Isolation and identification of arabinose mycolates of Cell Wall Skeleton (CWS) derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105).

A unique hydrolysis method using a two-layer solution, consisting of diluted hydrochloric acid and toluene was developed to isolate whole arabinose mycolates from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) in order to reveal its pivotal role in enhancing immune responses against tumors.

Morphological study on Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

Mycobacterial cell wall consists of rigid cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidiglycan complex, in which mycoloyl structure varies by the mycobacterial species diversely, whereas the arabinogalactan peptidoglycan structure is consistent comparatively. The CWS of Mycobacterium bovis BCG has long been expected as a potent adjuvant for immunotherapy of malignant tumor. Although the chemical structure of CWS has been established in the last few decades, the physicochemical properties of CWS having highly amphipathic micelle structure with very long mycoloyl and carbohydrate chains are not unveiled. In this study, the ultrastructure of CWS of M. bovis BCG Tokyo 172 (SMP-105), suspended in several solvents with different polarity, was investigated with a particle size analyzer, a transmission electron microscope (TEM) and other techniques. As a result, the particle size was about 4.7 to 67.8 microm in physiological saline, but it became smaller and more compact when suspended in hydrophobic solvents. TEM images showed two different morphological forms distinctively: double folded sheet structure in hydrophilic conditions and multilayered rolled sheet structure in hydrophobic conditions. These studies have revealed characteristic surface features of SMP-105, the hydrophobic moiety occupying dominant space and the hydrophilic moiety smaller space, respectively, which may lead to the acceleration of immunological studies on this product.

Separation and molecular characterization of mycolic acid from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) and BCG substrains by normal-phase high performance liquid chromatography and liquid chromatography/mass spectrometry.

Since mycolic acids, the most characteristic major lipid component in mycobacterial cell envelopes, play pivotal roles in the cell surface-based host immune responses, normal-phase HPLC has been developed to quantify and identify mycolic acids of the cell wall skeleton from Mycobacterium bovis BCG Tokyo 172 (SMP-105).

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).

Study on the cell wall skeleton derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105): establishment of preparation and analytical methods.

Mycobacterial cell walls have diverse adjuvant activities, and in particular, cell wall skeleton (CWS) of Mycobacterium bovis BCG has been expected as a drug for tumor immunotherapy. However, its molecular structure-biological activity relationship has not been fully elucidated despite more than 30 years of intensive research. Since it is important to secure purified CWS for such investigation, we established a preparation method of CWS from M. bovis BCG Tokyo 172 (SMP-105) and developed accurate, precise, and reliable analytical methods, based on previous reports. Furthermore, we confirmed that SMP-105 is composed of mycolic acids; arabinogalactan consisting of arabinose, galactose, and rhamnose; and peptidoglycan consisting of alanine, glutamic acid, diaminopimeric acid, muramic acid, glucosamine, and galactosamine. We also determined the levels of potential impurities that might be contaminated in the original bacterium or arise during the manufacturing process, such as glucose, mannose, non-constituted amino acids, as well as nucleic acid, trehaolse di-mycolate, and bacterial endotoxins. These results demonstrated that the prepared SMP-105 was of sufficient quality for research into the chemistry, bioactivity, and structure-activity relationship of CWS.

Molecular and supra-molecular structure related differences in toxicity and granulomatogenic activity of mycobacterial cord factor in mice.

To establish the structure biological activity relationship of cord factor (trehalose 6,6'-dimycolate, TDM), we compared the molecular or supra-molecular structure of TDM micelles with toxicity, thymic atrophy and granulomatogenicity in lungs and spleen of BALB/c mice. According to the difference in the mycolyl subclass composition, TDM was divided into two groups, one possessing alpha-, methoxy- and keto-mycolates in M. tuberculosis H37Rv, M. bovis BCG and M. kansasii (group A) and the other having alpha-, keto- and wax ester-mycolates in M. avium serotype 4, M. phlei and M. flavescens (group B), although mycolic acid molecular species composition differed in each group considerably. Supra-molecular structure of TDM micelle differed species to species substantially and the micelle size of TDM from M. bovis BCG Connaught was the largest. The highest toxicity was shown with TDM from M. tuberculosis H37Rv which possessed the highest amount of alpha- (47.3%) and methoxy-mycolates (40.8%), while TDM from M. phlei having the low amount of alpha-mycolate (11.6%) showed almost no toxicity with the given doses. The thymic atrophy was observed with TDM from group A, but not with TDM from group B. On the other hand, TDM from group B showed massive lung granulomatogenic activity based on the histological observations and organ indices. Taken together, group A TDM showed a wide variety of micelle sizes and specific surface areas, high to low toxicity and marked to moderate granulomatogenicity, while group B TDM showed smaller sizes of micelles and larger specific surface areas, lower toxicity but higher granulomatogenicity in lungs. Existence of higher amount of longer chain alpha-mycolates in TDM appeared to be essential for high toxicity and thymic apoptotic activity, whereas TDM possessing wax ester-mycolate with smaller sized micelles seemed to be less toxic, but more granulomatogenic in lungs in mice. Thus, the mycolic acid subclass and molecular species composition of TDM affect critically the micelle forms, toxicity and granulomatogenicity in mice, while the relative abundances and carbon chain length of alpha-mycolate affected the toxicity in mice.