Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), and its incidence and mortality are increasing. The BCG vaccine was developed in the early 20th century. As the most widely administered vaccine in the world, approximately 100 million newborns are vaccinated with BCG every year, which has saved tens of millions of lives. However, due to differences in region and race, the average protective rate of BCG in preventing tuberculosis in children is still not high in some areas. Moreover, because the immune memory induced by BCG will weaken with the increase of age, it is slightly inferior in preventing adult tuberculosis, and BCG revaccination cannot reduce the incidence of tuberculosis again. Research on the mechanism of Mtb and the development of new vaccines against TB are the main strategies for preventing and treating TB. In recent years, Pro-Glu motif-containing (PE) and Pro-Pro-Glu motif-containing (PPE) family proteins have been found to have an increasingly important role in the pathogenesis and chronic protracted infection observed in TB. The development and clinical trials of vaccines based on Mtb antigens are in progress. Herein, we review the immunological effects of PE/PPE proteins and the development of common PE/PPE vaccines.

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-1β, 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.

Background: Mycobacterium tuberculosisheat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant ofMycobacterium 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 inEscherichia coliby 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 quantifyMycobacteriumviability 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 byEscherichia coligenetic 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.

Tuberculosis is an important chronic and often fatal infectious disease mainly caused by the bacterium Mycobacterium tuberculosis (Mtb). Mtb is one of the most successful pathogens that harbors several potential virulence factors not found in nonpathogenic mycobacteria. As the Mtb cell envelope is closely associated with its virulence and resistance, it is very important to understand the cell envelope for better treatment of causative pathogen. There is increasing evidence that Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins are the major effectors of virulence and persistence encoded in the Mtb H37Rv genome. However, the function of PE8 has not been explored to date. In this study, we heterologously expressed PE8 in nonpathogenic, fast-growing M. smegmatis to investigate the interaction between PE8 and the host to determine its possible biological functions. We found that recombinant M. smegmatis cells expressing PE8 were less susceptible to sodium dodecyl sulfate-induced surface stress compared with those expressing the empty vector, suggesting that PE8 may be involved in stress responses. In addition, macrophages infected with PE8-expressing M. smegmatis produced obviously lower levels of the proinflammatory factor IL-1β, IL-6, and TNF-α and higher levels of the inhibitory factor IL-10. We further found that PE8 promoted M. smegmatis survival within macrophages by inhibiting late apoptosis of macrophages. Collectively, selective targeting of the PE/PPE protein family offers an untapped opportunity to the development of more effective and safer drugs against Mtb infection.

Tao Xu, Minying Li, Chutong Wang, Meili Yuan, Xianyou Chang, Zhongqing Qian, Baiqing Li, Meiqun Sun, and Hongtao Wang. J. Microbiol. Biotechnol. 2021;31:1583-90. https://doi.org/10.4014/jmb.2106.06006