Purification and biological analysis of specific antigens (ESAT6/CFP10) from Mycobacterium tuberculosis

Document Type : Research Articles

Authors

Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.

Abstract

The pathogenesis of Mycobacterium tuberculosis (Mtb) is related to its low molecular weight proteins mainly ESAT6 and CFP10 that are highly specific and potentially useful for the diagnosis of tuberculosis. This research focused on isolation, purification, and characterization of low molecular weight proteins from Mtb. Cultures of Mtb were inactivated by heating at 68 °C for 90 min and 100 °C for 3 hrs, respectively. Inactivated cultures were filtered and the proteins in the supernatant fluid precipitated with two rounds of ammonium sulfate, at 4 °C. The collected precipitates were dialyzed and subjected to gel chromatography (G-50) and the obtained fractions were analyzed for protein concentrations and molecular weight. ESAT6 and CFP10 protein complex in the purified fraction was confirmed by Western blotting. Guinea pig sensitization assay was used for estimating the potency of the purified fraction compared to the standard PPD. The maximum amount of low molecular weight proteins were precipitated by 20% ammonium sulfate. SDS-PAGE analysis revealed protein bands of approximately 10-15 kDa. The purity of the proteins was ≥95%, as confirmed by SDS-PAGE. The presence of the ESAT-6/CFP10 complex was confirmed by Western blot analysis. The purified fractions showed no cross-reaction with BCG or M. avium strain. ESAT-6/CFP-10 purified by the ammonium sulfate method appeared to be suitable for the development of a diagnostic kit for the detection of Mtb.

