Impacts of the Type I Toxin-Antitoxin System, SprG1/SprF1, on Staphylococcus aureus Gene Expression

Abstract

Type I toxin-antitoxin (TA) systems are widespread genetic modules in bacterial genomes. They express toxic peptides whose overexpression leads to growth arrest or cell death, whereas antitoxins regulate the expression of toxins, acting as labile antisense RNAs. The Staphylococcus aureus (S. aureus) genome contains and expresses several functional type I TA systems, but their biological functions remain unclear. Here, we addressed and challenged experimentally, by proteomics, if the type I TA system, the SprG1/SprF1 pair, influences the overall gene expression in S. aureus. Deleted and complemented S. aureus strains were analyzed for their proteomes, both intracellular and extracellular, during growth. Comparison of intracellular proteomes among the strains points to the SprF1 antitoxin as moderately downregulating protein expression. In the strain naturally expressing the SprG1 toxin, cytoplasmic proteins are excreted into the medium, but this is not due to unspecific cell leakages. Such a toxin-driven release of the cytoplasmic proteins may modulate the host inflammatory response that, in turn, could amplify the S. aureus infection spread.

Keywords: 2D-DIGE; RNA antitoxin; Staphylococcus aureus; peptide toxins; proteomics; toxin–antitoxin systems (type I).

Figure 1

The SprG1/SprF1 type 1 TA system does not influence S. aureus N315 growth in laboratory conditions. (A) Northern blot analysis of the expression of SprG1/SprF1 system components in S. aureus N315 (WT pCN35), the sprG1/sprF1 deletion mutant (ΔΔpCN35), and SprF1 antitoxin (over)expressing (ΔΔpCN35ΩsprF1) strains grown to logarithmic (L) and stationary (S) phases. (B–D) Growth curves in optimal and stress conditions.

Figure 2

Impacts of the SprG1/SprF1 type I TA system on the S. aureus N315 proteome. Differentiating proteins in the intracellular proteome at logarithmic (A) and stationary (B) growth phases and in the extracellular proteome at stationary growth phase (C), comparing: wild-type (WT pCN35) and the SprG1/SprF1-deleted module (ΔΔ pCN35) (blue circle); WT pCN35 and SprF1 (over)expressing strain (ΔΔ pCN35ΩsprF1) (green circle); ΔΔ pCN35 and ΔΔ pCN35sprF1 (red circle). Proteins upregulated in comparison to the second component from the pair are underlined. For the meaning of the protein acronyms, please refer to Table 2 and Table 3.

Figure 3

(Over)expression of SprF1 antitoxin decreases the level of gene transcripts expressing proteins identified as downregulated in the intracellular proteome. (A) RT-qPCR analysis of mRNAs isolated from S. aureus N315 (WT pCN35), sprG1/sprF1 deletion mutant (ΔΔ pCN35), and the sprG1/sprF1 deletion mutant with the SprF1 antitoxin expressing (ΔΔ pCN35ΩsprF1) plasmid. * and **, denote statistical significance at p < 0.05 and p < 0.01 level, respectively. (B) A model of in silico predicted interactions between the gene transcript (ppiB) for peptidyl-prolyl cis-trans isomerase and the SprF1 antitoxin (sRNA). (C) EMSA of radioactively labeled SprF1 (SprF1 *) with mRNA for ppiB. SprG1/SprF1 * complex serves as a positive control.

Figure 4

Expression of the SprG1-encoded peptides during the S. aureus stationary growth phase does not induce massive cell leakages. (A) RT-qPCR analysis of the transcript level for SprG1 toxin in S. aureus N315 (statistical significance at p < 0.01 level). (B) Western blot analysis with anti-GPF antibodies of cell lysates and spent growth medium of S. aureus N315 (WT) and the sprG1/sprF1 deletion mutant (ΔΔ) transformed with a GFP-expressing plasmid (pALCP2G). (C) Western blot with anti-Flag antibodies and SDS-PAGE of total cell lysates (intracellular proteins; upper panels) and culture medium (extracellular proteins; lower panels) of S. aureus N315 (WT pCN35), the sprG1/sprF1 deletion mutant (ΔΔ pCN35), SprF1 antitoxin (over)expressing (N315ΔΔ pCN35ΩsprF1), and Flagged SprG1 (over)expressing (N315ΔΔ pCN35ΩsprG1-Flag) strains grown to logarithmic and stationary phases.