Increased intracellular survival of Mycobacterium smegmatis containing the Mycobacterium leprae thioredoxin-thioredoxin reductase gene.

The thioredoxin (Trx) system of Mycobacterium leprae is expressed as a single hybrid protein containing thioredoxin reductase (TR) at its N terminus and Trx at its C terminus. This hybrid Trx system is unique to M. leprae, since in all other organisms studied to date, including other mycobacteria, both TR and Trx are expressed as two separate proteins. Because Trx has been shown to scavenge reactive oxygen species, we have investigated whether the TR-Trx gene product can inhibit oxygen-dependent killing of mycobacteria by human mononuclear phagocytes and as such could contribute to mycobacterial virulence. The gene encoding M. leprae TR-Trx was cloned into the apathogenic, fast-growing bacterium Mycobacterium smegmatis. Recombinant M. smegmatis containing the gene encoding TR-Trx was killed to a significantly lesser extent than M. smegmatis containing the identical vector with either no insert or a control M. leprae construct unrelated to TR-Trx. Upon phagocytosis, M. smegmatis was shown to be killed predominantly by oxygen-dependent macrophage-killing mechanisms. Coinfection of M. smegmatis expressing the gene encoding TR-Trx together with Staphylococcus aureus, which is known to be killed via oxygen-dependent microbicidal mechanisms, revealed that the TR-Trx gene product interferes with the intracellular killing of this bacterium. A similar coinfection with Streptococcus pyogenes, known to be killed by oxygen-independent mechanisms, showed that the TR-Trx gene product did not influence the oxygen-independent killing pathway. The data obtained in this study suggest that the Trx system of M. leprae can inhibit oxygen-dependent killing of intracellular bacteria and thus may represent one of the mechanisms by which M. leprae can deal with oxidative stress within human mononuclear phagocytes.     

Adjuvant Activity of Mycobacterium leprae

Mycobacterium leprae organisms isolated from infected spleen and liver tissue by zonal centrifugation were shown to possess adjuvant activity. Histochemical examination of the footpad macrophage epithelioid granuloma showed that macrophages contained large amounts of hemosiderin after the injection of M. leprae and much smaller amounts after the injection of M. tuberculosis.     

Protein antigens of Mycobacterium leprae

Protein antigens of Mycobacterium leprae have been identified by screening the lambda gt11, pYA626 and pHC79::M. leprae genomic libraries with pooled sera from leprosy patients and with antiserum to M. leprae cell wall protein (CWP) aggregate. Immunological screening of the lambda gt11 library with pooled sera from 21 lepromatous (LL) leprosy patients resulted in the identification of 19 antigens that are apparently different from previously identified M. leprae antigens. Five additional antigens were identified by screening the lambda gt11 library with pooled sera from 30 borderline tuberculoid or tuberculoid patients. Four other antigens were identified by screening the lambda gt11 library with anti-CWP. Two groups of recombinant cosmids were identified by screening the pHC79 library with LL patients' sera: one group specified proteins that reacted with monoclonal antibodies (mAb) against the 65-kDa protein and against the 18-kDa protein; the other group specified a 15-kDa protein that did not react with any of the mAb that were tested. One pYA626 clone also specified a 15-kDa protein that reacted with LL patients' sera, but did not react with any mAb. Genes specifying several of these antigens have been subcloned into the Asd+ plasmid vector pYA292 and have been introduced into a delta cya delta crp delta asd Salmonella typhimurium strain to evaluate the ability of individual M. leprae proteins to elicit immune responses against M. leprae infection.     

Secreted Proteins of Mycobacterium leprae

In mycobacteria, secreted proteins represent a distinct group, probably of particular importance for development of immune responses following infection. Quantification of individual proteins in culture fluid and corresponding disrupted bacilli permits determination of a localization index for identification of secreted proteins. This procedure cannot be applied to Mycobacterium leprae because secreted proteins are lost during isolation of bacilli from tissues. The DNA sequences of secreted proteins of Mycobacterium tuberculosis were compared with sequences of M. leprae . Genes for homologues of the 85a, 85b, 85c , mpt32 ( apa ), mpt51 , erp, mtc28, Rv2376c, Rv3354 and Rv0526 genes were identified. All of these contain signal sequences typical for secretion in M. leprae . In several instances the local distance between marker genes and occurrence on the same or the complementary DNA strand was similar in these two species. The genomic organisation of genes for secreted proteins is thus very similar in M. leprae and M. tuberculosis , the homology being higher for the mature polypeptide chains than for the corresponding signal peptides.     

