Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1.7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity--if representative of living M. leprae organisms--is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA 'elongase'. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.     

Fatty acid oxidation and the beta-oxidation complex in Mycobacterium leprae and two axenically cultivable mycobacteria that are pathogens

Intact, non-growing Mycobacterium leprae, M. avium and M. microti oxidized a wide range of 1-14C-labelled fatty acids (C8 to C24) to 14CO2. Laurate (C12) was oxidized most rapidly, and its oxidation by M. leprae was inhibited by the antileprosy agents Dapsone, clofazamine and rifampicin. Key enzymes of beta-oxidation were detected in extracts from all three mycobacteria. All these activities (both in intact mycobacteria and the enzymes) were stimulated in M. avium grown in Dubos medium plus palmitate but activities in M. microti or M. avium grown either in Dubos medium with added liposomes or triolein, or in vivo were similar to those detected in the same strain grown in Dubos medium alone. M. avium could be grown in medium in which 95% of its fatty acyl elongase activity is acetyl-CoA dependent. In this medium growing M. avium organisms oxidized [1-14C]palmitate to 14CO2 but simultaneously elongated palmitate to C24 acids and even longer. Acetyl-CoA-dependent elongase activity is similar but clearly not identical to reversed beta-oxidation, but the exact point(s) of difference have not yet been identified.     

Biosynthesis and scavenging of pyrimidines by pathogenic mycobacteria

Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 microM-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and--in organisms grown in Dubos medium with 50 microM-uridine-thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 microM-uracil, reflecting its more limited abilities in pyrimidine scavenging.     

Enzymes for purine synthesis and scavenging in pathogenic mycobacteria and their distribution in Mycobacterium leprae

Three enzymes catalysing the synthesis of four intermediates (phosphoribosylglycinamide, phosphoribosylaminoimidazole-succinocarboxamide, phosphoribosylaminoimidazole-carboxamide and AMP) in the purine biosynthetic pathway were detected in extracts of Mycobacterium microti and M. avium, even when the organisms had been grown in mice. However only one of the three enzymes, adenylosuccinate AMP-lyase (catalysing the synthesis of the last two of the four intermediates listed above) was detected in M. leprae. Phosphoribosyltransferases, which convert adenine, guanine and hypoxanthine to the corresponding nucleoside monophosphates, and adenosine kinase were the major enzymes for purine scavenging in all mycobacteria studied. In contrast to enzymes in the synthetic pathway, evidence for metabolic regulation of the purine-scavenging enzymes was obtained. In particular, 20-80-fold differences in the activities of guanine phosphoribosyltransferase and adenosine kinase were observed when M. microti was grown in media with or without purines, or in mice. In M. leprae, activities of all phosphoribosyltransferases were low in comparison with activities in M. microti and M. avium (specific activity less than 2% when comparisons were made between extracts of host-grown mycobacteria). However, activity of adenosine kinase was higher in host-grown M. leprae than in host-grown M. microti or M. avium.     

Use of carbon sources for lipid biosynthesis in Mycobacterium leprae: a comparison with other pathogenic mycobacteria

Carbon from glycerol and palmitate, but not significantly from five other carbon sources tested, was incorporated into lipids by suspensions of non-growing Mycobacterium leprae organisms. However, of the five other substrates three-citrate, glucose and pyruvate-were taken up. Nongrowing Mycobacterium microti and Mycobacterium avium incorporated carbon into lipids from most simple carbon sources tested unless they were obtained from growth media including palmitate or from experimentally infected animals, when incorporation of carbon into lipids from carbon sources except palmitate occurred up to 20 times more slowly. Thus, utilization of simple carbon appeared to be repressible while utilization of the one fatty acid tested, palmitate, appeared constitutive. In M. leprae, carbon from glycerol was incorporated into the glycerol moiety of acylglycerols but not into the fatty acid moieties or into free fatty acids. M. microti and M. avium incorporated carbon from simple carbon sources into fatty acids, even (though very slowly) when these organisms were obtained from host tissue. Isocitrate lyase, malate synthase and acetate kinase were detected in M. leprae. However acetyl-CoA synthetase was not detectable and phosphoacetylase was deficient; thus, M. leprae may be incapable of making acetyl-CoA from acetate. Phosphotransacetylase was readily detected in both host-grown M. avium and M. microti.     

Identification and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria

Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium , we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Myco-bacterium leprae and Mycobacterium tuberculosis . Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette–Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.     

Oxidation of carbon sources through the tricarboxylic acid cycle in Mycobacterium leprae grown in armadillo liver

All the enzymes of the tricarboxylic acid cycle have now been demonstrated in extracts of Mycobacterium leprae grown in armadillo liver. Many were also present in homogenates of host-tissue, but biochemical evidence is presented which indicates that all enzymes detected in extracts from M. leprae were authentic bacterial enzymes. Further evidence for a complete tricarboxylic acid cycle in M. leprae was obtained by first establishing that citrate could be taken up and catabolized by whole M. leprae organisms, then showing that oxidation of radioisotopically labelled pyruvate to CO2 by suspensions of M. leprae was stimulated by adding unlabelled citrate. Control of tricarboxylic acid cycle activity in M. leprae by the inactivation of fumarase by a protease is speculated upon.     

