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Multiple Acyl-Coa Dehydrogenase Deficiency Kills Mycobacterium Tuberculosis in Vitro and 2 During Infection

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Multiple Acyl-Coa Dehydrogenase Deficiency Kills Mycobacterium Tuberculosis in Vitro and 2 During Infection bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440201; this version posted April 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Multiple acyl-CoA dehydrogenase deficiency kills Mycobacterium tuberculosis in vitro and 2 during infection 3 Tiago Beites1, Robert S Jansen2#, Ruojun Wang1, Adrian Jinich2, Kyu Rhee1,2,, Dirk Schnappinger1, Sabine 4 Ehrt1. 5 Affiliations 6 1 Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065 USA. 7 2 Division of Infectious Diseases, Department of Medicine, Weill Cornell Medical College, New York, NY, 8 10065, USA. 9 # Current affiliation: Department of Microbiology, Radboud University, 6525 AJ, Nijmegen, the 10 Netherlands 11 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440201; this version posted April 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 12 ABSTRACT 13 The human pathogen Mycobacterium tuberculosis (Mtb) devotes a significant fraction of its genome to 14 fatty acid metabolism. Although Mtb depends on host fatty acids as a carbon source, fatty acid - 15 oxidation is mediated by genetically redundant enzymes, which has hampered the development of 16 antitubercular drugs targeting this metabolic pathway. Here, we identify rv0338c, referred to as etfDMtb, 17 to encode a membrane dehydrogenase essential for fatty acid -oxidation in Mtb. An etfD deletion 18 mutant (etfD) was incapable of growing on fatty acids in vitro, with long-chain fatty acids being 19 bactericidal, and failed to grow and survive in mice. The etfD metabolome revealed a block in - 20 oxidation at the step catalyzed by acyl-CoA dehydrogenases (ACADs). In many organisms, including 21 humans, ACADs are functionally dependent on an electron transfer flavoprotein (ETF) and cognate 22 dehydrogenase. Immunoprecipitation identified EtfD in complex with FixA (EtfBMtb). FixA (EtfBMtb) and 23 FixB (EtfAMtb) are homologous to the human ETF subunits. Our results demonstrate that EtfBAMtb 24 constitutes Mtb’s ETF, while EtfDMtb, although not homologous to human EtfD, functions as the 25 dehydrogenase. These findings identify Mtb’s fatty acid-oxidation as a novel potential target for TB 26 drug development. 27 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440201; this version posted April 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 28 MAIN 29 Maintenance of an energized membrane is essential for Mycobacterium tuberculosis (Mtb) to grow and 30 survive periods of non-replicating persistence1. This need has driven tuberculosis (TB) drug development 31 efforts towards Mtb’s energy metabolism. These efforts are supported by the first anti-TB drug approved 32 in over 40 years - the ATP synthase inhibitor bedaquiline2. 33 Mtb’s energy related pathways exhibit varying degrees of vulnerability to inhibition. Uptake of 34 its main carbon sources in vivo is performed by specialized transporters; the multi-subunit Mce1 complex 35 transports fatty acids3 and the Mce4 complex facilitates the uptake of cholesterol4. Inactivation of Mce1 36 can reduce intracellular growth5, while Mce4 was conclusively shown to be essential for survival during 37 the chronic phase of infection4. LucA, which acts as a regulator of both Mce1 and Mce4, is also required 38 for wild type levels of Mtb virulence3, further supporting that inhibiting the ability to import host lipids 39 affects Mtb’s pathogenicity. 40 Cholesterol degradation yields multiple products, including acetyl-CoA, propionyl-CoA, succinyl- 41 CoA and pyruvate that can be used for energy generation or lipid biosynthesis6. Deletion of genes 42 encoding cholesterol oxidation enzymes attenuated growth7 and caused survival defects of Mtb in 43 mice8,9. A screen against Mtb residing in the phagosomes of macrophages identified several compounds 44 that target cholesterol metabolism10. In contrast, fatty acids are degraded solely through -oxidation. 45 Mtb’s genome encodes multiple enzymes for each step of -oxidation, including thirty-four putative acyl- 46 CoA ligases, thirty-five putative acyl-CoA dehydrogenases, twenty-two putative enoyl-CoA dehydratase, 47 five putative -hydroxyacyl-CoA dehydrogenase and six putative thiolases11. Some of these enzymes are 48 necessary for infection, but they also have been shown to play roles in other pathways, such as complex 49 lipid biosynthesis12 and cholesterol degradation13. Due to the apparent redundancy of Mtb’s fatty acid - 50 oxidation machinery, this metabolic pathway has thus been presumed to be invulnerable to chemical 51 inhibition. