Massive gene decay in the leprosy bacillus.

Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.

The decaying genome of Mycobacterium leprae.

Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.

Sulphatide-binding properties are shared by serum amyloid P component and a polyreactive germ-line IgM autoantibody, the TH3 idiotype.

Serum amyloid P component (SAP) concentration was elevated in sera from leprosy patients, significantly so above endemic controls in lepromatous cases. In the sera of lepromatous leprosy (LL) patients who experienced an erythema nodosum leprosum (ENL) episode the SAP fell at the onset of ENL and remained low throughout, in two of three cases. Changes in SAP concentration parallel anti-sulphatide IgM concentrations. TH3, a monoclonal IgM germ-line antibody derived from a LL patient, and SAP share similar binding patterns. In this study we demonstrate binding to heparin and sulphatide. Moreover, SAP inhibited the binding of TH3 to sulphatide, as well as anti-sulphatide IgM found in a range of sera, and anti-sulphatide IgG in the only sera sample in which it was found. The observation that anti-TH3 idiotype monoclonal and polyclonal anti-SAP antibodies both inhibited the binding of TH3 and IgM in sera (but not IgG) to sulphatide without binding to sulphatide themselves further demonstrated similar binding specificities. The observations of similarity in binding reinforce ideas that SAP may function as a primitive opsonin, but the clear ability to inhibit binding of autoantibodies suggests that SAP may play a role in ameliorating tissue and particularly nerve damage in leprosy patients.

Autoantibodies to cerebroside sulphate (sulphatide) in leprosy.

Sera from 40 leprosy patients were screened for autoantibodies to cerebroside sulphate (sulphatide). Anti-sulphatide IgM in groups of patients with lepromatous (LL) and borderline (BL + BB + BT), but not with tuberculoid (TT) disease, were significantly elevated above the levels found in endemic control subjects. Eight-six percent (18 out of 21; mean 1.59 OD units) of LL, 33% (four out of 12; mean 1.08 OD units) of borderline and 13% (one out of eight; mean 0.69 OD units) of tuberculoid patients had anti-sulphatide IgM in their sera above a cut-off value of 2 s.d. above the mean value (0.66 OD units) for control sera. Elevated anti-sulphatide IgG was detected in only one patient's serum, an individual with LL disease. The level of anti-sulphatide IgM was strongly correlated to expression of the TH3 idiotype, an idiotype previously defined by a human MoAb that bound Mycobacterium leprae phenolic glycolipid, Klebsiella capsular polysaccharide, polynucleotides and human tissues. The purified, TH3 MoAb was found in this study to bind sulphatide, but not cholesterol-3-sulphate or cerebroside. It is suggested that anti-sulphatide IgM is elevated in leprosy, in relation to the bacterial load. Anti-sulphatide IgM fell at the onset of erythema nodosum leprosum (ENL) reaction, consistent with the deposition of serum antibodies, and thus may play a part in pathology during periods of inflammation, particularly in multibacillary patients.

Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.

Phospholipase activity of Mycobacterium leprae harvested from experimentally infected armadillo tissue.

Three types of phospholipase activity--phospholipase A1, A2, and lysophospholipase--were detected in Mycobacterium leprae harvested from armadillo tissue at about 25% of the specific activity found in a slowly growing mycobacterium, Mycobacterium microti, which was grown in medium to optimize its phospholipase activity. The highest activity found was lysophospholipase, which released fatty acid from 2-lyso-phosphatidylcholine. Phospholipase activity was detected by using phosphatidylcholine and phosphatidylethanolamine. Differences in relative activities with these three types of substrate distinguished phospholipase activity in M. leprae extracts from armadillo liver extracts. Furthermore, retention of activity in M. leprae after NaOH treatment showed that the activity associated with M. leprae was not host derived. The specific activity of phospholipase was 20 times higher in extracts of M. leprae than in intact M. leprae organisms. Diazotization, a treatment which abolishes activities of surface enzymes exposed to the environment by the formation of covalent azide bonds with exposed amino groups, did not affect M. leprae's phospholipase activity, with one exception: release of arachidonic acid from phosphatidylcholine, which was partially inhibited. Phenolic glycolipid I, the major excreted amphipathic lipid of M. leprae, inhibited phospholipase activity, including release of arachidonic acid, for both M. leprae- and armadillo-derived activity.

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.

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.

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.

Pyrimidine scavenging by Mycobacterium leprae.

Mycobacterium leprae incorporated exogenously supplied pyrimidines as bases or nucleosides, but not as a nucleotide, into its nucleic acids. Notably, thymine was incorporated approximately 5 times more rapidly than thymidine by both suspensions of, or intracellular M. leprae. Thymine incorporation was significantly inhibited by clofazamine and dapsone at near-pharmacological levels. Therefore, incorporation of thymine is preferable as an activity for assessing viability of M. leprae. Nucleosides were converted to nucleotides through kinases, bases through phosphoribosyltransferases. Alternatively, thymine and uracil could first be converted to nucleosides. Cytosine and uracil bases were interconvertible, and uracil alone could supply all the pyrimidine requirements of M. leprae, though conversion to the thymine base was extremely slow. Overall, pyrimidine scavenging occurs at a slower rate than, and appears not to be so important as purine scavenging in M. leprae.

Pyrimidine biosynthesis de novo in M. leprae.

Mycobacterium leprae can synthesise pyrimidines de novo. Although pyrimidine synthesis could not be detected in intact bacteria, extracts contained all four enzymes unique to the de novo pathway which are detectable in mycobacteria by the methods used. Inhibition of aspartate transcarbamylase by UTP and ATP suggested that lack of pyrimidine synthetic activity in whole M. leprae could be a result of strong feedback inhibition.

Biosynthetic pathways in Mycobacterium leprae.

The scope of this paper is biosynthetic pathways which are found generally in living organisms. Many are present in M. leprae but some are lacking, perhaps reflecting the parasitic mode of existence of this microbe. The biosynthesis of nucleotides (the immediate precursors of nucleic acids) and fatty acids (intermediates in the biosynthesis of most if not all mycobacterial lipids) has been studied in detail in M. leprae. For the former, M. leprae appears incapable of synthesising its own purines de novo and thus depends upon the host for a source of the purine ring. It can synthesise pyrimidines, though enzymes for this activity are inhibited in the M. leprae organisms obtained from host tissue. For the latter, M. leprae is capable of fatty acid biosynthesis de novo, probably at a repressed level. However, a key enzyme, phosphotransacetylase, appears to be deficient.