An Integrated approach for Proteomic and Immunological analysis of Cell wall and Cell membrane proteins of Mycobacterium tuberculosis
Main Article Content
Abstract
Mycobacterium cell wall and cell membrane proteins serve as key modulators in the host immune response. Some of these proteins, like MPT63, are known as immunodominant antigens, and others, such as LpqS and LpqH, are being explored as subunit vaccine candidates. However, due to their hydrophobic nature, conventional separation methods are not able to solubilize and isolate these proteins individually, leading to limited research on their systematic separation and immunological characterization.
In this study, we successfully employed a combination of Preparative Isoelectric focusing and Preparatory SDS-PAGE to separate mycobacterial cell wall and membrane proteins into 234 fractions. These fractions were pooled based on the proteins molecular weight, resulting in 24 pooled fractions labelled as Ag1 to Ag24. Immunological analyses on these pooled fractions were carried out in 3 different study groups (subjects with latent tuberculosis infection (LTBI, N=20), tuberculosis-diseased patients (PTB, N=20), and healthy subjects (HC, N=20). In these study groups, Interferon-γ and TNF-α responses against these antigens were analysed using the cytokine ELISA method.
Notably, one pooled fraction (Ag24 - Cell wall high molecular weight proteins pool) induced significantly higher IFN gamma response in LTBI compared to PTB, while also triggering significantly higher TNF-alpha response in PTB compared to LTBI. Additionally, three other pooled fractions (Ag6 - Cell Membrane Medium molecular weight proteins Pool, Ag11 - Cell Membrane high molecular weight proteins Pool, Ag23 - Cell wall high molecular weight proteins pool) induced significantly higher IFN gamma response in LTBI compared to PTB.
Proteomic analysis of the Ag24 fraction identified four proteins (ACN, KatG, Rv2623, and Rv0404). Among them, KatG and Rv0404 were previously reported as immunodominant T-cell antigens. On the other hand, ACN lacks immunological information, and limited information is available on protein Rv2623. In conclusion, extensive immunological studies on these four proteins could enhance their development as tuberculosis prognostic biomarkers, while our systematic 2-dimensional separation method shows promising potential for characterizing cell wall and membrane proteins in other pathogens
Keywords:
Mycobacterium tuberculosis, cell wall, cell membrane proteins, Protein purification, Preparative electrophoresis, Rotofor, Latent TB
Article Details
How to Cite
DEENADAYALAN, Anbarasu et al.
An Integrated approach for Proteomic and Immunological analysis of Cell wall and Cell membrane proteins of Mycobacterium tuberculosis.
Medical Research Archives, [S.l.], v. 11, n. 11, dec. 2023.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/4805>. Date accessed: 16 may 2024.
doi: https://doi.org/10.18103/mra.v11i11.4805.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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22. Rajavelu P, Das SD. Cell-mediated immune responses of healthy laboratory volunteers to sonicate antigens prepared from the most prevalent strains of Mycobacterium tuberculosis from South India harboring a single copy of IS6110. Clin Diagn Lab Immunol. 2003 Nov;10(6):1149-52
23. Raja A, Uma Devi KR, Ramalingam B, Brennan PJ. Immunoglobulin G, A, and M responses in serum and circulating immune complexes elicited by the 16-kilodalton antigen of Mycobacterium tuberculosis. Clin Diagn Lab Immunol. 2002 Mar; 9(2):308-12.
24. Deenadayalan A, Maddineni P, Raja A.Comparison of whole blood and PBMC assays for T-cell functional analysis. BMC Res Notes. 2013 Mar 27; 6:120. doi: 10.1186/ 1756-0500-6-120.
25. Anbarasu Deenadayalan, Jagadish Chandrabose Sundaramurthi, Alamelu Raja Immunological and proteomic analysis of preparative isoelectric-focusing separated Culture filtrate antigens of M. tuberculosis. Experimental and Molecular Pathology. 2010 Feb; 88(1):156-62.
26. Covert, B. A., Spencer, J. S., Orme, I. M., and Belisle, J. T. (2001). The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis. Proteomics 1, 574–586.
