Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the ‘nutrient sensitized’ Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged “bubble vacuoles” with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.

Toxoplasma infection causes serious complications in immunocompromised individuals and in the developing fetus. During infection, certain immune cells release a protein called interferon gamma that activates cells to destroy the parasite or inhibit its growth.

Coxiella burnetii is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of C. burnetii pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that nadB, encoding a putative L-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the WT, confirming the involvement of NadB in de novo NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of Escherichia coli NadB, and expression of WT and R275L GST-NadB fusion proteins in E. coli JM109, we found that purified recombinant WT GST-NadB has L-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii. This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections. Coxiella burnetii is a Gram-negative intracellular bacterium that causes the zoonotic disease Q fever (1), which primarily spreads to humans from livestock such as cattle and sheep. In humans, infection can present as an acute disease with systemic

SUMMARY Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram‐negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short‐chain dehydrogenase, is required for intracellular replication. Short‐chain dehydrogenases are NADP(H)‐dependent oxidoreductases, and SdrA contains a predicted NADP+ binding site, suggesting it may facilitate NADP(H) regeneration by C. burnetii, a key process for surviving oxidative stress. Purified recombinant 6×His‐SdrA was able to convert NADP+ to NADP(H) in vitro. Mutation to alanine of a conserved glycine residue at position 12 within the predicted NADP binding site abolished significant enzymatic activity. Complementation of the sdrA mutant (sdrA::Tn) with plasmid‐expressed SdrA restored intracellular replication to wild‐type levels, but expressing enzymatically inactive G12A_SdrA did not. The sdrA::Tn mutant was more susceptible in vitro to oxidative stress, and treating infected host cells with L‐ascorbate, an anti‐oxidant, partially rescued the intracellular growth defect of sdrA::Tn. Finally, stable isotope labelling studies demonstrated a shift in flux through metabolic pathways in sdrA::Tn consistent with the presence of increased oxidative stress, and host cells infected with sdrA::Tn had elevated levels of reactive oxygen species compared with C. burnetii NMII.

The zoonotic disease Q fever caused by the intracellular bacterium Coxiella burnetii remains a global health threat due to its high infectivity, environmental stability, the debilitating nature and the long duration of treatment. Designing new and potent drugs that target previously unexplored pathways is essential to shorten treatment time and minimise antibiotic resistance. Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous cofactor in all living organisms. NadB, an L-aspartate oxidase catalysing the first step of the prokaryotic-specific NAD de novo biosynthetic pathway, is required for C. burnetii growth and replication inside host cells. In this study, in vitro enzyme assays utilising recombinant glutathione S-transferase tagged NadB (GST-NadB) demonstrated inhibition of the L-aspartate oxidase activity of NadB by meso-tartrate. Furthermore, meso-tartrate inhibits intracellular growth and replication of C. burnetii inside host cells in a dose-dependent manner, and has no effect on the viability of mammalian cells. Unexpectedly, meso-tartrate also inhibited growth of C. burnetii in axenic medium, and further reduces replication of the nadB mutant inside host cells, suggesting it is acting more widely than simple inhibition of NadB. Overall, these results suggest that the antibacterial activity of meso-tartrate warrants further study, including investigation of its additional target(s).