Leprosy-derived Corynebacteria Research Articles

Analysis of Deoxyribonucleic Acids from Armadillo-Derived Mycobacteria

Armadillos are used for propagation of Mycobacterium leprae and are hosts of mycobacteria of the ADM (armadillo-derived mycobacteria) group. The deoxyribonucleic acids (DNAs) of isolates of the five phenetic subgroups of the ADM were analyzed and compared with the genomes of related bacteria. The guanine-pluscytosine (G+C) contents of the DNAs were 62.2 to 67.1 mol% for different ADM strains, 56 mol% for M. leprae and LDC (leprosy-derived corynebacteria), and 62 to 71 mol% for reference mycobacteria. Restriction analysis showed neither adenine methylation in the GATC site (a specific trait of LDC strains) nor cytosine methylation in the CCGG and GGCC sites. Hybridization higher than 80% was obtained with DNA from isolates within the same ADM subgroups, whereas 17 to 50% hybridizations were observed with organisms from different ADM subgroups. Genomes of ADM strains and reference mycobacteria were 15 to 40% homologous, except for subgroups IV and V, whose DNAs were 54 to 62% homologous with Mycobacterium lepraemurium DNA. Little or no homology between M. leprae and ADM genomes was found. We concluded that single ADM subgroups can be considered as distinct species within the genus Mycobacterium; they are genetically unrelated to the other leprosy-associated bacteria.

Biological, chemical, immunological and staining properties of bacteria isolated from tissues of leprosy patients

Two kinds of microorganisms are found in tissue of leprosy patients: Mycobacterium leprae (ML) and leprosy derived corynebacteria (LDC). ML from untreated patients has an alcohol-acid-fastness, which is lost upon treatment with antibiotics and immune response (tuberculoid leprosy). Vulnerable ML thus produced can be reversibly de-stained by organic solvent: in tissue sections from tuberculoid and treated patients, more bacteria are, thus, revealed by the Wade-Fite than by the Ziehl-Neelsen procedure. Organisms of genera Corynebacterium, Mycobacterium and Nocardia (CMN group), have DNA with %GC contents of 50-70, 69-72, and 68-70 respectively. GC values of DNA from ML and LDC are close to 56%. DNA from different LDC strains display high homology among them and low homology with reference corynebacteria. CMN cell wall consists of interconnected peptidoglycan and polysaccharide-mycolate complex. Peptidoglycan of LDC (and known CMN) has the polysaccharide backbone linked to a tetrapeptide of L-Ala, D-Glu, m-DAP (meso-diaminopimelate), D-Ala. In ML, L-Ala is replaced by glycine. Mycobacterial wall polysaccharides (that of ML is unknown) are branched arabinogalactans with end arabinoses linked to C70 to C90 mycolates. LDC peripheral polysaccharides are arabinogalactomannans with arabinose and mannose lateral strands. Mycolic acids of LDC are of corynomycolic type (C32, C34 and C36 with 1-4 double bonds) and those of ML are of mycobacterial type. Components of CMN wall and cytoplasm are immunologically active as antigens (polysaccharides, proteins), haptens (lipids) and adjuvants (peptidoglycans). Strong intrageneric and weak intergenera crossreactions are observed among CMN bacteria: LDC preparations, however, crossreact strongly with ML and mycobacteria, and weakly with reference corynebacteria. LDC in leprosy tissues can, thus, be revealed as well by fluorescent anti-LDC antisera as by anti-ML antisera. The main crossreacting component is antigen M1 of LDC, which corresponds to antigens Ag 7 of ML and Ag60 of BCG, the active components of lepromin and tuberculin (known reagents for cutaneous tests). Antigen M1 has a polysaccharide moiety crossreacting with the wall polysaccharide of LDC.(ABSTRACT TRUNCATED AT 400 WORDS)

Cross-reactivity of antigens from the cytoplasm and cell walls of some corynebacteria and mycobacteria.

Leprosy-derived corynebacteria (LDC) are non-acid-fast organisms isolated from leprosy lesions in humans. In this study 20 antigens of native LDC cytoplasm were identified by immunoelectrophoresis, and autoclaving yielded the M1 component, which strongly cross-reacted with antigen 60 of Mycobacterium bovis BCG (bacille Calmette-Guérin) and antigen 7 of Mycobacterium leprae. The polysaccharide moiety of M1 was immunologically related to the LDC cell wall polysaccharide previously characterized as arabinogalactomannan. The latter polysaccharide competitively inhibited the formation of immune complexes by labeled M1 and antisera to the LDC cell wall; cytoplasm and wall polysaccharides from other bacteria produced lower-level inhibition. In a radioimmunoassay with 125I-labeled antigen 7 of M. leprae, sera from patients with leprosy and antisera to the LDC cell wall yielded overlapping curves. Sera from patients with tuberculoid leprosy and those from patients with lepromatous leprosy afforded different levels of inhibition in this radioimmunoassay; this result indicated a difference in antibody specificity in the two forms of leprosy. In conclusion, the cell wall polysaccharide of LDC corresponds to the main thermostable cytoplasmic antigen M1, which strongly crossreacts with sera from patients with leprosy and, more specifically, with antigen 7 of M. leprae.