Effect of growth in biofilms on chlorine susceptibility of Mycobacterium avium and Mycobacterium intracellulare

Mycobacterium avium and Mycobacterium intracellulare were grown in suspension and in biofilms, and their susceptibilities to chlorine were measured. M. avium and M. intracellulare readily adhered within 2 h, and numbers increased 10-fold in 30 days at room temperature in biofilms on both polystyrene flasks and glass beads. The chlorine resistance of M. avium and M. intracellulare cells grown and exposed to chlorine in biofilms was significantly higher than that of cells grown in suspension. Survival curves showed no evidence of a resistant, persisting population after 6 h of exposure to 1 mug chlorine/ml. The chlorine susceptibility of cells grown in biofilms and exposed in suspension (cells detached from bead surfaces) was also significantly higher than that of cells grown and exposed in suspension (planktonic cells), although it was lower than that of cells grown and exposed in biofilms. The higher resistance of the detached biofilm-grown cells was reversed upon their growth in suspension. There was a strong correlation between the chlorine susceptibility of cells of both M. avium and M. intracellulare and cell surface hydrophobicity measured by contact angle for both biofilm- and suspension-grown cells.     

Factors influencing the chlorine susceptibility of Mycobacterium avium,Mycobacterium intracellulare,and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30 degrees C was increased when cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

Adenylate kinase activity in Mycobacterium leprae

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was detected in partially purified preparations of cell-free extracts of Mycobacterium leprae. The apparent Km values of M. leprae adenylate kinase for ADP and Mg2+ were 1 X 10(-4) M, respectively. The enzyme was heat-labile: loss of activity by 80% at 45 degrees C and over 90% at 60 degrees C occurred within 5 min. M. leprae adenylate kinase was distinct from armadillo adenylate kinase in respect of affinity for substrate and heat-sensitivity.     

Truncated structural variants of lipoarabinomannan in Mycobacterium leprae and an ethambutol-resistant strain of Mycobacterium tuberculosis

Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration.     

Characterization and expression of secA in Mycobacterium avium

Mycobacterium avium is both a pathogen that infects several hosts such as humans, pigs, and birds, as well as a microorganism that is encountered in environmental sources (soil and water). Protein secretion by the bacterium is likely to influence its ability to overcome adverse and competitive conditions both within or outside the host. Using a combination of cloning and information available in the databank, we characterized the secA gene from M. avium, encoding for a major preprotein translocase subunit associated with the secretion system of prokaryotics. In addition, we cloned the secA promoter sequence in a reporter construct upstream of a promoterless gfp. It was determined that the secA of M. avium shares large homology with the secA of Mycobacterium tuberculosis but not with secA of Mycobacterium leprae. secA expression was determined to be greater at logarithmic growth phase although it was also expressed at low levels during the stationary phase. secA expression was also observed when the bacteria were incubated in water as well as within human monocyte-derived macrophages and in conditions that are associated with biofilm formation. Future evaluation of the sec pathway in M. avium might provide important information about secreted proteins that are required for survival in different environments.     

Arylsulfatase activity of Mycobacterium avium, M. intracellulare, and M. scrofulaceum

A rapid (3-h) arylsulfatase assay for cell suspensions of mycobacteria, in which p-nitrophenyl sulfate is used as the substrate, was developed. Arylsulfatase activity was found in cell suspensions of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum grown without the substrate in either Middlebrook 7H9 medium containing 0.2% (wt/vol) glucose and 0.05% (vol/vol) Tween 80 or Dubos broth medium, but was absent in cells grown in a low-pH, minimal medium containing 1% (vol/vol) Tween 80 as the sole carbon source. The levels of arylsulfatase activity of representatives of all three species were equal whether the activity was measured at pH 5.5, 6.5, or 7.5 and whether the cells were suspended in phosphate or Tris buffer. The addition of high levels of sulfate (present in the low-pH, Tween 80-containing medium) to Middlebrook 7H9 medium resulted in significantly lower levels of arylsulfatase activity in strains of M. scrofulaceum, but did not affect the levels in either M. avium or M. intracellulare. The levels of arylsulfatase activity were highest in M. avium, intermediate in M. intracellulare, and lowest in M. scrofulaceum strains. Polyacrylamide gel electrophoresis of crude extracts from late-log-phase cells of representatives of each species produced activity bands of unique mobility (one in M. avium, three in M. intracellulare [82, 5, and 13%], and two in M. scrofulaceum [60 and 40%]).     

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.     

Conservation and cloning of CYP51: a sterol 14 alpha-demethylase from Mycobacterium smegmatis

The genetic locus encoding cytochrome P450 51 (CYP51; P450(14DM)) in Mycobacterium smegmatis is described here together with confirmation of activity in lanosterol 14 alpha-demethylation. The protein bound azole antifungals with high affinity and the rank order based on affinity matched the ranked order for microbiological sensitivity of the organism, thus supporting a possible role for CYP51 as a target in the antimycobacterial activity of these compounds. Non-saponifiable lipids were extracted from the bacteria grown on minimal medium. Unlike a previous report using growth on complex medium, no cholesterol was detected in two strains of M. smegmatis, but a novel lipid was detected. The genetic locus of CYP51 is discussed in relation to function; it is conserved as part of a putative operon in M. smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium bovis and consists of six open-reading frames including two CYPs and a ferredoxin under a putative Tet-R regulated promoter.     

Identification and semiquantitation of Mycobacterium avium using a competitive PCR method.

A competitive PCR method with standard DNA (MIMIC) was developed for the rapid detection and semiquantitation of Mycobacterium avium (M. avium) using primers specific for the alpha antigen sequence of the bacteria. DNA from both M. avium and Mycobacterium marinum was amplified by polymerase chain reaction (PCR), but only M. avium could be detected by subsequent blotting confirmation with a probe specific for the bacteria. With the PCR and subsequent dot blot hybridization, as little as 10 fg of the M. avium DNA could be detected, equivalent to about 2 cells of the mycobacteria. In addition, we could distinguish 10(5) CFUs of M. avium from 10(4) CFUs or less by competitive PCR using a MIMIC. The present competitive PCR test enabled rapid identification and semiquantitation of M. avium, and could be used clinically to monitor disease severity and response to treatment of human M. avium disease.