TDAG51 is a crucial regulator of maternal care and depressive-like behavior after parturition.

Postpartum depression is a severe emotional and mental disorder that involves maternal care defects and psychiatric illness. Postpartum depression is closely associated with a combination of physical changes and physiological stress during pregnancy or after parturition in stress-sensitive women. Although postpartum depression is relatively well known to have deleterious effects on the developing fetus, the influence of genetic risk factors on the development of postpartum depression remains unclear. In this study, we discovered a novel function of T cell death-associated gene 51 (TDAG51/PHLDA1) in the regulation of maternal and depressive-like behavior. After parturition, TDAG51-deficient dams showed impaired maternal behavior in pup retrieving, nursing and nest building tests. In contrast to the normal dams, the TDAG51-deficient dams also exhibited more sensitive depressive-like behaviors after parturition. Furthermore, changes in the expression levels of various maternal and depressive-like behavior-associated genes regulating neuroendocrine factor and monoamine neurotransmitter levels were observed in TDAG51-deficient postpartum brain tissues. These findings indicate that TDAG51 plays a protective role against maternal care defects and depressive-like behavior after parturition. Thus, TDAG51 is a maternal care-associated gene that functions as a crucial regulator of maternal and depressive-like behavior after parturition.

Recombinant Rv1654 protein of Mycobacterium tuberculosis induces mitochondria-mediated apoptosis in macrophage.

Mycobacterium tuberculosis contains diverse immunologically active components. This study investigated the biological function of a newly identified component, Rv1654, with the potential to induce apoptosis in macrophages. Recombinant Rv1654 induced macrophage apoptosis in a caspase-9/3-dependent manner through the production of reactive oxygen species (ROS) and interaction with Toll-like receptor 4. In addition, Rv1654 induced the production of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 through the mitogen-activated protein kinase pathway. Furthermore, Rv1654-induced c-Jun N-terminal kinase (JNK) activation was inhibited by the ROS scavenger and Rv1654-induced apoptosis was inhibited by the JNK inhibitor. Moreover, it was found that treatment of macrophages with Rv1654 led to the loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, and translocation of Bax into the mitochondria. Finally, Rv1654-mediated apoptosis was inhibited in macrophages transfected with Bax siRNA. These results suggest that Rv1654 induces macrophage apoptosis through a mitochondrial-dependent pathway and ROS-mediated JNK activation.

Mycobacterium tuberculosis RpfE-Induced Prostaglandin E2 in Dendritic Cells Induces Th1/Th17 Cell Differentiation.

Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Currently, there are no reports on the mycobacterial components that regulate PGE2 production. Previously, we have reported that RpfE-treated dendritic cells (DCs) effectively expanded the Th1 and Th17 cell responses simultaneously; however, the mechanism underlying Th1 and Th17 cell differentiation is unclear. Here, we show that PGE2 produced by RpfE-activated DCs via the MAPK and cyclooxygenase 2 signaling pathways induces Th1 and Th17 cell responses mainly via the EP4 receptor. Furthermore, mice administered intranasally with PGE2 displayed RpfE-induced antigen-specific Th1 and Th17 responses with a significant reduction in bacterial load in the lungs. Furthermore, the addition of optimal PGE2 amount to IL-2-IL-6-IL-23p19-IL-1ß was essential for promoting differentiation into Th1/Th17 cells with strong bactericidal activity. These results suggest that RpfE-matured DCs produce PGE2 that induces Th1 and Th17 cell differentiation with potent anti-mycobacterial activity.

Clitoromegaly, Vulvovaginal Hemangioma Mimicking Pelvic Organ Prolapse, and Heavy Menstrual Bleeding: Gynecologic Manifestations of Klippel-Trénaunay Syndrome.

Klippel-Trénaunay Syndrome (KTS) is a genetic vascular malformation involving the capillary, lymphatic, and venous channels. Prenatal sonographic diagnosis of KTS with an enlarged fetal limb is well-known; however, postnatal gynecologic manifestations are rarely reported. KTS can cause clitoromegaly, vulvovaginal hemangioma, and heavy menstrual bleeding. Somatic mosaicism of the PIK3CA gene is considered as responsible for KTS but reports based on whole-genome sequencing are limited. A 31-year-old woman with KTS presented with bulging of the clitoris and vagina. Analysis of whole-genome sequencing variant data revealed that gene ontology terms related to development and differentiation such as 'skeletal system morphogenesis', 'embryonic morphogenesis', and 'sensory organ development' were nominally significant in non-coding regions. Variants in non-coding genes may be responsible for this phenotype.

