Highly ordered supra-molecular organization of the mycobacterial lipoarabinomannans in solution. Evidence of a relationship between supra-molecular organization and biological activity

The complex mycobacterial mannosylated lipoarabinomannans (ManLAMs) are currently considered to be the major virulence factors of the pathogenic Mycobacterium tuberculosis. The recognition and the interaction of ManLAMs with immune system receptors have been shown to promote M.tuberculosis phagocytosis but also to down-regulate the bactericidal immune response of the host in favor of the survival of the pathogenic bacilli. To date these original biological activities were mainly associated to the presence of mannose residues capping the non-reducing ends of the ramified polysaccharide moiety of these complex lipoglycans. However, we demonstrated recently that the molecular recognition of ManLAM terminal mannose units by human pulmonary surfactant protein A (hSP-A) carbohydrate recognition domains depends on the presence of the lipid moiety of the ManLAMs as proposed by Sidobre et al. in 2002. Thus, we investigated the putative role of the ManLAM aglycon moiety. The data presented here, indicate that the hydrophobic aglycon part of ManLAM is associated to a characteristic concentration-dependent supra-molecular organization of these complex molecules. Furthermore, we observed that the deacylated ManLAMs or the lipid-free mannosylated arabinomannans, which do not exhibit characteristic ManLAM activities, do not display this supra-molecular organization. These observations strongly suggest that the ManLAMs immunomodulatory activities might be associated to their particular organization. Finally, the determination of the critical micellar concentration of ManLAMs obviously supports the notion that this supra-molecular organization may be responsible for the specific biological activities of these complex molecules.     

New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level

Lipoarabinomannans are key molecules of the mycobacterial envelopes involved in many steps of tuberculosis immunopathogenesis. Several of the biological activities of lipoarabinomannans are mediated by their ability to bind human C-type lectins, such as the macrophage mannose receptor, the mannose-binding protein and the surfactant proteins A and D. The lipoarabinomannan mannooligosaccharide caps have been demonstrated to be involved in the binding to the lectin carbohydrate recognition domains. We report an original analytical approach, based on capillary electrophoresis monitored by laser-induced fluorescence, allowing the absolute quantification, in nanomole quantities of lipoarabinomannan, of the number of mannooligosaccharide units per lipoarabinomannan molecule. Moreover, this analytical approach was successful for the glycosidic linkage determination of the mannooligosaccharide motifs and has been applied to the comparative analysis of parietal and cellular lipoarabinomannans of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv, H37Ra and Erdman strains. Significant differences were observed in the amounts of the various mannooligosaccharide units between lipoarabinomannans of different strains and between parietal and cellular lipoarabinomannans of the same strain. Nevertheless, no relationship was found between the number of mannooligosaccharide caps and the virulence of the corresponding strain. The results of the present study should help us to gain more understanding of the molecular basis of lipoarabinomannan discrimination in the process of binding to C-type lectins.     

Gene expression profiling of lipoarabinomannan-treated mouse macrophage cultures infected with Mycobacterium bovis BCG

The mannosylated lipoarabinomanan (ManLAM) from mycobacterial species possesses strong anti-apoptotic action. Here we examined the ability of ManLAM isolated from Mycobacterium tuberculosis H37Rv to alter expression profiles of apoptosis-related genes in mouse macrophages infected with Mycobacterium bovis BCG Danish strain. ManLAM suppressed BCG-induced apoptosis and activities of caspase-1, -3, -8 and 9. Mouse Apoptosis Gene Array showed that ManLAM significantly down-regulated pro-apoptotic and proinflammatory genes: caspase-1, -3, -7, -8 and -9, TNF-alpha/TNFSF2, Fas/TNFRSF6, Bax-alpha, as well as IL-12 p35 and iNOS simultaneously up-regulating anti-apoptotic genes such as Bcl-2 and Mcl-1. The effect of ManLAM was contrary to BCG-induced up-regulation of proapoptotic and pro-inflammatory genes and consistent with the functional data.     

Divergent effects of mycobacterial cell wall glycolipids on maturation and function of human monocyte-derived dendritic cells

Background

Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system.

Principal Findings

We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect.

Conclusions

These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.     

Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.     

