Unique structural features of the peptidoglycan of Mycobacterium leprae

The peptidoglycan structure of Mycobacterium spp. has been investigated primarily with the readily cultivable Mycobacterium smegmatis and Mycobacterium tuberculosis and has been shown to contain unusual features, including the occurrence of N-glycolylated, in addition to N-acetylated, muramic acid residues and direct cross-linkage between meso-diaminopimelic acid residues. Based on results from earlier studies, peptidoglycan from in vivo-derived noncultivable Mycobacterium leprae was assumed to possess the basic structural features of peptidoglycans from other mycobacteria, other than the reported replacement of l-alanine by glycine in the peptide side chains. In the present study, we have analyzed the structure of M. leprae peptidoglycan in detail by combined liquid chromatography and mass spectrometry. In contrast to earlier reports, and to the peptidoglycans in M. tuberculosis and M. smegmatis, the muramic acid residues of M. leprae peptidoglycan are exclusively N acetylated. The un-cross-linked peptide side chains of M. leprae consist of tetra- and tripeptides, some of which contain additional glycine residues. Based on these findings and genome comparisons, it can be concluded that the massive genome decay in M. leprae does not markedly affect the peptidoglycan biosynthesis pathway, with the exception of the nonfunctional namH gene responsible for N-glycolylmuramic acid biosynthesis.     

SOS induction in mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli . The recombinant protein bound to the promoter regions of the M. leprae lexA , M. leprae recA and M. smegmatis recA genes at sites with the sequences 5'-GAACACATGTTT and 5'-GAACAGGTGTTC, which belong to the 'Cheo box' family of binding sites recognized by the SOS repressor from Bacillus subtilis . Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis . Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti- M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA-damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.     

In vivo splicing and functional characterization of Mycobacterium leprae RecA

The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins. In contrast to the M. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here that M. leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.     

Comparative ultrastructure of Mycobacterium leprae and Mycobacterium lepraemurium cell envelopes.

The structural properties of the cell envelopes of Mycobacterium leprae and Mycobacterium lepraemurium were investigated by freeze-fracture, freeze-etching, and negative-staining techniques. Freeze-fracture split the cell wall and exposed the internal features of the peptidoglycolipid mycosidic filamentous network. The cell membrane was also split into two asymmetric faces. The external fracture face was characterized by linear arrays of intramembranous particles, whereas the protoplasmic fracture face showed randomly distributed clusters of particulate entities. Comparative analysis of the ultrastructural features observed in M. leprae and M. lepraemurium indicated that the organization of the cell envelope in these two species differed particularly with respect to the amount and complexity of the superficial peptidoglycolipid and mycosidic integument, which is poorly developed in the mycobacterium responsible for human disease.     

Identification of strong promoter elements of Mycobacterium smegmatis and their utility for foreign gene expression in mycobacteria

The isolation of elements driving high-level expression of foreign genes in mycobacteria would significantly aid characterization of mycobacterial antigens and recombinant vaccine development. Mycobacterium smegmatis is a widely employed host for recombinant mycobacterial gene expression. This report describes the identification of strong promoter elements of M. smegmatis. Fluorescence-activated cell sorting was employed to isolate DNA fragments permitting high-level expression of the Aequorea victoria green fluorescent protein within recombinant M. smegmatis. Ten postulated M. smegmatis promoters were identified which showed activity two to six times that of the strong beta-lactamase promoter of Mycobacterium fortuitum. The utility of one of these promoters for the over-expression of foreign genes in mycobacteria was demonstrated by the efficient purification of the Mycobacterium leprae 35-kDa antigen from recombinant M. smegmatis.     

