Cord Factors Research Articles

内毒素に類似する生物学的作用を示すＢＣＧ由来の免疫調節因子

An immunostimulator agent was partially purified from the phenol-water extracts of M. bovis BCG by fractionation with Sepharose 6B and 4B columns. The agents showed multifold bioactivities, many of which are common to endotoxins. They are pyrogenicity, lethality in galactosamine-loaded mice, induction of tumor-necrosis-factor and interferons α/β in primed mice and immunoadjuvancy enhancing antibody production following in vivo administration, activation of the clotting enzyme cascade of the horseshoe crab, complement activation, and stimulatory effects on murine splenocytes and guinea-pig macrophages in in vitro assay. Although the chemical entity responsible for the above bioactivities has not yet been fully understood, chemical analysis revealed that the bioactive agent is polysaccharide (ca 92%), whose main component sugars are mannose and arabinose, connected with fatty acids (ca 10%), amino acids and amino sugars (ca 3.5% together), containing no detectable amounts of such constitutes characteristic of known immunomodulators of bacterial origin as LPS, cord factors, cell wall peptidoglycans, lipoteichoic acids and biologically active nucleic acids.

A new synthesis of cord factors and analogs

Treatment of 6,6′-di- O-trityl-trehalose (1) [2] with benzyl chloride in dioxane followed by acid hydrolysis and chromatography gave the chromatographically pure 2,3,4,2′,3′,4′-hexa- O-benzyl trehalose (2). Compound 2 was converted into the corresponding 6,6′-di- O-methane-sulphonyl derivative 3 in quantitative yield. Treatment of the latter compound with the potassium salts of 4-[ p-(hexadecyloxy)-phenyl]butyric acid, corynomycolic acid and mycolic acid from Mycobacterium bovis afforded the corresponding benzylated-6,6′-di- O-acyl esters 4, 5 and 6 respectively. Catalytic hydrogenolysis of 4, 5, and 6 yielded 6,6′-di- O-4-[ p-(hexadecyloxy)-phenyl] butyryl-trehalose 7; 6,6′-di- O-corynomycolyl-trehalose 8; and 6,6′-di- O-bovi-mycolyl-trehalose 9 respectively.

Evidence for penetration in liposomes and in mitochondrial membranes of a fluorescent analogue of cord factor.

A fluorescent analogue of cord factor, a glycolipid toxin of mycobacteria, has been synthesized and its interactions with liposomes and isolated mitochondria have been studied. This compound, methyl alpha-D-6[12-(9-anthroyl)stearoyl]glucoside, is shown to be active against oxidative phosphorylation. When spread as a monolayer at the air-water interface, it forms a well organized phase and it strongly interacts with phosphatidylcholine. Addition of phosphatidylcholine liposomes or of isolated mitochondria to a water disperson of this fluorescent cord factor analogue results in a large increase of the fluorescence intensity. Moreover, the glycolipid probes for the temperature-dependent phase transition of the added suspensions. It is thus suggested that this cord factor analogue penetrates within mitochondrial membranes, a result which is discussed with respect to our previous conclusions concerning the way natural cord factors can interact with these organelles.

Pseudo cord factors: Derivatives of α- D-glucopyranuronosyl (1-1) α- D-glucopyranuronoside

We describe the synthesis of 6 pseudo cord factors (Ψ CF), analogs of the natural trehalose-6,6′-dimycolate, but based instead upon the dicarboxylic acid (TDA) that is obtained from trehalose by Pt-catalyzed oxidation. From TDA, several bis-amides (‘mirror amide’ Ψ CF) and a diester (‘mirror’ Ψ CF) of intermediate to high molecular weight were prepared. These superficially resemble cord factor, have similar infared spectra and, like the natural product, several have impressive toxicity in mice and tumor-regression activity; but the latter property does not depend upon the former. A curious abrogation of biological activities results from introduction of a hexamethylene diamine ‘spacer’ between the carbohydrate core and the lipid substitutents. The results suggest that (excepting the ‘spacer’ effect) the type of covalent linkage between the carbohydrate and lipid moieties may be relatively unimportant for expression of some of the biological activities of cord-factor-like glycolipids.

Property and Activity of Mycoloyl Esters of Methyl Glucoside and Trehalose

Methyl alpha-D-glucoside and alpha, alpha'-D-trehalose acylated on their primary hydroxyl group by alpha-alkyl-beta-hydroxy acids (mycolic acids) with 18, 32 and more than 80 carbon atoms were studied. These cord factors and analogues when dispersed in water gave hydrated and organized phases which bore resemblances and differences to phospholipid liposomes. Phase transition temperature was determined by fluorescence for the C32 derivatives and by differential scanning calorimetry for the C80 derivatives. A narrow temperature interval between the gel and the fluid states was observed, indicating that this transition was a strongly cooperative phenomenon. The activity on mitochondria of these diacyl trehaloses was studied with succinate as substrate. The fluid suspensions were more active than the rigid ones. There was no influence of the relative configuration of the mycoloyl residue. Acyl chains with 18, 32 and more than 80 carbon atoms had rather similar activities on phosphorylation, but differences appeared when considering effects on respiration. Electron microscopy suggested that cord factor weakened the peripheral part of the inner membrane, but cristae looked unchanged. These observations, together with the results of our previous monolayer studies of these glycolipids, led us to propose a model for interaction of cord factor with mitochondria.

