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Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

ABSTRACT: Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8- and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel ? strands stacked between ? helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely (127)ALDITLPMPPR(137) and (138)WTQVPDPNVPDAFVVIADR(156),are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping.

SUBMITTER: Kundu P

PROVIDER: S-EPMC4933155 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

Kundu Prasun P Biswas Rupam R Mukherjee Somnath S Reinhard Linda L Dutta Anirudha A Mueller-Dieckmann Jochen J Weiss Manfred S MS Pal Nishit Kumar NK Das Amit Kumar AK

The Journal of biological chemistry 20160504 27

Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8- and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel β strands stacked between α helices. Five probable epitopes have been located ...[more]

PMID: 27189947

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Project description:Lymphoproliferation and gamma interferon (IFN-gamma) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin- and lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-gamma production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-gamma response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-gamma secretion, respectively, in the majority of reactive subjects tested in this study. These results could be of value in the development of a subunit vaccine for tuberculosis and leprosy.

Dataset Information

Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

Publications

Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

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