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440201; this version posted April 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 52 In the present study, we define an enzyme complex that has previously not been recognized as 53 required for fatty acid degradation in Mtb. It consists of an electron transfer flavoprotein composed by 54 two subunits - FixA (Rv3029c) and FixB (Rv3028c) – and a membrane dehydrogenase (Rv0338c), which 55 we propose to re-name as EtfBMtb, EtfAMtb and EtfDMtb based on their human counterparts. Deletion of 56 Mtb’s EtfD causes multiple acyl-CoA dehydrogenase deficiency, which prevents utilization of fatty acids 57 as carbon sources and can kill Mtb in vitro and during mouse infection. 58 RESULTS 59 A possible role for EtfDMtb in Mtb’s energy metabolism 60 EtfDMtb (Rv0338) is a membrane protein of unknown function predicted to be essential for growth of Mtb 61 on agar plates14,15. To experimentally determine its topology, we fused E. coli alkaline phosphatase PhoA 62 to EtfD at specific residues of the predicted transmembrane helices. Transport of PhoA outside of the 63 cytoplasm enables reactivity with the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate p- 64 toluidine (BCIP). Based on this assay, the soluble portion of EtfDMtb faces the cytoplasm (Supplementary 65 Fig. 1a), and this topology agrees with the prediction generated by the MEMSAT3 algorithm16 66 (Supplementary Fig. 1b). 67 Next, we performed in silico analysis, which placed EtfDMtb into the cluster of orthologue groups 68 0247 (COG0247) composed of Fe-S oxidoreductases involved in energy production and conversion (Fig. 69 1a). COG0247 includes a variety of enzymes, including lactate dehydrogenase and the methanogenic- 70 related heterodisulfide reductase; however, most proteins in COG0247 remain uncharacterized and are 71 of unknown function. EtfDMtb is predicted to be a chimeric enzyme assembled from four domains: an N- 72 terminal domain, which is similar to the gamma subunit of nitrate reductases and putatively binds a 73 cytochrome b; a central 4Fe-4S di-cluster domain similar to succinate dehydrogenases; and two C- bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440201; this version posted April 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 74 terminal cysteine-rich domains (CCG), which are often found in heterodisulfide reductases (Fig. 1b). This 75 analysis led us to hypothesize that EtfD is a component of Mtb’s energy metabolism. 76 EtfDMtb is linked to fatty acid metabolism and it is essential in vivo 77 To analyze the impact of EtfDMtb depletion on Mtb’s growth in vitro, we generated a TetOFF strain, in 78 which EtfD levels are controlled by anhydrotetracycline (ATC)-inducible proteolysis17. Depletion of 79 EtfDMtb inhibited Mtb’s growth in regular medium supplemented with oleic acid, albumin, dextrose and 80 catalase (OADC), which is consistent with its predicted essentiality (Fig. 1c). Curiously, when the same 81 medium was supplemented with a fatty acid free enrichment (albumin, dextrose and sodium chloride, 82 ADN), depletion of EtfDMtb did not impact growth (Fig. 1c). This is similar to the fatty acid sensitive 83 phenotype observed in response to inactivation of Mtb’s type II NADH dehydrogenases18 which allowed 84 the genetic deletion of Ndh-2 by growth in a fatty acid free medium. We applied the same strategy to 85 etfDMtb and generated a deletion strain (etfD) (Supplementary Fig. 2a). The etfDMtb strain was 86 confirmed by whole genome sequencing (WGS) to not contain additional polymorphisms known to 87 affect growth (Supplementary Table 1). This knockout strain also phenocopied the fatty acid sensitivity 88 observed with the knockdown mutant (Supplementary Fig. 2b). 89 We next sought to investigate the importance of etfDMtb for pathogenesis in an aerosol model of 90 TB infection in mice. After aerosol infection, etfD was unable to grow in mouse lungs and declined in 91 viability from day 14 onwards (Fig. 2a, Supplementary Fig. 3a). In agreement with the CFU data, gross 92 lung pathology showed no lesions in the mice infected with etfD (Fig. 2b, Supplementary Fig. 3b). The 93 attenuation was even more pronounced in spleens, where no etfD CFU were recovered at any time point 94 (Fig. 2a, Supplementary Fig. 3a). All phenotypes were rescued by reintroducing an intact copy of etfDMtb. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.19.440201; this version posted April 19, 2021.
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