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Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp are strict aerobe and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli allowing the identification of fifteen Ubi-proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified UQ 8 as the major quinone found in the membranes of this bacterium. Then, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics and biochemical approaches. Our analysis disclosed the presence in Francisella of ten putative Ubi-proteins and we confirmed eight of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share a similar pattern. Ubiquinone and Francisella pathogenicity 2 However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp and homologs of the Ubi-proteins involved in the O 2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Then, via two approaches, i.e. the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, which both strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in a respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. Importance Francisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We showed that UQ 8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor, which strongly decreased the biosynthesis of UQ. Our demonstration of the crucial role of UQ for the respiratory metabolism of F. novicida and for the involving in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.

Summary: Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase, TgATS2, shows that the apicoplast provides (lyso)phosphatidic acid, required for the recruitment of a critical dynamin (TgDrpC) during parasite cytokinesis. Disruption of TgATS2 also leads parasites to shift metabolic lipid acquisition from de novo synthesis toward host scavenging. We show that both lipid scavenging and de novo synthesis pathways in wild-type parasites exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (1) upregulation of de novo fatty acid synthesis capacities in the apicoplast and (2) parasite-driven host remodeling to generate multi-membrane-bound structures from host organelles that are imported toward the parasite. : Apicoplast de novo lipid synthesis and lipid host scavenging are both critical for apicomplexan intracellular development. Amiar et al. show that the parasite adapts to the fluctuations of host nutritional content to regulate the metabolic activity of both apicoplast and scavenging pathways and maintain parasite development and division. Keywords: Apicomplexa, toxoplasmosis, malaria, apicoplast, lipid synthesis, phosphatidic acid, host-parasite interaction, lipidomics, host nutritional environment, cytokinesis

Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases including malaria and toxoplasmosis. Apicomplexan parasites must obtain and combine lipids both from host cell scavenging andde novosynthesis to maintain parasite propagation and survival within their human host. Major questions on the actual role for each lipid source or how these are regulated upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase TgATS2, shows that the apicoplast provides local (lyso)phosphatidic acid balance, which is required for the recruitment of a novel dynamin (TgDrpC) critical during parasite cytokinesis. Disruption of TgATS2 led parasites to shift metabolic lipid acquisition fromde novosynthesis towards host scavenging. Importantly, both lipid scavenging andde novosynthesis pathways exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (i) up-regulation ofde novofatty acid synthesis capacities in the apicoplast, and (ii) parasite-driven host cell remodelling to generate multi-membrane-bound structures from host organelles that are imported towards the parasite.