Antigenic Analysis of Mycobacterium leprae

About twenty distinct antigenic components have been demonstrated in Mycobacterium leprae (M. leprae) by crossed immunoelectrophoresis against a rabbit antiserum produced by immunization with concentrated M. leprae antigen. This system allows a more detailed analysis of the antigenic relationship between M. leprae and other mycobacteria and a better characterization of the antigenic content of various M leprae preparations than with previously available anstisera of the antigenic content of various M. leprae preparations than with previously available antisera which reacted with far fewer components. The antibody activity in sera of patients with lepromatous leprosy was studied by incorporating the sera into the intermediate gel of the M. leprae reference system. Antibodies were found against only seven of the components. Since those compared are all known to be cross-reacting widely with antigens of other mycobacteria, it is speculated that cross-immunization may influence the antibody response in lepromatous leprosy.     

麻风菌重组蛋白的抗原性分析

目的以ND-O-BSA、LID-1麻风菌特异抗原为参照,评价各类受检者对7种麻风菌新抗原的血清学反应性,从而筛选出可应用于麻风病诊断的抗原。方法 应用ND-O-BSA、LID-1、ML0098ss、ML0840、ML1632ss、ML2028、ML2044、ML2055ss和46f等9种合成或重组抗原建立ELISA方法,分别检测多菌型麻风病（Multibacillaryleprosy,MB）和少菌型麻风病（Paucibacillary leprosy,PB）患者以及来自非流行区、麻风病轻度和重度流行区的健康人血清中的相应抗体。结果 ELISA显示7种新抗原中ML0840与ND-O-BSA、LID-1抗原敏感性、特异性和准确性相近,在对PB患者的检测上,其准确性更显优势,高达72%。46f、ML0098ss、ML2028对MB患者的敏感性依次为86%、79%和76%,但对PB患者的检测只有ML0098ss敏感性达到55%。ML1632ss、ML2044、ML2055ss 3种抗原无论对MB还是PB患者的敏感性和特异性均较差。结论 7种麻风菌重组蛋白质均具有不同程度的抗原性,ML0840与LID-1、ND-O-BSA是目前有应用潜力的抗原,但需在血清学监测中证实其早期诊断麻风病的价值。     

Properties of lysophospholipase in Mycobacterium leprae

Lysophospholipids are key intermediates in the metabolism of phospholipids. Cytoplasmic membranes of both eukaryotes and prokaryotes are made of phospholipid bilayers. Phospholipases are activated during phagocytosis. Lysophospholipids generated by phospholipase A₂ or A₁ degrade cell membranes and can cause cell lysis. An active lysophospholipase, that hydrolyzes lysophospholipids, was detected by the radioisotope technique in Mycobacterium leprae. About two-thirds of the enzyme was particulate and one-third cytoplasmic. Optimum activity was at 37 °C, and at pH 6.0. Temperatures above 70 °C completely inactivated the enzyme. The compound AACOCF3, a trifluromethylketone analog of arachiodonic acid, inhibited the activity; the inhibition appeared to be of the uncompetetive type. The K_m of the enzyme was 2.5 × 10^?4 M, suggesting a fairly strong affinity for the substrate. Lysophospholipids have been shown to be microbicidal to invading organisms. Possession of lysophospholipase by M. leprae is apparently one of the methods by which the bacilli overcome the defense mechanisms of the host.     

Advances in Proteomics of Mycobacterium leprae

Although Mycobacterium leprae was the first bacterial pathogen identified causing human disease, it remains one of the few that is non-cultivable. Understanding the biology of M. leprae is one of the primary challenges in current leprosy research. Genomics has been extremely valuable, nonetheless, functional proteins are ultimately responsible for controlling most aspects of cellular functions, which in turn could facilitate parasitizing the host. Furthermore, bacterial proteins provide targets for most of the vaccines and immunodiagnostic tools. Better understanding of the proteomics of M. leprae could also help in developing new drugs against M. leprae. During the past nearly 15 years, there have been several developments towards the identification of M. leprae proteins employing contemporary proteomics tools. In this review, we discuss the knowledge gained on the biology and pathogenesis of M. leprae from current proteomic studies.     

Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay.

By using a set of four nested oligonucleotide primers, a two-step polymerase chain reaction assay for the detection and identification of Mycobacterium leprae that does not require the use of radioactivity labeled hybridization probes was developed. The nested-primer procedure amplified a 347-base-pair product from M. leprae genomic DNA. No amplification products were produced from DNAs of 19 other Mycobacterium species, 19 non-Mycobacterium species, mouse cells, or human cells. Minor amplification products were observed with three additional Mycobacterium species, i.e., "M. lufu", M. simiae, and M. smegmatis. These products were easily distinguished from the M. leprae product by size and restriction enzyme cleavage patterns. The assay could amplify the 347-base-pair product from samples containing as little as 3 fg of M. leprae genomic DNA--the amount of DNA in a single bacillus. The assay also amplified target sequences in crude lysates of M. leprae bacilli isolated from tissue biopsy specimens from infected animals and humans. The entire assay, from sample preparation to data analysis, can be completed in less than 8 h.     

