Brucella ovis cysteine biosynthesis contributes to peroxide stress survival and fitness in the intracellular niche

Brucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in axenic cultures during stationary phase. Studies of stationary phase may thus inform understanding of Brucella infection biology, yet the genes and pathways that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE 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 either cysE (ΔcysE) or both cysK1 and cysK2 (ΔcysK1 ΔcysK2) 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. ΔcysE and ΔcysK1 ΔcysK2 strains have no defect in host cell entry in vitro but have significantly diminished intracellular fitness between 2 and 24 hours post infection. Our study has uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis, and demonstrates that cysteine anabolism is a determinant of peroxide stress survival and fitness in the intracellular niche.

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts.

Ultrasound-assisted l-cysteine whole-cell bioconversion by recombinant Escherichia coli with tryptophan synthase

l-Cysteine is widely used in food, medicine, and cosmetics. In this study, a recombinant Escherichia coli whole-cell system with tryptophan synthase was used to complete the biological transformation of l-serine to l-cysteine, and bioconversion of l-cysteine was investigated by tryptophan synthase. The biotransformation of l-cysteine was optimized by response surface methodology. The optimal conditions obtained are 0.13 mol·L−1 l-serine, 75 min, 130 W ultrasound operation, where the V max of tryptophan synthase is 25.27 ± 0.16 (mmol·h−1·(g-cells)−1). The V max of tryptophan synthase for the biosynthesis without ultrasound is 12.91 ± 0.34 (mmol·h−1·(g-cells)−1). Kinetic analysis of the recombinant Escherichia coli whole-cell system with tryptophan synthase also showed that under the ultrasound treatment, the K m values of l-cysteine biosynthesis increase from 1.342 ± 0.11 mM for the shaking biotransformation to 2.555 ± 0.13 mM for ultrasound operation. The yield of l-cysteine reached 91% after 75 min of treatment after 130 W ultrasound, which is 1.9-fold higher than no ultrasound.

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

Brucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis 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 Brucella infection biology, yet the genes that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE 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 cysE or both cysK1 and cysK2 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 cysE results in sensitivity to exogenous hydrogen peroxide. Finally, we demonstrate that B. ovis ΔcysE 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 B. ovis, and demonstrated that cysteine anabolism is an important determinant of stationary phase entry in vitro and fitness in the intracellular niche.