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Decoupling Activation of Heme Biosynthesis from Anaerobic Toxicity in a Molecule Active in Staphylococcus aureus.

ABSTRACT: Small molecules active in the pathogenic bacterium Staphylococcus aureus are valuable tools for the study of its basic biology and pathogenesis, and many molecules may provide leads for novel therapeutics. We have previously reported a small molecule, 1, which activates endogenous heme biosynthesis in S. aureus, leading to an accumulation of intracellular heme. In addition to this novel activity, 1 also exhibits toxicity towards S. aureus growing under fermentative conditions. To determine if these activities are linked and establish what features of the molecule are required for activity, we synthesized a library of analogs around the structure of 1 and screened them for activation of heme biosynthesis and anaerobic toxicity to investigate structure-activity relationships. The results of this analysis suggest that these activities are not linked. Furthermore, we have identified the structural features that promote each activity and have established two classes of molecules: activators of heme biosynthesis and inhibitors of anaerobic growth. These molecules will serve as useful probes for their respective activities without concern for the off target effects of the parent compound.

SUBMITTER: Dutter BF

PROVIDER: S-EPMC5117894 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Decoupling Activation of Heme Biosynthesis from Anaerobic Toxicity in a Molecule Active in Staphylococcus aureus.

Dutter Brendan F BF Mike Laura A LA Reid Paul R PR Chong Katherine M KM Ramos-Hunter Susan J SJ Skaar Eric P EP Sulikowski Gary A GA

ACS chemical biology 20160304 5

Small molecules active in the pathogenic bacterium Staphylococcus aureus are valuable tools for the study of its basic biology and pathogenesis, and many molecules may provide leads for novel therapeutics. We have previously reported a small molecule, 1, which activates endogenous heme biosynthesis in S. aureus, leading to an accumulation of intracellular heme. In addition to this novel activity, 1 also exhibits toxicity towards S. aureus growing under fermentative conditions. To determine if th ...[more]

PMID: 26890615

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Project description:Staphylococcus aureus is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized de novo from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme homeostasis. In this work, we describe a posttranscriptional regulatory strategy in S. aureus heme biosynthesis. The first committed enzyme in the S. aureus heme biosynthetic pathway, glutamyl-tRNA reductase (GtrR), is regulated by heme abundance and the integral membrane protein HemX. GtrR abundance increases dramatically in response to heme deficiency, suggesting a mechanism by which S. aureus responds to the need to increase heme synthesis. Additionally, HemX is required to maintain low levels of GtrR in heme-proficient cells, and inactivation of hemX leads to increased heme synthesis. Excess heme synthesis in a ΔhemX mutant activates the staphylococcal heme stress response, suggesting that regulation of heme synthesis is critical to reduce self-imposed heme toxicity. Analysis of diverse organisms indicates that HemX is widely conserved among heme-synthesizing bacteria, suggesting that HemX is a common factor involved in the regulation of GtrR abundance. Together, this work demonstrates that S. aureus regulates heme synthesis by modulating GtrR abundance in response to heme deficiency and through the activity of the broadly conserved HemX.IMPORTANCEStaphylococcus aureus is a leading cause of skin and soft tissue infections, endocarditis, bacteremia, and osteomyelitis, making it a critical health care concern. Development of new antimicrobials against S. aureus requires knowledge of the physiology that supports this organism's pathogenesis. One component of staphylococcal physiology that contributes to growth and virulence is heme. Heme is a widely utilized cofactor that enables diverse chemical reactions across many enzyme families. S. aureus relies on many critical heme-dependent proteins and is sensitive to excess heme toxicity, suggesting S. aureus must maintain proper intracellular heme homeostasis. Because S. aureus provides heme for heme-dependent enzymes via synthesis from common precursors, we hypothesized that regulation of heme synthesis is one mechanism to maintain heme homeostasis. In this study, we identify that S. aureus posttranscriptionally regulates heme synthesis by restraining abundance of the first heme biosynthetic enzyme, GtrR, via heme and the broadly conserved membrane protein HemX.

Project description:To be a successful pathogen, Staphylococcus aureus has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, S. aureus uses glycolysis to generate ATP via substrate-level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by reoxidation of NADH equivalents. However, it is less clear how S. aureus proceeds under anoxic conditions and glucose limitation, likely representing the bona fide situation in the host. Using a combination of proteomic, transcriptional, and metabolomic analyses, we show that in the absence of an abundant glycolysis substrate, the available carbon source pyruvate is converted to acetyl coenzyme A (AcCoA) in a pyruvate formate-lyase (PflB)-dependent reaction to produce ATP and acetate. This process critically depends on derepression of the catabolite control protein A (CcpA), leading to upregulation of pflB transcription. Under these conditions, ethanol production is repressed to prevent wasteful consumption of AcCoA. In addition, our global and quantitative characterization of the metabolic switch prioritizing acetate over lactate fermentation when glucose is absent illustrates examples of carbon source-dependent control of colonization and pathogenicity factors.IMPORTANCE Under infection conditions, S. aureus needs to ensure survival when energy production via oxidative phosphorylation is not possible, e.g., either due to the lack of terminal electron acceptors or by the inactivation of components of the respiratory chain. Under these conditions, S. aureus can switch to mixed-acid fermentation to sustain ATP production by substrate level phosphorylation. The drop in the cellular NAD+/NADH ratio is sensed by the repressor Rex, resulting in derepression of fermentation genes. Here, we show that expression of fermentation pathways is further controlled by CcpA in response to the availability of glucose to ensure optimal resource utilization under growth-limiting conditions. We provide evidence for carbon source-dependent control of colonization and virulence factors. These findings add another level to the regulatory network controlling mixed-acid fermentation in S. aureus and provide additional evidence for the lifestyle-modulating effect of carbon sources available to S. aureus.

Dataset Information

Decoupling Activation of Heme Biosynthesis from Anaerobic Toxicity in a Molecule Active in Staphylococcus aureus.

Publications

Decoupling Activation of Heme Biosynthesis from Anaerobic Toxicity in a Molecule Active in Staphylococcus aureus.

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