Aspartate metabolism in Mycobacterium avium grown in host tissue and axenically and in Mycobacterium leprae

Aspartokinase activity was detected in extracts from Mycobacterium leprae (recovered from armadillo liver) and in Mycobacterium avium grown axenically and in vivo. Homoserine dehydrogenase activity was only detected in M. leprae and in M. avium grown axenically. Activities, when detected, were 50 to 70% lower in M. leprae or M. avium grown in vivo than in axenically grown M. avium. In these two pathogenic mycobacteria, aspartokinase and homoserine dehydrogenase are subject to feedback inhibition by methionine - an additional regulator over those observed for the enzymes from Mycobacterium smegmatis. Intact mycobacterium incorporated carbon from [U-14C]aspartate into the aspartate family of amino acids (threonine, isoleucine, methionine and lysine) though the rate of incorporation in M. avium grown in vivo was about half that in M. avium grown axenically.     

Oxidation of various substrates by host grown Mycobacteria leprae and M. lepraemurium.

Oxidation of various substrates by whole cell suspensions of M. Lepraemurium and M. leprae was investigated using manometric techniques. Yeast extract, L-cysteine, dithioerythritol, and DL-penicillamine were oxidized by both M. lepraemurium as well as by M. leprae. Although tween 80 was oxidized by M. lepraemurium cell suspensions, it was not by M. leprae. Succinate was readily oxidized by whole cells of M. leprae (without being frozen) whereas it was oxidized only by M. lepraemurium cells frozen at -40 degrees C for one minute. The results indicate that M. leprae and M. lepraemurium are capable of oxidizing some substrates without requiring any cofactor and are not dependent upon host cells for respiration.     

Study of DTH and resistance in Mycobacterium lepraemurium infection using a T-cell line isolated from mice infected with Mycobacterium bovis (BCG)

A T-cell line of mixed phenotype (60% L3T4+, 40% Lyt-2+) was isolated from mice infected with Mycobacterium bovis (BCG). This line responded to M. lepraemurium and BCG but not to M. leprae and produced TCGF spontaneously. It also produced factors which stimulated macrophages to secrete hydrogen peroxide and superoxide anion. In vivo studies showed that only L3T4+ cells were required to transfer DTH responses and that Lyt-2+ cells suppressed this response. Both L3T4+ and Lyt-2+ cells were required to inhibit M. lepraemurium multiplication in vivo.     

Mycobacterium leprae induces insulin-like growth factor and promotes survival of Schwann cells upon serum withdrawal

Peripheral nerve lesions are considered the most relevant symptoms of leprosy, a chronic infectious disease caused by Mycobacterium leprae . The strategies employed by M. leprae to infect and multiply inside Schwann cells (SCs), however, remain poorly understood. In this study, it is shown that treatment of SCs with M. leprae significantly decreased cell death induced by serum deprivation. Not displayed by Mycobacterium smegmatis or Mycobacterium bovis BCG , the M. leprae survival effect was both dose dependent and specific . The conditioned medium (CM) of M. leprae -treated cultures was seen to mimic the protective effect of the bacteria, suggesting that soluble factors secreted by SCs in response to M. leprae were involved in cell survival. Indeed, by quantitative RT-PCR and dot blot/ELISA, it was demonstrated that M. leprae induced the expression and secretion of the SC survival factor insulin-like growth factor-I. Finally, the involvement of this hormone in M. leprae -induced SC survival was confirmed in experiments with neutralizing antibodies. Taken together, the results of this study delineate an important strategy for the successful colonization of M. leprae in the nerve based on the survival maintenance of the host cell through induction of IGF-I production.     

Species variation in ATP-dependent protein degradation: protease profiles differ between mycobacteria and protease functions differ between Mycobacterium smegmatis and Escherichia coli