利用表型芯片系统高通量分析H37Ra与H37Rv差异表型

H37Rv是结核分枝杆菌标准有毒株,H37Ra是从H37Rv获得的稳定减毒株,但目前H37Ra毒力减弱原因尚不完全清楚。本研究利用表型芯片系统,高通量分析H37Ra生长表型,并与H37Rv表型比较,筛选两菌株表型差异,分析与H37Ra毒力减弱可能的相关表型及分子机制。结果发现,与H37Rv相比,H37Ra耐酸及耐渗透压能力显著下降,且不能利用丁二酸单甲酯和吐温40作为碳源。结核分枝杆菌耐酸能力直接影响其在吞噬体中的生存和代谢,耐高渗能力影响其必需营养物质的跨膜运输,代谢途径的改变影响其在宿主内的能量摄取,三者改变均可能与H37Ra毒力减弱相关。     

Cloning and characterization of GTP-binding proteins of Mycobacterium tuberculosis H(37)Rv

GTP-binding proteins (G-proteins) are highly conserved signaling molecules that participate in cellular signaling and bacterial pathogenesis by regulating the activity of cognate GTPases. However, the exact role of G-proteins in the pathogenesis of Mycobacterium tuberculosis is poorly understood. The complete genome sequence of M. tuberculosis H(37)Rv, suggests the presence of several homologs of bacterial G-proteins. In the present study, three G-proteins, Era, Obg and LepA of M. tuberculosis H(37)Rv were cloned and expressed in Escherichia coli. Purified proteins showed GTP-binding and hydrolyzing activities. A point mutation in the conserved GTP-binding motif, AspXXGly (Asp to Ala) in Era (Asp-258) and Obg (Asp-212) proteins resulted in the loss of the associated activities, confirming that known key residues in well-established G-proteins are also conserved in mycobacterial homologs. This study confirms that Era, Obg and LepA of M. tuberculosis H(37)Rv possess GTPase activity and provide a platform to understand the physiological significance of these proteins in associated pathogenesis.     

Dataset of potential targets for Mycobacterium tuberculosis H37Rv through comparative genome analysis

Mycobacterium tuberculosis is the causative agent of the disease, tuberculosis and H37Rv is the most studied clinical strain. We use comparative genome analysis of Mycobacterium tuberculosis H37Rv and human for the identification of potential targets dataset. We used DEG (Database of Essential Genes) to identify essential genes in the H37Rv strain. The analysis shows that 628 of the 3989 genes in Mycobacterium tuberculosis H37Rv were found to be essential of which 324 genes lack similarity to the human genome. Subsequently hypothetical proteins were removed through manual curation. This further resulted in a dataset of 135 proteins with essential function and no homology to human.     

A single‐stranded DNA aptamer against mannose‐capped lipoarabinomannan enhances anti‐tuberculosis activity of macrophages through downregulation of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ expression

Mannose‐capped lipoarabinomannan (ManLAM) is an immunomodulatory epitope of Mycobacterium tuberculosis (Mtb). An aptamer (ZXL1) that specifically binds to ManLAM from the virulent Mtb H37Rv strain was previously generated and it was found that ZXL1 functions as an antagonist, inhibiting the ManLAM‐induced immunosuppression of DCs. In the present study, it was found that ZXL1 inhibits Mtb entry into murine macrophages and that ZXL1 enhances IL‐1β and IL‐12 mRNA expression and cytokine production in ManLAM‐treated macrophages but decreases IL‐10 production. Inducible nitric oxide synthase expression in macrophages was upregulated in the presence of ZXL1 after stimulation with ManLAM. ZXL1 was also found to inhibit expression of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ (PPAR‐γ). These results suggest that ZXL1 promotes anti‐tuberculosis activity through downregulation of PPAR‐γ expression, which may contribute to M1 macrophage polarization and Mtb killing by macrophages.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan

