Effect of mycolic acid and its derivatives on the growth of Leptospira icterohaemorrhagiae.

Rabbit serum is the most important constituent in culture media for the growth of pathogenic leptospires. Many reports have shown that fatty acid or lipid fractions supported the growth of leptospires in place of rabbit serum [I. Mifuchi and T. Kawata, Med. Biol. (Tokyo) 28:128, 1953; H. Woratz, Zentr. Bakteriol. Parasitenk. Abt. I Orig. 162:106, 1955; 169:269, 1957; R. C. Johnson and J. B. Wilson, J. Bacteriol. 80:406, 1960; I. Mifuchi et al., Japan. J. Microbiol. 5:215, 1961; W. P. VenEseltine and S. A. Staples, J. Infect. Diseases 108:262, 1961; H. Vogel and S. H. Hunter, J. Gen. Microbiol. 26:223, 1961; R. C. Johnson and N. D. Gary, J. Bacteriol. 85:976, 1963]. More recently, we reported that heat-killed acid-fast bacterial cells or their lipid fractions could be used in place of rabbit serum for the growth of leptospires (I. Mifuchi et al., J. Bacteriol. 86:1128, 1963; Y. Yanagihara and I. Mifuchi, Japan. J. Microbiol. 9:1,1965; Y. Yanagihara et al., Japan. J. Microbiol., in press). Cells of Mycobacterium phlei, M. smegmatis, M. tuberculosis Aoyama B, and M. bovis Ushi 10 strains were fractionated by the method of Azuma and Yamamura (J. Biochem. 52:200, 1962), and the effect of each fraction on the growth of Leptospira icterohaemorrhagiae Mikawajima strain and L. canicola Utrecht strain was examined (Y. Yanagihara et al., Japan. J. Microbiol., in press). It was shown that the chloroform extract, purified wax, wax C, wax D, defatted bacilli, and bound lipid B and D fractions obtained from heat-killed mycobacterial cells replaced serum for the growth of the two strains. Chemical investigations showed that these active fractions contained mycolic acid which was a high molecular weight fatty acid of mycobacterial cells. These results suggest that mycolic acid or its derivatives is an active principle ofmycobacterial cells for the growth ofleptospira. In the present note, we describe the effect of mycolic acid and its derivatives purified from mycobacterial cells on the growth of L. icterohaemorrhagiae Mikawajima strain. Mycolic acid or its derivatives were added to the modified Korthof basal medium (Y. Yanagihara and I. Mifuchi, Japan. J. Microbiol. 9:1, 1965) in a concentration of 0.0008 to 2 mg of lipids. Each tube was inoculated with about 3.3 X 106 cells/ ml and incubated at 30 C. The growth of leptospires in the test medium was determined after 1 week by use ofPetroff-Hausser counting chamber, as described in a previous paper (Y. Yanagihara and I. Mifuchi, Japan. J. Microbiol. 9:161, 1965). The results are shown in Table 1. Mycolic acid preparations were purified from purified wax fraction by column chromatography on alumina after alkaline hydrolysis. More recently, Etemadi et al. established the chemical structure of mycolic acid by a mass-spectroscopic technique (J. Kremble and A. H. Etemadi, Tetrahedron 222:1113, 1965; A. H. Etemadi et al., Compt. Rend. 263:1257, 1966; J. Markovits et al., Comot. Rend. 263:960, 1966; A. H. Etemadi et al., Bull. Soc. Chim. France 195:1967). Wax D was a peptidoglycolipid containing mycolic acid, polysaccharide, and peptide. The hypothetical structure ofwax D was proposed by Lederer et al. (Biochim. Biophys. Acta 83:361, 1964). The chemical and immunological properties of the polysaccharide moiety of wax D fraction were investigated in our laboratory (I. Azuma et al., Am. Rev. Respirat. Diseases, submittedfor publication), and it was also shown that mycolic acid combined with the arabinose residue of polysaccharide, which was arabinogalactan in wax D, by ester linkage (I. Azuma et al., J. Biochem. 57:571, 1965). Arabinose mycolate was purified from the bound lipid B and D fractions, and its chemical structure was established as arabinose5-mycolate (I. Azuma and Y. Yamamura, J. Biochem. 52:200, 1962; 53:275, 1963). Cord factor which was a toxic glycolipid of mycobacteria was purified from wax C fraction by column chromatography on silicic acid. The chemical structure of cord factor was established as trehalose-6,6'-dimycolate (H. Noll et al., Biochim.