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The Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in mycobacteria plays a crucial role in pathogenesis and immune evasion. These proteins characterized by unique structures with conserved sequences. This study elucidated the specific immunological functions of MMAR_1296 from marine mycobacterium. Expressing MMAR_1296 in Mycobacterium smegmatis (M. smegmatis) led to significant alterations in bacterial morphology, as well as reduced survival of M. smegmatis under adverse in vitro conditions and within macrophages. Furthermore, transcriptome analysis of mouse macrophages indicated that natural immunity-related pathways were upregulated in the group infected with M. smegmatis recombinantly expressing MMAR_1296. Moreover, the mycobacterium Growth Inhibition Assays(MGIA)in mice demonstrated that M. smegmatis expressing MMAR_1296 exerted a significant inhibitory effect against Mycobacterium abscessus (M. abscessus) and Mycobacterium marinum (M. marinum) infections. Immunization challenge experiments in mice further confirmed its protective effects, showing a reduction in organ bacterial loads by 1 log10 value compared to the positive control group. These findings indicate that MMAR_1296 is a promising vaccine candidate for M. abscessus and M. marinum. Given that PE/PPE protein family is also a crucial component of Mycobacterium tuberculosis (M. tuberculosis) antigens, further exploration of sequence functions based on MMAR_1296 could reveal broader applications of PE/PPE proteins family for M. tuberculosis treatment. This study supported vaccine development targeting PE/PPE proteins in mycobacteria and paves the way for broader applications.

Mucosal-associated invariant T (MAIT) cells exhibit an intrinsic ability to recognize and respond to microbial infections. The semi-invariant antigen recognition receptor of MAIT cells specifically detects the non-polymorphic antigen-presenting molecule, major histocompatibility complex class I-related protein 1 (MR1), which primarily binds riboflavin-derived metabolites of microbial origin. To further interrogate the dependence of these antigens on riboflavin biosynthesis in mycobacteria, we deleted individual genes in the riboflavin biosynthesis pathways in Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) and evaluated the impact thereof on MAIT cell activation. Blocking the early steps of the pathway by deletion of RibA2 or RibG profoundly reduced, but did not completely ablate, MAIT cell activation by Msm or Mtb, whereas deletion of RibC, which catalyzes the last step in the pathway, had no significant effect. Interestingly, deletion of the lumazine synthase (RibH) specifically enhanced MAIT cell recognition of Mtb whereas loss of lumazine synthase activity had no impact on MAIT cell activation by Msm. MAIT cell activation by Msm was likewise unaffected by blocking the production of the MAIT cell antagonist, F o (by inhibiting its conversion from the riboflavin pathway intermediate, 5-amino-6-D-ribitylaminouracil (5-A-RU), through the deletion of fbiC ). Together, these results confirm a central role for 5-A-RU in generating mycobacterial MR1 ligands and reveal similarities and differences between Msm and Mtb in terms of the impact of riboflavin pathway disruption on MAIT cell activation.

Bovine tuberculosis (bTB) is a chronic, productivity-limiting infection in livestock with significant zoonotic potential that is caused by members of the Mycobacterium tuberculosis-complex (MTBC) which includes Mycobacterium bovis. Although the live, attenuated M. bovis bacillus Calmette-Guérin (BCG) vaccine licensed for use in people has been shown to protect cattle against bTB in experimental settings, its capacity to sensitize vaccinated animals to tuberculin used in bTB diagnosis is a major impediment to its use in livestock. As such, a bTB vaccine that will allow for the differentiation of M. bovis-infected animals among vaccinated animals (DIVA) is preferred. In a previous study, we reported that a recombinant Mycobacterium smegmatis strain engineered to express a functional MTBC type-7 ESX-1 secretion system called MSX-1 protects C57BL/6 mice against the causative agent of human TB, M. tuberculosis, without sensitizing the vaccinated mice to tuberculin. In this study, we wanted to determine if MSX-1 will also protect mice against M. bovis. Accordingly, we found that C57BL/6 mice vaccinated with MSX-1 and challenged with M. bovis had reduced burdens of bTB bacilli in their lungs and spleens and presented with reduced lung pathology. Furthermore, MSX-1 vaccination reduced bTB-mediated weight-loss and lethality in challenged mice. Consistent with previous observations, we found that mice vaccinated with MSX-1 did not become sensitized to tuberculin nor to a peptide fragment of EsxA, a potent T-cell antigen and secreted protein of the M. tuberculosis ESX-1 system. While the lack of sensitization in mice by MSX-1 to tuberculin and EsxA underscores its promise as a DIVA bTB vaccine, it suggests the mechanism of protection of MSX-1 may be CD4+ and CD8+ T-cell and IFN-γ independent and will require further investigation. Nevertheless, our results indicate MSX-1 is an effective bTB vaccine that deserves further development for use in livestock.

Mycobacterium tuberculosis ( M. tb ) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-α, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2α, IRE1α, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. Recombinant Mycobacterium smegmatis ( M. smegmatis ) containing pe6 displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host–pathogen interaction during M. tb  infection.

New vaccine candidates are urgently needed for the control and prevention of Mycobacterium tuberculosis (Mtb) infection. Kari Sweeney and her colleagues now report that an attenuated strain of Mycobacterium smegmatis expressing the esx-3 genes from Mtb induces effective CD4 + T cell dependent immunity against infection with Mtb in mice. The study offers a new avenue for the identification of protective immunogens in Mtb infection and a candidate vaccine platform warranting further study. We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δ esx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis . Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4 + memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4 + cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis .

