Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression

Abstract

Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. Importance: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact on S. aureus virulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

FIG 1

Characteristics and phenotypes of S. aureus SH1000 wild type, ΔthyA mutant, and complemented mutant. Phenotypes of the wild-type strain SH1000 (A), the ΔthyA mutant (B), and the complemented mutant (C) are shown on Columbia blood agar (row I). Susceptibility testing for TMP-SMX was performed on Columbia blood agar (row II). Light microscopy (row III) and transmission electron microscopy (row IV) revealed the typical features of the respective strains.

FIG 2

Growth phase analysis and cocci size of SH1000 wild type, ΔthyA mutant, and complemented mutant. (a) Growth phase analysis. The strains were cultured at 37°C in BHI at 160 rpm in baffled flasks (50 ml cultures in 500-ml flasks). Samples were taken every hour to determine the optical density. For the mutant, growth analysis was also performed in BHI supplemented with 100 µg/ml thymidine. ●, SH1000, WT; ▲, ΔthyA-C, complemented mutant; ■, ΔthyA mutant and thymidine; ♦, ΔthyA mutant. RNA isolation time points for DNA microarray analysis are indicated by black arrows. (b) Size of individual cocci. Five hundred cocci for every phenotype were measured after Gram staining by light microscopy of overnight cultures in BHI and for the mutant also in BHI supplemented with 100 µg/ml thymidine. ●, SH1000, WT; ▲, ΔthyA-C, complemented mutant; ■, ΔthyA mutant and thymidine; ♦, ΔthyA mutant.

FIG 3

Differentially regulated genes in SH1000 compared to ΔthyA SH1000. Transcriptional changes were grouped according to the comprehensive microbial resource (CMR) database of the Craig Venter Institute (CVI). Statistical filters (fold change of ±2.5 and P value of <0.05). Transcriptional changes between SH1000 wild type and ΔthyA SH1000 at late exponential phase of growth were analyzed. Red bars indicate downregulation of genes (a total of 303), and green bars indicate upregulation of genes (a total of 490) in the ΔthyA mutant.

FIG 4

S. aureus pyrimidine metabolic pathway and the transcriptional activities of respective genes in the ΔthyA mutant. Upregulated genes are labeled in green, while unregulated genes are labeled in black. (I) Pyrimidine ribonucleotide biosynthesis pathway; (II) salvage pathways of nucleotides; (III) external sources of nucleosides. Gene identifier and gene names were reported according to S. aureus COL, accession number NC_002951.

FIG 5

Transcriptional analysis of nupC, carA, pyrB, and purL under different conditions. Wild type (JE2), ΔthyA mutant (JE2 ΔthyA mutant), ΔthyA-C mutant (JE2 ΔthyA pNXR thyA complemented mutant), and ΔthyA-C_empty mutant (JE2 ΔthyA complemented with empty vector pNXR100) were analyzed under different conditions: in BHI, in BHI and thymidine, in BHI and TMP-SMX, and in BHI, TMP-SMX, and thymidine. Quantification of expression of nupC, carA, pyrB, and purL in late exponential growth phase. The data (means ± standard errors of the means [SEM]) were normalized using three internal control genes (gmk, aroE, gyrB) and expressed as n-fold expression relative to the values of the wild-type phenotype. The analysis is based on 3 independent biological replicates analyzed in triplicate.

FIG 6

S. aureus wild type (a) and nupC mutant (b) challenged with TMP-SMX in an agar disk diffusion experiment. The black arrow indicates the inhibition zone with tiny SCV-like colonies. Disk with 25 µg TMP-SMX were placed on a Columbia blood agar plate, and respective isolates were incubated for 24 h at 37°C. JE2 (S. aureus USA300 wild type) and NE544 (S. aureus nupC mutant of JE2) were kindly obtained through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) program.

FIG 7

Virulence of the SH1000 wild type, the ΔthyA mutant, and the complemented mutant. (a) Caenorhabditis elegans infection model. The wild type, ΔthyA mutant, and complemented mutant (ΔthyA-C) were streaked on TA plates, and C. elegans was placed on the plates to assess virulence of the strains by enumeration of surviving nematodes after 24 h, 48 h, 72 h, and 96 h. (b) Acute murine pneumonia model. Cumulative survival rate after infection of C57Bl/6 mice with S. aureus. Mice were infected with 5 × 10⁸ CFU/lung with the SH1000 wild type, the ΔthyA mutant, and the complemented mutant and monitored for mortality over 1 week. Mortality of mice infected with the wild type was significantly greater than that for the ΔthyA mutant and the complemented mutant. Significant differences were detected also between wild type and complemented mutant. The asterisks indicate statistical significance by log rank (Mantel-Cox) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 9 mice). (c) Murine lung histology and localization of S. aureus. Mice were infected with 5 × 10⁸ CFU/lung with the SH1000 wild type, the ΔthyA mutant, and the complemented mutant (ΔthyA-C). Lungs recovered after 4 h (A to C, F to H) or 24 h (D, E, I, J) postinfection. The lungs were stained with H&E (A to E) or with a specific antibody against S. aureus strains (red). Counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bars in panels A to E, 200 µm (top) and 50 µm (bottom); bars in panels F to J, 100 µm (top) and 25 µm (bottom).

FIG 8

Cytotoxicity and virulence gene regulation under different conditions. Wild type (JE2), ΔthyA mutant (JE2 ΔthyA mutant), ΔthyA-C mutant (JE2 ΔthyA pNXR thyA complemented mutant), and ΔthyA-C_empty (JE2 ΔthyA complemented with empty vector pNXR100) were analyzed under different conditions: in BHI, in BHI and thymidine, in BHI and TMP-SMX, and in BHI, TMP-SMX, and thymidine. (a) The supernatant of the respective strains was used to challenge A549 cells. LDH release was determined to assess cytotoxicity. The data (means ± standard deviations [SD]) were normalized against the positive control (completely lysed cells) and were performed in 3 biological triplicates. *** indicate statistical significant differences (P < 0.0001) tested by unpaired t test. (b) Quantification of expression of hld in late exponential growth phase. The data (means ± SEM) were normalized using three internal control genes (gmk, aroE, gyrB) and presented as n-fold expression relative to the values of the wild-type phenotype. The analysis is based on 3 independent biological replicates analyzed in triplicate.