Characterization of Metabolically Quiescent Parasites in Murine Lesions Using Heavy Water Labeling

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Microbial pathogens can adapt to changing conditions in their hosts by switching between different growth and physiological states. However, current methods for measuring microbial physiology in vivo are limited, hampering detailed dissection of host-pathogen interactions. Here we have used heavy water labeling to measure the growth rate and physiological state of Leishmania parasites in murine lesions. Based on the rate of in situ labeling of parasite DNA, RNA, protein, and lipids, we show that the growth rate of intracellular parasite stages is very slow, and that these stages enter a semi-quiescent state characterized by very low rates of RNA, protein, and membrane turnover. These changes in parasite growth and physiology are more pronounced than in in vitro differentiated parasites, suggesting that they are induced in part by the lesion environment. Despite their slow growth, the parasite burden in these lesions progressively increases as a result of low rates of parasite death and host cell turnover. We propose that these changes in Leishmania growth and physiology contribute to the development of a relatively benign tissue environment that is permissive for long term parasite expansion. This approach is suitable for studying the dynamics of other host-pathogen systems.

Vyšlo v časopise: Characterization of Metabolically Quiescent Parasites in Murine Lesions Using Heavy Water Labeling. PLoS Pathog 11(2): e32767. doi:10.1371/journal.ppat.1004683
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004683

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Zdroje

1. Tischler AD, McKinney JD (2010) Contrasting persistence strategies in Salmonella and Mycobacterium. Curr Opin Microbiol 13: 93–99. doi: 10.1016/j.mib.2009.12.007 20056478

2. Gengenbacher M, Kaufmann SHE (2012) Mycobacterium tuberculosis: success through dormancy. FEMS Microbiol Rev 36: 514–532. doi: 10.1111/j.1574-6976.2012.00331.x 22320122

3. Dembélé L, Franetich J-F, Lorthiois A, Gego A, Zeeman A-M, et al. (2014) Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nat Med 20: 307–312. doi: 10.1038/nm.3461 24509527

4. Rittershaus ESC, Baek S-H, Sassetti CM (2013) The normalcy of dormancy: common themes in microbial quiescence. Cell Host Microbe 13: 643–51. doi: 10.1016/j.chom.2013.05.012 23768489

5. Srikanta D, Santiago-Tirado FH, Doering TL (2014) Cryptococcus neoformans: historical curiosity to modern pathogen. Yeast 31: 47–60. doi: 10.1002/yea.2997 24375706

6. Gill WP, Harik NS, Whiddon MR, Liao RP, Mittler JE, Sherman DR (2009) A replication clock for Mycobacterium tuberculosis. Nat Med 15: 211–214. doi: 10.1038/nm.1915 19182798

7. Murray HW, Berman JD, Davies CR, Saravia NG (2005) Advances in leishmaniasis. Lancet 366: 1561–1577. 16257344

8. McConville MJ, Naderer T (2011) Metabolic pathways required for the intracellular survival of Leishmania. Annu Rev Microbiol 65: 543–561. doi: 10.1146/annurev-micro-090110-102913 21721937

9. Kaye P, Scott P (2011) Leishmaniasis: complexity at the host-pathogen interface. Nat Rev Microbiol 9: 604–615. doi: 10.1038/nrmicro2608 21747391

10. Beattie L, Peltan A, Maroof A, Kirby A, Brown N, et al. (2010) Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells. PLoS Pathog 6: e1000805. doi: 10.1371/journal.ppat.1000805 20300603

11. Moore JWJ, Moyo D, Beattie L, Andrews PS, Timmis J, Kaye PM (2013) Functional complexity of the Leishmania granuloma and the potential of in silico modeling. Front Immunol 4: 35. doi: 10.3389/fimmu.2013.00035 23423646

12. Guirado E, Schlesinger LS. (2013) Modeling the Mycobacterium tuberculosis granuloma—the critical battlefield in host immunity and disease. Front Immunol 4: 98. doi: 10.3389/fimmu.2013.00098 23626591

13. Sacks D, Noben-Trauth N (2002) The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2: 845–858. 12415308

14. McMahon-Pratt D, Alexander J (2004) Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol Rev 201: 206–24. 15361243

15. Lang T, Goyard S, Lebastard M, Milon G (2005) Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice. Cell Microbiol 7: 383–392. 15679841

