RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus

Abstract

In Staphylococcus aureus, metabolism is intimately linked with virulence determinant biosynthesis, and several metabolite-responsive regulators have been reported to mediate this linkage. S. aureus possesses at least three members of the RpiR family of transcriptional regulators. Of the three RpiR homologs, RpiRc is a potential regulator of the pentose phosphate pathway, which also regulates RNAIII levels. RNAIII is the regulatory RNA of the agr quorum-sensing system that controls virulence determinant synthesis. The effect of RpiRc on RNAIII likely involves other regulators, as the regulators that bind the RNAIII promoter have been intensely studied. To determine which regulators might bridge the gap between RpiRc and RNAIII, sarA, sigB, mgrA, and acnA mutations were introduced into an rpiRc mutant background, and the effects on RNAIII were determined. Additionally, phenotypic and genotypic differences were examined in the single and double mutant strains, and the virulence of select strains was examined using two different murine infection models. The data suggest that RpiRc affects RNAIII transcription and the synthesis of virulence determinants in concert with σ(B), SarA, and the bacterial metabolic status to negatively affect virulence.

FIG 1

Effect of rpiRc mutation on transcription of RNAIII (A) and genes encoding the virulence determinants alpha-toxin (B), protein A (C), and capsule (D) throughout the growth cycle. Quantitative real-time RT-PCR analyses of total RNA isolated from S. aureus strain SA564, isogenic single mutants of acnA, agr, mgrA, sarA, and sigB (black bars), and their respective rpiRc double mutants (white bars) cultivated for the indicated times were performed. The relative transcript levels of target genes were normalized to gyrB and 16S rRNA. Shown are the mean and standard deviation (SD) for at least three independent experiments performed in duplicate.

FIG 2

Phenotypic characterization of virulence determinants in S. aureus regulatory mutants. S. aureus strain SA564, isogenic single mutants of acnA, agr, mgrA, sarA, and sigB, and their respective rpiRc double mutants were assayed for hemolytic activity (A), protein A accumulation (B), and capsule (C). In panel A, the means and standard deviations (SD) for at least three independent experiments are shown. In panel B, the Western blot is representative of at least three independent experiments. In panel C, the capsule immunoblot is representative of at least three independent experiments.

FIG 3

Effect of the deletion of rpiRc and sarA on the agr quorum-sensing system in S. aureus strain SA564. S. aureus strain SA564 or sarA single mutant (■) and their isogenic rpiRc mutants (□) were cultivated for 2 h prior to harvest. (A) Luciferase activities (means ± the SD) of strains harboring the agr P3 promoter reporter plasmid pSB2035 from at least three independent experiments. (B) Total RNA was isolated from bacterial cells and subjected to quantitative real-time RT-PCR, and the relative transcript levels of agrBD were normalized to gyrB and 16S rRNA. Means and SD for at least three independent experiments performed in duplicate are shown.

FIG 4

Positive effect of rpiRc inactivation on transcript levels of sarA. Northern blot analysis of sar transcripts from S. aureus strain SA564, the rpiRc deletion mutant (SA-rpiRc), and the rpiRc mutant carrying the cis-integrated plasmid pEC4-rpiRc for complementation (SA-rpiRc_rpiRc) cultivated for the indicated times was performed. The sar locus originates from three different promoters (P1, P3, and P2) resulting in transcripts that differ in size (0.56, 0.8, and 1.2 kb, respectively). The blot is representative of at least two independent experiments.

FIG 5

Inactivation of rpiRc results in reduced survival (A) and increased bacterial burden (B and C) of S. aureus in a murine pneumonia model. Wild-type S. aureus strain SA564, the rpiRc deletion mutant (SA-rpiRc), the rpiRc mutant carrying the cis-integrated plasmid pEC4-rpiRc for complementation (SA-rpiRc_rpiRc), and the sarA single (SA-sarA) and rpiRc double (SA-sarA rpiRc) mutants were grown to exponential growth phase (2 h), washed, and intranasally administered to 8- to 9-week-old C57/BL6N mice. (A) The survival of mice using an infectious dose of 2.5 × 10⁸ CFU was monitored. (B and C) After infection with 10⁸ CFU, the bacterial loads in BAL samples (B) and lung tissue homogenates (C) were determined at 24 h postinfection. Each symbol represents an individual animal, and the mean values per group are depicted by horizontal lines. For statistical analysis, nonparametric Mann-Whitney tests were performed (*, P < 0.05; **, P < 0.01).

FIG 6

Inactivation of rpiRc results in more abscesses (A and B) and higher bacterial burdens (C) in a murine abscess formation model. S. aureus wild-type strain SA564, the rpiRc deletion mutant (SA-rpiRc), the rpiRc mutant carrying the cis-integrated complementation plasmid pEC4-rpiRc (SA-rpiRc_rpiRc), and the sarA single (SA-sarA) and rpiRc double (SA-sarA rpiRc) mutants were grown to exponential growth phase (2 h) and washed, and 10⁷ CFU were administered via retroorbital injection to C57BL/6N mice. At 4 days postinfection, the mice were euthanized, and the livers were removed. Representative H&E staining of liver sections from the wild type and the rpiRc mutant showing abscess lesions (arrows) is shown. In panel A, the magnification bar represents 500 μm (20 μm in each inset). (B) The number of abscesses was enumerated and normalized to the observed tissue area. (C) Bacterial loads in homogenized liver. Each symbol represents an individual animal, and the mean values per group are depicted by horizontal lines. For statistical analysis, nonparametric Mann-Whitney tests were performed (*, P < 0.05; **, P < 0.01; ***, P < 0.001).