1887

Abstract

. ML1899 is conserved in all mycobacterium sp. and is a middle member of operon involved in mycolic acid modification.

. In the present study attempts were made to characterize ML1899 in detail.

. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.

. analysis predicted ML1899 as a member of / hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM min catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. , and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min ml and 27 U mg , respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.

. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

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2019-09-25
2019-10-19
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References

  1. Bhat RM, Prakash C. Leprosy: an overview of pathophysiology. Interdiscip Perspect Infect Dis 2012;2012:16 [CrossRef]
    [Google Scholar]
  2. Scollard DM, Adams LB, Gillis TP, Krahenbuhl JL, Truman RW et al. The continuing challenges of leprosy. Clin Microbiol Rev 2006;19:338–381 [CrossRef]
    [Google Scholar]
  3. Vissa VD, Brennan PJ. The genome of Mycobacterium leprae: a minimal mycobacterial gene set. Genome Biol 2001;2:1021 [CrossRef]
    [Google Scholar]
  4. Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR et al. Massive gene decay in the leprosy Bacillus. Nature 2001;409:10071011 [CrossRef]
    [Google Scholar]
  5. Cole ST. Comparative mycobacterial genomics. Curr Opin Microbiol 1998;1:567–571 [CrossRef]
    [Google Scholar]
  6. Patil KN, Singh P, Harsha S, Muniyappa K. Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein. Biochim Biophys Acta 2011;1814:1802–1811 [CrossRef]
    [Google Scholar]
  7. Elamin AA, Stehr M, Singh M. Lipid Droplets and Mycobacterium leprae Infection. J Pathog 2012;2012:110 [CrossRef]
    [Google Scholar]
  8. Kaur G, Kaur J. Multifaceted role of lipids in Mycobacterium leprae. Future Microbiol 2017;12:315–335 [CrossRef]
    [Google Scholar]
  9. Kaur G, Saini V, Kumari B, Kaur J, Kaur J. Characterization of an extracellular protein, Rv1076 from M. tuberculosis with a potential role in humoral response. Int J Biol Macromol 2017;101:621–629 [CrossRef]
    [Google Scholar]
  10. Nath I, Saini C, Valluri VL. Immunology of leprosy and diagnostic challenges. Clin Dermatol 2015;33:90–98 [CrossRef]
    [Google Scholar]
  11. Walker SL, Lockwood DNJ. The clinical and immunological features of leprosy. Br Med Bull 2006;77-78:103–121 [CrossRef]
    [Google Scholar]
  12. Jadeja D, Dogra N, Arya S, Singh G, Singh G et al. Characterization of LipN (Rv2970c) of Mycobacterium tuberculosis H37Rv and its probable role in xenobiotic degradation. J Cell Biochem 2016;117:390–401 [CrossRef]
    [Google Scholar]
  13. Shen G, Singh K, Chandra D, Serveau-Avesque C, Maurin D et al. Lipc (Rv0220) is an immunogenic cell surface esterase of Mycobacterium tuberculosis. Infect Immun 2012;80:243–253 [CrossRef]
    [Google Scholar]
  14. Singh G, Arya S, Kaur J, Kaur J. Rv2485c, a putative lipase of M. tuberculosis: expression, purification and biochemical characterization. Int J Trop Dis Health 2014a;4:1–17 [CrossRef]
    [Google Scholar]
  15. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537544 [CrossRef]
    [Google Scholar]
  16. Singh G, Singh G, Jadeja D, Kaur J. Lipid hydrolizing enzymes in virulence: Mycobacterium tuberculosis as a model system. Crit Rev Microbiol 2010;36:259–269 [CrossRef]
    [Google Scholar]
  17. Talati S, Mahadevan PR. Lipase activity in Mycobacterium leprae--an indicator of metabolic function. Indian J Lepr 1986;58:367–372
    [Google Scholar]
  18. Taboada B, Ciria R, Martinez-Guerrero CE, Merino E. ProOpDB: prokaryotic operon database. Nucleic Acids Res 2012;40:D627–D631 [CrossRef]
    [Google Scholar]
  19. Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD et al. "Protein identification and analysis tools on the ExPASy server," The proteomics protocols handbook Springer: 2005; pp571–607
    [Google Scholar]
