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Bacterial itaconate degradation promotes pathogenicity

Abstract

Itaconate (methylenesuccinate) was recently identified as a mammalian metabolite whose production is substantially induced during macrophage activation. This compound is a potent inhibitor of isocitrate lyase, a key enzyme of the glyoxylate cycle, which is a pathway required for the survival of many pathogens inside the eukaryotic host. Here we show that numerous bacteria, notably many pathogens such as Yersinia pestis and Pseudomonas aeruginosa, have three genes for itaconate degradation. They encode itaconate coenzyme A (CoA) transferase, itaconyl-CoA hydratase and (S)-citramalyl-CoA lyase, formerly referred to as CitE-like protein. These genes are known to be crucial for survival of some pathogens in macrophages. The corresponding enzymes convert itaconate into the cellular building blocks pyruvate and acetyl-CoA, thus enabling the bacteria to metabolize itaconate and survive in macrophages. The itaconate degradation and detoxification pathways of Yersinia and Pseudomonas are the result of convergent evolution. This work revealed a common persistence factor operating in many pathogenic bacteria.

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Figure 1: Itaconate degradation pathway and the corresponding gene clusters in Y. pestis and P. aeruginosa.
Figure 2: In vitro reconstitution of itaconate degradation pathway with the heterologously produced and purified enzymes from Y. pestis.
Figure 3: In vitro reconstitution of itaconate degradation pathway with the heterologously produced and purified enzymes from P. aeruginosa.

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References

  1. Nathan, C.F. Secretory products of macrophages. J. Clin. Invest. 79, 319–326 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Strelko, C.L. et al. Itaconic acid is a mammalian metabolite induced during macrophage activation. J. Am. Chem. Soc. 133, 16386–16389 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sugimoto, M. et al. Non-targeted metabolite profiling in activated macrophage secretion. Metabolomics 8, 624–633 (2011).

    Article  CAS  Google Scholar 

  4. Michelucci, A. et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc. Natl. Acad. Sci. USA 110, 7820–7825 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Williams, J.O., Roche, T.E. & McFadden, B.A. Mechanism of action of isocitrate lyase from Pseudomonas indigofera. Biochemistry 10, 1384–1390 (1971).

    Article  CAS  PubMed  Google Scholar 

  6. Hillier, S. & Charnetzky, W.T. Glyoxylate bypass enzymes in Yersinia species and multiple forms of isocitrate lyase in Yersinia pestis. J. Bacteriol. 145, 452–458 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Höner Zu Bentrup, K., Miczak, A., Swenson, D.L. & Russell, D.G. Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis. J. Bacteriol. 181, 7161–7167 (1999).

    PubMed  PubMed Central  Google Scholar 

  8. Adler, J., Wang, S.F. & Lardy, H.A. The metabolism of itaconic acid by liver mitochondria. J. Biol. Chem. 229, 865–879 (1957).

    CAS  PubMed  Google Scholar 

  9. Wang, S.F., Adler, J. & Lardy, H.A. The pathway of itaconate metabolism by liver mitochondria. J. Biol. Chem. 236, 26–30 (1961).

