Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system

Abstract

The overuse of antibiotics in humans and livestock has driven the emergence and spread of antimicrobial resistance and has therefore prompted research on the discovery of novel antibiotics. Complestatin (Cm) and corbomycin (Cb) are glycopeptide antibiotics with an unprecedented mechanism of action that is active even against methicillin-resistant and daptomycin-resistant Staphylococcus aureus. They bind to peptidoglycan and block the activity of peptidoglycan hydrolases required for remodeling the cell wall during growth. Bacterial signaling through two-component transduction systems (TCSs) has been associated with the development of S. aureus antimicrobial resistance. However, the role of TCSs in S. aureus susceptibility to Cm and Cb has not been previously addressed. In this study, we determined that, among all 16 S. aureus TCSs, VraSR is the only one controlling the susceptibility to Cm and Cb. Deletion of vraSR increased bacterial susceptibility to both antibiotics. Epistasis analysis with members of the vraSR regulon revealed that deletion of spdC, which encodes a membrane protein that scaffolds SagB for cleavage of peptidoglycan strands to achieve physiological length, in the vraSR mutant restored Cm and Cb susceptibility to wild-type levels. Moreover, deletion of either spdC or sagB in the wild-type strain increased resistance to both antibiotics. Further analyses revealed a significant rise in the relative amount of peptidoglycan and its total degree of cross-linkage in ΔspdC and ΔsagB mutants compared to the wild-type strain, suggesting that these changes in the cell wall provide resistance to the damaging effect of Cm and Cb. IMPORTANCE Although Staphylococcus aureus is a common colonizer of the skin and digestive tract of humans and many animals, it is also a versatile pathogen responsible for causing a wide variety and number of infections. Treatment of these infections requires the bacteria to be constantly exposed to antibiotic treatment, which facilitates the selection of antibiotic-resistant strains. The development of new antibiotics is, therefore, urgently needed. In this paper, we investigated the role of the sensory system of S. aureus in susceptibility to two new antibiotics: corbomycin and complestatin. The results shed light on the cell-wall synthesis processes that are affected by the presence of the antibiotic and the sensory system responsible for coordinating their activity.

Keywords: Staphylococcus aureus; VraSR; autolysins; complestatin; corbomycin; two-component system.

Fig 1

Absence of all non-essential TCSs increases S. aureus susceptibility to complestatin and corbomycin. (A) The optical density at 595 nm was recorded during growth of the wild-type MW2 strain and the ΔXV mutant, lacking all 15 non-essential TCSs, in MHB supplemented with Cm (left panel) or Cb (right panel) at the ΔXV MIC for 20 h at 37°C with shaking. (B) Comparison of the susceptibility to bacitracin (1 µg mL⁻¹) of the wild-type MW2 strain and the ΔXV mutant with that of a collection of 15 MW2 single mutants in each non-essential TCS (left panel) and with that of a second collection of 15 strains, derivative of ΔXV, each complemented with a plasmid expressing a single TCS (right panel). In the latter case, the wild-type and ΔXV strains carried an empty pCN51 plasmid. Use of both collections allows the identification of BraRS as the specific TCS that controls S. aureus susceptibility to bacitracin. The average and SD of three technical replicates from one representative experiment of at least three independent experiments are shown.

Fig 2

The VraSR TCS is the only non-essential TCS involved in S. aureus susceptibility to Cm and Cb. (A) Comparison of the susceptibility to Cm (left panel) or Cb (right panel) of the wild-type MW2 strain and the ΔXV mutant with that of a collection of 15 MW2 single mutants in each non-essential TCS. The ΔvraSR mutant strain is the only mutant unable to grow in the presence of the antibiotics. (B) Results obtained with a second collection of 15 strains, derivative of ΔXV, each complemented with a plasmid expressing a single TCS. In this case, the wild-type and ΔXV strains carried an empty pCN51 plasmid. The ΔXV derivative expressing vraSR (ΔXV + vra) is the only one showing a significantly increased resistance to Cm and Cb. The optical density at 595 nm was recorded during growth in MHB supplemented with the antibiotics at the ΔXV MIC for 20 h at 37°C with shaking. Average and SD of three technical replicates from one representative experiment of at least three independent experiments are shown.

Fig 3

Complementation of the ΔXV mutant with a constitutively active form of WalR does not increase resistance levels of ΔXV to complestatin and corbomycin. (A) The optical density at 595 nm was recorded during growth of the wild-type MW2 strain, the ΔXV mutant, and the ΔXV strain complemented with a constitutively active form of the response regulator WalR* (WalR D52E) or the response regulator VraR* (VraR D55E), in MHB supplemented with Cm (left panel) or Cb (right panel) at the ΔXV MIC for 20 h at 37°C with shaking. The wild-type and ΔXV strains carried an empty pRMC2 plasmid. The experiments were performed in the presence of anhydrotetracycline at a concentration of 0.1 µg mL⁻¹ to induce expression from the pRMC2 plasmid. The wild-type 15981 strain and the 15981 ΔvraSR mutant carried an empty pCN51 plasmid. The ΔvraSR mutant is unable to grow in the presence of both antibiotics. (B) The VraSR TCS determines Cm and Cb susceptibility in the methicillin-sensitive S. aureus 15981 strain. The optical density at 595 nm was recorded during growth of the wild-type 15981 strain, the 15981 ΔvraSR mutant, and the ΔvraSR mutant complemented with a pCN51 plasmid carrying the complete vraSR TCS (15981 Δvra + vra), in MHB supplemented with Cm (left panel) or Cb (right panel) at the MW2 ΔXV MIC for 20 h at 37°C with shaking. Average and SD of three technical replicates from one representative experiment of at least three independent experiments are shown.

Fig 4

Role of SpdC and SagB in S. aureus susceptibility to complestatin and corbomycin. (A) Comparison of the susceptibility to Cm (left panel) or Cb (right panel) of the wild-type MW2, ΔvraSR, ΔvraSR complemented with a plasmid expressing the mgt gene (Δvra + mgt) and the double mutants ΔvraSR ΔspdC,ΔvraSR ΔssaA, and ΔvraSR ΔisaA. Deletion of spdC in the ΔvraSR background increases resistance to both Cm and Cb. The optical density at 595 nm was recorded during growth in MHB supplemented with Cm or Cb at the ΔXV MIC for 20 h at 37°C with shaking. (B) Comparison of the susceptibility to Cm (left panel) or Cb (right panel) of the wild-type MW2, and the single mutants ΔspdC and ΔsagB. The optical density at 595 nm was recorded during growth for 20 h, at 37°C, in MHB supplemented with Cm or Cb at a concentration above the MIC of the wild-type strain. Average and SD of three technical replicates from one representative experiment of at least three independent experiments are shown.

Fig 5

Changes in the peptidoglycan structure of spdC and sagB mutants provide resistance to Cm and Cb. (A) Peptidoglycan profile obtained for S. aureus MW2 ΔspdC mutant strain grown in TSB medium and identity of muropeptides (see Fig. S1). M, disaccharide NAG-NAM; numbers, length of stem peptides or glycine bridges. (B) Representative peptidoglycan chromatograms obtained for the wild-type, ΔvraSR, ΔvraSR ΔspdC, ΔspdC, and ΔsagB mutant strains grown in TSB medium. Obtained chromatograms (from three biological replicates) were used to calculate the relative amount of peptidoglycan per OD (C) and the degree of cross-linkage (D). Error bars in (C) and (D) correspond to the standard deviation of three biological replicates. ns, not significant; *P < 0.05, unpaired t test.