Nonspecific immunostimulant activities of synthetic trehalose-6,6'-diesters (lower homologs of cord factor)

Mycobacterial cord factors (6,6'-diesters of trehalose with mycolic acids ranging from C80 to C90) have been shown to protect mice effectively against infection with Klebsiella pneumoniae or with Listeria monocytogenes. Our present findings indicate that the low-molecular-weight cord factor of Corynebacterium diphtheriae (with corynomycolic acids ranging from C28 PTO C36) is equally active. Moreover, its synthetic analog (with synthetic C32 mycolic acid) has the same activity. Two lower synthetic 6,6'-diesters of trehalose with C22 acids, which are described here for the first time, as well as dipalmitate and a dioleate of sucrose, were found inactive. The synthetic C76 trehalose diesters, which are capable of enhancing nonspecific resistance to infection, increase the immune response in mice, even when injected in metabolizable oil. They induce in the injected paws an inflammatory process weaker and more transient than the natural cord factor.

Sur la structure des diesters de tréhalose (“cord factors”) produits par Nocardia asteroides et Nocardia rhodochrous

The free lipids of Nocardia asteroides contain a series of corynomycolic acids ranging from C 28H 54O 3 to C 36H 68O 3. The cell walls contain “bound lipids” which liberate after alkaline hydrolysis a series of saturated, mono-, di- and tri-unsaturated nocardic acids ranging from C 50H 100O 3 to C 58H 114O 3. From the free lipids a “cord factor” (diester of trehalose) has been isolated (m.p. 76–78°C [ α] D = +62°) which contains only corynomycolic acids ranging from C 32H 66O 3. to C 36H 72O 3. Thus the nocardic acids seem to be incorporated exclusively into the cell wall. The free lipids of Nocardia rhodochrous contain a “cord factor” (m.p. 59–60°C [ α] D = +50°) which is a diester of trehalose containing nocardic acids ranging from C 38H 76O 3 to C 46H 90O 3. The structures of the various corynomycolic and nocardic acids as well as those of the glycolipids (“cord factors”) have been studied mainly by mass spectrometry of the permethylated compounds.

Mass spectrometry of glycolipids. 2. Natural and synthetic diesters of trehalose

This paper describes mass spectrometric studies of the peracetylated derivatives of synthetic and natural 6,6′‐diesters of trehalose containing acyl radicals varying from C22 to C90.The peracetylated synthetic 6,6′‐dieicosnoyl trehalose (I) gave a molecular ion at m/e 1238. The synthetic 2,3,4,2′,3′,4′‐hexaacetyl‐6,6′‐di(α‐eicosanyl‐β‐acetoxy tetracosanoyl)‐trehalose (IIb) showed no molecular ion but a peak (M‐60) at m/e 1910 (Fig. 1). The cleavage of the glycosidic bond leads to typical oxonium ions which are at m/e 611 (A) for (I) and at m/e 977 (B2) for (IIb).The three natural trehalose 6,6′‐diesters examined are the “cord factors” of a human virulent strain of Mycobacterium tuberculosis (strain Prévois), of a bovine attenuated strain (BCG) and of Mycobacterium butyricum.The mycolic acids isolated after saponification of these compounds have also been studied by mass spectrometry. Formulae III, IV, V and VI are proposed for their methyl esters.The molecular weights of the paracetylated “cord factors” are above 3000; no molecular ion could be detected. The oxonium ions, however, give significant peaks which are useful for structural determination. Oxonium ions for the glycolipid of the human virulent strain Prévois are found at m/e 1360–1528 (Fig.2). The peracetylated glycolipid of BCG contains two different series of homologous mycolic acids (acyl ions at m/e 1144–1284 and 1072–1184) and gives oxonium ions at m/e 1432–1572 for one series and at m/e 1360–1472 for the second one. The “cord factor” of M. butyricum gives homologous oxonium ions from m/e 1361 to 1445 corresponding to acyl ions from m/e 1073 to 1157.

Mass spectometry of glycolipids. I. Structure of the "cord factor" of Corynebacterium diphtheriae

“Cord factors” are diesters of α,α‐trehalose produced by Mycobacteriae and Corynebacteriae. Saponification of the “cord factor” of Corynebacterium diphtheriae gives an equimolecular mixture of homologues of the saturated corynomycolic and of the unsaturated corynomycolenic acids (I and II, R = H). Máss spectrometry of their methyl esters confirms their structures (see Fig. 1, R = CH3).The peracetylated cord factor (IV, a, b, c) gave very weak molecular ions at m/e 1630, 1632 and 1634 showing apparently that it is a mixture of the diesters having respectively two saturated, one saturated and one unsaturated, and two unsaturated acyl radicals. Strong peaks at m/e 809 and 807 are attributed to the oxonium ions (V a and V b).These results as well as chemical degradations show that the “cord factor” of C. diphtheriae is a mixture of glycolipids (III a, b, c).

Glycolipids of mycobacteria and related microorganisms

The following groups of glycolipids are reviewed: 1. (1)|The toxic cord factors (6,6′-diesters of trehalose), 2. (2)|Wax D, a macromolecular peptidoglycolipid having adjuvant activity, 3. (3)|The mycosides A, B and C, which are deoxyhexose containing “type-specific” glycolipids of mycobacterial origin, 4. (4)|The phosphatidyl myo-inositol mannosides, 5. (5)|Carotenol-glycosides. Wherever possible, chemical structure, biosynthesis and biological activities are considered.