258. Clinical Rel Mutations in Staphylococcus aureus Primes Pathogen Expansion Under Nutrient Stress

Abstract Background Nutritional immunity is an important component of host defense whereby nutrients are sequestered to limit pathogen replication. The bacterial stringent response (SR) enables cells to modulate their growth, metabolism, and virulence in accordance with available nutrition. By analyzing within-host evolution of MRSA in patients with persistent bacteremia, we recently identified 5 protein-altering mutations in rel. Rel synthesizes or degrades ‘alarmone’ molecules to regulate the SR in response to stress conditions. Here, we characterized the phenotypes caused by these mutations and linked them to a mechanism for evading nutritional immunity. Rel domain architecture and location of clinical mutations. (A) The N-terminal enzymatic region containing the hydrolase (Hd) and synthetase (Syn) domains are shown in blue. The C-terminal regulatory region containing the TGS (ThrRs, GTPase, SpoT), AH (Alpha-Helical), RIS (Ribosome Inter Subunit), and ACT (Aspartate kinase, Chorismate mutase, TyrA) domains are shown in green. The Hd domain catalyzes the hydrolysis of (p)ppGpp to yield inorganic pyrophosphate (PPi) and either GTP or GDP. The Syn domain catalyzes the transfer of pyrophosphate from ATP to either GTP or GDP to form (p)ppGpp. The mutations examined in this study are denoted with red markers. Previously identified mutations are denoted with purple markers. (B) Clinical Rel mutations mapped onto a structure of a Hdon/Synoff RelΔRIS-CT from B. subtilis (PDB 6YXA) with the CTD of a Hdoff/Synon Rel from E. coli (PDB 5KPV) superimposed. Domain colorings: hydrolase, green; central 3-helix bundle, yellow; synthetase, blue; TGS-AH, orange; RIS-ACT, tan. Rel mutations focused in this study are denoted by red spheres. Previously identified mutations are denoted by purple spheres. Methods Mutations were introduced into the rel locus of MRSA strain JE2 by allelic exchange. The growth, fitness, and antibiotic profiles (vancomycin, daptomycin, ceftaroline, mupirocin) of the mutant strains were compared to wildtype in different nutrient conditions. Mass spectrometry (MS) was used to quantify intracellular alarmone levels during different phases of growth. Results We found that the rel mutations caused increased SR signaling and conveyed a survival advantage during stationary phase growth. The mutants additionally displayed a fitness advantage when subsequently seeded in nutrient-limiting media. This suggested that the cells become primed for survival and re