Metals are essential nutrients that all living organisms acquire from their environment. While metals are necessary for life, excess
metal uptake can be toxic; therefore, intracellular
metal levels are tightly regulated in bacterial cells. Staphylococcus aureus, a Gram-positive bacterium, relies on
metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of
metal homeostasis in S. aureus will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit S. aureus growth in a
metal-dependent manner as a strategy to uncover pathways that maintain
metal homeostasis. Here, we demonstrate that
VU0026921 kills S. aureus through disruption of
metal homeostasis.
VU0026921 activity was characterized through cell culture assays, transcriptional sequencing, compound structure-activity relationship,
reactive oxygen species (ROS) generation assays,
metal binding assays, and
metal level analyses.
VU0026921 disrupts
metal homeostasis in S. aureus, increasing intracellular accumulation of metals and leading to toxicity through mismetalation of
enzymes, generation of
reactive oxygen species, or disruption of other cellular processes.
Antioxidants partially protect S. aureus from
VU0026921 killing, emphasizing the role of
reactive oxygen species in the mechanism of killing, but
VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is not solely
oxygen dependent.
VU0026921 disrupts
metal homeostasis in multiple Gram-positive bacteria, leading to increased
reactive oxygen species and cell death, demonstrating the broad applicability of these findings. Further, this study validates
VU0026921 as a probe to further decipher mechanisms required to maintain
metal homeostasis in Gram-positive bacteria.IMPORTANCEStaphylococcus aureus is a leading agent of
antibiotic-resistant
bacterial infections in the world. S. aureus tightly controls
metal homeostasis during
infection, and disruption of
metal uptake systems impairs staphylococcal virulence. We identified small molecules that interfere with
metal handling in S. aureus to develop chemical probes to investigate metallobiology in this organism. Compound
VU0026921 was identified as a small molecule that kills S. aureus both aerobically and anaerobically. The activity of
VU0026921 is modulated by
metal supplementation, is enhanced by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular
reactive oxygen species. Treatment with
VU0026921 causes accumulation of multiple metals within S. aureus cells and concomitant upregulation of genes involved in
metal detoxification. This work defines a small-molecule probe for further defining the role of
metal toxicity in S. aureus and validates future
antibiotic development targeting
metal toxicity pathways.