The Biosynthetic Pathway of Ubiquinone Contributes to Pathogenicity of Francisella novicida

Abstract

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. 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.

Recent advances in the metabolic pathways and microbial production of coenzyme Q

“…UbiX produces the prenylated FMN used as a cofactor by UbiD (Marshall et al 2017 , 2019 ). Although widely conserved in many bacterial species, the UbiD-UbiX system is absent in some, suggesting that alternative systems exist, as recently proposed for Xanthomonas campestris and Francisella tularensis (Zhou et al 2019 ; Kazemzadeh et al 2021 ). In eukaryotes, the C1-decarboxylation step remains genetically and biochemically uncharacterized (Fernández-del-Río and Clarke 2021 ).…”

Galleria mellonella–intracellular bacteria pathogen infection models: the ins and outs

“…However, dosages, methods, and strains differed, complicating any meaningful comparison between the two studies. G. mellonella has also been used to evaluate F. novicida mutants, demonstrating the importance for virulence of ubiquinone biosynthesis (Kazemzadeh et al 2021 ) and the type VI secretion system and three of its effectors (Brodmann et al 2021 ). Similarly, an F. novicida qseC mutant was attenuated in G. mellonella , showing that the cognate protein QseC (a two-component system sensor kinase) is a virulence factor (Dean and van Hoek 2015 ).…”

Diversification of Ubiquinone Biosynthesis via Gene Duplications, Transfers, Losses, and Parallel Evolution

“…Given its broad conservation across Pseudomonadota and its likely vertical transmission, we hypothesized that Coq7 might harbor a conserved C6-hydroxylation activity. In accord with this hypothesis, six bacterial Coq7 sequences were previously confirmed to possess a C6-hydroxylation activity ( Kwon et al 2000 ; Stenmark et al 2001 ; Pelosi et al 2016 ; Jiang et al 2019 ; Zhou et al 2019 ; Kazemzadeh et al 2021 ) ( Table S3 ). Their capacity to hydroxylate C1 or C5 in addition to C6 was nevertheless not systematically tested.…”