Recombinant Rv3261 protein of Mycobacterium tuberculosis induces apoptosis through a mitochondrion-dependent pathway in macrophages and inhibits intracellular bacterial growth.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen known to persist in host cells. The apoptotic response of macrophages serves as a defense mechanism to inhibit the growth of intracellular bacteria, the failure of which can favor the spread of the pathogen to new cells. However, the mycobacterial components that regulate cell death and the related underlying mechanisms remain poorly understood. In this study, we investigated protein Rv3261, isolated from an Mtb culture filtrate, for its apoptotic potential using multidimensional fractionation. Rv3261 was found to induce macrophage apoptosis through the caspase-3/-9-dependent pathway. Furthermore, the ROS-dependent JNK activation pathway was found to be critical in Rv3261-mediated apoptosis. Rv3261 inhibited the growth of intracellular Mtb, which was significantly abrogated by pre-treatment with the ROS scavenger N-acetylcysteine (NAC), suggesting that Rv3261-mediated apoptosis may act as a host defense response. These findings suggest that Rv3261 is involved in the apoptotic modulation of Mtb-infected macrophages.

Mycobacterium tuberculosis protein Rv2220 induces maturation and activation of dendritic cells.

Tuberculosis remains a serious health problem worldwide. Characterization of the dendritic cell (DC)-activating mycobacterial proteins has driven the development of effective TB vaccine candidates besides improving the understanding of immune responses. Some studies have emphasized the essential role of protein Rv2220 from M. tuberculosis in mycobacterial growth. Nonetheless, little is known about cellular immune responses to Rv2220. In this study, our aim was to test whether protein Rv2220 induces maturation and activation of DCs. Rv2220-activated DCs appeared to be in a mature state with elevated expression of relevant surface molecules and proinflammatory cytokines. DC maturation caused by Rv2220 was mediated by MAPK and NF-κB signaling pathways. Specifically, Rv2220-matured DCs induced the expansion of memory CD62LlowCD44highCD4+ T cells in the spleen of mycobacteria-infected mice. Our results suggest that Rv2220 regulates host immune responses through maturation of DCs, a finding that points to a new vaccine candidate against tuberculosis.

Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis.

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) negatively regulates the TRAF2 ubiquitin-dependent pathway by suppressing the TRAF2-sphingosine 1-phosphate (S1P) interaction.

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys(63)-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.

Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

Secretion of a truncated osteopetrosis-associated transmembrane protein 1 (OSTM1) mutant inhibits osteoclastogenesis through down-regulation of the B lymphocyte-induced maturation protein 1 (BLIMP1)-nuclear factor of activated T cells c1 (NFATc1) axis.

Genetic mutations in osteoclastogenic genes are closely associated with osteopetrotic bone diseases. Genetic defects in OSTM1 (osteopetrosis-associated transmembrane protein 1) cause autosomal recessive osteopetrosis in humans. In particular, OSTM1 mutations that exclude the transmembrane domain might lead to the production of a secreted form of truncated OSTM1. However, the precise role of the secreted form of truncated OSTM1 remains unknown. In this study, we analyzed the functional role of truncated OSTM1 in osteoclastogenesis. Here, we showed that a secreted form of truncated OSTM1 binds to the cell surface of osteoclast (OC) precursors and inhibits the formation of multinucleated OCs through the reduction of cell fusion and survival. Truncated OSTM1 significantly inhibited the expression of OC marker genes through the down-regulation of the BLIMP1 (B lymphocyte-induced maturation protein 1)-NFATc1 (nuclear factor of activated T cells c1) axis. Finally, we demonstrated that truncated OSTM1 reduces lipopolysaccharide-induced bone destruction in vivo. Thus, these findings suggest that autosomal recessive osteopetrosis patients with an OSTM1 gene mutation lacking the transmembrane domain produce a secreted form of truncated OSTM1 that inhibits osteoclastogenesis.

D-chiro-inositol negatively regulates the formation of multinucleated osteoclasts by down-regulating NFATc1.

PURPOSE: Osteoclasts (OCs) are multinucleated giant cells that resorb bone matrix. Accelerated bone destruction by OCs might cause several metabolic bone-related diseases, such as osteoporosis and inflammatory bone loss. D-pinitol (3-O-methyl-D-chiro-inositol) is a prominent component of dietary legumes and is actively converted to D-chiro-inositol, which is a putative insulin-like mediator. In this study, we analyzed the effect of D-chiro-inositol on OC differentiation. METHODS: To analyze the role of D-chiro-inositol on OC differentiation, we examined OC differentiation by the three types of osteoclastogenesis cultures with tartrate-resistant acid phosphatase (TRAP) staining and solution assay. Then, we carried out cell fusion assay with purified TRAP(+) mononuclear OC precursors. Finally, we analyzed the effect of D-chiro-inositol on OC maker expression in response to the regulation of nuclear factor of activated T cells c1 (NFATc1). RESULTS: We demonstrated that D-chiro-inositol acts as an inhibitor of receptor activator of NF-κB ligand-induced OC differentiation. The formation of multinucleated OCs by cell-cell fusion is reduced by treatment with D-chiro-inositol in a dose-dependent manner. In addition, we demonstrated that D-chiro-inositol inhibits the expression of several osteoclastogenic genes by down-regulating NFATc1. CONCLUSIONS: We have shown that D-chiro-inositol is negatively involved in osteoclastogenesis through the inhibition of multinucleated OC formation by cell-cell fusion. The expression of NFATc1 was significantly down-regulated by D-chiro-inositol in OCs and consequently, the expression of OC marker genes was significantly reduced. Hence, these results show that D-chiro-inositol might be a good candidate to treat inflammatory bone-related diseases or secondary osteoporosis in diabetes mellitus.