    CAS  PubMed  Google Scholar 

  10. Cooper, R.A., Itiaba, K. & Kornberg, H.L. The utilization of aconite and itaconate by Micrococcus sp. Biochem. J. 94, 25–31 (1965).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Martin, W.R., Frigan, F. & Bergman, E.H. Noninductive metabolism of itaconic acid by Pseudomonas and Salmonella species. J. Bacteriol. 82, 905–908 (1961).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Cooper, R.A. & Kornberg, H.L. The utilization of itaconate by Pseudomonas sp. Biochem. J. 91, 82–91 (1964).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Deng, W. et al. Genome sequence of Yersinia pestis KIM. J. Bacteriol. 184, 4601–4611 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pujol, C., Grabenstein, J.P., Perry, R.D. & Bliska, J.B. Replication of Yersinia pestis in interferon γ–activated macrophages requires ripA, a gene encoded in the pigmentation locus. Proc. Natl. Acad. Sci. USA 102, 12909–12914 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chaturvedi, K.S., Hung, C.S., Crowley, J.R., Stapleton, A.E. & Henderson, J.P. The siderophore yersiniabactin binds copper to protect pathogens during infection. Nat. Chem. Biol. 8, 731–736 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Eriksson, S., Lucchini, S., Thompson, A., Rhen, M. & Hinton, J.C. Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol. Microbiol. 47, 103–118 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Shi, L. et al. Proteomic analysis of Salmonella enterica serovar Typhimurium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar Typhimurium inside macrophages. J. Biol. Chem. 281, 29131–29140 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Zhao, Y. et al. Identification of genes affecting Salmonella enterica serovar enteritidis infection of chicken macrophages. Infect. Immun. 70, 5319–5321 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Santiviago, C.A. et al. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice. PLoS Pathog. 5, e1000477 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Periaswamy, B. et al. Live attenuated S. Typhimurium vaccine with improved safety in immuno-compromised mice. PLoS ONE 7, e45433 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Torres, R. et al. Biochemical, structural and molecular dynamics analyses of the potential virulence factor RipA from Yersinia pestis. PLoS ONE 6, e25084 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Torres, R. et al. Structural insights into RipC, a putative citrate lyase β subunit from a Yersinia pestis virulence operon. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 2–7 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Goulding, C.W. et al. The structure and computational analysis of Mycobacterium tuberculosis protein CitE suggest a novel enzymatic function. J. Mol. Biol. 365, 275–283 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Hisano, T. et al. Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis. J. Biol. Chem. 278, 617–624 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Erb, T.J. et al. Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. Proc. Natl. Acad. Sci. USA 104, 10631–10636 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zarzycki, J., Brecht, V., Müller, M. & Fuchs, G. Identifying the missing steps of the autotrophic 3-hydroxypropionate CO2 fixation cycle in Chloroflexus aurantiacus. Proc. Natl. Acad. Sci. USA 106, 21317–21322 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Khomyakova, M., Bükmez, Ö., Thomas, L.K., Erb, T.J. & Berg, I.A. A methylaspartate cycle in haloarchaea. Science 331, 334–337 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Dimroth, P., Buckel, W., Loyal, R. & Eggerer, H. Isolation and function of the subunits of citramalate lyase and formation of hybrids with the subunits of citrate lyase. Eur. J. Biochem. 80, 469–477 (1977).

    Article  CAS  PubMed  Google Scholar 

  29. Stover, C.K. et al. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959–964 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Jacobs, M.A. et al. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 100, 14339–14344 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chávez-Avilés, M., Díaz-Pérez, A.L., Reyes-de la Cruz, H. & Campos-García, J. The Pseudomonas aeruginosa liuE gene encodes the 3-hydroxy-3-methylglutaryl coenzyme A lyase, involved in leucine and acyclic terpene catabolism. FEMS Microbiol. Lett. 296, 117–123 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Schürmann, M., Wübbeler, J.H., Grote, J. & Steinbüchel, A. Novel reaction of succinyl coenzyme A (succinyl-CoA) synthetase: activation of 3-sulfinopropionate to 3-sulfinopropionyl-CoA in Advenella mimigardefordensis strain DPN7T during degradation of 3,3′-dithiodipropionic acid. J. Bacteriol. 193, 3078–3089 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. McKinney, J.D. et al. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735–738 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Lorenz, M.C. & Fink, G.R. Life and death in a macrophage: role of the glyoxylate cycle in virulence. Eukaryot. Cell 1, 657–662 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. He, Y. Analyses of Brucella pathogenesis, host immunity, and vaccine targets using systems biology and bioinformatics. Front. Cell. Infect. Microbiol. 2, 2 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Lamberti, Y.A., Hayes, J.A., Perez Vidakovics, M.L., Harvill, E.T. & Rodriguez, M.E. Intracellular trafficking of Bordetella pertussis in human macrophages. Infect. Immun. 78, 907–913 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Valvano, M.A., Keith, K.E. & Cardona, S.T. Survival and persistence of opportunistic Burkholderia species in host cells. Curr. Opin. Microbiol. 8, 99–105 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Dunn, M.F., Ramírez-Trujillo, J.A. & Hernández-Lucas, I. Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology 155, 3166–3175 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Fang, F.C., Libby, S.J., Castor, M.E. & Fung, A.M. Isocitrate lyase (AceA) is required for Salmonella persistence but not for acute lethal infection in mice. Infect. Immun. 73, 2547–2549 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. van Schaik, E.J., Tom, M. & Woods, D.E. Burkholderia pseudomallei isocitrate lyase is a persistence factor in pulmonary melioidosis: implications for the development of isocitrate lyase inhibitors as novel antimicrobials. Infect. Immun. 77, 4275–4283 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lindsey, T.L., Hagins, J.M., Sokol, P.A. & Silo-Suh, L.A. Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase. Microbiology 154, 1616–1627 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Shin, J.-H. et al. 1H NMR–based metabolomic profiling in mice infected with Mycobacterium tuberculosis. J. Proteome Res. 10, 2238–2247 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Mittal, R., Aggarwal, S., Sharma, S., Chhibber, S. & Harjai, K. Contribution of macrophage secretory products to urovirulence of Pseudomonas aeruginosa. FEMS Immunol. Med. Microbiol. 57, 156–164 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Fuchs, T.M., Eisenreich, W., Heesemann, J. & Goebel, W. Metabolic adaptation of human pathogenic and related nonpathogenic bacteria to extra- and intracellular habitats. FEMS Microbiol. Rev. 36, 435–462 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Okabe, M., Lies, D., Kanamasa, S. & Park, E.Y. Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terreus. Appl. Microbiol. Biotechnol. 84, 597–606 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Vosloo, A., Van Aardt, W.J. & Mienie, L.J. Presence of itaconic acid in the hemolymph and tissues of the freshwater crab, Potamonautes warreni Calman. Comp. Biochem. Physiol. 113, 823–825 (1996).

