Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals' blood cells in combination with CpG-DNA.

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.

Inhibition of mycobacteria proliferation in macrophages by low cisplatin concentration through phosphorylated p53-related apoptosis pathway.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: DDP may be a new host-directed therapy for tuberculosis treatment, as well as the p53 promoter Kevetrin hydrochloride.

A Bumpy Ride of Mycobacterial Phagosome Maturation: Roleplay of Coronin1 Through Cofilin1 and cAMP.

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.

Mycobacterium tuberculosis Protein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival.

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.

Mycobacterium tuberculosis effector PPE36 attenuates host cytokine storm damage via inhibiting macrophage M1 polarization.

Tuberculosis caused by Mycobacterium tuberculosis remains a serious global public health threat. Macrophage polarization is crucial for the innate immunity against M. tuberculosis. However, how M. tuberculosis interferes with macrophage polarization is elusive. We demonstrated here that M. tuberculosis PPE36 (Rv2108) blocked macrophage M1 polarization, preventing the cytokine storm, and alleviating inflammatory damage to mouse immune organs. PPE36 inhibited the polarization of THP-1 cell differentiation to M1 macrophages, reduced mitochondrial dehydrogenase activity, inhibited the expression of CD16, and repressed the expression of pro-inflammatory cytokines IL-6 and TNF-α, as well as chemokines CXCL9, CXCL10, CCL3, and CCL5. Intriguingly, in the mouse infection model, PPE36 significantly alleviated the inflammatory damage of immune organs caused by a cytokine storm. Furthermore, we found that PPE36 inhibited the polarization of macrophages into mature M1 macrophages by suppressing the ERK signaling. The study provided novel insights into the function and mechanism of action of M. tuberculosis effector PPE36 both at the cellular and animal level.

Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling.

Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.

Host phospholipase C-γ1 impairs phagocytosis and killing of mycobacteria by J774A.1 murine macrophages.

Macrophages represent the first line of defense against invading Mycobacterium tuberculosis (Mtb). In order to enhance intracellular survival, Mtb targets various components of the host signaling pathways to limit macrophage functions. The outcome of Mtb infection depends on various factors derived from both host and pathogen. A detailed understanding of such factors operating during interaction of the pathogen with the host is a prerequisite for designing new approaches for combating mycobacterial infections. This work analyzed the role of host phospholipase C-γ1 (PLC-γ1) in regulating mycobacterial uptake and killing by J774A.1 murine macrophages. Small interfering RNA mediated knockdown of PLC-γ1 increased internalization and reduced the intracellular survival of both Mtb and Mycobacterium smegmatis (MS) by macrophages. Down-regulation of the host PLC-γ1 was observed during the course of mycobacterial infection within these macrophages. Finally, Mtb infection also suppressed the expression of pro-inflammatory cytokine tumor necrosis factor-α and chemokine (C-C motif) ligand 5 (RANTES) which was restored by knocking down PLC-γ1 in J774A.1 cells. These observations suggest a role of host PLC-γ1 in the uptake and killing of mycobacteria by murine macrophages.

Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems.

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

Natural T Cell Epitope Containing Methyl Lysines on Mycobacterial Heparin-Binding Hemagglutinin.

T cell epitopes are mostly nonmodified peptides, although posttranslationally modified peptide epitopes have been described, but they originated from viral or self-proteins. In this study, we provide evidence of a bacterial methylated T cell peptide epitope. The mycobacterial heparin-binding hemagglutinin (HBHA) is a protein Ag with a complex C-terminal methylation pattern and is recognized by T cells from humans latently infected with Mycobacterium tuberculosis By comparing native HBHA with recombinant HBHA produced in Mycobacterium smegmatis (rHBHA-Ms), we could link antigenic differences to differences in the methylation profile. Peptide scan analyses led to the discovery of a peptide containing methyl lysines recognized by a mAb that binds to native HBHA ∼100-fold better than to rHBHA-Ms This peptide was also recognized by T cells from latently infected humans, as evidenced by IFN-γ release upon peptide stimulation. The nonmethylated peptide did not induce IFN-γ, arguing that the methyl lysines are part of the T cell epitope.

Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

HMGN2 regulates non-tuberculous mycobacteria survival via modulation of M1 macrophage polarization.