Genetic predisposition to leprosy: A major gene reveals novel pathways of immunity to Mycobacterium leprae

The elucidation of the genetic control of susceptibility to common infectious diseases is expected to provide new and more effective tools for prevention and control of some of the most pressings health needs on a global scale. A major advantage of whole genome based genetic approaches is that no a priori assumptions about mechanisms of pathogenesis need to be made in these studies. Hence, genetic studies can identify previously unrecognized pathways of disease susceptibility and tag critical pathogenic events for further biochemical, immunological or physiological analysis. We have applied this strategy to leprosy, a disease that still claims 400,000 new cases each year. We identified genetic variants in the shared promoter region of the PARK2 and PACRG genes as major risk factors of leprosy susceptibility. Both encoded proteins are part of the cellular ubiquitination system. Specifically, PARK2, the cause of early onset Parkinson's disease, is an E3 ligase that likely is involved in controlled proteolysis, the cellular anti-oxidants response and the regulation of innate immune responsiveness. In addition, numerous E3 ligases have recently been shown to be critical regulators of immunity. While the specific role of PARK2/PACRG in leprosy pathogenesis remains unknown, a number of experimentally testable scenarios can be developed to further explore the role of these proteins in anti-Mycobacterium leprae host responsiveness.     

Heat stability of Mycobacterium leprae immunogenicity.

The protection provided to mice by vaccines administered intradermally was measured after footpad challenge with Mycobacterium leprae. The protection offered by M. leprae suspensions was not decreased when the vaccines were killed by 60 degrees C heat or at the higher temperatures tested, which included 215 degrees C (autoclave). Even highly purified suspensions retained their immunogenicity. In contrast, the vaccine protection provided by intradermal M. bovis (strain BCG) was markedly reduced when heated to 60 degrees C. The enlargement of the lymph nodes regional to the intradermal vaccines was measured and found generally to parallel the vaccine protection provided by M. leprae and by BCG.     

Expression of Mycobacterium tuberculosis and Mycobacterium leprae proteins by vaccinia virus.

Eight Mycobacterium tuberculosis and M. leprae genes were inserted into the vaccinia virus genome by in vivo recombination. The resulting virus recombinants were shown to express five different M. tuberculosis proteins (71, 65, 35, 19, and 12 kDa) and three M. leprae proteins (65 and 18 kDa and a biotin-binding protein) by Western immunoblot analysis, radioimmunoprecipitation, or black-plaque assay. When injected into BALB/c mice, the recombinants expressing the M. tuberculosis 71-, 65-, or 35-kDa protein and the M. leprae 65-kDa protein or the biotin-binding protein elicited antibodies against the appropriate M. tuberculosis or M. leprae protein. These vaccinia virus recombinants are being tested for the ability to elicit immune protection against M. tuberculosis or M. leprae challenge in animal model systems. The recombinants are also useful in generating target cells for assays aimed at elucidating the cellular immune responses to mycobacterial proteins in leprosy and tuberculosis. Furthermore, the M. tuberculosis 65-kDa protein and four of the other mycobacterial proteins share homology with known eucaryotic and procaryotic stress proteins, some of which may play a role in autoimmunity.     

DNA methylation in leprosy-associated bacteria: Mycobacterium leprae and Corynebacterium tuberculostearicum

The DNAs of two kinds of microorganisms from human leprosy lesion, Mycobacterium leprae and Corynebacterium tuberculostearicum (also known as leprosy-derived Corynebacterium or LDC), have been analysed and compared with the genomes of reference bacteria of the CMN group (genera Corynebacterium, Mycobacterium and Nocardia). The guanine-plus-cytosine content (% GC) of DNA was determined by a double-labelling procedure, which is unaffected by the presence of modified and unusual bases (that alter both buoyant density and mid-melting-point determinations). Accordingly, the DNAs of seven LDC strains had GC values of 54–56 mol %, and that of armadillo-grown M. leprae a value of 54.8±0.9 mol %. Restriction patterns disclosed no methylated cytosine in the DNA sequences CCGG, GGCC, AGCT and GATC of either LDC or M. leprae DNA. N⁶-methyl adenine was present in the sequence GATC of all LDC strains, but was missing from the genomes of all others CMN organisms analysed, including M. leprae. By HPLC analysis of LDC-DNA hydrolysates, it was found that N⁶-methyladenine amounted to 1.8% of total DNA adenine, and was present exclusively within GATC sequences, which appeared all to be methylated. It is concluded that LDC represent a group of corynebacteria endowed with high genetic homogeneity and a unique restriction pattern, whereby their genome is easily distinguished from that of M. leprae, which has a similar base composition.Abbreviations %GC guanine-plus-cytosine content of DNA (mol/100 mol) - T _m midpoint of optical absorption (A_260nm) melting curve of DNA - MeA N⁶ methyl-adenine - MeC 5 methyl-cytosine - HPLC high performance liquid chromatography Enzyme Code DNA poll deoxynucleotide triphosphate - DNA deoxynucleotidyltransferase (EC 2.7.7.7) - RNAase I ribonuclease A (EC 3.1.27.5)     

Metabolism of Mycobacterium leprae in macrophages.

