Small Alarmone Synthetases RelP and RelQ of Staphylococcus aureus Are Involved in Biofilm Formation and Maintenance Under Cell Wall Stress Conditions

Abstract

The stringent response is characterized by the synthesis of the alarmone (p)ppGpp. The phenotypic consequences resulting from (p)ppGpp accumulation vary among species, and for several pathogenic bacteria, it has been shown that the activation of the stringent response strongly affects biofilm formation and maintenance. In Staphylococcus aureus, (p)ppGpp can be synthesized by the RelA/SpoT homolog Rel upon amino acid deprivation or by the two small alarmone synthetases RelP and RelQ under cell wall stress. We found that relP and relQ increase biofilm formation under cell wall stress conditions induced by a subinhibitory vancomycin concentration. However, the effect of (p)ppGpp on biofilm formation is independent of the regulators CodY and Agr. Biofilms formed by the strain HG001 or its (p)ppGpp-defective mutants are mainly composed of extracellular DNA and proteins. Furthermore, the induction of the RelPQ-mediated stringent response contributes to biofilm-related antibiotic tolerance. The proposed (p)ppGpp-inhibiting peptide DJK-5 shows bactericidal and biofilm-inhibitory activity. However, a non-(p)ppGpp-producing strain is even more vulnerable to DJK-5. This strongly argues against the assumption that DJK-5 acts via (p)ppGpp inhibition. In summary, RelP and RelQ play a major role in biofilm formation and maintenance under cell wall stress conditions.

Keywords: (p)ppGpp; Staphylococcus aureus; biofilm; cell wall stress; stringent response.

FIGURE 1

(p)ppGpp synthases effect biofilm formation under cell wall stress conditions. (A) Biofilm formation in TSB (+3% NaCl, +0.5% glucose), BM2 and CDM after 24 h. (B) Biofilm formation under uninduced and vancomycin-stress (subinhibitory vancomycin 0.78 μg/ml) conditions in CDM after 24 h. Three separate experiments were performed with biological triplicates each. Error bars represent the standard deviation, statistical significance based on ordinary one-way ANOVA (ns: not significant, ****: P < 0.0001). (C) Representative plate stained with crystal violet. (D) CFU was determined from resolved biofilm and planktonic bacteria after 24 h of static incubation.

FIGURE 2

RelP and RelQ affect biofilm formation synergistically. Biofilm formation under vancomycin-stress (0.78 μg/ml vancomycin) in CDM. Strains: HG001 wild type, relP and relQ single mutants, relPQ double mutant and complemented strains. Three separate experiments were performed with biological triplicates each. Error bars represent the standard deviation, statistical significance based on ordinary one-way ANOVA (ns: not significant, *: P < 0.1, ****: P < 0.0001).

FIGURE 3

Biofilm composition is not affected by (p)ppGpp. (A) Preformed biofilms of the wild type and the isogenic (p)ppGpp⁰ strain were treated with either DNase or proteinase K for 4 h at 37°C or with sodium periodate for 24 h at 4°C. The remaining biofilm was stained with crystal violet and quantified by OD₆₀₀ measurement. Three separate experiments were performed with biological triplicates each. Error bars represent the standard deviation. (B) Representative plate with wild type, rel_syn, relPQ and the (p)ppGpp⁰ strain stained with crystal violet.

FIGURE 4

Biofilm formation under stringent conditions is independent of CodY and Agr. Biofilm formation under non-stressed and vancomycin-stress (0.78 μg/ml vancomycin) in CDM, 24 h. Three separate experiments were performed with biological triplicates each. Error bars represent the standard deviation, statistical significance based on ordinary one-way ANOVA (ns: P > 0.05).

FIGURE 5

(p)ppGpp contributes to biofilm related antibiotic tolerance. (A) Preformed biofilms (8 h) were exposed to increasing concentrations of vancomycin for 16 h. Three separate experiments were performed with biological triplicates each. Error bars represent the standard deviation, statistical significance based on ordinary one-way ANOVA (ns: not significant, **: P < 0.01). (B) Representative plate stained with crystal violet.

FIGURE 6

DJK-5 interferes with biofilm formation under relaxed and stringent conditions. (A) Biofilms grown with or without vancomycin (0.78 μg/ml) and/or the anti-biofilm peptide DJK-5 (5 μg/ml) in CDM for 24 h. (B) Representative plate stained with crystal violet. (C) CFU of planktonic and biofilm bacteria determined in parallel after static growth for 24 h. Three separate experiments were performed with biological triplicates each. Error bars represent the standard deviation, statistical significance based on ordinary one-way ANOVA (ns: not significant, *: P < 0.1, ****: P < 0.0001).