Golden pigment production and virulence gene expression are affected by metabolisms in Staphylococcus aureus

Abstract

The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of approximately 400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival.

FIG. 1.

Pigmentation of representative S. aureus pigment mutants and complementation experiments. (A) Pigmentation display of S. aureus strains that were grown on a TSA plate at 37°C for 24 h. (B) Measurement of the carotenoid pigment of different S. aureus strains by methanol extraction. The relative optical density units at 465 nm were normalized to the wild-type Newman strain, which was set at 100. Results are means + standard error of the means (errors bars) of a triplicate experiment. (C) Complementation experiments of representative S. aureus mutants, as indicated. Pigmentation of S. aureus strains grown on TSA plates at 37°C for 24 h is shown. Wild-type Newman, mutant strains, and mutant strains harboring complementation plasmids (-C) are shown.

FIG. 2.

Virulence of wild-type strain Newman and ctaA, qoxB, purH, and purA mutants. The murine abscess model of infection was used to compare the virulence of strain Newman and purH, ctaA, and qoxB mutants (A and B) and of a purA mutant (C). S. aureus colonization in murine liver or kidney was measured by tissue homogenization, dilution, and colony formation on TSA plates after 5 days of infection following retro-orbital injection. Each circle indicates one mouse. Ten animals were tested for each strain. The horizontal black line represents the mean log₁₀ CFU (y axis). The statistical difference between mutant and wild-type strains was determined by a Student's two-tailed t test. The limit of detection for organ infection was 100 CFU/organ.

FIG. 3.

Analysis of purA mutants. (A) Bacterial strains were inoculated onto TSA plates, TSA-2% skim milk plates, or TSA-5% sheep blood plates and incubated at 37°C for 48 h. (B) Northern blot analysis of the transcripts of sarA, sigB, and RNAIII when S. aureus strains were grown on TSA plates at 37°C for 24 h. (C) The transcripts of sigB and RNAIII were measured for the Newman and purA mutant strains grown on TSA plates (48 h) in the absence or presence of exogenous adenine (final concentration of 0.36 mM). (D) Pigmentation displays of S. aureus strains grown in TSB medium (48 h) with or without exogenous adenine, as indicated. Newman and purA mutant strains were grown on TSA-5% sheep blood plates or TSA-2% skim milk plates with or without exogenous adenine at 37°C for 48 h. Adenine concentration was set at 0.9 mM. For Northern blot analysis, 5 μg of total cellular RNA was used in each experiment, and the ethidium bromide-stained gel of the loaded RNA sample is shown below each lane.

FIG. 4.

Northern blot analysis of the transcripts of crtM, sigB, and RNAIII. The overnight culture was diluted 1,000-fold in fresh TSB medium. Bacteria were harvested after incubation at 37°C with shaking at 250 rpm for 3, 6, 12, 24, and 48 h and then subjected to total RNA isolation. Lanes 1, Newman/pYJ335; lanes 2, purA/pYJ335; lanes 3, purA/pYJ335::purA.

FIG. 5.

Inhibition of the purine biosynthetic pathway in S. aureus. (A) Pigmentation displays of the Newman strain grown on TSA or TSA with 6-TG plates (TSA plates supplied with 10 μg/ml of 6-TG dissolved in DMSO) at 37°C for 48 h. TSA plates were supplied with the same amount of DMSO as a control. (B) Measurement of the carotenoid pigment by methanol extraction. Newman cells were harvested after growth at 37°C for 48 h on TSA plates, TSA-MPA (TSA plates supplied with 400 μg/ml of MPA dissolved in DMSO) plates, or TSA plates plus 6-TG. TSA plates were supplied with the same amount of DMSO as a control. Results are means + standard errors of the means (errors bars) of a triplicate experiment. (C) Northern blot analysis of sigB, sarA, and RNAIII. The transcripts were measured for S. aureus strains grown on TSA plates (48 h) in the absence or presence of 6-TG or MPA as described in panel B. The effect of 6-TG on S. aureus survival in the liver (D) and kidney (E) of tested mice after 5 days of intraperitoneal infection is shown. The limit of detection for organ infection was 100 CFU/organ. 6-TG was dissolved in 0.1 M NaOH, followed by neutralization with HCl. The same amount of NaOH/HCl was used as a control. The statistical difference was determined by using a Student's two-tailed t test.

FIG. 6.

Cluster analysis and Northern blot analysis. (A) Cluster analysis showing that ∼400 genes affected by a purA mutation also show similar expression profiles in a purH mutant compared with those in wild-type Newman. The ratios are log₂ transformed. The scale of gene activities is represented from green (downregulated) to red (upregulated). (B) Northern blot analysis of eight genes which show different expression levels between wild-type Newman and purA, purH, and guaB mutants.

FIG. 7.

Analysis of representative S. aureus pigment mutants. (A) The transcripts of sigB, sarA, and RNAIII were measured for S. aureus strains grown on TSA plates at 37°C for 24 h. (B) Northern blot analysis of the transcripts of sigB and RNAIII. S. aureus strains were grown on TSA plates for 24 h before total RNA was harvested. (C and D) Hemolytic activity assay. Bacterial strains were inoculated onto plates with a toothpick and incubated at 37°C for 36 h.