The incorporation of 14C-labeled palmitic acid ( [U-14C]PA) into the phenolic glycolipid-I (PGL-I) fraction of Mycobacterium leprae was studied in a murine macrophage system in vitro. Peritoneal macrophages from Swiss Webster mice were infected with fresh viable or Formalin-killed M. leprae harvested from infected footpads of nu/nu mice, and [U-14C]PA was added to the culture medium. Labeled glycolipid synthesized by live M. leprae was fractionated on a Florisil-silicic acid column and identified as PGL-I by using thin-layer chromatography and localization on a polysulfone membrane with an anti-PGL-I monoclonal antibody. Increased incorporation of [U-14C]PA into the PGL-I fraction was observed in macrophages infected with only live M. leprae. Treatment of the infected macrophages with rifampin caused a significant reduction in the incorporation of palmitic acid into PGL-I. These preliminary studies suggest that PGL-I synthesis can be used to quantitate the metabolism of M. leprae in macrophages in vitro.     

DNA methylation in leprosy-associated bacteria: Mycobacterium leprae and Corynebacterium tuberculostearicum

The DNAs of two kinds of microorganisms from human leprosy lesion, Mycobacterium leprae and Corynebacterium tuberculostearicum (also known as "leprosy-derived corynebacterium" or LDC), have been analysed and compared with the genomes of reference bacteria of the CMN group (genera Corynebacterium, Mycobacterium and Nocardia). The guanine-plus-cytosine content (% GC) of DNA was determined by a double-labelling procedure, which is unaffected by the presence of modified and unusual bases (that alter both buoyant density and mid-melting-point determinations). Accordingly, the DNAs of seven LDC strains had GC values of 54-56 mol %, and that of armadillo-grown M. leprae a value of 54.8 +/- 0.9 mol %. Restriction patterns disclosed no methylated cytosine in the DNA sequences CCGG, GGCC, AGCT and GATC of either LDC or M. leprae DNA. N6-methyl adenine was present in the sequence GATC of all LDC strains, but was missing from the genomes of all others CMN organisms analysed, including M. leprae. By HPLC analysis of LDC-DNA hydrolysates, it was found that N6-methyladenine amounted to 1.8% of total DNA adenine, and was present exclusively within GATC sequences, which appeared all to be methylated. It is concluded that LDC represent a group of corynebacteria endowed with high genetic homogeneity and a unique restriction pattern, whereby their genome is easily distinguished from that of M. leprae, which has a similar base composition.     

Viability of Mycobacterium leprae after multiplication in mice

To measure the rate at which Mycobacterium leprae are killed in the course of the mouse footpad infection after the maximum of multiplication has been achieved, M. leprae were harvested shortly before and at intervals after multiplication had reached the level of 10(6) organisms per footpad, serially diluted, and inoculated into the footpads of passage mice. Beginning 1 year later, foot-by-foot harvests of M. leprae were performed from passage mice, and the proportion of viable organisms in the passage inocula was calculated by means of a most-probable-number calculation. In addition, the proportion of solidly staining M. leprae was measured in the passage inocula. The proportion of viable M. leprae in the passage inocula was found to decrease with the time after multiplication to 10(6) organisms per footpad of donor mice; the half-time of loss of viable M. leprae was 25 days. The proportion of solidly staining organisms appeared to be directly related to the proportion of viable organisms, as measured by mouse passage, and inversely proportional to the time after multiplication to 10(6) organisms per footpad.     

Biophysical optima for metabolism of Mycobacterium leprae.

The metabolic response of freshly harvested, nude-mouse-derived Mycobacterium leprae to biophysical parameters was studied to facilitate an understanding of axenic culture requirements. Quantitation of intracellular ATP and the rate of [U-14C]palmitic acid incorporation into phenolic glycolipid I (PGL-I) were used as metabolic indicators after axenic incubation in modified Dubos medium under various biophysical conditions. PGL-I synthesis was optimal at 33 degrees C, whereas ATP was optimally maintained at less than or equal to 33 degrees C. Both metabolic indices showed sharp reductions at 37 degrees C. After 5 days of incubation, PGL-I synthesis and ATP maintenance showed pH optima of 5.1 to 5.6, with the higher value appearing optimal for ATP maintenance after extended incubation. Metabolic activity was negatively affected by strong reducing agents, and ATP maintenance was optimal when the gaseous environment was maintained at 2.5 to 10% oxygen. The results may partially explain the failure to cultivate the leprosy bacillus in vitro.