16. Romero I, Téllez J, Suárez Y, Cardona M, Figueroa R, et al. (2010) Viability and burden of Leishmania in extralesional sites during human dermal leishmaniasis. PLoS Negl Trop Dis 4: e819. doi: 10.1371/journal.pntd.0000819 20856851

17. Michel G, Ferrua B, Lang T, Maddugoda MP, Munro P, et al. (2011) Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro. PLoS Negl Trop Dis 5: e1323. doi: 10.1371/journal.pntd.0001323 21931877

18. Müller AJ, Aeschlimann S, Olekhnovitch R, Dacher M, Späth GF, Bousso P (2013) Photoconvertible pathogen labeling reveals nitric oxide control of Leishmania major infection in vivo via dampening of parasite metabolism. Cell Host Microbe. 14: 460–467. doi: 10.1016/j.chom.2013.09.008 24139402

19. Kramer S (2012) Developmental regulation of gene expression in the absence of transcriptional control: The case of kinetoplastids. Mol Biochem Parasitol 181: 61–72. doi: 10.1016/j.molbiopara.2011.10.002 22019385

20. Lynn MA, Marr AK, McMaster WR (2013) Differential quantitative proteomic profiling of Leishmania infantum and Leishmania mexicana density gradient separated membranous fractions. J Proteomics 82: 179–192. doi: 10.1016/j.jprot.2013.02.010 23466312

21. Previs SF, Fatica R, Chandramouli V, Alexander JC, Brunengraber H, Landau BR (2004) Quantifying rates of protein synthesis in humans by use of 2H2O: application to patients with end-stage renal disease. Am J Physiol Endocrinol Metab 286: E665–672. 14693509

22. Neese RA, Misell LM, Turner S, Chu A, Kim J, et al. (2002) Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA. Proc Natl Acad Sci U S A 99: 15345–15350. 12424339

23. Busch R, Neese RA, Awada M, Hayes GM, Hellerstein MK (2007) Measurement of cell proliferation by heavy water labeling. Nat Protoc 2: 3045–3057. 18079703

24. Saunders EC, Ng WW, Kloehn J, Chambers JM, Ng M, McConville MJ (2014) Induction of a Stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism. PLoS Pathog 10: e1003888. doi: 10.1371/journal.ppat.1003888 24465208

25. Warner JR (1999) The economics of ribosome biosynthesis in yeast. Trends Biochem Sci 24: 437–440. 10542411

26. Busch R, Kim Y-K, Neese RA, Schade-Serin V, Collins M, et al. Measurement of protein turnover rates by heavy water labeling of nonessential amino acids. Biochim Biophys Acta 1760: 730–744. 16567052

27. Ramakrishnan S, Serricchio M, Striepen B, Bütikofer P (2013) Lipid synthesis in protozoan parasites: a comparison between kinetoplastids and apicomplexans. Prog Lipid Res. 52: 488–512. doi: 10.1016/j.plipres.2013.06.003 23827884

28. Real F, Florentino PTV, Reis LC, Ramos-Sanchez EM, Veras PS, et al. (2014) Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions. Cell Microbiol 16: 1549–1564 doi: 10.1111/cmi.12311 24824158

29. van Zandbergen G, Solbach W, Laskay T (2007) Apoptosis driven infection. Autoimmunity 40: 349–52. 17516227

30. Donovan MJ, Maciuba BZ, Mahan CE, McDowell MA (2009) Leishmania infection inhibits cycloheximide-induced macrophage apoptosis in a strain-dependent manner. Exp Parasitol. 123: 58–64. doi: 10.1016/j.exppara.2009.05.012 19500578

31. Petersen HJ, Smith AM (2013) The role of the innate immune system in granulomatous disorders. Front Immunol 4: 120. doi: 10.3389/fimmu.2013.00120 23745122

32. Ramakrishnan L (2012) Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol 12: 352–366. doi: 10.1038/nri3211 22517424

33. Davis JM, Ramakrishnan L (2009) The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell 136: 37–49. doi: 10.1016/j.cell.2008.11.014 19135887

34. Lahav T, Sivam D, Volpin H, Ronen M, Tsigankov P, et al. (2010) Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania. FASEB J 25: 515–525. doi: 10.1096/fj.10-157529 20952481

35. Chow C, Cloutier S, Dumas C, Chou M-N, Papadopoulou B (2011) Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation. Cell Microbiol 13: 1059–1077. doi: 10.1111/j.1462-5822.2011.01602.x 21624030

