Elucidation of the Role of VraTSR and Lipid Metabolism in the Development of Resistance Phenotypes in Staphylococcus aureus

Abstract

Staphylococcus aureus is a gram-positive bacterium, which has developed resistance to many antimicrobials. Methicillin-resistant S. aureus (MRSA) was first isolated in 1961. Since then, glycopeptides (e.g. vancomycin), lipopeptides (e.g. daptomycin), and long-acting lipoglycopeptides (e.g. dalbavancin) have been developed. MRSA, however, has developed resistance against all three types of antimicrobials over time. VraSR is one two-component system (TCS) out of the 16 prototypical TCSs in S. aureus, which is activated in response to cell-envelope-targeting antimicrobials, such as vancomycin and β-lactams. A third component, VraT, has been shown to be essential for VraSR full activation, making it a three-component regulatory system VraTSR. The molecular inducer of VraTSR has been proposed to be the inhibition of transglycosylation in the peptidoglycan layer, and hundreds of genes have been shown to be in the VraR regulon, including those related to cell wall synthesis. On the other hand, cell wall synthesis has been hypothesized to crosstalk with cell membrane metabolism via three potential ways: lipoteichoic acids (LTAs), acetyl-CoA, and lipid II. In fact, overall decreased levels of lipid abundance have been observed in multiple S. aureus strains that have developed resistance against vancomycin, daptomycin, or dalbavancin, some of which harbor mutations in VraTSR along with other mutations. None of the strains, nevertheless, have mutations only in VraTSR until we isolated S7-D2 from the parent S7 strain by serial passage against dalbavancin. S7-D2 has a non-synonymous mutation in vraT (c. 377C>T; p. P126L) compared to S7. We hypothesized this mutation to be gain-of-function. By using this strain pair and vraTSR loss-of-function mutants, we aim to elucidate the contribution of vraTSR to the remodeling of the cell envelope and the modulation of antimicrobial susceptibility. Chapter 1 provides background on S. aureus and bacterial TCSs and their relation to antimicrobial susceptibility. In Chapter 2, we applied a multi-omics methodology (transcriptomics, metabolomics, and lipidomics), along with various phenotypic characterization, to assess the role of VraTSR. We found that the loss-of-function mutations in VraTSR resulted in a general increase in antimicrobial susceptibility and that an increase in only limited lipid species was unexpectedly observed. On the contrary, the gain-of-function mutant S7-D2 (confirmed by transcriptomics) exhibited characteristic decreased levels of lipids. We also showed from the multi-omics studies several aspects that could have implications in resistance modulation, e.g., decreased membrane fluidity and upregulated arginine deiminase pathway and betaine biosynthesis pathway, and in crosstalk between cell wall and cell membrane, e.g. LTAs and acetyl-CoA. We proposed several future experiments to follow up on the observations from the multi-omics studies. From a practical standpoint, inhibiting the lipid synthesis with AFN-1252 and the VraTSR with histidine kinase inhibitors appear promising in modulating resistance to cell-envelope-targeting antimicrobials, and more studies are warranted. The agr system is another TCS in S. aureus that plays critical roles in quorum sensing and virulence. The phenol-soluble modulins (PSMs) produced from Agr and released by S. aureus have been suggested to antagonize the lipid shedding mechanism of daptomycin inactivation by binding to the released lipids. Others have shown that S. aureus survival in the presence of daptomycin is enhanced with loss of the Agr function. In Chapter 3, we examined several pairs of S. aureus strains with dysfunctional Agr (KO or mutants) for their survival with exposure to daptomycin and their lipid profile (released and membrane lipids) in static time-kill experiments and in vitro pharmacokinetic/pharmacodynamic (PK/PD) modeling. We found that the contribution of dysfunctional Agr to enhanced survival varied depending on the genetic background or the type of mutations and that the enhanced survival did not correlate with the released lipids. We proposed that PSMs might not be the only molecules released by S. aureus that contributed to the antagonizing effects. Studying other released factors might help shed light on the variations among the Agr dysfunctional mutants in terms of enhanced survival against daptomycin. Chapter 4 summarizes the overall findings and proposes future directions.