Varesio Lydia M, Fiebig Aretha, Crosson Sean
Committee on Microbiology, The University of Chicago, Chicago, Illinois, USA.
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
Infect Immun. 2021 May 17;89(6). doi: 10.1128/IAI.00808-20.
is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. The same conditions are often encountered in axenic cultures during stationary phase. Studies of stationary phase may thus inform our understanding of infection biology, yet the genes and pathways that are important in stationary-phase physiology remain poorly defined. We measured fitness of a barcoded pool of Tn- mutants as a function of growth phase and identified as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine, and we provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis at steps downstream of CysE. Deleting (Δ) or both and (Δ Δ) results in premature entry into stationary phase, reduced culture yield, and sensitivity to exogenous hydrogen peroxide. These phenotypes can be chemically complemented by cysteine or glutathione. Δ and Δ Δ strains have no defect in host cell entry but have significantly diminished intracellular fitness between 2 and 24 h postinfection. Our study has uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in and demonstrates that cysteine anabolism is a determinant of peroxide stress survival and fitness in the intracellular niche.
是一种对雄性生殖道具有嗜性的绵羊细胞内病原体。为了建立和维持感染，必须在宿主细胞内的应激条件下存活，包括低pH值、营养限制和活性氧。在静止期的无细胞培养中经常会遇到相同的条件。因此，对静止期的研究可能有助于我们理解感染生物学，但在静止期生理学中重要的基因和途径仍不清楚。我们测量了Tn-突变体条形码文库的适应性作为生长阶段的函数，并确定为静止期适应性的一个决定因素。CysE催化从丝氨酸合成半胱氨酸的第一步，我们提供了遗传证据，表明两种相关酶CysK1和CysK2在CysE下游的步骤中发挥冗余作用以催化半胱氨酸合成。缺失（Δ）或同时缺失和（ΔΔ）会导致过早进入静止期、培养产量降低以及对外源过氧化氢敏感。这些表型可以通过半胱氨酸或谷胱甘肽进行化学互补。Δ和ΔΔ菌株在宿主细胞进入方面没有缺陷，但在感染后2至24小时内细胞内适应性显著降低。我们的研究揭示了在半胱氨酸生物合成的CysK步骤中意外的冗余，并证明半胱氨酸合成代谢是过氧化物应激存活和细胞内生态位适应性的一个决定因素。
