Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

Introduction: The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. Methods: The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. Results: We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Discussion: Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps.

Introduction: Neutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence. Methods: Here, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy. Results: For the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation. Discussion: These findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Mycobacterium tuberculosis PPE25 and PPE26 proteins expressed in Mycobacterium smegmatis modulate cytokine secretion in mouse macrophages and enhance mycobacterial survival.

PPE25 and PPE26, the Mycobacterium tuberculosis proline-proline-glutamic acid (PPE) family proteins, are members of the M. tuberculosis ESX-5 system associated with virulence of M. tuberculosis. To investigate the roles of PPE25 and PPE26 during M. tuberculosis infection, we expressed them in non-pathogenic fast-growing Mycobacterium smegmatis, respectively, and used these recombinant strains to infect ANA-1 macrophages and BALB/c mice. We observed that both PPE25 and PPE26 enhanced survival of M. smegmatis in ANA-1 macrophages, and prolonged the persistence of M. smegmatis in mouse tissues. M. smegmatis-expressed PPE25 and PPE26 induced a significantly higher level of TNF-α and a slightly higher amount of IL-1ß, which was found to be mediated by the NF-κB, ERK and p38 pathways in ANA-1 macrophages. In addition, M. smegmatis-expressed PPE26 inhibited synthase of inducible nitric oxide and induced stronger cell necrosis. In summary, our data suggest that PPE25 and PPE26 enhance non-pathogenic M. smegmatis to survive in ANA-1 macrophages and persistence in mice, modify expression of multiple cytokines and affect host cell necrosis. Our results could help to understand the complex interactions between the host and M. tuberculosis.

PPE26 induces TLR2-dependent activation of macrophages and drives Th1-type T-cell immunity by triggering the cross-talk of multiple pathways involved in the host response.

The pathophysiological functions and the underlying molecular basis of PE /PPE proteins of M. tuberculosis remain largely unknown. In this study, we focused on the link between PPE26 and host response. We demonstrated that PPE26 can induce extensive inflammatory responses in macrophages through triggering the cross-talk of multiple pathways involved in the host response, as revealed by iTRAQ-based subcellular quantitative proteomics. We observed that PPE26 is able to specifically bind to TLR2 leading to the subsequent activation of MAPKs and NF-κB signaling. PPE26 functionally stimulates macrophage activation by augmenting pro-inflammatory cytokine production (TNF-α, IL-6 and IL-12 p40) and the expression of cell surface markers (CD80, CD86, MHC class I and II). We observed that PPE26-treated macrophages effectively polarizes naïve CD4(+) T cells to up-regulate CXCR3 expression, and to secrete IFN-γ and IL-2, indicating PPE26 contributes to the Th1 polarization during the immune response. Importantly, rBCG::PPE26 induces stronger antigen-specific TNF-α and IFN-γ activity, and higher levels of the Th1 cytokines TNF-α and IFN-γ comparable to BCG. Moreover, PPE26 effectively induces the reciprocal expansion of effector/memory CD4(+)/CD8(+) CD44(high)CD62L(low) T cells in the spleens of mice immunized with this strain. These results suggest that PPE26 may be a TLR2 agonist that stimulates innate immunity and adaptive immunity, indicating that PPE26 is a potential antigen for the rational design of an efficient vaccine against M. tuberculosis.