    Article  Google Scholar 

  47. Costa-Ramos, C. & Rowley, A.F. Effect of extracellular products of Pseudoalteromonas atlantica on the edible crab Cancer pagurus. Appl. Environ. Microbiol. 70, 729–735 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Sakai, A., Kusumoto, A., Kiso, Y. & Furuya, E. Itaconate reduces visceral fat by inhibiting fructose 2,6-bisphosphate synthesis in rat liver. Nutrition 20, 997–1002 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Furuya, E. & Uyeda, K. A novel enzyme catalyzes the synthesis of activation factor from ATP and d-fructose-6-P. J. Biol. Chem. 256, 7109–7112 (1981).

    CAS  PubMed  Google Scholar 

  50. Morikawa, J., Nishimura, Y., Uchida, A. & Tanaka, T. Molecular cloning of novel mouse and human putative citrate lyase β-subunit. Biochem. Biophys. Res. Commun. 289, 1282–1286 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Simon, E.J. & Shemin, D. The preparation of S-succinyl coenzyme-A. J. Am. Chem. Soc. 75, 2520 (1953).

    Article  CAS  Google Scholar 

  52. Stadtman, E.R. Preparation and assay of acyl coenzyme A and other thiol esters; use of hydroxylamine. Methods Enzymol. 3, 931–941 (1957).

    Article  Google Scholar 

  53. Buckel, W., Ziegert, K. & Eggerer, H. Acetyl-CoA–dependent cleavage of citrate on inactivated citrate lyase. Eur. J. Biochem. 37, 295–304 (1973).

    Article  CAS  PubMed  Google Scholar 

  54. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1989).

  55. Studier, F.W. & Moffatt, B.A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130 (1986).

    Article  CAS  PubMed  Google Scholar 

  56. Ausubel, F.M. et al. Current Protocols in Molecular Biology (Wiley, New York, 1987).

  57. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  58. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  PubMed  Google Scholar 

  59. Zehr, B.D., Savin, T.J. & Hall, R.E. A one-step, low background Coomassie staining procedure for polyacrylamide gels. Anal. Biochem. 182, 157–159 (1989).

    Article  CAS  PubMed  Google Scholar 

  60. Bradford, M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was funded by the Deutsche Forschungsgemeinschaft (BE 4822/2-1 and Heisenberg fellowship to I.A.B.). We thank B. Alber, R. Teufel, J. Zarzycki and M. Carter for critical reading the manuscript; W. Buckel for the discussions of the Ich mechanism and G. Fuchs for constant support and discussions during the work and for critically reading the manuscript.

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J.S. performed all of the experiments with Y. pestis proteins. M.Z. performed the experiments with PaCcl and P. aeruginosa cells and cell extracts. P.K.Z. performed the experiments with H. sapiens Ccl. A.F. performed the experiments with PaIct and PaIch and an in vitro reconstruction of P. aeruginosa itaconate conversion. I.A.B. designed research, analyzed data and wrote the paper.

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Correspondence to Ivan A Berg.

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Sasikaran, J., Ziemski, M., Zadora, P. et al. Bacterial itaconate degradation promotes pathogenicity. Nat Chem Biol 10, 371–377 (2014). https://doi.org/10.1038/nchembio.1482

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