Brucella ovis Cysteine Biosynthesis Contributes to Peroxide Stress Survival and Fitness in the Intracellular Niche

Abstract

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.

Mining the Flavoproteome of Brucella ovis, the Brucellosis Causing Agent in Ovis aries

“…The sequence identity of the B. ovis FAD dependent glutathione-disulphide reductase (EC 1.8.1.7) with the S. meliloti homologue indicates that it must contribute to maintain high levels of reduced glutathione to control redox homeostasis. This agrees with a recent report where disruptions in the gene encoding for it in B. ovis produce a significant disadvantage in bacterial growth ( 54 , 55 ). The three predicted dihydrolipoyl dehydrogenases (ldpA-1, ldpA-2, ldpA-3, EC 1.8.1.4) are highly conserved in Brucella and alphaproteobacteria, with the exception of ldpA-1 poorly represented in alphaproteobacteria.…”

The Retrospective on Atypical Brucella Species Leads to Novel Definitions

“…Likewise, H 2 S, which is produced by certain classical Brucella and most novel Brucella isolates (Figure 3), may support an important self-protective mechanism through scavenging harmful oxidants [65,66], in particular the reactive oxygen species (ROS) superoxide anion (O 2 − ) and H 2 O 2 , generated by phagocytic immune cells to combat Brucella. H 2 S generation is facilitated by the uptake of sulfate, its assimilatory reduction to sulfite, and subsequently to H 2 S, which can be released or alternatively used by Brucella for the conversion of serine into cysteine, the latter contributing to the peroxide stress defense and survival inside macrophages [67]. Furthermore, H 2 S might be released through a 3-mercaptopyruvate sulfurtransferase SseA during cysteine degradation [68], when Brucella persists in an environment with sufficient proteinaceous, cysteine-containing substrates.…”

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens

“…Furthermore, cysE deletion in the sheep pathogen Brucella ovis resulted in poor growth in rich media and an early entry into stationary phase [ 32 ]. Brucella ovis ΔcysE strains were more susceptible to oxidative stress, shown through increased sensitivity to hydrogen peroxide.…”

“…Brucella ovis ΔcysE strains were more susceptible to oxidative stress, shown through increased sensitivity to hydrogen peroxide. Cell invasion assays revealed that deletion of cysE did not affect cell infection but did significantly reduce replication within macrophages [ 32 ]. While the deletion of cysE is non-lethal, it imposes a fitness cost on B. ovis during intracellular growth, demonstrating the requirement for cysteine biosynthesis for survival within the host.…”