Role of apoptosis-regulating signal kinase 1 in innate immune responses by Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and ROS generation in a TLR2- and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellular-regulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-alpha and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate.     

The PGRS domain is responsible for translocation of PE_PGRS30 to cell poles while the PE and the C-terminal domains localize it to the cell wall

PE_PGRS proteins localize in the mycobacterial cell wall and the cell wall localization of PE_PGRS33 has been shown to be attributed to its PE domain. In this study, we expressed deletion mutants of PE_PGRS30 in Mycobacterium smegmatis to characterize the role of its domains in protein localization. It was revealed that, apart from the PE domain, the C-terminal domain present in few PE_PGRS proteins carries individual cell wall localization signals. Proteinase K sensitivity assay showed that PE_PGRS30 is exposed on the mycobacterial surface through its PGRS domain. PGRS domain was also shown to be responsible for polar localization of PE_PGRS30.     

Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV(+) persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-alpha. In contrast, apoptosis of differentiated HIV(+) human U1 macrophages (HIV(+) U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-alpha release, whereas apoptosis and TNF-alpha release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-alpha release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-kappaB nuclear translocation were reduced in HIV(+) U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV(+) persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-alpha release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV(+) persons.     

Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure

Identification of the novel PE multigene family was an unexpected finding of the genomic sequencing of Mycobacterium tuberculosis. Presently, the biological role of the PE and PE_PGRS proteins encoded by this unique family of mycobacterial genes remains unknown. In this report, a representative PE_PGRS gene (Rv1818c/PE_PGRS33) was selected to investigate the role of these proteins. Cell fractionation studies and fluorescence analysis of recombinant strains of Mycobacterium smegmatis and M. tuberculosis expressing green fluorescent protein (GFP)-tagged proteins indicated that the Rv1818c gene product localized in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu. Further analysis of this PE_PGRS protein showed that the PE domain is necessary for subcellular localization. In addition, the PGRS domain, but not PE, affects bacterial shape and colony morphology when Rv1818c is overexpressed in M. smegmatis and M. tuberculosis. Taken together, the results indicate that PE_PGRS and PE proteins can be associated with the mycobacterial cell wall and influence cellular structure as well as the formation of mycobacterial colonies. Regulated expression of PE genes could have implications for the survival and pathogenesis of mycobacteria within the human host and in other environmental niches.     

Functional dissection of the PE domain responsible for translocation of PE_PGRS33 across the mycobacterial cell wall

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PE(Rv1818c)), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PE(Rv1818c) domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation.     

Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses

We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-kappa B activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-kappa B activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-alpha production in both RAW 264.7 cells and primary human alveolar macrophages (AM phi). E5531 inhibited Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-alpha production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-alpha production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.     

Acylation determines the toll-like receptor (TLR)-dependent positive versus TLR2-, mannose receptor-, and SIGNR1-independent negative regulation of pro-inflammatory cytokines by mycobacterial lipomannan

Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria.     

Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation

Macrophage apoptosis is an important component of the innate immune defense machinery (against pathogenic mycobacteria) responsible for limiting bacillary viability. However, little is known about the mechanism of how apoptosis is executed in mycobacteria-infected macrophages. Apoptosis signal-regulating kinase 1 (ASK1) was activated in Mycobacterium avium-treated macrophages and in turn activated p38 mitogen-activated protein (MAP) kinase. M. avium-induced macrophage cell death could be blocked in cells transfected with a catalytically inactive mutant of ASK1 or with dominant negative p38 MAP kinase arguing in favor of a central role of ASK1/p38 MAP kinase signaling in apoptosis of macrophages challenged with M. avium. ASK1/p38 MAP kinase signaling was linked to the activation of caspase 8. At the same time, M. avium triggered caspase 8 activation, and cell death occurred in a Fas-associated death domain (FADD)-dependent manner. The death signal induced upon caspase 8 activation linked to mitochondrial death signaling through the formation of truncated Bid (t-Bid), its translocation to the mitochondria and release of cytochrome c. Caspase 8 inhibitor (z-IETD-FMK) could block the release of cytochrome c as well as the activation of caspases 9 and 3. The final steps of apoptosis probably involved caspases 9 and 3, since inhibitors of both caspases could block cell death. Of foremost interest in the present study was the finding that ASK1/p38 signaling was essential for caspase 8 activation linked to M. avium-induced death signaling. This work provides the first elucidation of a signaling pathway in which ASK1 plays a central role in innate immunity.     

