Evolution and adaptation of Staphylococcus aureus to nutrient stress

ABSTRACT Cellular adaption to nutrient stress is a fundamental biological process. In prokaryotes, the stringent response (SR) regulates growth, survival, and virulence to adapt to nutrient availability within the environment. This is particularly consequential for Staphylococcus aureus, where SR activation also impacts the CodY regulon and accessory gene regulator (agr) systems, which harbor critical pathogenicity determinants. In S. aureus, the SR is controlled by the RelA-SpoT Homolog (RSH) family enzyme Rel, which synthesizes and hydrolyzes the second-messenger alarmone molecule (p)ppGpp. Under amino acid limitation, the enzyme switches to a net synthesis state and elevated (p)ppGpp levels effect downstream targets to induce a state of metabolic quiescence and modulate virulence factor production. Although S. aureus is a human commensal that has evolved to colonize skin, these features give it an extraordinary ability to adapt to new environments and cause severe, invasive disease. After invasion, it is no longer in its ecological niche and must adapt to different host immune and nutrient stresses, as well as antibiotics. By analyzing within-host evolution during persistent clinical S. aureus infections, we recently identified five mutations in rel. Remarkably, these mutations heightened SR signaling via a net increase in (p)ppGpp production, leading to increased growth and survival during nutrient limitation, strongly suggesting they evolved as adaptions to the nutrient stress imposed by the pathogenic host environment. The focus this proposal is to understand how S. aureus is impacted by this stress. We propose to do this by determining how the bacteria adapt and evolve to it, using the tools of molecular evolution. The long- term vision of the laboratory is to use this knowledge to develop novel therapies that block this adaptation. In this proposal, we will address the following three knowledge gaps: Gap 1. Understanding the role of Rel’s ACT domain. Rel’s ACT domain is poorly characterized, however, direct regulation of (p)ppGpp hydrolysis activity by BCAA binding to the ACT domain was reported in the bacterium Rhodobacter capsulatus. Recently, we uncovered two S. aureus clinical rel mutations that map to the ACT domain near the proposed BCAA binding site and these caused elevated (p)ppGpp levels. In Project 1, we will determine whether direct BCAA binding to the S. aureus Rel ACT domain regulates (p)ppGpp levels. Gap 2. Understanding how adaptation to nutrient stress impacts mutagenesis. SR activation has been implicated in modulating bacterial mutation rates to hasten adaptive evolution. In Project 2, we will determine the genome-wide effects of the SR on mutational phenomena using whole genome sequencing. Gap 3. Understanding evolutionary trade-offs between growth and virulence. The agr system is the major virulence module in S. aureus, yet often evolves loss of function mutations (agrˉ) during infection. Our preliminary data shows that amino acid stress rapidly selects for agrˉ mutants, suggesting a fitness cost to agr activity that is SR/CodY-dependent. In Project 3, we will determine how nutrient stress affects the evolution of virulence.