Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen's Specific Memory T Cells in Healthy Household Contacts.
In vitro mimicking conditions are thought to reflect the environment experienced by Mycobacterium tuberculosis inside the host granuloma. The majority of in vitro dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4(+) central memory T cells in the circulation of HHC compared to PTB (p < 0.005 for ESAT-6 and p < 0.0005 for Lpd). This shows proteins that are predicted to be up regulated during in vitro hypoxia in most prevalent clinical strains would indicate possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms.
Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India.
Santhi
S
Devasundaram
Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India.
Akilandeswari
A
Gopalan
Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India.
Sulochana D
SD
Das
Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India.
Alamelu
A
Raja
M. tuberculosis; hypoxia; mass spectrometry; multicolor flow cytometry; prevalent clinical strains; two-dimensional electrophoresis
PLoS Pathog. 2010 Nov 11;6(11):e1001181
21085601
Tuberculosis (Edinb). 2004;84(3-4):218-27
15207491
Microbiology. 2011 Nov;157(Pt 11):3024-35
21816881
J Exp Med. 1934 Sep 30;60(4):515-40
19870319
Tuber Lung Dis. 1995 Dec;76(6):550-4
8593378
J Bacteriol. 2001 Apr;183(8):2672-6
11274129
Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7534-9
11416222
Proteomics. 2006 Apr;6(8):2485-94
16548060
Proteome Sci. 2010 Nov 18;8:59
21083941
Proteomics. 2001 Apr;1(4):574-86
11681210
J Infect Dis. 2011 Nov 15;204 Suppl 4:S1168-78
21996699
Nature. 2000 Aug 17;406(6797):683-5
10963578
Biotechnol Appl Biochem. 2014 Mar-Apr;61(2):153-64
23826872
J Bacteriol. 2001 Sep;183(18):5311-6
11514514
Vet Microbiol. 2009 Apr 14;136(1-2):82-90
19019575
Infect Immun. 1996 Jun;64(6):2062-9
8675308
Science. 2002 Feb 8;295(5557):1073-7
11799204
J Infect Dis. 2013 Sep 1;208(5):739-48
23715661
Cell Microbiol. 2009 Aug;11(8):1151-9
19388905
Hum Immunol. 2013 Jan;74(1):45-51
23079237
Eur J Biochem. 2003 Feb;270(4):625-34
12581202
J Biotechnol. 2015 Sep 20;210:70-80
26001906
BMC Res Notes. 2013 Mar 27;6:120
23531281
Nat Rev Microbiol. 2009 Dec;7(12):845-55
19855401
Mol Cell Proteomics. 2010 Mar;9(3):538-49
20031926
Proteomics. 2010 May;10(10):1966-84
20217870
PLoS One. 2010 Jan 19;5(1):e8769
20098737
Tuberculosis (Edinb). 2010 Jan;90(1):60-9
19944647
J Proteomics. 2013 Dec 6;94:68-77
24036035
J Infect Dis. 2013 Sep;208(6):952-68
23966657
J Biol Chem. 2001 Jun 8;276(23):19845-54
11279114
FEMS Microbiol Lett. 2015 Feb;362(4):null
25724777
J Infect Dis. 2009 Sep 1;200(5):774-82
19622047
Mol Cell Proteomics. 2013 May;12(5):1180-91
23345537
Trends Microbiol. 2007 Jan;15(1):7-13
17141504
J Biol Chem. 2004 Oct 8;279(41):42584-92
15292265
PLoS Comput Biol. 2012;8(9):e1002688
23028286
Tuberculosis (Edinb). 2010 Jul;90(4):236-44
20541977
Biochemistry. 2001 Sep 25;40(38):11353-63
11560483
Cell Host Microbe. 2011 Jan 20;9(1):21-31
21238944
Crit Rev Microbiol. 2011 Nov;37(4):349-57
21919701
IUBMB Life. 2002 Apr-May;53(4-5):239-42
12121002
Tuberculosis (Edinb). 2004;84(3-4):239-46
15207493
FEBS J. 2007 Apr;274(8):1957-69
17371506
FEMS Immunol Med Microbiol. 2005 Jul 1;45(1):95-102
15985228
PLoS Pathog. 2005 Nov;1(3):269-78
16322769
Microbiology. 2004 Nov;150(Pt 11):3821-9
15528667
J Bacteriol. 2010 Mar;192(6):1662-70
20023019
Vaccine. 2010 Dec 10;29(1):51-7
20974305
Immunol Invest. 2014;43(2):137-59
24467664
Microbiology. 2000 Dec;146 Pt 12:3205-16
11101678