Description of Corynebacterium tuberculostearicum sp. nov., a leprosy-derived corynebacterium

Leprosy-derived corynebacteria (LDC) have been extensively studied over the past decade. A composite of their biological properties (cell morphology, staining reactions, cellular inclusions and guanine-plus-cytosine content of their deoxyribonucleic acid; 16 strains studied) and their chemical structures (peptidoglycan type, major cell wall polysaccharide, major glycolipid as well as characteristic mycolic acids) appears to define them as members of the genus Corynebacterium. In relation to other corynebacteria found in humans, including "JK corynebacteria", they seem to be distinct. They are here named Corynebacterium tuberculostearicum sp. nov. because they produce a 10-methyloctadecanoic (tuberculostearic) acid (8 strains studied). This and some of their other attributes are considered in relation to properties of leprosy bacilli and Mycobacterium leprae.

Cell envelope architectures of leprosy-derived corynebacteria,Mycobacterium leprae, and related organisms: a comparative study

Cell-envelope architectures of three strains of leprosy-derived corynebacteria (LDC) named Kim, FPSA, and 43LL were compared withMycobacterium leprae andM. avium as well as with related organisms (Nocardia asteroides andCorynebacterium psuedotuberculosis). Cytochemical studies were performed at the ultrastructural level after the lead citrate, silver proteinate, acidic phosphotungstic, and ruthenium red colorations. This study showed that, while the organisms belonging to theCorynebacterium-Mycobacterium-Nocardia (CMN) group had only the cytoplasmic membrane but not the cell wall reacting with the silver proteinate coloration, the LDC organisms had both the cell wall and the cytoplasmic membrane reacting with this coloration method. Moreover, the three strains of the LDC organisms differed from one another at the level of their exopolymer content.Mycobacterium leprae, on the other hand, gave a cytochemical response common to other mycobacteria and the members of the CMN group studied. Consequently, the envelopes of the LDC organisms were not identical toM. leprae, neither morphologically nor cytochemically.

Properties of microorganisms isolated from human leprosy lesions.

Diphtheroids, which in addition to Mycobacterium leprae are present in human leprosy lesions, were identified as true corynebacteria by DNA and cell wall analysis. Peptidoglycan (adjuvant) of these leprosy-derived corynebacteria (LDC) consists of N-acetylglycosaminyl-N-acetyl(glycolyl)-muramic acid and L-Ala-D-Glu(NH2)-(L)-meso-A2pm-(L)-D-Ala (A2pm = diaminopimelic acid). (The amino group of the tetrapeptide is attached to the carboxyl group of the muramate). Peripheral polysaccharide (antigen) is arabinogalactomannan with lateral chains of mannofuranose and arabinofuranose. To the latter are linked mycolic acids containing groups of isomers with 24-36 carbon atoms and containing between zero and four double bonds. DNAs of LDC isolates have a guanine + cytosine content of 56% and demonstrate a high degree of homology. LDC ribosomes cross-react with antisera against mycobacteria and with sera from patients with leprosy. Thermostable antigen M of LDC cross-reacts with the main antigens of tuberculin and lepromin. LDC thus represents a homogeneous and unique group of corynebacteria immunologically related to M. leprae. Leprosy might be the result of a pathogenic cooperation between both organisms, as suggested by the enhancement of M. leprae growth rate promoted in mice by living LDC.

Chemical composition of cell-wall polysaccharides from leprosy-derived corynebacteria

Chemical composition of cell-wall polysaccharides from leprosy-derived corynebacteria

Chemical composition of cell-wall polysaccharides from leprosy-derived corynebacteria