Non-tuberculous mycobacteria (NTM), also known as an environmental and atypical mycobacteria, can cause the chronic pulmonary infectious diseases. Macrophages have been suggested as the main host cell to initiate the innate immune responses to NTM infection. However, the molecular mechanism to regulate the antimicrobial immune responses to NTM is still largely unknown. Current study showed that the NTM clinical groups, Mycobacterium abscessus and Mycobacterium smegmatis, significantly induced the M1 macrophage polarization with the characteristic production of nitric oxide (NO) and marker gene expression of iNOS, IFNγ, TNF-α, IL1-ß and IL-6. Interestingly, a non-histone nuclear protein, HMGN2 (high-mobility group N2), was found to be spontaneously induced during NTM-activated M1 macrophage polarization. Functional studies revealed that HMGN2 deficiency in NTM-infected macrophage promotes the expression of M1 markers and the production of NO via the enhanced activation of NF-κB and MAPK signalling. Further studies exhibited that HMGN2 knock-down also enhanced IFNγ-induced M1 macrophage polarization. Finally, we observed that silencing HMGN2 affected the survival of NTM in macrophage, which might largely relevant to enhanced macrophage polarization into M1 phenotype under the NTM infection. Collectively, current studies thus suggested a novel function of HMGN2 in regulating the anti-non-tuberculous mycobacteria innate immunity of macrophage.

Short communication: Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, causing pyogranulomatous mastitis and its cross-reactivity in bovine (para)tuberculosis testing.

Different mycobacterial species are encountered in bovine medicine. The fastidiously growing mycobacteria (Mycobacterium bovis as the cause of bovine tuberculosis, and Mycobacterium avium ssp. paratuberculosis, MAP, as the cause of paratuberculosis) are well known and targeted in eradication/control or monitoring programs in different countries, whereas the rapidly growing species is only rarely identified from bovine disease. The latter have occasionally been reported as the cause of bovine clinical mastitis, but recent reports are scarce. In this study, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis) was identified as cause of granulomatous, relapsing clinical mastitis in 2 cows from one Belgian dairy herd. Milk, blood, and fecal samples were collected, as well as tissue samples after the cows were culled. Serological analysis conducted on milk and serum samples resulted in positive reactions for MAP, but negative for Mycobacterium bovis. Production of IFN-γ showed sensitization with mycobacteria or similar organisms, other than M. bovis, in one cow. Detection of MAP by bacteriological culture and IS900-based quantitative PCR on milk and feces remained negative. In conclusion, this paper describes M. smegmatis as a cause of bovine clinical mastitis in Belgium and suggests cross-reactivity of the intramammary M. smegmatis infection with routinely used serological tests for MAP.

Mannosylated structures of mycobacterial lipoarabinomannans facilitate the maturation and activation of dendritic cells.

Lipoarabinomannan (LAM) is an important virulent factor secreted by mycobacteria, which generally elicit a strong immune response in the host. In this study, the structural difference of LAMs from three mycobacterial strains, Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155 and a newly discovered clinical isolate, M. sp. QGD101, was analyzed and further evaluated whether these LAMs can induce DC maturation and promote the immunomodulatory properties. The results reveal that the major structural difference of these LAMs is the amount of mannosyl residues, especially at the terminal end of LAM, which play a key role in determining the divergent response of DCs after mycobacterial infection. Also, this study indicates an important relevance between the glycosylated structure of LAM and its immunomodulatory property, which is helpful to develop a potential approach for identification of different mycobacteria and also lays a foundation for the development of a novel polysaccharide immunological strategy against tuberculosis.

Mycobacterium smegmatis Induces Neurite Outgrowth and Differentiation in an Autophagy-Independent Manner in PC12 and C17.2 Cells.

Both pathogenic and non-pathogenic Mycobacteria can induce the differentiation of immune cells into dendritic cells (DC) or DC-like cells. In addition, pathogenic Mycobacteria is found to stimulate cell differentiation in the nerves system. Whether non-pathogenic Mycobacteria interacts with nerve cells remains unknown. In this study, we found that co-incubation with fast-growing Mycobacteria smegmatis induced neuron-like morphological changes of PC12 and C17.2 cells. Moreover, the M. smegmatis culture supernatant which was ultrafiltrated through a membrane with a 10 kDa cut-off, induced neurite outgrowth and differentiation in an autophagy-independent pathway in PC12 and C17.2 cells. Further analysis showed that IFN-γ production and activation of the PI3K-Akt signaling pathway were involved in the neural differentiation. In conclusion, our finding demonstrated that non-pathogenic M. smegmatis was able to promote neuronal differentiation by its extracellular proteins, which might provide a novel therapeutic strategy for the treatment of neurodegenerative disorders.

LpqT improves mycobacteria survival in macrophages by inhibiting TLR2 mediated inflammatory cytokine expression and cell apoptosis.

