Cultivation of leptospires: fatty acid requirements.
Both the parasitic and the saprophytic leptospires grow well on a pair of fatty acids (one saturated, the other unsaturated) if they contain at least 15 carbon atoms.
Biomedical subjects
Publications and source records attributed to R C Johnson.
Both the parasitic and the saprophytic leptospires grow well on a pair of fatty acids (one saturated, the other unsaturated) if they contain at least 15 carbon atoms.
Spherical forms of Leptospira interrogans serotype canicola Hond Utrecht IV were induced with 1 m NaCl. Electron micrographs of these salt-altered cells (SAC) revealed that the outer envelope or sheath had pulled away from the protoplasmic cylinder. Treatment of SAC with 0.02% sodium lauryl sulfate solubilized the sheath and released the protoplasmic cylinder. Further processing of the solubilized sheath yielded a pellet which displayed a membrane structure in electron micrographs. The released protoplasmic cylinder showed loss of intracellular organization and the outer envelope present in normal cells. Immunization of hamsters with whole formalized cells, SAC, or sheath in doses as low as 10 mug/animal protected them from death upon challenge with virulent canicola 27.
Free fatty acids, aldehydes, alcohols, and 1-O-alkyl and alk-1-enyl glycerols were identified and quantified in lipid extracts from bovine cardiac muscle. Although a number of components present in the free fatty aldehydes were also noted in the fatty chains in the 1-O-alk-1-enyl glycerols, a direct qualitative similarity did not exist as would be expected if the free fatty aldehydes were artifactual in origin. Also, a qualitative similarity did not exist between the fatty chains of the 1-O-alkyl and alk-1-enyl glycerols. This latter observation would suggest a mechanism other than biodehydrogenation of the alkyl ethers for the origin of the alk-1-enyl glycerols. Free fatty aldehydes were distributed evenly between the 105,000 g supernatant and particulate fractions of cardiac muscle, while the 1-O-alk-1-enyl glycerols were associated primarily with the particulate fraction. Free fatty alcohols were noted only in the supernatant fraction, while the 1-O-alkyl glycerols were present in both fractions.
Specific immunofluorescence staining was used for the detection of leptospires in soil and water under laboratory and field conditions.
The fatty acid requirements of two avirulent treponemes were investigated by using a "lipid-poor" albumin-thioglycolate medium. The Kazan 5 and the Reiter stains of Treponema pallidum required a pair of fatty acids for growth. One member of the pair was saturated and the other was unsaturated. The saturated fatty acids could contain either an odd or even number of carbon atoms, but a chain length of at least 14 carbon atoms was necessary. Unsaturated fatty acids with one, two, or three double bonds were satisfactory if they contained 15 or more carbon atoms. The pair of fatty acids could be replaced with a single 18-carbon monounsaturated fatty acid if it was in the trans configuration rather than the naturally occurring cis form.
The utilization of glucose, amino acids, fatty acids, bicarbonate, purines, and pyrimidines by the Reiter treponeme was studied by using carbon 14-labeled substrates. The distribution of carbon from the substrates into various cell components was determined. Radioactivity from labeled bicarbonate in the cellular protein was restricted to aspartic acid. The Reiter treponeme is capable of synthesizing glycine, serine, alanine, aspartic acid, glutamic acid, proline, and possibly ornithine. Phenylalanine, arginine, lysine, leucine, isoleucine, valine, threonine, and histidine do not appear to be synthesized by this treponeme. The Reiter treponeme cannot synthesize fatty acids, and thymine is not incorporated. Glucose is a major carbon and energy source. Arginine, histidine, serine, threonine, and glutamic acid are degraded by the Reiter treponeme and may serve as energy sources. It was calculated that exogenously supplied amino acids contribute 41 to 54% of the cellular material; fatty acids, 18%; and glucose, 28 to 43%.
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The lipid composition of five parasitic and six saprophytic leptospires was compared. Lipids comprise 18 to 26% of the dry weight of the cells after chloroform-methanol extraction. No residual (bound) lipid was found after acid or alkaline hydrolysis of the extracted residue. The total lipid was composed of 60 to 70% phospholipid, and the remaining lipid was free fatty acids. The phospholipid fraction contained phosphatidylethanolamine as the major component, and phosphatidylglycerol and diphosphatidylglycerol were minor components with traces of lysophatidylethanolamine sometimes found. The major fatty acids of leptospires were hexadecanoic, hexadecenoic, and octadecenoic acids. Both the unusual cis-11-hexadecenoic acid and the more common cis-9-hexadecenoic acid were synthesized by the leptospires. Neither the parasitic nor the saprophytic leptospires can chain elongate fatty acids. However, they were capable of beta-oxidation of fatty acids. Both groups of leptospires desaturate fatty acids by an aerobic pathway. When the parasite canicola was cultivated on octadecanoic acid, 87% of the hexadecenoic acid was the 11 isomer, whereas the saprophyte semeranga consisted of 10% of this isomer. In addition, the saprophytic leptospires contained more tetradecanoic acid than the parasites. No differences were observed in the lipid composition of virulent and avirulent strains of canicola.
The lipid composition of Treponema pallidum Kazan 5 cultivated in a lipid-defined medium was investigated. Lipids comprised 18 to 20% of the dry weight of the treponeme. Glycolipid and phospholipids accounted for 90 to 95% of the total lipids and free fatty acids made up the remaining 5 to 10%. The major polar lipids were the glycolipid, 1-(O-alpha-d-galactopyranosyl)-2,3-diglyceride (45 to 55%), and phosphatidylcholine (30 to 40%). Phosphatidylethanolamine (5 to 10%), an unidentified compound (1 to 2%), and occasional trace amounts of diphosphatidylglycerol (cardiolipin) were also found. The monogalactosyldiglyceride was also a major component (50%) of the lipids of the Reiter, Noguchi, and Nichols strains of T. pallidum. The fatty acid composition of Kazan 5 usually consisted of saturated and unsaturated fatty acids ranging from 14 to 18 carbons depending upon the fatty acids added to the culture medium. When the cells were cultivated on elaidic acid (trans-9-octadecenoic acid), their lipids contained only elaidic acid.
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