Rv3080c regulates the rate of inhibition of mycobacteria by isoniazid through FabD

The mycobacterial FASII multi-enzyme complex has been identified to be a target of Ser/Thr protein kinases (STPKs) of Mycobacterium tuberculosis (MTB), with substrates, including the malonyl-CoA:ACP transacylase (FabD) and the β-ketoacyl-ACP synthases KasA and KasB. These proteins are phosphorylated by various kinases in vitro. The present study links the correlation of FASII pathway with serine threonine protein kinase of MTB. In the preliminary finding, we have shown that mycobacterial protein Rv3080c (PknK) phosphorylates FabD and the knockdown of PknK protein in mycobacteria down regulates FabD expression. This event leads to the differential inhibition of mycobacteria in the presence of isoniazid (INH), as the inhibition of growth of mycobacteria in the presence of INH is enhanced in PknK deficient mycobacteria.     

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.     

Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species

Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain. In cell-free assays, extracts from M. smegmatis overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M. smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria.     

Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression

Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.     

Cell wall core galactofuran synthesis is essential for growth of mycobacteria

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In Mycobacterium tuberculosis the glf gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that glf can be knocked out in Mycobacterium smegmatis by allelic replacement only in the presence of two rescue plasmids carrying functional copies of glf and Rv3808c. The glf rescue plasmid was designed with a temperature-sensitive origin of replication and the M. smegmatis glf knockout mutant is unable to grow at the higher temperature at which the glf-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and the glf-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both glf and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.     

Studies on transfer RNA from mycobacteria.

Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity. The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M. smegmatis and M. tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases. The analytical separation of the different amino acid specific tRNAs from M. smegmatis resembles the pattern found in other bacteria. Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques. Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria.     

Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.     

Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556

The normal growth of mycobacteria attributes to the integrity of cell wall core which consists of peptidoglycan (PG), arabinogalactan (AG) and mycolic acids. N-acetyl glucosamine (GlcNAc) is an essential component in both PG and AG of mycobacterial cell wall. The biosynthetic pathway for UDP-N-acetylglucosamine (UDP-GlcNAc), as a sugar donor of GlcNAc, is different in prokaryotes and eukaryotes. The conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is catalyzed by phosphoglucosamine mutase (GlmM), is unique to prokaryotes. Bioinformatic analysis showed that Msm MSMEG_1556 and Mtb Rv3441c are homologous to Ec GlmM. In this study, soluble Msm MSMEG_1556 protein and Mtb Rv3441c protein were expressed in E. coli BL21(DE3) and their phosphoglucosamine mutase activity were detected. In order to further investigate the essentiality of MSMEG_1556 for the growth of M. smegmatis, we generated a conditional MSMEG_1556 knockout mutant, which harbored thermo-sensitive rescue plasmid carrying Mtb Rv3441c. As the rescue plasmid was unable to complement MSMEG_1556 deficiency at 42°C, MSMEG_1556 knockout mutant did not grow. The dramatic morphological changes of MSMEG_1556 knockout mutant after temperature shift from 30°C to 42°C have been observed by scanning electron microscope. These results demonstrated that MSMEG_1556 is essential for growth of M. smegmatis. This study provided evidence that GlmM enzyme could be as a potential target for developing anti-tuberculosis drugs.     

ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria

The RecA-dependent DNA damage response pathway (SOS response) appears to be the major DNA repair mechanism in most bacteria, but it has been suggested that a RecA-independent mechanism is responsible for controlling expression of most damage-inducible DNA repair genes in Mycobacterium tuberculosis. The specific reparative responses and molecular mediators involved in the DNA repair mechanism remain largely unclear in this pathogen and its related species. In this study, a mycobacterial ClpR-like regulator, corresponding to Rv2745c in M. tuberculosis and to Ms2694 in M. smegmatis mc(2)155, was found to interact with the promoter regions of multiple damage-inducible DNA repair genes. Specific binding of the ClpR-like factor to the conserved RecA-independent promoter RecA-NDp motif was then confirmed using in vitro electrophoretic mobility shift assays as well as in vivo chromatin immunoprecipitation experiments. The ClpR knock-out experiments, in combination with quantitative real time PCR assays, demonstrated that the expression of these RecA-independent genes were significantly down-regulated in the mutant strain of M. smegmatis in response to a DNA-damaging agent compared with the wild type strain. Furthermore, the ClpR-like factor was shown to contribute to mycobacterial genomic stability. These results enhance our understanding of the function of the ClpR regulator and the regulatory mechanism of RecA-independent DNA repair in mycobacteria.     

Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan-peptidoglycan linker, leads to irreversible loss of viability

Temperature-sensitive mutant 2-20/32 of Mycobacterium smegmatis mc(2)155 was isolated and genetically complemented with a Mycobacterium tuberculosis H37Rv DNA fragment that contained a single open reading frame. This open reading frame is designated Rv3265c in the M. tuberculosis H37Rv genome. Rv3265c shows homology to the Escherichia coli gene wbbL, which encodes a dTDP-Rha:alpha-D-GlcNAc-pyrophosphate polyprenol, alpha-3-L-rhamnosyltransferase. In E. coli this enzyme is involved in O-antigen synthesis, but in mycobacteria it is required for the rhamnosyl-containing linker unit responsible for the attachment of the cell wall polymer mycolyl-arabinogalactan to the peptidoglycan. The M. tuberculosis wbbL homologue, encoded by Rv3265c, was shown to be capable of restoring an E. coli K12 strain containing an insertionally inactivated wbbL to O-antigen positive. Likewise, the E. coli wbbL gene allowed 2-20/32 to grow at higher non-permissive temperatures. The rhamnosyltransferase activity of M. tuberculosis WbbL was demonstrated in 2-20/32 as was the loss of this transferase activity in 2-20/32 at elevated temperatures. The wbbL of the temperature-sensitive mutant contained a single-base change that converted what was a proline in mc(2)155 to a serine residue. Exposure of 2-20/32 to higher non-permissive temperatures resulted in bacteria that could not be recovered at the lower permissive temperatures.     

VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.     

Gene replacement and expression of foreign DNA in mycobacteria.

A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms.     

Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

A fragment of 21 ORFs around the direct repeat (DR) region of Mycobacterium tuberculosis is absent from the other sequenced mycobacterial genomes: implications for the evolution of the DR region

The direct repeat (DR) region is a singular locus of the Mycobacterium tuberculosis complex genome. This region consists of 36 bp repetitive sequences separated by non-repetitive unique spacer sequences. Around this region there are several genes coding for proteins of unknown function. To determine whether the M. smegmatis, M. avium, M. marinum and M. leprae genomes contain sequences and ORFs similar to those of the DR locus of the M. tuberculosis complex, we analysed the corresponding regions in these species. As a first step, some conserved genes that flank the DR genes [Rv2785c (rpsO), Rv2786c (ribF), Rv2790c (ltp1 ), Rv2793c (truB), Rv2800, Rv2825, Rv2828, Rv2831 (echA16 ), Rv2838 (rbfA) and Rv2845 (proS )] were used as markers to locate the corresponding orthologues in M. smegmatis, M. avium, M. marinum and M. leprae in silico. Most of these M. tuberculosis marker genes have highly similar orthologues located in the same order and orientation in the other mycobacteria. In contrast, no orthologues were found for ORFs Rv2801-Rv2824, suggesting that these genes are unique to M. tuberculosis within the genus Mycobacterium.We observed that in M. smegmatis and M. avium, Rv2800 and Rv2825 are adjacent. This observation was experimentally confirmed by PCR. In conclusion, as the DR locus and the ORFs around it are absent in M. smegmatis and M. avium and, as it is possible that these species are older than M. tuberculosis, we postulated that the DR locus was acquired by the M. tuberculosis complex species or by an ancestor bacterium.     

Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages

Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.     

Aminoglycoside 2'-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2 ')-Ic gene from Mycobacterium tuberculosis and the aac(2 ')-Id gene from Mycobacterium smegmatis

The genus Mycobacterium comprises clinically important pathogens such as M. tuberculosis , which has re-emerged as a major cause of morbidity and mortality world-wide especially with the emergence of multidrug-resistant strains. The use of fast-growing species such as Mycobacterium smegmatis has allowed important advances to be made in the field of mycobacterial genetics and in the study of the mechanisms of resistance in mycobacteria. The isolation of an aminoglycoside-resistance gene from Mycobacterium fortuitum has recently been described. The aac(2 ' )-Ib gene is chromosomally encoded and is present in all isolates of M. fortuitum . The presence of this gene in other mycobacterial species is studied here and genes homologous to that of M. fortuitum have been found in all mycobacterial species studied. In this report, the cloning of the aac(2 ' )-Ic gene from M. tuberculosis H37Rv and the aac(2 ' )-Id gene from M. smegmatis mc²155 is described. Southern blot hybridizations have shown that both genes are present in all strains of this species studied to date. In addition, the putative aac(2 ' )-Ie gene has been located in a recent release of the Mycobacterium leprae genome. The expression of the aac(2 ' )-Ic and aac(2 ' )-Id genes has been studied in M. smegmatis and only aac(2 ' )-Id is correlated with aminoglycoside resistance. In order to elucidate the role of the aminoglycoside 2'- N -acetyltransferase genes in mycobacteria and to determine whether they are silent resistance genes or whether they have a secondary role in mycobacterial metabolism, the aac(2 ' )-Id gene from M. smegmatis has been disrupted in the chromosome of M. smegmatis mc²155. The disruptant shows an increase in aminoglycoside susceptibility along with a slight increase in the susceptibility to lysozyme.     

Comparative analysis of iron regulated genes in mycobacteria

Iron dependent regulator, IdeR, regulates the expression of genes in response to intracellular iron levels in M. tuberculosis. Orthologs of IdeR are present in all the sequenced genomes of mycobacteria. We have used a computational approach to identify conserved IdeR regulated genes across the mycobacteria and the genes that are specific to each of the mycobacteria. Novel iron regulated genes that code for a predicted 4-hydroxy benzoyl coA hydrolase (Rv1847) and a protease dependent antibiotic regulatory system (Rv1846c, Rv0185c) are conserved across the mycobacteria. Although Mycobacterium natural-resistance-associated macrophage protein (Mramp) is present in all mycobacteria, it is, as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-regulated exochelin biosynthetic operon, which is present only in non-pathogenic Mycobacterium, M. smegmatis.     

Differential expression of NF-kappaB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-kappaB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-kappaB, pCMV-IkappaBalphaM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IkappaBalphaM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-alpha production. Increase in apoptosis of infected THP-1-IkappaBalphaM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-kappaB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-kappaB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-kappaB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-kappaB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.