The wag31 gene of Mycobacterium tuberculosis is positively regulated by the stringent response

The stringent response of Mycobacterium tuberculosis is coordinated by Rel and is required for full virulence in animal models. A serological-based approach identified Wag31(Mtb) as a protein that is upregulated in M. tuberculosis in a rel-dependent manner. This positive regulation was confirmed by analysis of M. tuberculosis mRNA expression. Mycobacterium smegmatis was used to confirm that the expression of wag31(Mtb) from its native promoter is positively regulated by the stringent response. Furthermore, elevated wag31(Mtb) expression in M. smegmatis drastically alters the cell-surface hydrophobic properties.     

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.     

A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis

The Snm protein secretion system is a critical determinant of Mycobacterium tuberculosis virulence. However, genes encoding components of this pathway are conserved among all mycobacteria, including the nonpathogenic saprophyte Mycobacterium smegmatis. We show that the Snm system is operational in M. smegmatis and that secretion of its homologous ESAT-6 and CFP-10 substrates is regulated by growth conditions. Importantly, we show that Snm secretion in M. smegmatis requires genes that are homologous to those required for secretion in M. tuberculosis. Using a gene knockout strategy in M. smegmatis, we have also discovered four new gene products that are essential for Snm secretion, including the serine protease mycosin 1. Despite the evolutionary distance between M. smegmatis and M. tuberculosis, the M. smegmatis Snm system can secrete the M. tuberculosis ESAT-6 and CFP-10 proteins, suggesting that substrate recognition is also conserved between the two species. M. smegmatis, therefore, represents a powerful system to study the multicomponent Snm secretory machine and to understand the role of this conserved system in mycobacterial biology.     

Macrophage's proinflammatory response to a mycobacterial infection is dependent on sphingosine kinase-mediated activation of phosphatidylinositol phospholipase C, protein kinase C, ERK1/2, and phosphatidylinositol 3-kinase

Previous studies have shown that the ability of Mycobacterium tuberculosis to block a Ca(2+) flux is an important step in its capacity to halt phagosome maturation. This affect on Ca(2+) release results from M. tuberculosis inhibition of sphingosine kinase (SPK) activity. However, these studies did not address the potential role of SPK and Ca(2+) in other aspects of macrophage activation including production of proinflammatory mediators. We previously showed that nonpathogenic Mycobacterium smegmatis and to a lesser extent pathogenic Mycobacterium avium, activate Ca(2+)-dependent calmodulin/calmodulin kinase and MAPK pathways in murine macrophages leading to TNF-alpha production. However, whether SPK functions in promoting MAPK activation upon mycobacterial infection was not defined in these studies. In the present work we found that SPK is required for ERK1/2 activation in murine macrophages infected with either M. avium or M. smegmatis. Phosphoinositide-specific phospholipase C (PI-PLC) and conventional protein kinase C (cPKC) were also important for ERK1/2 activation. Moreover, there was increased activation of cPKC and PI3K in macrophages infected with M. smegmatis compared with M. avium. This cPKC and PI3K activation was dependent on SPK and PI-PLC. Finally, in macrophages infected with M. smegmatis compared with M. avium, we observed enhanced secretion of TNF-alpha, IL-6, RANTES, and G-CSF and found production of these inflammatory mediators to be dependent on SPK, PI-PLC, cPKC, and PI3K. These studies are the first to show that the macrophage proinflammatory response following a mycobacterial infection is regulated by SPK/PI-PLC/PKC activation of ERK1/2 and PI3K pathways.     

FbpA-Dependent biosynthesis of trehalose dimycolate is required for the intrinsic multidrug resistance, cell wall structure, and colonial morphology of Mycobacterium smegmatis

Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating alpha,alpha'-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.     

A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.     

Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C

The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in 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.     

Functional complementation of the essential gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but not Escherichia coli fabG

Mycolic acids are a key component of the mycobacterial cell wall, providing structure and forming a major permeability barrier. In Mycobacterium tuberculosis mycolic acids are synthesized by type I and type II fatty acid synthases. One of the enzymes of the type II system is encoded by fabG1. We demonstrate here that this gene can be deleted from the M. tuberculosis chromosome only when another functional copy is provided elsewhere, showing that under normal culture conditions fabG1 is essential. FabG1 activity can be replaced by the corresponding enzyme from the closely related species Mycobacterium smegmatis but not by the enzyme from Escherichia coli. M. tuberculosis carrying FabG from M. smegmatis showed no phenotypic changes, and both the mycolic acids and cell wall permeability were unchanged. Thus, M. tuberculosis and M. smegmatis enzymes are interchangeable and do not control the lengths and types of mycolic acids synthesized.     

A protein kinase inhibitor as an antimycobacterial agent

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.     

The proteomic response of Mycobacterium smegmatis to anti-tuberculosis drugs suggests targeted pathways

Mycobacterium smegmatis is a fast-growing model mycobacterial system that shares many features with the pathogenic Mycobacterium tuberculosis while allowing practical proteomics analysis. With the use of shotgun-style mass spectrometry, we provide a large-scale analysis of the M. smegmatis proteomic response to the anti-tuberculosis (TB) drugs isoniazid, ethambutol, and 5-chloropyrazinamide and elucidate the drugs' systematic effects on mycobacterial proteins. A total of 2550 proteins were identified with approximately 5% false-positive identification rate across 60 experiments, representing approximately 40% of the M. smegmatis proteome ( approximately 6500 proteins). Protein differential expression levels were estimated from the shotgun proteomics data, and 485 proteins showing altered expression levels in response to drugs were identified at a 99% confidence level. Proteomic comparison of anti-TB drug responses shows that translation, cell cycle control, and energy production are down-regulated in all three drug treatments. In contrast, systems related to the drugs' targets, such as lipid, amino acid, and nucleotide metabolism, show specific protein expression changes associated with a particular drug treatment. We identify proteins involved in target pathways for the three drugs and infer putative targets for 5-chloropyrazinamide.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

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.     

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.     

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 two Mycobacterium smegmatis lipoproteins exported by a SecA2-dependent pathway

The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and DeltasecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a DeltasecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.