We report here that the existence of the potentially broad substrate specificity protease Lon (also called La), is evolutionarily discontinuous within the order Actinomycetales. Lon homologues were identified in the fast-growing species Mycobacterium smegmatis, and the slow-growing species Micobacterium avium and Mycobacterium intracellulare. However, Lon homologues were not detected in the slow-growing species Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium leprae; or in the non-mycobacterial Actinomycetale Corynebacterium glutamica. To characterize the function of the Lon protease within the Actinomycetales, a viable M. smegmatis Deltalon strain was constructed, demonstrating that lon is not essential under certain conditions. Surprisingly, lon was also dispensable in M. smegmatis cells already lacking intact 20S proteasome alpha- and beta-subunit genes (called prcA and prcB, respectively). Creation of the later double deletion strain (prcBA::kan Deltalon) necessitated use of a novel gene deletion strategy that does not require an antibiotic resistance marker. The M. smegmatis prcBA::kan Deltalon double mutants displayed wild type (wt) growth rates and wt stress tolerances. In addition, the M. smegmatis prcBA::kan Deltalon double mutants degraded at wt rates the broad spectrum of truncated proteins induced by treating cells with puromycin. This later result was in sharp contrast to those in Escherichia coli, where either lon or hslUV single mutants are strongly impaired in their degradation of puromycyl peptides (hslV is a prcB homologue). Overall these data suggested that mycobacterial species contain additional ATP-dependent proteases that have broad substrate specificity. Consistent with this suggestion, M. smegmatis and M. tuberculosis each contain at least one homologue of ClpP, the proteolytic subunit common to the ClpAP and ClpXP proteases.     

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.     

Determination and evolutionary significance of nucleotide sequences near to the 3''-end of 16S ribosomal RNA of mycobacteria

The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.     

Human phagocyte oxidative burst activation by BCG, M. leprae, and atypical mycobacteria: defective activation by M. leprae is not reversed by interferon gamma

The activation of the phagocyte oxidative respiratory burst by various mycobacteria was evaluated in an in vitro assay, by measuring the chemiluminescence, associated to the release of oxidizing species, generated by normal human whole blood phagocytes. All mycobacterium species, except Mycobacterium leprae, induced a significant chemiluminescence response. The strongest stimulus was provided by BCG, followed by M. triviale, M. chelonei, and M. fortuitum. M. kansasii, M. intracellulare, and M. lepraemurium elicited a weak response, although higher than that triggered by M. leprae. Both polymorphonuclear and mononuclear cells contributed to the whole blood cell chemiluminescence stimulated by mycobacteria, mononuclear cells being more efficient on a per cell basis. Phagocyte activation by recombinant interferon gamma did not improve M. leprae ability to trigger a significant chemiluminescence response. The failure of M. leprae and of some atypical mycobacteria to stimulate a strong phagocyte oxidative respiratory burst may have some relevance to their pathogenicity.     

The 16S rRNA nucleotide sequence of Mycobacterium leprae: phylogenetic position and development of DNA probes

The almost complete 16S rRNA sequence from Mycobacterium leprae was determined by direct sequencing of the chromosomal gene amplified by the polymerase chain reaction. The primary sequence revealed an insertion of 12 nucleotides at the 5' end of the 16S rRNA gene, which consists of an A-T stretch and appears to be unique for M. leprae. Within the mycobacteria M. leprae branches off with a group of slow-growing species comprising M. scrofulaceum, M. kansasii, M. szulgai, M. malmoense, M. intracellulare and M. avium. A systematic comparison of the nucleotide sequence resulted in the characterization of oligonucleotide probes which are highly specific for M. leprae. The probes hybridized exclusively to 16S rRNA nucleic acids from M. leprae, but not to nucleic acids from 20 cultivable fast- and slow-growing mycobacteria.     

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.     

Detection of trehalose monomycolate in Mycobacterium leprae grown in armadillo tissues

Trehalose-6-monomycolate (TMM) was isolated from the lipids of armadillo-derived Mycobacterium leprae. Only meagre amounts of this glycolipid were recovered, but its structure was unequivocally established. Only alpha-mycolates were detected in the TMM by 252Cf plasma desorption mass spectrometry. Electron impact mass spectrometry showed the alpha branch to be principally C20. Trehalose dimycolate (cord factor) was not detectable. Since we have also found TMM in M. lepraemurium and in every Mycobacterium species so far examined, we suggest that this glycolipid is truly ubiquitous amongst mycobacteria.     

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.