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1.    Mattow J, Jungblut PR, Müller EC, Kaufmann SH. Identification of acidic, low molecular mass proteins of Mycobacterium tuberculosis strain H37Rv by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Proteomics. 2001; 1:494-507.
2.    Elston JW, Thaker HK. Co-infection with human immunodeficiency virus and tuberculosis. Indian J Dermatol Venereolleprol. 2008; 74:194-199.
3.    Seibert FB, Glen JT. PPD-S was comprised of approximately 92.1% protein, 5.9% polysaccharides and 1.2% nucleic acid. Am Rev Tuberc. 1941; 44:9-24.
4.    Behr MA, Wilson MA, Gill WP, Salamon H, Schoolnik GK, Rane S, et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science. 1999; 284:1520-1523.
5.    Huebner RE, Schein MF, Bass JB. The tuberculin skin test. Clin Infect Dis. 1993; 17:968-975.
6.    Farhat M, Greenaway C, Pai M, Menzies D. False-positive tuberculin skin tests: what is the absolute effect of BCG and non-tuberculous mycobacteria? Int J Tuberc Lung Dis. 2006; 10:1192-1204.
7.    Shingadia D, Novelli V. The tuberculin skin test: a hundred, not out? Arch Dis Child. 2008; 93:189-190.
8.    Coler RN, Skeiky YA, Ovendale PJ, Vedvick TS, Gervassi L, Guderian J, et al. Cloning of a Mycobacterium tuberculosis gene encoding a purifed protein derivative protein that elicits strong tuberculosis-specific delayed-type hypersensitivity. J Infect Dis. 2000; 182:224-233.
9.    Kumar GSS, Venugopal AK, Selvan LDN, Marimuthu A, Keerthikumar S, Pathare S, et al. Gene expression profiling of tuberculous meningitis co-infected with HIV. J Proteomics Bioinform. 2011; 5:235-244.
10.    Brodin P, Majlessi L, Marsollier L, De Jonge MI, Bottai D, Demangel C, et al. Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect Immun. 2006; 74:88-98.
11.    Meher AK, Bal NC, Chary KV, Arora A. Mycobacterium tuberculosis H37Rv ESAT-6/CFP-10 complex formation confers thermodynamic and biochemical stability. FEBS J. 2006; 273:1445-1462.
12.    Alito A, McNair J, Girvin RM, Zumarraga M, Bigi F, Pollock JM, et al. Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain. Braz J Med Biol Res. 2003; 36 (11):1523-1531.
13.    Lewinsohn DM, Grotzke JE, Heinzel AS, Zhu L, Ovendale PJ, Johnson M, et al. Secreted proteins from Mycobacterium tuberculosis gain access to the cytosolic MHC class-1 antigen-processing pathway. J Immunol. 2006; 177(1):437-442.
14.    Sørensen AL, Nagai S, Houen, G, Andersen P, Andersen AB. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun. 1995; 63(5):1710-1717.
15.    Vordermeier HM, Whelan A, Cockle PJ, Farrant L, Palmer N, Hewinson RG. Use of synthetic peptides derived from the antigens ESAT-6 and CFP-10 for differential diagnosis of bovine tuberculosis in cattle. Clin Diagn Lab Immunol. 2001; 8:571-578.
16.    Babaie M, Ghaempanah A, Mehrabi Z, Mollaei A. Partial purification and characterization of antimicrobial effects from snake (Echis carinatus), scorpion (Mesosobuthus epues) and bee (Apis mellifera) venoms. Iran J Med Microbiol. 2020; 14(5):460-477.
17.    Babaie M, Zolfagharian H, Jamili S, Zolfaghari M. Biochemical, hematological effects and complications of Pseudosynanceia melanostigma envenoming. J Pharmacopuncture. 2019; 22(3):140-146.
18.    Chevallet M, Luche S, Rabilloud T. Silver staining of proteins in polyacrylamide gels. Nat Protoc. 2006; 1(4):1852-1858.
19.    Babaie M, Zolfagharian H, Salmanizadeh H, Mirakabadi AZ, Alizadeh H. Isolation and partial purification of anticoagulant fractions from the venom of the Iranian snake Echis carinatus. Acta Biochim Pol. 2013; 60(1):17-20.
20.    Franco J, Camarena J, Nogueira J, Blanquer R, Ruiz M, Marin J. Serological response )Western blot( to fractions of Mycobacterium tuberculosis sonicate antigen in tuberculosis patients and contacts. Int J Tuberc Lung Dis. 2001; 5(10):958-962.
21.    Weldingh K, Andersen P. ESAT-6/CFP10 Skin Test Predicts Disease in M. tuberculosis-Infected Guinea Pigs. PLoS One. 2008; 3(4):1-8.
22.    Mahmoudi S, Mamishi S, Ghazi M, Sadeghi RH, Pourakbari B. Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens. Iran J Microbiol. 2013; 5(4):374-378.
23.    Van Pinxteren LA, Ravn P, Agger EM, Pollock J, Andersen P. Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10. Clin Diagn Lab Immunol. 2000; 7:155-160.
24.    Andersen P, Andersen AB, Sørensen AL, Nagai S. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol. 1995; 154(7):3359-3372.
25.    Long ER, Seibert FB. The chemical composition of the active principle of tuberculin. Am Rev Tubercc. 1926; 13:393-397.
26.    Seibert FB, Pedersen KO, Tiselius A. Molecular weight, electro chemical and biological properties of tuberculin protein and polysaccharide molecules. J Exp Med. 1938; 68:413-438.
27.    Landi S, Held HR. Physicochemical and biological studies on various preparations of tuberculin purified protein derivative. Appl Microbiol. 1965; 13(2):132-139.
28.    Orme IM, McMurray DN, Belisle JT. Tuberculosis vaccine development: recent progress. Trends Microbiol. 2001; 9:115-118.
29.    Behar SM, Dascher CC, Grusby MJ, Wang CR, Brenner MB. Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med. 1999; 189:1973-1980.
30.    Thaveekarn W, Pakmanee N, Suntrarachun S, Khow O, Udomsantisuk N. ESAT-6 protein for specific Mycobacterium tuberculosis in guinea pigs skin test. Sch Acad J Biosci. 2013; 1(7):357-362.
31.    Wu X, Feng S, Duan H, Zhang L, Zhang J, Yang Y, et al. Use of recombinant CFP-10 protein for a skin test specific for Mycobacterium tuberculosis infection. Afr J Biotechnol. 2010; 9(42):810-7185.
32.    Wu X, Zhang L, Zhang J, Zhang C, Zhu L, Shi Y. Recombinant early secreted antigen target 6 protein as a skin test antigen for the specific detection of Mycobacterium tuberculosis infection. Clin Exp Immunol. 2008; 152(1):81-87.
33.    Tebianian M, Hoseini AZ, Ebrahimi SM, Memarnejadian A, Mokarram AR, Mahdavi M, et al. Cloning, expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70. Biologicals. 2011; 39(3):143-148.  
34.    Arend SM, Ottenhoff THM, Andersen P, Dissel JTV. Uncommon presentation of tuberculosis: the potential value of a noveldiagnostic assay based on the Mycobacterium tuberculosis-specificantigens ESAT-6 and CPF-10. Int J Tuberc Lung Dis. 2001; 7:680-686.
35.    Renshaw PS, Lightbody KL, Veverka V, Muskett FW, Kelly G, Frenkiel TA, et al. Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO J. 2005; 24: 2491–2498.

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