27. Cruz MIO, de Janeiro R: Whole blood assay to access T cell-immune responses to mycobacterium tuberculosis antigens in healthy Brazilian individuals. Immunology and immunotherapy 2004, 99(1):53–55.
28. Anbarasu D, Raja CP, Raja A. Multiplex analysis of cytokines/chemokines as biomarkers that differentiate healthy contacts from tuberculosis patients in high endemic settings. Cytokine. 2013 Mar;61(3):
29. Sinha S, Kosalai K, Arora S, Namane A, Sharma P, Gaikwad AN, Brodin P, Cole ST.Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics. Microbiology (Reading). 2005 Jul;151(Pt 7):2411-2419.
30. Walzl G, McNerney R, du Plessis N, Bates M, McHugh TD, Chegou NN, et al. Tuberculosis: Advances and Challenges in Development of New Diagnostics and Biomarkers. Lancet Infect Dis (2018) 18:e199–210. doi: 10.1016/s1473-3099(18)30111-7.
31. Pathakumari B, Prabhavathi M, Raja A. Evaluation of cytokine and chemokine response elicited by Rv2204c and Rv0753c to detect latent tuberculosis infection..Cytokine. 2015 Dec; 76(2):496-504. doi: 10.1016/j.cyto. 2015.07.028. Epub 2015 Aug 19.
32. Lucas M, Ryan JM, Watkins J, Early K, Kruh-Garcia NA, Mehaffy C, Dobos KM. Extraction and Separation of Mycobacterial Proteins. Methods Mol Biol. 2021; 2314:77-107.
33. Rabodoarivelo MS, Aerts M, Vandamme P, Palomino JC, Rasolofo V, Martin A. Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry. J Microbiol Methods. 2016 Dec; 131:144-147.
34. Kumar G, Kapoor S. Targeting mycobacterial membranes and membrane proteins: Progress and limitations. Bioorg Med Chem. 2023 Mar 1; 81:117212. doi: 10. 1016/j.bmc.2023.117212. Epub 2023 Feb 11.
35. Manca C, Paul S, Barry CE 3rd, Freedman VH, Kaplan G. Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro. Infect Immun. 1999 Jan; 67(1):74-9. doi: 10.1128/IAI.67.1.74-79.1999.
36. Wu X, Yang Y, Zhang J, Li B, Liang Y, Zhang C, Dong M. Comparison of antibody responses to seventeen antigens from Mycobacterium tuberculosis. Clin Chim Acta. 2010 Oct 9; 411(19-20):1520-8. doi: 10.1016/ j.cca.2010.06.014. Epub 2010 Jun 22.
37. Rosenkrands I, Slayden RA, Crawford J, Aagaard C, Barry CE 3rd, Andersen P.Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins. J Bacteriol. 2002 Jul;184(13):3485-91
38. Shi L, Jung YJ, Tyagi S, Gennaro ML, North RJ. Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence. Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):241-6
39. Drumm JE, Mi K, Bilder P, Sun M, Lim J, Bielefeldt-Ohmann H, Basaraba R, So M, Zhu G, Tufariello JM, Izzo AA, Orme IM, Almo SC, Leyh TS, Chan J. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection. PLoS Pathog. 2009 May; 5(5):e1000460.
40. Provvedi R, Boldrin F, Falciani F, Palù G, Manganelli R. Global transcriptional response to vancomycin in Mycobacterium tuberculosis. Microbiology (Reading). 2009 Apr; 155(Pt 4):1093-1102.
41. Lata M, Sharma D, Kumar B, Deo N, Tiwari PK, Bisht D, Venkatesan K. Proteome analysis of ofloxacin and moxifloxacin induced mycobacterium tuberculosis isolates by proteomic approach. Protein Pept Lett. 2015; 22(4):362-71.
42. Jain RK, Nayak AR, Husain AA, Panchbhai MS, Chandak N, Purohit HJ, Taori GM, Daginawala HF, Kashyap RS. Mycobacterial dormancy regulon protein Rv2623 as a novel biomarker for the diagnosis of latent and active tuberculous meningitis. Dis Markers. 2013;35(5):311-6
43. Wang Y, X, Zhang J, Liang J, Zhang Teng X, Yuan X, Fan X. Protection against Mycobacterium tuberculosis infection offered by a new multistage subunit vaccine correlates with increased number of IFN-γ+ IL-2+ CD4+ and IFN-γ+ CD8+ T cells. PLoS One. 2015 Mar 30; 10(3):e0122560.
