_tac-37-rsaL-GST were grown to an OD₆₀₀ of 1.0 in M9 medium with glucose or itaconate as the sole carbon source at 37°C. The RsaL-GST levels were determined by Western blotting. Data represent the results from three independent experiments.

FIG 7

Schematic diagram of the CspC-mediated regulation on rsaL and the Las QS system. LasI synthesizes the signal molecule 3-oxo-C12-HSL. After binding to 3-oxo-C12-HSL, LasR binds to the intergenic region between lasI and rsaL and activates the transcription of both of the genes. Meanwhile, RsaL binds to the intergenic region between lasI and rsaL and represses the transcription of both genes. CspC binds to the 5′UTR of the rsaL mRNA and represses its translation. Acetylation of CspC reduces the affinity between CspC and the rsaL mRNA, resulting in upregulation of rsaL. The presence of itaconate causes deacetylation of CspC, which represses the translation of the rsaL mRNA and subsequently activates the Las system. The 5′ and 3′UTRs of the rsaL mRNA are shown in green, and the coding region is shown in gray. Ac, acetylation.