Mycobacterial acyl carrier protein suppresses TFEB activation and upregulates miR-155 to inhibit host defense.

Mycobacterial acyl carrier protein (AcpM; Rv2244), a key protein involved in Mycobacterium tuberculosis (Mtb) mycolic acid production, has been shown to suppress host cell death during mycobacterial infection. This study reports that mycobacterial AcpM works as an effector to subvert host defense and promote bacterial growth by increasing microRNA (miRNA)-155-5p expression. In murine bone marrow-derived macrophages (BMDMs), AcpM protein prevented transcription factor EB (TFEB) from translocating to the nucleus in BMDMs, which likely inhibited transcriptional activation of several autophagy and lysosomal genes. Although AcpM did not suppress autophagic flux in BMDMs, AcpM reduced Mtb and LAMP1 co-localization indicating that AcpM inhibits phagolysosomal fusion during Mtb infection. Mechanistically, AcpM boosted the Akt-mTOR pathway in BMDMs by upregulating miRNA-155-5p, a SHIP1-targeting miRNA. When miRNA-155-5p expression was inhibited in BMDMs, AcpM-induced increased intracellular survival of Mtb was suppressed. In addition, AcpM overexpression significantly reduced mycobacterial clearance in C3HeB/FeJ mice infected with recombinant M. smegmatis strains. Collectively, our findings point to AcpM as a novel mycobacterial effector to regulate antimicrobial host defense and a potential new therapeutic target for Mtb infection.

T-Cell Death Associated Gene 51 Is a Novel Negative Regulator of PPARγ That Inhibits PPARγ-RXRα Heterodimer Formation in Adipogenesis

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.

A Dendritic Cell-Activating Rv1876 Protein Elicits Mycobacterium Bovis BCG-Prime Effect via Th1-Immune Response.

The widely administered tuberculosis (TB) vaccine, Bacillus Calmette-Guerin (BCG), is the only licensed vaccine, but has highly variable efficiency against childhood and pulmonary TB. Therefore, the BCG prime-boost strategy is a rational solution for the development of new TB vaccines. Studies have shown that Mycobacterium tuberculosis (Mtb) culture filtrates contain proteins that have promising vaccine potential. In this study, Rv1876 bacterioferritin was identified from the culture filtrate fraction with strong immunoreactivity. Its immunobiological potential has not been reported previously. We found that recombinant Rv1876 protein induced dendritic cells' (DCs) maturation by MAPK and NF-κB signaling activation, induced a T helper type 1 cell-immune response, and expanded the population of the effector/memory T cell. Boosting BCG with Rv1876 protein enhanced the BCG-primed Th1 immune response and reduced the bacterial load in the lung compared to those of BCG alone. Thus, Rv1876 is a good target for the prime-boost strategy.

Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation.

Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

Mycobacterium tuberculosis Rv2005c Induces Dendritic Cell Maturation and Th1 Responses and Exhibits Immunotherapeutic Activity by Fusion with the Rv2882c Protein.

Immunotherapy represents a promising approach for improving current antibiotic treatments through the engagement of the host's immune system. Latency-associated antigens have been included as components of multistage subunit tuberculosis vaccines. We first identified Rv2005c, a DosR regulon-encoded protein, as a seroreactive protein. In this study, we found that Rv2005c induced dendritic cell (DC) maturation and Th1 responses, and its expression by Mycobacterium tuberculosis (Mtb) within macrophages was enhanced by treatment with CoCl2, a hypoxia-mimetic agent. T cells activated by Rv2005c-matured DCs induced antimycobacterial activity in macrophages under hypoxic conditions but not under normoxic conditions. However, Rv2005c alone did not exhibit any significant vaccine efficacy in our mouse model. The fusion of Rv2005c to the macrophage-activating protein Rv2882c resulted in significant activation of DCs and antimycobacterial activity in macrophages, which were enhanced under hypoxic conditions. Furthermore, the Rv2882c-Rv2005c fusion protein showed significant adjunctive immunotherapeutic effects and led to the generation of long-lasting, antigen-specific, multifunctional CD4+ T cells that coproduced TNF-α, IFN-γ and IL-2 in the lungs of our established mouse model. Overall, these results provide a novel fusion protein with immunotherapeutic potential as adjunctive chemotherapy for tuberculosis.