44. : Artymiuk PJ, Green J. The double life of aconitase. Structure. 2006 Jan;14(1):2-4
45. Banerjee S, Nandyala AK, Raviprasad P, Ahmed N, Hasnain SE. Iron-dependent RNA-binding activity of Mycobacterium tuberculosis aconitase. J Bacteriol. 2007 Jun; 189(11):4046-52.
46. Banerjee S, Nandyala AK, Raviprasad P, Ahmed N, Hasnain SE. Iron-dependent RNA-binding activity of Mycobacterium tuberculosis aconitase. J Bacteriol. 2007 Jun; 189(11):4046-52.
2. Deenadayalan A, Heaslip D, Rajendiran AA, Velayudham BV, Frederick S, Yang HL, Dobos K, Belisle JT, Raja A. Immunoproteomic identification of human T cell antigens of Mycobacterium tuberculosis that differentiate healthy contacts from tuberculosis patients. Mol Cell Proteomics. 2010 Mar; 9(3):538-49. Epub 2009 Dec 22.
3. Prabhavathi M, Pathakumari B, Raja A. FN-γ/TNF-α ratio in response to immuno proteomically identified human T-cell antigens of Mycobacterium tuberculosis - The most suitable surrogate biomarker for latent TB infection. J Infect. 2015 Aug; 71(2):238-49.
4. Seshadri C, Turner MT, Lewinsohn DM, Moody DB, Van Rhijn I. Lipoproteins are major targets of the polyclonal human T cell response to Mycobacterium tuberculosis. J Immunol. 2013 Jan 1; 190(1):278-84.
5. Kunnath-Velayudhan S, Salamon H, Wang HY, Davidow AL, Molina DM, Huynh VT, Cirillo DM, Michel G, Talbot EA, Perkins MD, Felgner PL, Liang X, Gennaro ML. Dynamic antibody responses to the Mycobacterium tuberculosis proteome.Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14703-8.
6. Noss EH, Pai RK, Sellati TJ, Radolf JD, Belisle J, Golenbock DT, Boom WH, Harding CV. Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19 kD lipoprotein of Mycobacterium tuberculosis. J Immunol. 2001; 167:910–918.
7. Pai RK, Convery M, Hamilton TA, Boom WH, Harding CV. Inhibition of IFN-gamma-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion. J Immunol. 2003;171:175–184.
8. Pai RK, Pennini ME, Tobian AA, Canaday DH, Boom WH, Harding CV. Prolonged toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages. Infect Immun. 2004;72:6603–14
9. Sutcliffe IC, Harrington DJ. Lipoproteins of Mycobacterium tuberculosis: an abundant and functionally diverse class of cell envelope components. FEMS Microbiol Rev. 2004; 28:645–59.
10. Gehring AJ, Dobos KM, Belisle JT, Harding CV, Boom WH. Mycobacterium tuberculosis LprG (Rv1411c): A novel TLR-2 ligand that inhibits human macrophage class II MHC antigen processing. J Immunol. 2004; 173:2660–8.
11. Pecora ND, Gehring AJ, Canaday DH, Boom WH, Harding CV. Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function. J Immunol. 2006; 177:422–429.
12. Jung SB, Yang CS, Lee JS, Shin AR, Jung SS, Son JW, Harding CV, Kim HJ, Park JK, Paik TH, Song CH, Jo EK. The mycobacterial 38-kilodalton glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes. Infect Immun. 2006; 74:2686–96.
13. A Kovacs-Simon , R W Titball, S L Michell. Lipoproteins of bacterial pathogens. Infect Immun. 2011 Feb; 79(2):548-61.