ASK1-p38 MAPK-p47phox activation is essential for inflammatory responses during tuberculosis via TLR2-ROS signalling

The roles of intracellular reactive oxygen species (ROS) and related signalling pathways in mycobacterial infection are largely unknown. Here we show that tuberculin purified protein derivative (PPD)/Toll-like receptor (TLR) 2/ROS signalling through activation of apoptosis-regulating signal kinase (ASK) 1 and p47phox pathways is responsible for the induction of proinflammatory responses during tuberculosis (TB) infection. Tuberculin PPD stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs) and an early burst of ROS in monocytes/macrophages in a TLR2-dependent manner. PPD-induced ROS production led to robust activation of ASK1 upstream of p38 MAPK, via TLR2. Interestingly, phosphorylation of the cytosolic NADPH oxidase subunit p47phox and ASK1 activation are mutually dependent on PPD/TLR2-mediated signalling. Furthermore, active pulmonary TB patients showed upregulated ROS generation, as well as enhanced activation of ASK1/p38/p47phox pathways in their primary monocytes compared with healthy controls, which suggests a systemic primed status during TB. Taken together, these results indicate that activation of the ASK1/p38 MAPK/p47phox cascade plays a central role in PPD/TLR2-induced ROS generation and suggests the existence of a 'ROS/ASK1' inflammatory amplification feedback loop in monocytes/macrophages. The altered regulation of this axis with an increasing free-radical burden may contribute to the immunopathogenesis of human TB.     

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome–lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C‐terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non‐pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.     

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.     

Gliadin stimulation of murine macrophage inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the innate immune response in Celiac disease

Recent studies have demonstrated the importance of TLR signaling in intestinal homeostasis. Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. In this study, we sought to test the hypothesis that gliadin initiates this response by stimulating the innate immune response to increase intestinal permeability and by up-regulating macrophage proinflammatory gene expression and cytokine production. To this end, intestinal permeability and the release of zonulin (an endogenous mediator of gut permeability) in vitro, as well as proinflammatory gene expression and cytokine release by primary murine macrophage cultures, were measured. Gliadin and its peptide derivatives, 33-mer and p31-43, were found to be potent inducers of both a zonulin-dependent increase in intestinal permeability and macrophage proinflammatory gene expression and cytokine secretion. Gliadin-induced zonulin release, increased intestinal permeability, and cytokine production were dependent on myeloid differentiation factor 88 (MyD88), a key adapter molecule in the TLR/IL-1R signaling pathways, but were neither TLR2- nor TLR4-dependent. Our data support the following model for the innate immune response to gliadin in the initiation of CD. Gliadin interaction with the intestinal epithelium increases intestinal permeability through the MyD88-dependent release of zonulin that, in turn, enables paracellular translocation of gliadin and its subsequent interaction with macrophages within the intestinal submucosa. There, the interaction of gliadin with macrophages elicits a MyD88-dependent proinflammatory cytokine milieu that facilitates the interaction of T cells with APCs, leading ultimately to the Ag-specific adaptive immune response seen in patients with CD.     

Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages

The beta-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized that dectin-1 has potent synergistic effects with both TLR2 and TLR4 in human PBMCs and macrophages. Human PBMCs and monocyte-derived macrophages were stimulated with curdlan, a linear beta-1,3-glucan-polymer derived from Alcaligenes faecalis with specific ligand affinity for dectin-1, in combination with the synthetic TLR2 ligand Pam3Cys and the ultrapure TLR4 ligand LPS. TNF-alpha and IL-10 production was measured in the supernatants with ELISA. Curdlan is a specific dectin-1 ligand without TLR2- or TLR4-stimulating properties. Human primary monocytes and macrophages express dectin-1 on the cell membrane. Stimulation of human PBMCs with curdlan in combination with Pam3Cys or LPS leads to synergistic increase in TNF-alpha production that was inhibited by GE2, a neutralizing dectin-1 antibody. Dectin-1-dependent synergy between curdlan and TLR agonists was also apparent in human monocyte-derived macrophages. Conclusively, dectin-1 synergizes with both TLR2 and TLR4 pathways for the production of TNF-alpha in human primary PBMCs and in monocyte-derived macrophages.     