Fusion of Dendritic Cells Activating Rv2299c Protein Enhances the Protective Immunity of Ag85B-ESAT6 Vaccine Candidate against Tuberculosis.

In Mycobacterium tuberculosis infection, naïve T cells that encounter mycobacterial antigens through dendritic cells (DCs) induce various CD4+ T-cell responses; therefore, appropriate DC activation is the key for protective immunity against tuberculosis. We previously found that Rv2299c-matured DCs induce Th1 differentiation with bactericidal activity. In this study, to prove that Rv2299c could enhance the protective immunity of other vaccine candidates comprising T-cell-stimulating antigens, Ag85B-ESAT6, a well-known vaccine candidate, was selected as a fusion partner of Rv2299c. Recombinant Rv2299c-Ag85B-ESAT6 protein induced DC maturation and activation. Furthermore, fusion of Rv2299c enhanced the protective efficacy of the Ag85B-ESAT6 vaccine in a mouse model and significantly higher production of TNF-α and IL-2 was detected in the lungs, spleen, and lymph nodes of the group immunized with the Rv2299c-fused protein than with Ag85B-ESAT6. In addition, fusion of Rv2299c enhanced the Ag85B-ESAT6-mediated expansion of multifunctional CD4+ T cells. These data suggested that the DC-activating protein Rv2299c may potentiate the protective immunity of the vaccine candidate comprising T cell antigens.

Antigen-Specific IFN-γ/IL-17-Co-Producing CD4+ T-Cells Are the Determinants for Protective Efficacy of Tuberculosis Subunit Vaccine.

The antigen-specific Th17 responses in the lungs for improved immunity against Mycobacterium tuberculosis (Mtb) infection are incompletely understood. Tuberculosis (TB) vaccine candidate HSP90-ESAT-6 (E6), given as a Bacillus Calmette-Guérin (BCG)-prime boost regimen, confers superior long-term protection against the hypervirulent Mtb HN878 infection, compared to BCG or BCG-E6. Taking advantage of protective efficacy lead-out, we found that ESAT-6-specific multifunctional CD4+IFN-γ+IL-17+ T-cells optimally correlated with protection level against Mtb infection both pre-and post-challenge. Macrophages treated with the supernatant of re-stimulated lung cells from HSP90-E6-immunised mice significantly restricted Mtb growth, and this phenomenon was abrogated by neutralising anti-IFN-γ and/or anti-IL-17 antibodies. We identified a previously unrecognised role for IFN-γ/IL-17 synergism in linking anti-mycobacterial phagosomal activity to enhance host control against Mtb infection. The implications of our findings highlight the fundamental rationale for why and how Th17 responses are essential in the control of Mtb, and for the development of novel anti-TB subunit vaccines.

Mycobacterium tuberculosis acyl carrier protein inhibits macrophage apoptotic death by modulating the reactive oxygen species/c-Jun N-terminal kinase pathway.

Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host-mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms_AcpM) and a strain transformed with an empty vector (Ms_Vec). Ms_AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms_AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms_Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection.

Cell wall skeleton of Mycobacterium bovis BCG enhances the vaccine potential of antigen 85B against tuberculosis by inducing Th1 and Th17 responses.

A safe and effective adjuvant is necessary to induce reliable protective efficacy of the protein-based vaccines against tuberculosis (TB). Mycobacterial components, such as synthetic cord factor and arabinogalactan, have been used as one of the adjuvant components. Mycobacterium bovis bacillus Calmette- Guérin cell-wall skeleton (BCG-CWS) has been used as an effective immune-stimulator. However, it is not proven whether BCG-CWS can be an effective adjuvant for the subunit protein vaccine of TB. In this study, we demonstrated that the BCG-CWS effectively coupled with Ag85B and enhanced the conjugated Ag85B activity on the maturation of dendritic cells (DCs). Ag85B-BCG-CWS-matured DCs induced significant Th1 and Th17 responses when compared to BCG-CWS or Ag85B alone. In addition, significant Ag85B-specific Th1 and Th17 responses were induced in Ag85B-BCG-CWS-immunized mice before infection with M. tuberculosis and maintained after infection. Moreover, Ag85B-BCG-CWS showed significant protective effect comparable to live BCG at 6 weeks after infection and maintained its protective efficacy at 32 weeks post-challenge, whereas live BCG did not. These results suggest that the BCG-CWS may be an effective adjuvant candidate for a protein-based vaccine against TB.