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

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 αß 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.

Transcriptional analysis of human peripheral blood mononuclear cells stimulated by Mycobacterium tuberculosis antigen.

Background: Mycobacterium tuberculosis antigen (Mtb-Ag) is a polypeptide component with a molecular weight of 10-14 kDa that is obtained from the supernatant of the H37Ra strain after heat treatment. It stimulates the activation and proliferation of γδT cells in the blood to produce an immune response against tuberculosis. Mtb-Ag is therefore crucial for classifying and detecting the central genes and key pathways involved in TB initiation and progression. Methods: In this study, we performed high-throughput RNA sequencing of peripheral blood mononuclear cells (PBMC) from Mtb-Ag-stimulated and control samples to identify differentially expressed genes and used them for gene ontology (GO) and a Kyoto Encyclopedia of Genomes (KEGG) enrichment analysis. Meanwhile, we used PPI protein interaction network and Cytoscape analysis to identify key genes and qRT-PCR to verify differential gene expression. Single-gene enrichment analysis (GSEA) was used further to elucidate the potential biological functions of key genes. Analysis of immune cell infiltration and correlation of key genes with immune cells after Mtb-Ag-stimulated using R language. Results: We identified 597 differentially expressed genes in Mtb-Ag stimulated PBMCs. KEGG and GSEA enrichment analyzed the cellular pathways related to immune function, and DEGs were found to be primarily involved in the TNF signaling pathway, the IL-17 signaling pathway, the JAK-STAT signaling pathway, cytokine-cytokine receptor interactions, and the NF-κB signaling pathway. Wayne analysis using GSEA, KEGG, and the protein-protein interaction (PPI) network showed that 34 genes, including PTGS2, IL-1ß, IL-6, TNF and IFN-γ et al., were co-expressed in the five pathways and all were up-regulated by Mtb-Ag stimulation. Twenty-four DEGs were identified using qRT-PCR, including fourteen up-regulated genes (SERPINB7, IL20, IFNG, CSF2, PTGS2, TNF-α, IL36G, IL6, IL10, IL1A, CXCL1, CXCL8, IL4, and CXCL3) and ten down-regulated genes (RTN1, CSF1R CD14, C5AR1, CXCL16, PLXNB2, OLIG1, EEPD1, ENG, and CCR1). These findings were consistent with the RNA-Seq results. Conclusion: The transcriptomic features associated with Mtb-Ag provide the scientific basis for exploring the intracellular immune mechanisms against Mtb. However, more studies on these DEGs in pathways associated with Mtb-Ag stimulation are needed to elucidate the underlying pathologic mechanisms of Mtb-Ag during Mtb infection.