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ABSTRACTAmong the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.IMPORTANCEThe opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

Healthcare associated infections caused by vancomycin-resistant Enterococcus faecium (VREfm) have a major impact on health outcomes. VREfm is difficult to treat because of intrinsic and acquired resistance to many clinically used antimicrobials, with daptomycin being one of the few last line therapeutic options for treating multidrug-resistant VREfm. The emergence of daptomycin-resistant VREfm is therefore of serious clinical concern. Despite this, the impact that daptomycin-resistant VREfm have on patient health outcomes is not clearly defined and knowledge on the mechanisms and genetic signatures linked with daptomycin resistance in VREfm remains incomplete. To address these knowledge gaps, phenotypic daptomycin susceptibility testing was undertaken on 324 E. faecium isolates from Australia and New Zealand. Approximately 15% of study isolates were phenotypically resistant to daptomycin. Whole genome sequencing revealed a strong association between vanA-VREfm and daptomycin resistance, with 95% of daptomycin-resistant study isolates harbouring vanA. Genomic analyses showed that daptomycin-resistant VREfm isolates were polyclonal and carried several previously characterised mutations in the liaR and liaS genes as well as several novel mutations within the rpoB, rpoC, and dltC genes. Overall, 70% of daptomycin-resistant study isolates were found to carry mutations within the liaR, rpoB, rpoC, or dltC genes. Finally, in a mouse model of VREfm bacteraemia, infection with the locally dominant daptomycin-resistant clone led to reduced daptomycin treatment efficacy in comparison to daptomycin-susceptible E. faecium. These findings have important implications for ongoing VREfm surveillance activities and the treatment of VREfm infections.

Multidrug-resistant Staphylococcus and vancomycin-resistant Enterococcus (VRE) are important human pathogens that are resistant to most clinical antibiotics. Treatment options are limited and often require the use of ‘last-line’ antimicrobials such as linezolid, daptomycin, and in the case of Staphylococcus, also vancomycin. The emergence of resistance to these last-line antimicrobial agents is therefore of considerable clinical concern. This mini-review provides an overview of resistance to last-line antimicrobial agents in Staphylococcus and VRE, with a particular focus on how genomics has provided critical insights into the emergence of resistant clones, the molecular mechanisms of resistance, and the importance of mobile genetic elements in the global spread of resistance to linezolid.