Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.     

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.     

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.     

DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG

DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.     

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.     

分枝杆菌所致家兔皮肤液化病理模型研究

目的 建立卡介苗( BCG) 、H37Ra 和耻垢分枝杆菌感染的新西兰兔皮肤模型, 为肺结核干酪样坏死和继而发生的液化提供研究模型。方法 新西兰兔皮内分别注射BCG、H37Ra 和耻垢分枝杆菌的5 ×10⁶CFU、5 ×10⁴CFU、5 ×10²CFU/ml 菌液, 6 周后在病灶周围再次以相同剂量皮内注射,14 d 后病变明显时取材, 制作切片, 行HE 染色, 显微镜下观察。结果新西兰兔分别经皮内接种BCG、H37Ra 或耻垢分枝杆菌后, 高剂量组观察到明显的炎症反应和脓肿液化、破溃等改变。再次免疫可观察到郭霍现象。引起病变的严重程度依次为BCG 强于H37Ra, 后者又强于耻垢分枝杆菌。显微改变可具典型的结核结节样病灶。皮肤模型处取材, 行细菌抗酸染色, 结果阳性。BCG 中、低剂量组再次免疫可诱导小结节样病变, 但不发生液化溃疡, 其余中剂量组及低剂量组没有观察到明显改变。结论 BCG、H37Ra 和耻垢分枝杆菌均可引起皮肤干酪样坏死和液化,病理损伤与感染细菌剂量密切相关, 5 ×10⁶CFU/ml 浓度的分枝杆菌可有效诱导液化和坏死, 其中BCG 引起的病理改变最明显。     

Processing and presentation of a mycobacterial antigen 85B epitope by murine macrophages is dependent on the phagosomal acquisition of vacuolar proton ATPase and in situ activation of cathepsin D

Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.     

Biochemical characterization of three mycobacterial ribosomal fractions

The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity.     

Immunogenicity of Cell Walls from Various Mycobacteria Against Airborne Tuberculosis in Mice

Protective potency of oil-treated cell walls of various mycobacteria against airborne infection of mice with a few cells of Mycobacterium tuberculosis H37Rv was compared with that of viable BCG. Although less potent than BCG cell walls, the cell walls of atypical mycobacteria of Runyon's groups I to IV protected against challenge by aerosol to some degree. Protection afforded by cell walls of H37Rv and of the avirulent mutants H37Ra and Washington II was comparable to that provided by BCG cell walls. However, cell walls of a highly virulent strain of M. bovis (Bovinus I) provided the best protection yet achieved. Present evidence suggests that protective substances are shared by all mycobacteria but in differing amounts; the relationship between virulence and immunogenicity has yet to be clarified.     

Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis

The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis.     

Characterization of the functional replication origin of Mycobacterium tuberculosis.

The gene order in the 5kb Mycobacterium tuberculosis dnaA region is rnpA, rpmH, dnaA, dnaN and recF. We show that M. tuberculosis DNA fragment containing the dnaA-dnaN intergenic region functioned as oriC, i.e., allowed autonomous replication to otherwise nonreplicative plasmids, in M. tuberculosis H37Ra (H37Ra), avirulent strain of M. tuberculosis, and in Mycobacterium bovis BCG (BCG), a closely related, slowly growing mycobacterial strain. Removal of Escherichia coli plasmid replication origin (ColE1) from the M. tuberculosis oriC plasmids did not abolish their ability to function as oriC, confirming that the autonomous replication activity of these plasmids is due to the presence of the DNA fragment containing the dnaA-dnaN intergenic region. Deletion analyses revealed that the minimal oriC DNA fragment is 814bp. The copy number of M. tuberculosis oriC plasmids containing ColE1 ori relative to chromosomal oriC is one and the 5' flanking region of minimal oriC contains features that support stable autonomous replication. The M. tuberculosis oriC did not function in rapidly growing mycobacterial species such as M. smegmatis. M. smegmatis oriC functioned only in M. fortuitum, but not in any of the slowly growing mycobacterial species such as M. tuberculosis and BCG. Together these data suggest that the replication initiation mechanisms in the slowly growing Mycobacteria are similar and probably different from those in the rapidly growing Mycobacteria and vice versa.     

Mycobactins and exochelins of Mycobacterium tuberculosis, M. bovis, M. africanum and other related species

Following iron-deficient growth, mycobactins and exochelins were isolated from 11 strains of Mycobacterium tuberculosis (including type strains of the virulent H37Rv and avirulent H37Ra organisms) as well as from M. bovis (one strain), M. bovis BCG (two strains), M. africanum (eight strains) and M. xenopi (one strain) but not from M. microti (one strain). The mycobactins from the tuberculosis group (M. tuberculosis, M. bovis and M. africanum) were identical and could each be resolved into four compounds by thin-layer chromatography (TLC) and into several fractions by high-performance liquid chromatography, supporting previous claims that this group is a single taxon. Exochelins were all chloroform-soluble and showed no species specificity; no single exochelin was recognized by TLC that had not been previously seen in M. avium or a related species.     