  20. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal omega. Mol Syst Biol 2011;7:539 [CrossRef]
    [Google Scholar]
  21. DeLano WL. PyMOL. 2002
  22. Krieger E, Joo K, Lee J, Lee J, Raman S et al. Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: four approaches that performed well in CASP8. Proteins 2009;77:114–122 [CrossRef]
    [Google Scholar]
  23. Yao B, Zhang L, Liang S, Zhang C. SVMTriP: a method to predict antigenic epitopes using support vector machine to integrate tri-peptide similarity and propensity. PLoS One 2012;7:e45152 [CrossRef]
    [Google Scholar]
  24. Kaur J, Kumar A, Kaur J. Strategies for optimization of heterologous protein expression in E. coli: roadblocks and reinforcements. Int J Biol Macromol 2018;106:803–822 [CrossRef]
    [Google Scholar]
  25. Dosanjh NS, Kaur J. Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp. Protein Expr Purif 2002;24:71–75 [CrossRef]
    [Google Scholar]
  26. Price JA. A colorimetric assay for measuring phospholipase A2 degradation of phosphatidylcholine at physiological pH. J Biochem Biophys Methods 2007;70:441–444 [CrossRef]
    [Google Scholar]
  27. Sarkar M, Varshney R, Chopra M, Sekhri T, Adhikari JS et al. Flow-Cytometric analysis of reactive oxygen species in peripheral blood mononuclear cells of patients with thyroid dysfunction. Cytometry B Clin Cytom 2006;70:20–23 [CrossRef]
    [Google Scholar]
  28. Yamamoto K, Akbar SM, Masumoto T, Onji M. Increased nitric oxide (NO) production by antigen-presenting dendritic cells is responsible for low allogeneic mixed leucocyte reaction (MLR) in primary biliary cirrhosis (pBC). Clin Exp Immunol 1998;114:94–101 [CrossRef]
    [Google Scholar]
  29. Santucci P, Point V, Poncin I, Guy A, Crauste C et al. LipG a bifunctional phospholipase/thioesterase involved in mycobacterial envelope remodeling. Biosci Rep 2018;38:BSR20181953 [CrossRef]
    [Google Scholar]
  30. Galli CL, Viviani B, Marinovich M. Cell cultures: a tool for the study of mechanisms of toxicity. Toxicology in Vitro 1993;7:559–568 [CrossRef]
    [Google Scholar]
  31. Swathi M, Tagore R. Study of oxidative stress in different forms of leprosy. Indian J Dermatol 2015;60:321 [CrossRef]
    [Google Scholar]
  32. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997;15:323–350 [CrossRef]
    [Google Scholar]
  33. Hu S, He W, Du X, Yang J, Wen Q et al. Il-17 production of neutrophils enhances antibacteria ability but promotes arthritis development during Mycobacterium tuberculosis infection. EBioMedicine 2017;23:88–99 [CrossRef]
    [Google Scholar]
  34. Lin Y, Li Q, Xie L, Xie J. Mycobacterium tuberculosis rv1400c encodes functional lipase/esterase. Protein Expr Purif 2017;129:143–149 [CrossRef]
    [Google Scholar]
  35. Singh G, Arya S, xKumar S, Narang D, Kaur J. Molecular characterization of oxidative stress-inducible LipD of Mycobacterium tuberculosis H37Rv. Curr Microbiol 2014b;68:387–396 [CrossRef]
    [Google Scholar]
  36. Singh G, Kumar A, Arya S, Gupta UD, Singh K et al. Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating β-lactamase activity. Enzyme and microbial technology 2016;82:180–190
    [Google Scholar]
  37. Rastogi R, Kumar A, Kaur J, Saini V, Kaur J et al. Rv0646c, an esterase from M. tuberculosis, up-regulates the host immune response in THP-1 macrophages cells. Mol Cell Biochem 2018;447:189–202 [CrossRef]
    [Google Scholar]
  38. Tanigawa K, Degang Y, Kawashima A, Akama T, Yoshihara A et al. Essential role of hormone-sensitive lipase (HSL) in the maintenance of lipid storage in Mycobacterium leprae-infected macrophages. Microb Pathog 2012;52:285–291 [CrossRef]
    [Google Scholar]
  39. Agita A, Alsagaff MT. Inflammation, immunity, and hypertension. Acta Med Indones 2017;49:158–165
    [Google Scholar]
  40. Shin D-M, Jeon B-Y, Lee H-M, Jin HS, Yuk J-M et al. Mycobacterium tuberculosis EIS regulates autophagy, inflammation, and cell death through redox-dependent signaling. PLoS Pathog 2010;6:e1001230 [CrossRef]
    [Google Scholar]