Tuberculosis is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb). LpqT is a lipoprotein of Mtb identified as a candidate virulence factor by a high-throughput screen searching for genes important for mycobacteria intracellular survival. To investigate its function, we constructed M. smegmatis strains deficient of LpqT or overexpressing LpqT. Wildtype or LpqT modified M. smegmatis strains were used to infect macrophages and mice, and intracellular survival of mycobacteria was measured. We found that LpqT can improve M. smegmatis survival in macrophage cell line, bone marrow derived macrophages (BMDMs), and murine lungs. This survival promoting effect is dependent on TLR2 and Myd88. Western blot analysis of M. smegmatis infected macrophages showed that LpqT suppressed M. smegmatis induced NF-κB and MAPK phosphorylation, indicating that LpqT hampered TLR2 signal activation. In consistent with this, LpqT inhibited M. smegmatis induced inflammatory cytokine expression and cell apoptosis in macrophages, thus supported mycobacteria intracellular survival.

MptpB Promotes Mycobacteria Survival by Inhibiting the Expression of Inflammatory Mediators and Cell Apoptosis in Macrophages.

Tuberculosis is a severe contagious disease caused by Mycobacterium tuberculosis (Mtb). To develop new vaccines and medicine against TB, there is an urgent need to provide insights into the mechanisms by which Mtb induces tuberculosis. In this study, we found that secreted Mtb virulence factor MptpB significantly enhanced the survival of H37Rv in macrophages. MptpB suppressed the production of iNOS, the expression of inflammatory factors IL-1ß and IL-6, as well as the apoptosis of the macrophage in Mtb infected RAW264.7 cells. Mechanism investigation showed that MptpB simultaneously hampered the NF-κB and MAPK signal pathways, evidenced by its blocking of p65, IKKα, Erk1/2, and p38 phosphorylation induced by Mtb infection. MptpB also inhibited host cell p53 expression. The results demonstrated that MptpB contributed to the survival of H37Rv by inhibiting host inflammatory responses and apoptosis through impeding the NF-κB and MAPK signal pathways and p53 expression in the macrophage.

Expression of Rv2031c-Rv2626c fusion protein in Mycobacterium smegmatis enhances bacillary survival and modulates innate immunity in macrophages.

Dormancy-associated antigens encoded by the dormancy survival regulon (DosR) genes are required for survival of Mycobacterium tuberculosis (Mtb) in macrophages. However, mechanisms underlying survival of Mtb in macrophages remains to be elucidated. A recombinant Mycobacterium smegmatis strain (rMs) expressing a fusion protein of two dormancy­associated antigens Rv2031c and Rv2626c from Mtb was constructed in the present study. In an in vitro culture, growth rate of rMs was lower compared with Ms. A total of 24 h following infection of murine macrophages with rMs or Ms, percentage of viable cells decreased and the number of bacteria in viable cells increased compared with Ms, demonstrating that virulence and intracellular survival of rMs were enhanced. Compared with macrophages infected with Ms, necrosis of macrophages infected with rMs was increased, while apoptosis was inhibited. Macrophages infected with rMs secreted more interferon­Î³ and interleukin­6, but fewer nitric oxide and tumor necrosis factor­α, compared with macrophages infected with Ms. The present study demonstrated that the fusion protein composed of dormancy­associated antigens Rv2031c and Rv2626c in Ms serves a physiological function of a dormancy­associated antigen and modulates innate immunity of host macrophages, therefore favoring intracellular bacillary survival.

Identification of antigenic proteins from Mycobacterium avium subspecies paratuberculosis cell envelope by comparative proteomic analysis.

Johne's disease (JD) is a contagious, chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The aim of this study was to identify antigenic proteins from the MAP cell envelope (i.e. cell wall and cytoplasmic membranes) by comparing MAP, M. avium subsp. hominissuis (MAH) and M. smegmatis (MS) cell envelope protein profiles using a proteomic approach. Composite two-dimensional (2D) difference gel electrophoresis images revealed 13 spots present only in the image of the MAP cell envelope proteins. Using serum from MAP-infected cattle, immunoblot analysis of 2D gels revealed that proteins in the 13 spots were antigenic. These proteins were identified by liquid chromatography tandem mass spectrometry as products of the following genes: sdhA, fadE25_2, mkl, citA, gapdh, fadE3_2, moxR1, mmp, purC, mdh, atpG, fbpB and desA2 as well as two proteins without gene names identified as transcriptional regulator (MAP0035) protein and hypothetical protein (MAP1233). Protein functions ranged from energy generation, cell wall biosynthesis, protein maturation, bacterial replication and invasion of epithelial cells, functions considered essential to MAP virulence and intracellular survival. Five MAP cell envelope proteins, i.e. SdhA, FadE25_2, FadE3_2, MAP0035 and DesA2 were recombinantly expressed, three of which, i.e. SdhA, FadE25_2 and DesA2, were of sufficient purity and yield to generate polyclonal antibodies. Immunoblot analysis revealed antibodies reacted specifically to the respective MAP cell envelope proteins with minimal cross-reactivity with MAH and MS cell envelope proteins. Identification and characterization of MAP-specific proteins and antibodies to those proteins may be useful in developing new diagnostic tests for JD diagnosis.