14. Zheng J, Wei C, Leng W, Dong J, Li R, Li W, Wang J, Zhang Z, Jin Q Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette-Guérin.. Proteomics. 2007 Nov;7(21):3919-31
15. Wolfe LM, Mahaffey SB, Kruh NA, Dobos KM. Proteomic definition of the cell wall of Mycobacterium tuberculosis. J Proteome Res. 2010 Nov 5;9(11):5816-26
16. Wallin, E., and von Heijne, G. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 1998, 7, 1029–1038.
17. Almén MS, Nordström KJ, Fredriksson R, Schiöth HB. Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin. BMC Biol. 2009 Aug 13; 7:50.
18. Almeida JG, Preto AJ, Koukos PI, Bonvin AMJJ, Moreira IS.Membrane proteins structures: A review on computational modeling tools. Biochim Biophys Acta Biomembr. 2017 Oct; 1859(10):2021-2039. doi: 10.1016/j.bbamem.2017.07.008. Epub 2017 Jul 15.
19. Bluhm, W. F., Bourne, P. E., Burkhardt, K., Feng, Z., Gilliland, G. L., Iype, L., Jain, S., Berman, H. M., Battistuz, T., Bhat, T. N.,Fagan, P., Marvin, J., Padilla, D., Ravichandran, V., Schneider, B., Thanki, N., Weissig, H.,Westbrook, J. D., and Zardecki, C. (2002). The Protein Data Bank. Acta Crystallogr. DBiol. Crystallogr. 58, 899–907.
20. Smith.SM Strategies for the Purification of Membrane Proteins, Methods Mol Biol, 2017; 1485:389-400.
21. Narayanan S, Das S, Garg R, Hari L, Rao VB, Frieden TR, Narayanan PR Narayanan S, Das S, Garg R, Hari L, Rao VB, Frieden TR, Narayanan PR Molecular epidemiology of tuberculosis in a rural area of high prevalence in South India: implications for disease control and prevention. J Clin Microbiol. 2002 Dec;40(12):4785-8
22. Rajavelu P, Das SD. Cell-mediated immune responses of healthy laboratory volunteers to sonicate antigens prepared from the most prevalent strains of Mycobacterium tuberculosis from South India harboring a single copy of IS6110. Clin Diagn Lab Immunol. 2003 Nov;10(6):1149-52
23. Raja A, Uma Devi KR, Ramalingam B, Brennan PJ. Immunoglobulin G, A, and M responses in serum and circulating immune complexes elicited by the 16-kilodalton antigen of Mycobacterium tuberculosis. Clin Diagn Lab Immunol. 2002 Mar; 9(2):308-12.
24. Deenadayalan A, Maddineni P, Raja A.Comparison of whole blood and PBMC assays for T-cell functional analysis. BMC Res Notes. 2013 Mar 27; 6:120. doi: 10.1186/ 1756-0500-6-120.
25. Anbarasu Deenadayalan, Jagadish Chandrabose Sundaramurthi, Alamelu Raja Immunological and proteomic analysis of preparative isoelectric-focusing separated Culture filtrate antigens of M. tuberculosis. Experimental and Molecular Pathology. 2010 Feb; 88(1):156-62.
26. Covert, B. A., Spencer, J. S., Orme, I. M., and Belisle, J. T. (2001). The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis. Proteomics 1, 574–586.
27. Cruz MIO, de Janeiro R: Whole blood assay to access T cell-immune responses to mycobacterium tuberculosis antigens in healthy Brazilian individuals. Immunology and immunotherapy 2004, 99(1):53–55.
28. Anbarasu D, Raja CP, Raja A. Multiplex analysis of cytokines/chemokines as biomarkers that differentiate healthy contacts from tuberculosis patients in high endemic settings. Cytokine. 2013 Mar;61(3):
29. Sinha S, Kosalai K, Arora S, Namane A, Sharma P, Gaikwad AN, Brodin P, Cole ST.Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics. Microbiology (Reading). 2005 Jul;151(Pt 7):2411-2419.
30. Walzl G, McNerney R, du Plessis N, Bates M, McHugh TD, Chegou NN, et al. Tuberculosis: Advances and Challenges in Development of New Diagnostics and Biomarkers. Lancet Infect Dis (2018) 18:e199–210. doi: 10.1016/s1473-3099(18)30111-7.