The importance of cell-wall components for bacterial taxonomy has been repeatedly stressed [1,2]. Microorganisms of genera Corynebacterium, Mycobacterium and Nocardia (CMN group) have envelopes of similar structure and contain 3 kinds of polymers: peptidoglycan, arabinogalactan and mycolates [3,4]. Lechevalier and Lechevalier [5] have classified different genera of Actinomyces into 9 cell-wall types (I-IX) differing in the major amino acid constituents of their cell walls and 4 whole-cell sugar patterns (A-D) by analysis of cell hydrolysates. Cell-wall type IV and whole-cell sugar pattern type A, which are characteristic of the CMN group of microorganisms, are known to contain arabinose and galactose in the approximate ratio of 5 : 2. Two kinds of microorganisms have been recognized in human leprosy lesions: (a) Mycobacterium leprae, acid-fast Gram-positive, which does not replicate in vitro; and (b) some non-acid-fast Gram-positive bacteria which multiply in axenic culture and were previously indicated as 'diphtheroids' because of their resemblance to Corynebacterium diphtheriae (cf. refs. 6 and 7 for review). These microorganisms were recently characterized as true corynebacteria according to their DNA base composition (56 to 58% GC) [8] and mycolic acid structure (polyunsaturated corynomycolic acids) [10,13]. Consequently, 'diphtheroids' were renamed as 'leprosy derived corynebacteria' (LDC) according to Barksdale [11]. The aim of the present study was the purification and analysis of cell-wall polysaccharides of 3 LDC and 2 reference microorganisms, Corynebacterium hoffmannii and Mycobacterium smegmatis.

Analysis of mycolic acids from a group of corynebacteria by capillary gas chromatography and mass spectrometry.

The cell wall of leprosy-derived corynebacteria (a group of 'diphtheroids' isolated from human leprosy lesions and patients' blood) was previously shown to contain, in addition to peptidoglycan and arabinogalactan, mycolic acids. These alpha-branched beta-hydroxy fatty acids were attributed to the corynomycolic group, according to their RF in monodimensional thin-layer chromatography. In the present work, mycolic acids from leprosy-derived and reference corynebacteria have been fractionated by monodimensional and bidimensional thin-layer chromatography and by gas chromatography. Pyrolyzed mycolic acids have been analyzed on conventional packed columns, whereas intact methyl esters of mycolic acids with free and silylated beta-hydroxyl group have been analyzed on capillary columns, and their structure has been established by mass spectrometry. In all leprosy-derived corynebacteria, some 20 components containing 24-36 carbon atoms and 0-4 double bonds were obtained. The three major groups had 32, 34 and 36 carbons, and the frequency of unsaturated versus saturated chains increased proportionally to the molecular weight. For comparison, the main components of a reference corynebacterium. Corynebacterium diphtheriae PW8, had 30 and 32 carbons, and their hydrocarbon chains were essentially saturated. This work confirms the relative chemical homogeneity of different leprosy-derived corynebacteria and describes some peculiar traits in the chemical structure of this group of organisms. In addition, it shows the complexity of the mycolic acid fraction of corynebacterial cell wall and suggests that the mycolic acid pattern is a sort of fingerprint of each bacterial strain grown under standard conditions. Finally, the fractionation of intact corynomycolic acid methyl esters with free or silylated beta-hydroxyl group by capillary gas chromatography proved to be the best analytical procedure at present available for resolving this complex mixture of corynomycolate isomers. Structural determination of silylated samples by mass spectrometry is preferred because they have more diagnostic fragments.

Base Compositions and Homologies of Deoxyribonucleic Acids of Corynebacteria Isolated from Human Leprosy Lesions and of Related Microorganisms

The deoxyribonucleic acids (DNAs) of 25 strains of leprosy-derived corynebacteria (LDC)—non-acid-fast, gram-positive bacteria independently isolated from human leprosy lesions and propagated in axenic culture—were purified and analyzed. The guanine plus cytosine content, by buoyant density determination, was 54 to 59 mol% for most LDC strains, a range that corresponds to that (50 to 60 mol%) of corynebacteria which multiply in animal cells. These values were checked by chromatographic analyses of acid digests of the DNAs. The taxonomic position of the LDC as determined by DNA base composition was confirmed by the results of the corynomycolic acid determinations of the cell walls of the LDC. The results of the hybridization of the DNAs from the LDC strains suggest the occurrence of two high-homology groups, in which most of the strains were accommodated. In contrast, little homology was observed between the DNAs of the LDC and the reference corynebacteria employed. From these data, it can be inferred that the LDC represent a homogeneous and unique cluster of organisms within the genus Corynebacterium, more specifically within the group of corynebacteria pathogenic for humans.

Primary structure of the wall peptidoglycan of leprosy-derived corynebacteria.

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan strands of the wall peptidoglycan are essentially composed of N-acetylglycosaminyl-N-acetylmuramic acid disaccharide units. Small amounts of N-acetylglycosaminyl-N-glycolylmuramic acid (less than 10%) were also detected. The muramic acid residues of adjacent glycan strands are substituted by amidated tetrapeptide units which, in turn, are cross-linked through direct linkages extending between the C-terminal D-alanine residue of one tetrapeptide and the mesodiaminopimelic acid residue of another tetrapeptide. Such a structure is very similar to that of the wall peptidoglycan found in the taxonomically related microorganisms of the Corynebacterium, Mycobacterium, and Nocardia groups.