Identification of a 75 kDa highly immunodominant antigen from Mycobacterium smegmatis and cross-reactivity with other species

Three monospecific antibodies MSAb 1, MSAb 2 and MSAb 3 were raised in BALB/C mice against respective antigens. M. smegmatis whole cell lysate was first separated on SDS-PAGE and randomly chosen bands were cut and then used for immunization. Antibodies were collected as ascites by injecting mice with myeloma cell line P3X63 Ag 658.4. All the three antibodies showed high reactivity with denatured antigens compared to native. Different extent of cross-reactivity was observed as evident from ELISA. MSAb1 recognized a 75 kDa immunodominant antigen from M. smegmatis and 66 kDa from M. tuberculosis (H37Ra), respectively. An apparently similar molecular weight antigen shown to be present in M. tuberculosis (H37Ra) an avirulent strain and BCG, but not recognized by MSAb1. The 75 kDa antigen has a stimulatory effect on T-cell proliferation.     

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.     

Isolation and Characterization of Acyl Coenzyme A Carboxylases from Mycobacterium tuberculosis and Mycobacterium bovis, Which Produce Multiple Methyl-Branched Mycocerosic Acids

Mycobacterium tuberculosis H37Ra and M. bovis BCG produce multiple methyl-branched fatty acids called mycocerosic acids, presumably from methyl-malonyl coenzyme A (CoA). An acyl-CoA carboxylase was isolated from these organisms at a 30 to 50% yield by a purification procedure involving ammonium sulfate fractionation, gel filtration, and affinity chromatography with a monomeric avidin–Sepharose 4B-CL gel with d-biotin as the eluant. Sodium dodecyl sulfate electrophoresis and avidin binding indicate that each enzyme is probably composed of two dissimilar subunits with a covalently bound biotin in the larger subunit. The enzyme preparations from H37Ra and BCG had specific activities of 2.1 and 5.5 μmol min⁻¹ mg⁻¹, respectively, when propionyl-CoA was the substrate. The enzymes from the two species displayed striking similarities in their kinetic parameters. They showed maximal activity at pH 8.0 when propionyl-CoA was the substrate, but displayed a relatively broad pH-activity profile when acetyl-CoA was the substrate. With both substrates, potassium phosphate buffer gave maximal activity. Apparent K_m values for propionyl-CoA, ATP, Mg²⁺, and NaHCO₃ were 70 μM, 100 μM, 5.4 mM, and 2.2 mM, respectively. The enzyme also carboxylated acetyl-CoA and butyryl-CoA, and high-performance liquid chromatography showed the expected products of carboxylation. However, with these substrates, the K_m was higher and the V_max was lower than those of propionyl-CoA. The enzyme was shown to be stereospecific, synthesizing exclusively (S)-methylmalonyl-CoA from propionyl-CoA. No other acyl-CoA carboxylase was observed during the purification procedure, indicating that the present carboxylase may provide malonyl-CoA for the synthesis of n-fatty acids as well as methylmalonyl-CoA for the synthesis of mycocerosic acids.     

Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 2'-'up' fluoro (or hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium inhibitors

Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mechanisms of action is urgently required for the treatment of mycobacterial infections due to the emerging global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy) methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 2'-'up' fluoro (or hydroxy) nucleosides (1, 2, 4-6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among 2'-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-β-d-arabinofuranosyl)-5-ethyluracil (13) exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with isoniazid. The most active compound emerging from these studies, 1-(β-d-arabinofuranosyl)-4-thio-5-hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC(50)=0.5 μg/mL) and M. bovis (MIC(50)=0.5 μg/mL) at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine (MIC(50)=5.0 μg/mL), a second line drug. It also showed an additive effect when combined with isoniazid. Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC(50)=1 μg/mL) at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21 (MIC(50)=10 μg/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested (CC(50)>100 μg/mL). These observations offer promise for a new drug treatment regimen to augment and complement the current chemotherapy of TB.     

Bacterial luciferase is naturally destabilized in Mycobacterium tuberculosis and can be used to monitor changes in gene expression

Reporter systems efficient at monitoring temporal gene expression in slow-growing mycobacteria would significantly aid the characterization of gene expression in specific environments. Bacterial luciferase is a reporter that has not been widely used to study gene expression in mycobacteria. This report describes the determination of the degradation of bacterial luciferase in Mycobacterium tuberculosis H37Ra and its utility as a reporter of temporal gene expression in this slow-growing mycobacterium. The inducible/repressible alanine dehydrogenase promoter of M. tuberculosis H37Rv was used to track the decay kinetics of Vibrio harveyi luciferase in both mid-log phase and stationary phase grown M. tuberculosis H37Ra, which proved to be highly similar during both phases of growth. The luciferase reporter was then used to detect changes in expression from the heat-shock promoter, phsp60, of M. bovis BCG during M. tuberculosis H37Ra growth in culture. Quantitative real-time PCR analysis of groEL2, the hsp60 homologue in M. tuberculosis, displayed a similar pattern of expression to phsp60-driven luciferase. These results strongly suggest that the luciferase reporter can be used to monitor temporal changes in gene expression in M. tuberculosis and may serve as a novel system to examine gene expression under specific conditions.