The success of Mycobacterium tuberculosis (M. tuberculosis ) as a pathogen is largely contributes to its ability to manipulate the host immune responses. The genome of M. tuberculosis encodes multiple immune-modulatory proteins, including several members of the multi-genic PE_PPE family. Despite of intense research, the roles of PE_PGRS proteins in mycobacterial pathogenesis remain elusive. The function of M. tuberculosis PE_PGRS41, characterized by an extended and unique C-terminal domain, was studied. Expression of PE_PGRS41 in Mycobacterium smegmatis , a non-pathogenic species intrinsically deficient of PE_PGRS, severely impaired the resistance of the recombinant to multiple stresses via altering the cell wall integrity. Macrophages infected by M. smegmatis harboring PE_PGRS41 decreased the production of TNF-α, IL-1β and IL-6. In addition, PE_PGRS41 boosted the survival of M. smegmatis within macrophage accompanied with enhanced cytotoxic cell death through inhibiting the cell apoptosis and autophagy. Taken together, these results implicate that PE_PGRS41 is a virulence factor of M. tuberculosis and sufficient to confer pathogenic properties to M. smegmatis .

Drug resistance is a prominent problem in the treatment of tuberculosis, so it is urgent to develop new anti- tuberculosis drugs. Here, we investigated the effects and mechanisms of cisplatin (DDP) on intracellular Mycobacterium smegmatis to tap the therapeutic potential of DDP in mycobacterial infection. Macrophages infected with Mycobacterium smegmatis were treated with DDP alone or combined with isoniazid or rifampicin. The results showed that the bacterial count in macrophages decreased significantly after DDP (≤ 6 μg/mL) treatment. When isoniazid or rifampicin was combined with DDP, the number of intracellular mycobacteria was also significantly lower than that of isoniazid or rifampicin alone. Apoptosis of infected cells increased after 24 h of DDP treatment, as shown by flow cytometry and transmission electron microscopy detection. Transcriptome sequencing showed that there were 1161 upregulated and 645 downregulated differentially expressed genes (DEGs) between the control group and DDP treatment group. A Trp53-centered protein interaction network was found based on the top 100 significant DEGs through STRING and Cytoscape software. The expression of phosphorylated p53, Bax, JAK, p38 MAPK and PI3K increased after DDP treatment, as shown by Western blot analysis. Inhibitors of JAK, PI3K or p38 MAPK inhibited the increase in cell apoptosis and the reduction in the intracellular bacterial count induced by DDP. The p53 promoter Kevetrin hydrochloride scavenges intracellular mycobacteria. If combined with DDP, Kevetrin hydrochloride could increase the effect of DDP on the elimination of intracellular mycobacteria. In conclusion, DDP at low concentrations could activate the JAK, p38 MAPK and PI3K pathways in infected macrophages, promote the phosphorylation of p53 protein, and increase the ratio of Bax to Bcl-2, leading to cell apoptosis, thus eliminating intracellular bacteria and reducing the spread of mycobacteria. DDP may be a new host-directed therapy for tuberculosis treatment, as well as the p53 promoter Kevetrin hydrochloride.

The innate immune system provides the first response to infection and is now recognized to be partially pathogen-specific. Mycobacterium tuberculosis (MTB) is able to subvert the innate immune response and survive inside macrophages. Curiously, only 5–10% of otherwise healthy individuals infected with MTB develop active tuberculosis (TB). We do not yet understand the genetic basis underlying this individual-specific susceptibility. Moreover, we still do not know which properties of the innate immune response are specific to MTB infection. To identify immune responses that are specific to MTB, we infected macrophages with eight different bacteria, including different MTB strains and related mycobacteria and studied their transcriptional response. We identified a novel subset of genes whose regulation was affected specifically by infection with mycobacteria. This subset includes genes involved in phagosome maturation, superoxide production, response to vitamin D, macrophage chemotaxis and sialic acid synthesis. We suggest that genetic variants that affect the function or regulation of these genes should be considered candidate loci for explaining TB susceptibility.

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Summary Toll‐like receptor (TLR)‐mediated interactions of Mycobacterium tuberculosis (M. tb) with macrophages are major determinant in the outcome of innate immune defence and subsequent adaptive immune responses. Here we report a novel interaction of the M. tb protein pair PE9 (Rv1088)–PE10 (Rv1089) with the macrophage TLR4 leading to apoptosis and modulation of cytokine levels. We demonstrate that the two proteins physically interact, and that PE9 is required for the cell wall localization of PE10 in Mycobacterium smegmatis. Interaction of the PE9–PE10 complex with TLR4 in THP‐1 macrophages was associated with increased levels of phospho‐IRF‐3, which correlated with an increase in transcript levels of its target gene interferon‐β. THP‐1 macrophages treated with PE9–PE10 complex showed multiple hallmarks of apoptosis and modulation of interleukin (IL)‐1b and IL‐10 levels. All of these effects were abrogated when cells were treated either with an antibody to PE10 or an anti‐TLR4 antibody, indicating that the complex specifically interacts with TLR4 through PE10, establishing this protein pair as a TLR4 ligand. This novel observation of two proline‐glutamate (PE) proteins forming functional heterodimers represents a considerable expansion of the PE_PPE repertoire in the context of receptor engagement and the concomitant modulation of host responses by this unique class of proteins.