  41. Nahirnyj A, Livne-Bar I, Guo X, Sivak JM. Ros detoxification and proinflammatory cytokines are linked by p38 MAPK signaling in a model of mature astrocyte activation. PLoS One 2013;8:e83049 [CrossRef]
    [Google Scholar]
  42. Jin S-H, Ahn KJ, An S. Importance of the immune response to Mycobacterium leprae in the skin. Biomedical Dermatology 2018;2:1 [CrossRef]
    [Google Scholar]
  43. Cassirer-Costa F, Medeiros NI, Chaves AT, Lyon S, Coelho-Dos-Reis JGA et al. Cytokines as biomarkers to monitoring the impact of multidrug therapy in immune response of leprosy patients. Cytokine 2017;97:42–48 [CrossRef]
    [Google Scholar]
  44. Sykam A, Gutlapalli VR, Tenali SP, Meena AK, Chandran P et al. Association of tumor necrosis factor-alpha and interferon gamma gene polymorphisms and their plasma levels in leprosy, HIV and other peripheral neuropathies. Cytokine 2015;76:473–479 [CrossRef]
    [Google Scholar]
  45. Tohyama M, Shirakara Y, Yamasaki K, Sayama K, Hashimoto K. Differentiated keratinocytes are responsible for TNF-alpha regulated production of macrophage inflammatory protein 3alpha/CCL20, a potent chemokine for Langerhans cells. J Dermatol Sci 2001;27:130–139 [CrossRef]
    [Google Scholar]
  46. Polycarpou A, Walker SL, Lockwood DNJ. A systematic review of immunological studies of erythema nodosum leprosum. Front Immunol 2017;8:233 [CrossRef]
    [Google Scholar]
  47. Lockwood DNJ, Suneetha L, Sagili KD, Chaduvula MV, Mohammed I et al. Cytokine and protein markers of leprosy reactions in skin and nerves: baseline results for the North Indian INFIR cohort. PLoS Negl Trop Dis 2011;5:e1327 [CrossRef]
    [Google Scholar]
  48. Aktan F. iNOS-mediated nitric oxide production and its regulation. Life Sci 2004;75:639–653 [CrossRef]
    [Google Scholar]
  49. Hagge DA, Scollard DM, Ray NA, Marks VT, Deming AT et al. Il-10 and NOS2 modulate antigen-specific reactivity and nerve infiltration by T cells in experimental leprosy. PLoS Negl Trop Dis 2014;8:e3149 [CrossRef]
    [Google Scholar]
  50. Andrade PR, Jardim MR, da Silva ACC, Manhaes PS, Antunes SLG et al. Inflammatory cytokines are involved in focal demyelination in leprosy neuritis. J Neuropathol Exp Neurol 2016;75:272–283 [CrossRef]
    [Google Scholar]
  51. Pires CAA, Quaresma JAS, de Souza Aarão TL, de Souza JR, Macedo GMM et al. Expression of interleukin-1β and interleukin-6 in leprosy reactions in patients with human immunodeficiency virus coinfection. Acta Trop 2017;172:213–216 [CrossRef]
    [Google Scholar]
  52. Sales-Marques C, Cardoso CC, Alvarado-Arnez LE, Illaramendi X, Sales AM et al. Genetic polymorphisms of the IL6 and NOD2 genes are risk factors for inflammatory reactions in leprosy. PLoS Negl Trop Dis 2017;11:e0005754 [CrossRef]
    [Google Scholar]
  53. Fulya I, Mehmet O, Handan A, Vedat B. Cytokine measurement in lymphocyte culture supernatant of inactive lepromatous leprosy patients. Indian J Med Microbiol 2006;24:121 [CrossRef]
    [Google Scholar]
  54. Choudhary RK, Pullakhandam R, Ehtesham NZ, Hasnain SE. Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis. Protein Expr Purif 2004;36:249–253 [CrossRef]
    [Google Scholar]
  55. Mishra KC, de Chastellier C, Narayana Y, Bifani P, Brown AK et al. Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY. Infect Immun 2008;76:127–140 [CrossRef]
    [Google Scholar]
  56. Bhamidi S, Scherman MS, Jones V, Crick DC, Belisle JT et al. Detailed structural and quantitative analysis reveals the spatial organization of the cell walls of in vivo grown Mycobacterium leprae and in vitro grown Mycobacterium tuberculosis. J Biol Chem 2011;286:23168–23177 [CrossRef]
    [Google Scholar]
  57. Dautin N, de Sousa-d'Auria C, Constantinesco-Becker F, Labarre C, Oberto J et al. Mycoloyltransferases: a large and major family of enzymes shaping the cell envelope of Corynebacteriales. Biochim Biophys Acta Gen Subj 2017;1861:3581–3592 [CrossRef]
    [Google Scholar]
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