Analysis of genetic requirements and nutrient availability forStaphylococcus aureusgrowth in cystic fibrosis sputum

Abstract

Staphylococcus aureusis 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 forS. aureusgrowth in media prepared fromex vivoCF 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 metabolismcymR. To investigate the mechanisms by which these genes contribute toS. aureusgrowth 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 theS. aureusrequirement for metal-starvation genes. Further, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements ofS. aureus. Deletion of the cysteine importer genestcyAandtcyPin the ΔcymRbackground restored growth to wild-type levels in CF sputum, suggesting that the mechanism by whichcymRis 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 limitS. aureusgrowth in CF sputum.