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

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.

Applying microbial ecology to antimicrobial discovery.

Introduction of antibiotics into clinical use has contributed to some of the greatest improvements to public health in the 20th century. Most antibiotics are based on antimicrobials that were isolated from environmental microorganisms over 50 years ago, but emerging resistance requires discovery of new molecules and development of these molecules into therapeutics. Bioinformatic analyses of microbial genomes indicate that many more microbial bioactive molecules remain undiscovered. Understanding when, where, and why these molecules are produced informs efforts to tap into the hidden unexplored chemical diversity. Expanding the search to undersampled ecological niches and improving culturing techniques will ensure discovery of new antibiotics.

Staphylococcus aureus growth using human hemoglobin as an iron source.

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm. This pathway has been dissected through numerous in vitro studies. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.