36. Gosline SJC, Nascimento M, McCall L-I, Zilberstein D, Thomas DY, et al. (2011) Intracellular eukaryotic parasites have a distinct unfolded protein response. PLoS One 6: e19118. doi: 10.1371/journal.pone.0019118 21559456

37. Wilson J, Huynh C, Kennedy KA, Ward DM, Kaplan J, et al. (2008) Control of parasitophorous vacuole expansion by LYST/Beige restricts the intracellular growth of Leishmania amazonensis. PLoS Pathog 4: e1000179. doi: 10.1371/journal.ppat.1000179 18927622

38. Cortez M, Huynh C, Fernandes MC, Kennedy KA, Aderem A, Andrews NW (2011) Leishmania promotes its own virulence by inducing expression of the host immune inhibitory ligand CD200. Cell Host Microbe. 9: 463–471. doi: 10.1016/j.chom.2011.04.014 21669395

39. Lira R, Doherty M, Modi G, Sacks D (2000) Evolution of lesion formation, parasitic load, immune response, and reservoir potential in C57BL/6 mice following high- and low-dose challenge with Leishmania major. Infect Immun 68: 5176–5182. 10948141

40. Belkaid Y, Mendez S, Lira R, Kadambi N, Milon G, Sacks D (2000) A natural model of Leishmania major infection reveals a prolonged “silent” phase of parasite amplification in the skin before the onset of lesion formation and immunity. J Immunol 165: 969–977. 10878373

41. Sacks D, Kamhawi S (2001) Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. Annu. Rev. Microbiol 55: 453–483. 11544364

42. Mukherjee S, Sen Santara S, Das S, Bose M, Roy J, Adak S (2012) NAD(P)H cytochrome b5 oxidoreductase deficiency in Leishmania major results in impaired linoleate synthesis followed by increased oxidative stress and cell death. J Biol Chem 287: 34992–35003. doi: 10.1074/jbc.M112.389338 22923617

43. Alloatti A, Gupta S, Gualdrón-López M, Igoillo-Esteve M, Nguewa PA, et al. (2010) Genetic and chemical evaluation of Trypanosoma brucei oleate desaturase as a candidate drug target. PLoS One 5: e14239. doi: 10.1371/journal.pone.0014239 21151902

44. Winter G, Fuchs M, McConville MJ, Stierhof YD, Overath P (1994) Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid. J Cell Sci 107: 2471–2482. 7844164

45. Zhang O, Wilson MC, Xu W, Hsu F-F, Turk J, et al. (2009) Degradation of host sphingomyelin is essential for Leishmania virulence. PLoS Pathog 5: e1000692. doi: 10.1371/journal.ppat.1000692 20011126

46. Naderer T, Heng J, McConville MJ (2010) Evidence that intracellular stages of Leishmania major utilize amino sugars as a major carbon source. PLoS Pathog. 6: e1001245. doi: 10.1371/journal.ppat.1001245 21203480

47. Shah V, Herath K, Previs SF, Hubbard BK, Roddy TP (2010) Headspace analyses of acetone: a rapid method for measuring the 2H-labeling of water. Anal Biochem 404: 235–237. doi: 10.1016/j.ab.2010.05.010 20488158

48. Saunders EC, Ng WW, Chamber JM, Ng M, Naderer T, et al. (2011) Isoptopomer profiling of Leishmania mexicana promastigotes reveals important roles for succinate fermentation and aspartate uptake in TCA cycle anaplerosis, glutamate synthesis and growth. J Biol Chem 286: 27706–27717. doi: 10.1074/jbc.M110.213553 21636575

49. Rotureau B, Gego A, Carme B (2005) Trypanosomatid protozoa: a simplified DNA isolation procedure. Exp Parasitol. 111: 207–209. 16139269

50. Wu M-Y, Chen B-G, Chang CD, Huang M-H, Wu T-G, et al. (2008) A novel derivatization approach for simultaneous determination of glyoxal, methylglyoxal, and 3-deoxyglucosone in plasma by gas chromatography-mass spectrometry. J Chromatogr A 1204: 81–86. doi: 10.1016/j.chroma.2008.07.040 18692194

51. Neese RA, Siler SQ, Cesar D, Antelo F, Lee D, et al. (2001) Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation. Anal Biochem 298: 189–95. 11700973