Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
mBio. 2020 Oct 27;11(5):e02555-20. doi: 10.1128/mBio.02555-20.
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. , a Gram-positive bacterium, relies on metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of metal homeostasis in will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit growth in a metal-dependent manner as a strategy to uncover pathways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills 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 , 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 from VU0026921 killing, emphasizing the role of reactive oxygen species in the mechanism of killing, but VU0026921 also kills 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. is a leading agent of antibiotic-resistant bacterial infections in the world. 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 to develop chemical probes to investigate metallobiology in this organism. Compound VU0026921 was identified as a small molecule that kills 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 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 and validates future antibiotic development targeting metal toxicity pathways.
金属是所有生物体从环境中获取的必需营养素。虽然金属是生命所必需的，但过量的金属摄入可能是有毒的；因此，细菌细胞内的金属水平受到严格调节。作为一种革兰氏阳性菌，它依赖于金属的摄取和代谢来定殖脊椎动物。因此，我们假设对 的金属稳态的深入了解将导致发现可以用未来的抗菌药物靶向的途径。我们试图确定以依赖金属的方式抑制 生长的小分子，作为发现维持金属稳态途径的策略。在这里，我们证明 VU0026921 通过破坏金属稳态来杀死 。通过细胞培养测定、转录测序、化合物结构-活性关系、活性氧 (ROS) 生成测定、金属结合测定和金属水平分析来表征 VU0026921 的活性。VU0026921 破坏了 的金属稳态，导致金属细胞内积累增加，并通过酶的错金属化、活性氧的产生或其他细胞过程的破坏导致毒性。抗氧化剂部分保护 免受 VU0026921 的杀伤，这强调了活性氧在杀伤机制中的作用，但 VU0026921 也在厌氧条件下杀死 ，表明观察到的毒性不仅仅是氧气依赖性的。VU0026921 破坏了多种革兰氏阳性菌的金属稳态，导致活性氧增加和细胞死亡，证明了这些发现的广泛适用性。此外，这项研究验证了 VU0026921 作为一种探针，以进一步破译维持革兰氏阳性菌金属稳态所需的机制。 是世界上导致抗生素耐药性细菌感染的主要病原体。 在感染过程中严格控制金属稳态，破坏金属摄取系统会损害葡萄球菌的毒力。我们鉴定了干扰 中金属处理的小分子，以开发化学探针来研究该生物体中的金属生物学。化合物 VU0026921 被鉴定为一种既能在有氧条件下又能在厌氧条件下杀死 的小分子。VU0026921 的活性受金属补充的调节，Mn 稳态基因的遗传失活增强了其活性，并与细胞内活性氧的增加相关。用 VU0026921 处理会导致 细胞内多种金属的积累，并伴随参与金属解毒的基因的上调。这项工作定义了一种小分子探针，用于进一步定义金属毒性在 中的作用，并验证了针对金属毒性途径的未来抗生素开发。