Interactions between dendritic cells (DCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, most likely play a key role in anti-mycobacterial immunity. We have recently shown that M. tuberculosis binds to and infects DCs through ligation of the DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and that M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) inhibits binding of the bacilli to the lectin, suggesting that ManLAM might be a key DC-SIGN ligand. In the present study, we investigated the molecular basis of DC-SIGN ligation by LAM. Contrary to what was found for slow growing mycobacteria, such as M. tuberculosis and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin, our data demonstrate that the fast growing saprophytic species Mycobacterium smegmatis hardly binds to DC-SIGN. Consistent with the former finding, we show that M. smegmatis-derived lipoarabinomannan, which is capped by phosphoinositide residues (PILAM), exhibits a limited ability to inhibit M. tuberculosis binding to DC-SIGN. Moreover, using enzymatically demannosylated and chemically deacylated ManLAM molecules, we demonstrate that both the acyl chains on the ManLAM mannosylphosphatidylinositol anchor and the mannooligosaccharide caps play a critical role in DC-SIGN-ManLAM interaction. Finally, we report that DC-SIGN binds poorly to the PILAM and uncapped AraLAM-containing species Mycobacterium fortuitum and Mycobacterium chelonae, respectively. Interestingly, smooth colony-forming Mycobacterium avium, in which ManLAM is capped with single mannose residues, was also poorly recognized by the lectin. Altogether, our results provide molecular insight into the mechanisms of mycobacteria-DC-SIGN interaction, and suggest that DC-SIGN may act as a pattern recognition receptor and discriminate between Mycobacterium species through selective recognition of the mannose caps on LAM molecules.     

Synthesis of isonicotinic acid N'-arylidene-N-[2-oxo-2-(4-aryl-piperazin-1-yl)-ethyl]-hydrazides as antituberculosis agents

A new series of antituberculosis agents 6-9 was designed, synthesized and evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv and clinical isolates in an agar dilution method. Compound 9h showed comparable in vitro activity (MIC) to isoniazid against M. tuberculosis H37Rv and clinical isolates (sensitive strains) and superior activity against resistant strains of M. tuberculosis.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

Secretory proteins of Mycobacterium habana induce a protective immune response against experimental tuberculosis

The capacity of proteins of Mycobacterium habana TMC 5135 secreted into culture medium during the mid-exponential growth phase (secretory proteins, SPs) to induce protective immunity against Mycobacterium tuberculosis H37Rv was studied in the mouse model. Mice immunized with SPs followed by a challenge with M. tuberculosis H37Rv showed lesser M. tuberculosis bacilli in their lung and spleen and survived longer than unimmunized controls. The findings suggest that SP antigens of M. habana are protective against tuberculosis infection.     

Antigens secreted from Mycobacterium tuberculosis: identification by proteomics approach and test for diagnostic marker

Tuberculosis caused by mycobacteria, mainly Mycobacterium tuberculosis, is a major infectious disease of the respiratory system. An early diagnosis followed by chemotherapy is the major control strategy. In an effort to identify the antigens suitable for immunodiagnosis and vaccines, the proteins secreted in a culture medium from the M. tuberculosis K-strain, which is the most prevalent among the clinical isolates in Korea and belongs to the Beijing family, were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and compared with those from the M. tuberculosis H37Rv and CDC1551 strains. Eight proteins, Rv0652, Rv1636, Rv2818c, Rv3369, Rv3865, Rv0566c, MT3304, and Rv3160, were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and found to be relatively abundant in the culture medium from the M. tuberculosis K-strain but less so from the CDC1551 or H37Rv strains. In addition, Rv3874 (CFP-10), Rv-0560c and Rv3648c, which were expressed increasingly in the K and CDC1551 strains, were also identified using the same proteomics technology. All proteins were prepared by molecular cloning, expression in Escherichia coli followed by affinity purification. Among them, three proteins, rRv3369, rRv0566c, and rRv3874, were selected by prescreening and examined for their potential as serodiagnostic antigens using an enzyme-linked immunosorbent assay. When 100 sera from tuberculosis patients and 100 sera from the healthy controls were analyzed, rRV3369, rRv3874, and rRv0566c showed a sensitivity of 60%, 74%, and 43%, and a specificity of 96%, 97%, and 84%, respectively. These results suggest that the rRv3369 and rRv3874 proteins, which were expressed more abundantly in the more recently obtained clinical isolates of M. tuberculosis than in the laboratory-adapted H37Rv strain, are promising for use in the serodiagnosis of tuberculosis.     

tRNA genes in mycobacteria: organization and molecular cloning.