31. Pathakumari B, Prabhavathi M, Raja A. Evaluation of cytokine and chemokine response elicited by Rv2204c and Rv0753c to detect latent tuberculosis infection..Cytokine. 2015 Dec; 76(2):496-504. doi: 10.1016/j.cyto. 2015.07.028. Epub 2015 Aug 19.
32. Lucas M, Ryan JM, Watkins J, Early K, Kruh-Garcia NA, Mehaffy C, Dobos KM. Extraction and Separation of Mycobacterial Proteins. Methods Mol Biol. 2021; 2314:77-107.
33. Rabodoarivelo MS, Aerts M, Vandamme P, Palomino JC, Rasolofo V, Martin A. Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry. J Microbiol Methods. 2016 Dec; 131:144-147.
34. Kumar G, Kapoor S. Targeting mycobacterial membranes and membrane proteins: Progress and limitations. Bioorg Med Chem. 2023 Mar 1; 81:117212. doi: 10. 1016/j.bmc.2023.117212. Epub 2023 Feb 11.
35. Manca C, Paul S, Barry CE 3rd, Freedman VH, Kaplan G. Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro. Infect Immun. 1999 Jan; 67(1):74-9. doi: 10.1128/IAI.67.1.74-79.1999.
36. Wu X, Yang Y, Zhang J, Li B, Liang Y, Zhang C, Dong M. Comparison of antibody responses to seventeen antigens from Mycobacterium tuberculosis. Clin Chim Acta. 2010 Oct 9; 411(19-20):1520-8. doi: 10.1016/ j.cca.2010.06.014. Epub 2010 Jun 22.
37. Rosenkrands I, Slayden RA, Crawford J, Aagaard C, Barry CE 3rd, Andersen P.Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins. J Bacteriol. 2002 Jul;184(13):3485-91
38. Shi L, Jung YJ, Tyagi S, Gennaro ML, North RJ. Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence. Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):241-6
39. Drumm JE, Mi K, Bilder P, Sun M, Lim J, Bielefeldt-Ohmann H, Basaraba R, So M, Zhu G, Tufariello JM, Izzo AA, Orme IM, Almo SC, Leyh TS, Chan J. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection. PLoS Pathog. 2009 May; 5(5):e1000460.
40. Provvedi R, Boldrin F, Falciani F, Palù G, Manganelli R. Global transcriptional response to vancomycin in Mycobacterium tuberculosis. Microbiology (Reading). 2009 Apr; 155(Pt 4):1093-1102.
41. Lata M, Sharma D, Kumar B, Deo N, Tiwari PK, Bisht D, Venkatesan K. Proteome analysis of ofloxacin and moxifloxacin induced mycobacterium tuberculosis isolates by proteomic approach. Protein Pept Lett. 2015; 22(4):362-71.
42. Jain RK, Nayak AR, Husain AA, Panchbhai MS, Chandak N, Purohit HJ, Taori GM, Daginawala HF, Kashyap RS. Mycobacterial dormancy regulon protein Rv2623 as a novel biomarker for the diagnosis of latent and active tuberculous meningitis. Dis Markers. 2013;35(5):311-6
43. Wang Y, X, Zhang J, Liang J, Zhang Teng X, Yuan X, Fan X. Protection against Mycobacterium tuberculosis infection offered by a new multistage subunit vaccine correlates with increased number of IFN-γ+ IL-2+ CD4+ and IFN-γ+ CD8+ T cells. PLoS One. 2015 Mar 30; 10(3):e0122560.
44. : Artymiuk PJ, Green J. The double life of aconitase. Structure. 2006 Jan;14(1):2-4
45. Banerjee S, Nandyala AK, Raviprasad P, Ahmed N, Hasnain SE. Iron-dependent RNA-binding activity of Mycobacterium tuberculosis aconitase. J Bacteriol. 2007 Jun; 189(11):4046-52.
46. Banerjee S, Nandyala AK, Raviprasad P, Ahmed N, Hasnain SE. Iron-dependent RNA-binding activity of Mycobacterium tuberculosis aconitase. J Bacteriol. 2007 Jun; 189(11):4046-52.