The RND efflux pump FemT encoded by SAUSA300_2213 of Staphylococcus aureus USA300 is co-transcribed with femX which has an essential role in synthesizing the Lipid II precursor of peptidoglycan. Anticipating that this arrangement reflects a critical accessory role for femT, we constructed USA300ΔfemT to assess its function. Although growth of USA300ΔfemT in tryptic soy broth (TSB) was not impaired, transcriptomic data revealed a mild cellular stress response, accompanied by reduced expression of ohyA and crt genes involved in fatty acid metabolism and carotenoid lipid synthesis respectively. Accordingly, USA300ΔfemT exhibited impaired growth on exposure to saturated and unsaturated fatty acids, and exposure to subinhibitory 50 µM palmitic acid promoted accumulation of reactive oxygen species, reduced respiratory activity, and altered membrane function and morphology. The transcriptome of cells grown under this condition revealed strongly attenuated expression of ohyA and crt, and several genes required for oxidative and anaerobic respiration, concomitant with strongly enhanced expression of several stress response pathways. Cellular metabolites were also profoundly altered. Finally, lipidomic analysis of USA300ΔfemT exposed to oleic acid revealed increased incorporation of oleic acid into phosphatidylglycerol, accompanied by a significant reduction in undecaprenol C55 lipid carrier, and respiratory quinones MK-7 and MK-8. Our data are consistent with a role for FemT in maintaining cellular lipid homeostasis by promoting efflux of isoprenoid and carotenoid lipids that are prone to oxidative damage, including C55 and menaquinones that undergo cyclic reactions in peptidoglycan synthesis and electron transport. IMPORTANCE The FemT efflux pump of S. aureus is co-expressed in an operon with femX encoding an essential enzyme needed to complete the synthesis of peptidoglycan precursor Lipid II. Although this alluded to a specific role for FemT in supporting peptidoglycan synthesis, our data are instead consistent with a general role in efflux of cellular isoprenoids and carotenoid lipids that are susceptible to oxidation during routine cellular functions. Consequently, S. aureus became strongly dependent on FemT function when exogenous host-derived fatty acids were being actively metabolized. This represents a significant advance in our understanding of the role of an RND efflux pump in supporting routine growth-related functions of S. aureus and exposes a function that could be targeted to impair S. aureus growth on exposure to host-derived fatty acids.
A. Thukral, Camryn Bonn Dunbar, Jamie N Halucha et al.· bioRxiv· 0 citations
ABSTRACT Staphylococcus aureus is a globally prevalent gram-positive pathogen that can cause numerous types of infection. Due to host nutritional immunity and iron (Fe) sequestration, S. aureus experiences Fe limitation during infection. To overcome this, S. aureus expresses an arsenal of Fe acquisition systems whose expression is coordinated through the Fe-binding transcriptional regulator, Fur. Here, from a screen to identify S. aureus mutants defective for Fe-restricted growth, we identified several with mutations in perR, encoding a transcriptional regulator involved in resistance to oxidative stress. RNA-seq identified that the most downregulated genes in a perR mutant growing in Fe-restriction are those from the sbn operon that encodes staphyloferrin B biosynthesis. In agreement, perR mutants grew poorly in Fe-deficient media due to deficient staphyloferrin B production. In a subcutaneous model of S. aureus skin infection, S. aureus perR caused significantly smaller lesions, consistent with our finding that this mutant had decreased alpha-hemolysin expression during Fe-restricted growth. These findings are consistent with the hypothesis that PerR acts to fine-tune access to Fe ostensibly to avoid Fe-dependent toxicity. The importance of the PerR function to S. aureus was further highlighted by examination of over 8,000 human bloodstream isolates of S. aureus, showing that the PerR sequence was highly conserved. Together, these findings demonstrate the importance of PerR to S. aureus in providing an additional level of regulation of Fe homeostasis beyond Fur-dependent Fe sensing. IMPORTANCE Staphylococcus aureus is a notoriously antibiotic-resistant human pathogen and has the potential to cause a myriad of potentially life-threatening infections. Understanding the intricacies of S. aureus pathogenesis in the host will underpin the development of novel therapeutic approaches that target bacterial virulence mechanisms rather than essential cellular processes. The significance of our research lies in identifying how S. aureus regulates Fe acquisition in response to environmental signals, providing further insight into nutrient sensing and acquisition at the host–pathogen interface to facilitate the development of therapeutics that may target this process. Staphylococcus aureus is a notoriously antibiotic-resistant human pathogen and has the potential to cause a myriad of potentially life-threatening infections. Understanding the intricacies of S. aureus pathogenesis in the host will underpin the development of novel therapeutic approaches that target bacterial virulence mechanisms rather than essential cellular processes. The significance of our research lies in identifying how S. aureus regulates Fe acquisition in response to environmental signals, providing further insight into nutrient sensing and acquisition at the host–pathogen interface to facilitate the development of therapeutics that may target this process.
Alexander A Sheikh, Ronald S. Flannagan, Nathan J Nicholson et al.· Journal of Bacteriology· 0 citations