Analysis of genetic requirements and nutrient availability for <i>Staphylococcus aureus</i> growth in cystic fibrosis sputum

<i>Staphylococcus aureus</i> 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 transposon-sequencing (Tn-seq) screen to identify genes necessary for <i>S. aureus</i> growth in media prepared from <i>ex vivo</i> 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, <i>cymR</i>. To investigate the mechanisms by which these genes contribute to <i>S. aureus</i> 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 <i>S. aureus</i> requirement for metal-starvation genes. Furthermore, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements of <i>S. aureus</i>. Deletion of the cysteine importer genes <i>tcyA</i> and <i>tcyP</i> in the ∆<i>cymR</i> background restored growth to wild-type levels in CF sputum, suggesting that the mechanism by which <i>cymR</i> 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 <i>S. aureus</i> growth in CF sputum.IMPORTANCE<i>Staphylococcus aureus</i> is a major cause of lung infections in people with cystic fibrosis (CF). This work identifies genes required for <i>S. aureus</i> 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.