A Small-Molecule Modulator of Metal Homeostasis in Gram-Positive Pathogens

Abstract

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. IMPORTANCE Staphylococcus 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.

Coordination Complexes to Combat Bacterial Infections: Recent Developments, Current Directions and Future Opportunities

“…demonstrated the efficacy of a small molecule, VU0026921 exhibiting enhanced activity in a S. aureus strain lacking high‐affinity Mn import systems. The conjugate is proposed to dysregulate Mn homeostasis in S. aureus and other Gram‐positive pathogens, causing substantial toxicity [77] …”

“…The conjugate is pro- posed to dysregulate Mn homeostasis in S. aureus and other Gram-positivep athogens, causing substantial toxicity. [77] Zinc also plays an important role in the mammalian immune response to invading pathogens. In humans, Zn 2 + homeostasis is controlled by Zn 2 + transporters and metallothionein.…”

Copper intoxication in group B Streptococcus triggers transcriptional activation of the cop operon that contributes to enhanced virulence during acute infection

“…It would be of interest to analyze the effect of Cu transport deficiency in GBS in other relevant models of infection, including in vaginal colonization (58). Additional to defining the effects of metal homeostasis in GBS on the nature of infection and disease caused by this organism, small-molecules probes might hold promise for the identification of other molecular mechanisms of metal homeostasis in GBS, as reported for Gram-positive bacteria (59).…”

“…Additional to defining the effects of metal homeostasis in GBS on the nature of infection and disease caused by this organism, small-molecules probes might hold promise for the identification of other molecular mechanisms of metal homeostasis in GBS, as reported for Gram-positive bacteria (61).…”

Identification of Three Small Molecules That Can Selectively Influence Cellular Manganese Levels in a Mouse Striatal Cell Model

“…We have previously validated in vivo activity for two molecules of the Mn toolbox (VU0063088 and VU0026921), that were shown to modulate Mn levels in C. elegans , through mechanisms independent of the species DMT-2 homologue, and induce protection from Mn neurotoxicity [ 37 ]. Likewise, VU0026921 was also found to be a modifier of metal homeostasis in gram-positive bacteria [ 38 ]. Here, we set out to test the hypothesis that cell-level homeostatic processes selectively regulate intracellular Mn to avoid hazardous concentrations in the brain, and thus small molecules could be found that specifically disrupt these processes as compared to other Mn-altering small molecules that may show affinity for other divalent metals.…”