Mycobacterium tuberculosis Lipoprotein MPT83 Induces Apoptosis of Infected Macrophages by Activating the TLR2/p38/COX-2 Signaling Pathway.

Tuberculosis caused by Mycobacterium tuberculosis continues to pose a serious global health threat. The attenuated Mycobacterium bovis bacillus Calmette-Guérin, as the only licensed vaccine, has limited protective efficacy against TB. The development of more effective antituberculosis vaccines is urgent and demands for further identification and understanding of M. tuberculosis Ags. MPT83 (Rv2873), a secreted mycobacterial lipoprotein, has been applied into subunit vaccine development and shown protective effects against M. tuberculosis infection in animals; however, the understanding of the underlying mechanism is limited. In present study, we systematically studied the effect of MPT83 on macrophage apoptosis by constructing Mycobacterium smegmatis strain overexpressing MPT83 (MS_MPT83) and purifying rMPT83 protein. We found that MPT83 induced apoptosis in both human and mouse macrophages. MPT83 induced cyclooxygenase-2 (COX-2) expression at both the transcriptional and protein levels in macrophages, whereas silencing or inhibiting COX-2 blocked rMPT83-induced apoptosis or the enhanced apoptotic response to MS_MPT83 in comparison with M. smegmatis transfected with pMV261 vector (MS_Vec), indicating that COX-2 is required for MPT83-induced apoptosis. Additionally, tlr2 deficiency led to significant reduction of COX-2 expression, accompanied by less apoptosis in macrophages stimulated with rMPT83 or infected with MS_MPT83. Moreover, the activation of p38 accounted for MPT83-induced COX-2 expression. Finally, lower bacteria burdens in the lungs and spleens and enhanced survival were observed in mice i.v. infected with MS_MPT83 compared with MS_Vec. Taken together, our results established a proapoptotic effect of MPT83 and identified the TLR2/p38/COX-2 axis in MPT83-induced macrophage apoptosis.

The R753Q polymorphism in Toll-like receptor 2 (TLR2) attenuates innate immune responses to mycobacteria and impairs MyD88 adapter recruitment to TLR2.

Toll-like receptor 2 (TLR2) plays a critical role in host defenses against mycobacterial infections. The R753Q TLR2 polymorphism has been associated with increased incidence of tuberculosis and infections with non-tuberculous mycobacteria in human populations, but the mechanisms by which this polymorphism affects TLR2 signaling are unclear. In this study, we determined the impact of the R753Q TLR2 polymorphism on macrophage sensing of Mycobacterium smegmatis Upon infection with M. smegmatis, macrophages from knock-in mice harboring R753Q TLR2 expressed lower levels of TNF-α, IL-1ß, IL-6, and IL-10 compared with cells from WT mice, but both R753Q TLR2- and WT-derived macrophages exhibited comparable bacterial burdens. The decreased cytokine responses in R753Q TLR2-expressing macrophages were accompanied by impaired phosphorylation of IL-1R-associated kinase 1 (IRAK-1), p38, ERK1/2 MAPKs, and p65 NF-κB, suggesting that the R753Q TLR2 polymorphism alters the functions of the myeloid differentiation primary response protein 88 (MyD88)-IRAK-dependent signaling axis. Supporting this notion, HEK293 cells stably transfected with YFP-tagged R753Q TLR2 displayed reduced recruitment of MyD88 to TLR2, decreased NF-κB activation, and impaired IL-8 expression upon exposure to M. smegmatis Collectively, our results indicate that the R753Q polymorphism alters TLR2 signaling competence, leading to impaired MyD88-TLR2 assembly, reduced phosphorylation of IRAK-1, diminished activation of MAPKs and NF-κB, and deficient induction of cytokines in macrophages infected with M. smegmatis.