DNAs from nine mycobacteria cleaved with restriction endonucleases were hybridized with cDNA probes synthesized to tRNAs from Mycobacterium tuberculosis and Mycobacterium smegmatis. The tRNA genes are conserved, but their gross genomic organization has diverged in six of the nine species examined. Organisms of the M. tuberculosis H37Ra and H37Rv-M. bovis BCG complex appeared to have identical tRNA gene organization and were indistinguishable from each other. M. tuberculosis and M. smegmatis tRNA-derived cDNA probes hybridized differentially to tRNA-coding DNA segments in five of the species examined, suggesting the existence of qualitatively different tRNA pools in these slow- and fast-growing species. Mycobacterial DNAs hybridized with cDNA synthesized to 23S plus 16S rRNAs from Escherichia coli, and the data suggested that the tRNA genes map close to the rRNA genes. A gene bank of M. tuberculosis H37Rv DNA was constructed, and a recombinant plasmid, pSB2, coding for tRNA(s) and rRNA(s) was partially characterized. Plasmid pSB2 recognized a SalI restriction fragment length polymorphism (RFLP) in M. tuberculosis H37Rv and H37Ra; however, the RFLP is not linked to the tRNA-coding region. To the best of our knowledge, this is the first report of an RFLP which distinguishes the pathogenic strain M. tuberculosis H37Rv from its avirulent derivative H37Ra.     

Differential regulation of protein kinase C isoforms of macrophages by pathogenic and non-pathogenic mycobacteria

Given the fact that Mycobacterium tuberculosis (Mtb) may respond to the intracellular milieu of the macrophage with the induction of environmentally regulated genes required for survival and growth of the bacteria we assumed that the protein kinases may also be the factors in Mycobacterium-macrophage interaction. Since, protein kinases play a major role in various critical cellular processes including regulation of immune responses, we describe the fate of expression and phosphorylation of protein kinase C in macrophage cell lines exposed to Mtb H37Rv and raised the question whether the change in the events of expression and phosphorylation are the results of direct interaction of bacilli with macrophages and/or, are also indirectly mediated by specific cytokines that are induced in response to exposure. Our results show that only novel PKCs are phosphorylated during infection of macrophages by pathogenic and non-pathogenic mycobacteria and the alteration is a result of direct host-bacilli association which is independent of cytokines as mediators. Expression of PKC-alpha (conventional PKC isoform) was down regulated by Mtb H37Rv. In contrast the non-pathogenic fast grower Mycobacterium smegmatis (MS) increased the expression and phosphorylation of PKC-alpha. PKC-alpha was also increased in macrophages treated with serum of mice immunized with Mtb H37Rv. The study has shown that pathogenic and non-pathogenic mycobacteria categorically select the type of protein kinases C for activation/deactivation.     

Survival of virulent Mycobacterium tuberculosis involves preventing apoptosis induced by Bcl-2 upregulation and release resulting from necrosis in J774 macrophages

Macrophage apoptosis plays a role in mycobacterial infection. To define the mechanism by which virulent Mycobacterium tuberculosis escapes apoptosis and killing in macrophages, J774 macrophages were infected with virulent M. tuberculosis H37Rv and attenuated H37Ra strains. H37Rv induced less apoptosis than H37Ra, and caspase 3 was activated in H37Ra- and H37Rv-infected macrophages. Intracellular H37Rv bacilli were released at a higher rate into the supernatant than were H37Ra by the sixth day of infection, and this was simultaneously accompanied by the increased necrosis of infected cells showing lactate dehydrogenase (LDH) release. Fas mRNA expression was downregulated and FasL was upregulated in H37Ra- and H37Rv-infected macrophages, while Bcl-2 was upregulated in H37Rv-infected macrophages but downregulated in H37Ra-infected macrophages as seen by real-time PCR. These results indicate that M. tuberculosis H37Ra and H37Rv proliferate in macrophages by preventing them from inducing apoptosis during the early phase of infection, and that M. tuberculosis H37Rv-infected macrophages are found to express Bcl-2 mRNA, which leads to anti-apoptotic activity, and that relatively distinct necrosis might occur during the later phase of infection.