Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function

Abstract

Background: Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown.

Methods: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.

Results: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.

Conclusion: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

Keywords: Mycobacterium tuberculosis heat resistant antigen (Mtb-HAg); IFN-γ; TNF-α; γδ T cell.

Fig. 1

Workflow for analysing the components of Mtb-HAg and their regulation of γδ T-cell function

Fig. 2

Electrophoresis analyses of PCR products. M: DNA Marker; 1: HtpG; 2: DnaK; 3: GroEL2; 4: GroEL1; 5: HspX; 6: GroES; 7: HbhA; 8: Mpt63; 9: EsxB; 10: EsxJ; 11: EsxA; 12: EsxN

Fig. 3

SDS‒PAGE analysis of the recombinant proteins expressed in Escherichia coli. M: Protein marker; 1: Escherichia coli BL21 without transformation; 2: Recombinant cells before induction with IPTG; 3: Recombinant cells after induction with IPTG; 4: Supernatant of lysate; 5: Precipitate of lysate. A HtpG; B DnaK; C GroEL2; D GroEL1; E HspX; F GroES; G HbhA; H Mpt63; I EsxB; J EsxJ; K EsxA; L EsxN

Fig. 4

The purified recombinant proteins were analysed by SDS‒PAGE and Western blotting. A SDS‒PAGE analysis of the purified recombinant proteins. M: Protein marker; 1: HtpG; 2: DnaK; 3: GroEL2; 4: GroEL1; 5: HspX; 6: GroES; 7: EsxN; 8: HbhA; 9: Mpt63; 10: EsxB; B Western blot analysis of the purified recombinant proteins. M: Protein Marker; 1: HtpG; 2: DnaK; 3: GroEL2; 4: GroEL1; 5: HspX; 6: GroES; 7: EsxN; 8: HbhA; 9: Mpt63; 10: EsxB

Fig. 5

Analysis of Mtb-HAg and recombinant proteins stimulated peripheral blood γδ T-cell proliferation. A Flow chart of γδ T-cell proliferation. B Statistical analysis of γδ T-cell proliferation. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6

Proportion of secreted TNF-α-producing cells in Mtb-HAg and recombinant protein-stimulated peripheral blood γδ T cells and αβ T cells. A Flow cytometric analysis of TNF-α production by specifically expanded γδ T cells. B Statistical analysis of TNF-α production by specifically expanded γδ T cells (***P < 0.001, **P < 0.01). C Flow cytometric analysis of TNF-α production by specifically expanded αβ T cells. D Statistical analysis of TNF-α production by specifically expanded αβ T cells (***P < 0.001, **P < 0.01)

Fig. 7

Proportion of secreted IFN-γ-producing cells in Mtb-HAg and component-stimulated peripheral blood γδ T cells and αβ T cells. A Flow cytometric analysis of IFN-γ production by specifically expanded γδ T cells. B Statistical analysis of IFN-γ production by specifically expanded γδ T cells (***P < 0.001, **P < 0.01). C Flow cytometric analysis of IFN-γ production by specifically expanded αβ T cells. D Statistical analysis of IFN-γ production by specifically expanded αβ T cells (***P < 0.001, **P < 0.01)

Fig. 8

Expanded γδ T by GroES, GroEL1, HspX cells inhibit intracellular H37Ra growth in THP-1 cells, respectively (**P < 0.01)