Wolter, D. J. | Search bioRxiv

bioRxiv2026

A c-di-AMP-controlled glutamine synthesis pathway promotes persistence of Staphylococcus aureus thymidine-dependent small colony variants in the lung

Children with cystic fibrosis (CF) commonly harbor Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs), which are associated with reduced lung function and increased respiratory exacerbations. How TD-SCVs survive in the thymidine-limited CF lung is unknown. Here, we show that TD-SCVs exhibit impaired glutamine uptake and depend on c-di-AMP-regulated de novo glutamine synthesis for survival in the murine lung. We found that transcription of the glutamine synthetase gene glnA is cooperatively repressed by the transcriptional regulator GlnR, the c-di-AMP-binding protein PstA, and GlnA itself. Glutamine starvation elevates c-di-AMP levels, relieving repression by this ternary complex and promoting glutamine synthesis. Reducing c-di-AMP levels causes a profound growth defect in TD-SCVs under low-thymidine conditions, which is rescued by glnA overexpression. Moreover, pharmacological inhibition of GlnA markedly impairs TD-SCV growth in murine lung. These findings elucidate the molecular mechanism underlying S. aureus TD-SCV survival during infection and identify glutamine synthesis as a promising therapeutic target for treating infections caused by antifolate-resistant bacteria.

microbiology↗

bioRxiv2024

Analysis of genetic requirements and nutrient availability for Staphylococcus aureus growth in cystic fibrosis sputum

Staphylococcus aureus is one of the most common pathogens isolated from the lungs of people with cystic fibrosis (CF), but little is known about its ability to colonize this niche. We performed a Tn-seq screen to identify genes necessary for S. aureus growth in media prepared from ex vivo CF sputum. We identified 19 genes that were required for growth in all sputum media tested and dozens more that were required for growth in at least one sputum medium. Depleted mutants of interest included insertions in many genes important for surviving metal starvation as well as the primary regulator of cysteine metabolism cymR. To investigate the mechanisms by which these genes contribute to S. aureus growth in sputum, we quantified low-molecular-weight thiols, nutrient transition metals, and the host metal-sequestration protein calprotectin in sputum from 11 individuals with CF. In all samples, the abundance of calprotectin exceeded nutrient metal concentration, explaining the S. aureus requirement for metal-starvation genes. Further, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements of S. aureus. Deletion of the cysteine importer genes tcyA and tcyP in the {Delta}cymR background restored growth to wild-type levels in CF sputum, suggesting that the mechanism by which cymR is required for growth in sputum is to prevent uncontrolled import of cysteine or cystine from this environment. Overall, this work demonstrates that calprotectin and cysteine limit S. aureus growth in CF sputum. IMPORTANCEStaphylococcus aureus is a major cause of lung infections in people with cystic fibrosis (CF). This work identifies genes required for S. aureus growth in this niche, which represent potential targets for anti-Staphylococcal treatments. We show that genes involved in surviving metal starvation are required for growth in CF sputum. We also found that the primary regulator of cysteine metabolism, CymR, plays a critical role in preventing cysteine intoxication during growth in CF sputum. To support these models, we analyzed sputum from 11 individuals with CF to determine concentrations of calprotectin, nutrient metals, and low-molecular-weight thiols, which have not previously been quantified together in the same samples.

microbiology↗