Staphylococcus aureus is a significant human pathogen due to its capacity to cause a multitude of diseases. As such, S. aureus efficiently pillages vital nutrients from the host; however, the molecular mechanisms that support sulfur acquisition during infection have not been established. One of the most abundant extracellular sulfur-containing metabolites within the host is cysteine, which acts as the major redox buffer in the blood by transitioning between reduced and oxidized (cystine) forms. We therefore hypothesized that S. aureus acquires host-derived cysteine and cystine as sources of nutrient sulfur during systemic infection. To test this hypothesis, we used the toxic cystine analogue selenocystine to initially characterize S. aureus homologues of the Bacillus subtilis cystine transporters TcyABC and TcyP. We found that genetic inactivation of both TcyA and TcyP induced selenocystine resistance. The double mutant also failed to proliferate in medium supplemented with cystine, cysteine, or N-acetyl cysteine as the sole sulfur source. However, only TcyABC was necessary for proliferation in defined medium containing homocystine as the sulfur source. Using a murine model of systemic infection, we observed tcyP-dependent competitive defects in the liver and heart, indicating that this sulfur acquisition strategy supports proliferation of S. aureus in these organs. Phylogenetic analyses identified TcyP homologues in many pathogenic species, implying that this sulfur procurement strategy is conserved. In total, this study is the first to experimentally validate sulfur acquisition systems in S. aureus and establish their importance during pathogenesis.
Extra Cytoplasmic Function (ECF) σ factors are a diverse group of alternate σ factors bacteria use to respond to changes in the environment. The Bacillus subtilis ECF σ factor σV responds to lysozyme. In the absence of lysozyme, σV is held inactive by the anti-σ factor, RsiV. In the presence of lysozyme RsiV is degraded via regulated intramembrane proteolysis, which results in the release of σV and thus activation of lysozyme resistance genes. Signal peptidase is required to initiate degradation of RsiV. Previous work indicated that RsiV only becomes sensitive to signal peptidase upon direct binding to lysozyme. We have identified a unique domain of RsiV that is responsible for protecting RsiV from cleavage by signal peptidase in the absence of lysozyme. We provide evidence that this domain contains putative amphipathic helices. Disruption of the hydrophobic surface of these helices by introducing positively charged residues results in constitutive cleavage of RsiV by signal peptidase and thus constitutive σV activation. We provide further evidence that this domain contains amphipathic helices using a membrane-impermeable reagent. Finally, we show that upon lysozyme binding to RsiV, the hydrophobic face of the amphipathic helix becomes accessible to a membrane-impermeable reagent. Thus, we propose the amphipathic helices protect RsiV from cleavage in the absence of lysozyme. Additionally, we propose the amphipathic helices rearrange to form a suitable signal peptidase substrate upon binding of RsiV to lysozyme leading to the activation of σV.
Sulfur is a requirement for life. Therefore, both the host and colonizing bacteria must regulate sulfur metabolism in a coordinated fashion to meet cellular demands.
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