Pre-Exposure of Mycobacterium tuberculosis-Infected Macrophages to Crystalline Silica Impairs Control of Bacterial Growth by Deregulating the Balance between Apoptosis and Necrosis

Inhalation of crystalline silica (CS) particles increases the risk of pulmonary tuberculosis; however, the precise mechanism through which CS exposure facilitates Mycobacterium tuberculosis (Mtb) infection is unclear. We speculate that macrophage exposure to CS deregulates the cell death pathways that could explain, at least in part, the association observed between exposure to CS and pulmonary tuberculosis. We therefore established an in vitro model in which macrophages were exposed to CS and then infected with Mtb. Expression of surface markers was analyzed by flow cytometry, JNK1/2, ASK1, caspase 9, P-p38, Bcl-2 and Mcl-1 were analyzed by Western blot, and cytokines by ELISA. Our results show that exposure to CS limits macrophage ability to control Mtb growth. Moreover, this exposure reduced the expression of TLR2, Bcl-2 and Mcl-1, but increased that of JNK1 and ASK1 molecules in the macrophages. Finally, when the pre-exposed macrophages were infected with Mtb, the concentrations of TNFα, IL-1β and caspase-9 expression increased. This pro-inflammatory profile of the macrophage unbalanced the apoptosis/necrosis pathway. Taken together, these data suggest that macrophages exposed to CS are sensitized to cell death by MAPK kinase-dependent signaling pathway. Secretion of TNF-α and IL-1β by Mtb-infected macrophages promotes necrosis, and this deregulation of cell death pathways may favor the release of viable bacilli, thus leading to the progression of tuberculosis.     

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4(+) or CD8(+) T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-gamma-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-kappaB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-kappaB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-kappaB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL(+)-specific cytotoxic lymphocytes.     

Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9

Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.     

Expression of the PE_PGRS 33 protein in Mycobacterium smegmatis triggers necrosis in macrophages and enhanced mycobacterial survival

Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence and the infection of host cells by mycobacteria. In the studies presented here, we have expressed the PE_PGRS 33 gene in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that it survives better in macrophage cultures, in vitro as well as in mice after intraperitoneal administration, than the parental strain containing the vector only or a strain expressing only the PE domain of PE_PGRS 33. In macrophages, enhanced colonization by the M. smegmatis expressing PE_PGRS 33 was associated with macrophage aggregation and clearance of macrophage monolayers, visible cell necrosis and significantly greater levels of TNF (TNF-alpha) in the cultures compared with controls. The presence of macrophage cell necrosis was confirmed by measurement of significantly greater levels of lactate dehydrogenase and nucleosomes in the supernatants of the macrophage cultures infected with M. smegmatis expressing PE_PGRS 33. Antibodies directed against TNF partially reduced cytolysis, suggesting that this cytokine is critical but not sufficient for the observed macrophage necrosis and enhanced mycobacterial survival. These results extend earlier observations, which suggested that PE_PGRS proteins may have a role in the pathogenesis of mycobacterial disease and that there may be a specific role for these proteins in influencing host cell responses to infection.     

Activation of Ca2+-dependent signaling by TLR2

Upon contact with airway epithelial cells, bacterial products activate Ca(2+) fluxes that are required for induction of NF-kappaB-dependent gene expression. TLR2 is apically displayed on airway cells, making it a likely transducer linking bacterial stimuli and kinases that affect Ca(2+) release. Using biochemical and genetic approaches, we demonstrate that TLR2 ligands stimulate release of Ca(2+) from intracellular stores by activating TLR2 phosphorylation by c-Src, and recruiting PI3K and phospholipase Cgamma to affect Ca(2+) release through inositol (1,4,5) trisphosphate receptors. In the absence of TLR2, murine macrophages as well as airway cells do not generate Ca(2+) fluxes or induce proinflammatory signaling. Thus, Ca(2+) participates as a second messenger in TLR2-dependent signaling and provides another target to modulate proinflammatory responses to bacterial infection.