Cysteine biosynthesis is a determinant of Brucella ovis stress survival and fitness in the intracellular niche release_4qgyfj7vsbdunlekmka2ukgwri

Abstract

<jats:italic>Brucella ovis</jats:italic> is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, <jats:italic>B. ovis</jats:italic> must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in stationary phase cultures. Studies of stationary phase may thus inform understanding of <jats:italic>Brucella</jats:italic> infection biology, yet the genes that are important in <jats:italic>Brucella</jats:italic> stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of <jats:italic>B. ovis</jats:italic> Tn-<jats:italic>himar</jats:italic> mutants as a function of growth phase and identified <jats:italic>cysE</jats:italic> as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine. We provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis downstream of CysE. Deleting either <jats:italic>cysE</jats:italic> or both <jats:italic>cysK1</jats:italic> and <jats:italic>cysK2</jats:italic> leads to premature entry into stationary phase and reduced culture yield. These phenotypes are rescued by addition of cysteine or glutathione to the medium. We further show that deletion of <jats:italic>cysE</jats:italic> results in sensitivity to exogenous hydrogen peroxide. Finally, we demonstrate that <jats:italic>B. ovis</jats:italic> Δ<jats:italic>cysE</jats:italic> has no defect in host cell entry but is attenuated in macrophage-like cells and in ovine testis epithelial cells at one- and two-days post infection. Our study uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in <jats:italic>B. ovis</jats:italic>, and demonstrated that cysteine anabolism is an important determinant of stationary phase entry in vitro and fitness in the intracellular niche.
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