Essential Amino Acids in the Culture of Leishmania tarentolae

Abstract

The following amino acids may be singly deleted from Trager's Defined Medium C without inhibiting the continuous culture of Leishmania tarentolae promastigotes: L-glycine, L-alanine, L-aspartic acid, L-methionine, L-proline, L-glutamic acid, and L-isoleucine. Deletion of L-proline prevents continuous culture of L. tarentolae when either L-methionine, L-alanine, L-glutamic acid, L-aspartic acid, or L-isoleucine is removed as well. Lack of glucose in Medium C is detrimental when either L-glutamic acid or L-proline is missing. The following amino acids are considered essential for the continuous culture of L. tarentolae in Medium C: L-arginine, L-histidine, L-tryptophan, DL-phenylalanine, DL-serine, L-tyrosine, L-threonine, L-valine, L-leucine, and L-lysine. L-proline may also be considered an essential amino acid because continuous culture in its absence is impossible if any other amino acid, except Lglycine, is deleted from Medium C as well. Cultivation of parasitic organisms in chemically defined media is an important prerequisite for determining their nutritional requirements, the understanding of which is of value in the development of rational methods of chemotherapy. In 1957 Trager devised one of the first chemically defined media for a parasitic protozoan, Defined Medium C, which was used to culture promastigotes (leptomonads) of the hemoflagellate Leishmania tarentolae. Trager's Medium C has been used in studies involving L. tarentolae metabolism (see review by Trager, 1968), transformation of L. donovani (Simpson, 1968), and culture of parasitic nematodes (Jackson, 1962). Since the amino acid requirements of L. tarentolae are complex (Trager, 1968), a study was undertaken to determine which ones were essential for continuous culture by singly deleting each of the amino acids in Medium C. This paper reports the results of deletion tests on all of the amino acids in Medium C. The effect of additional deletion of the energy sources D-glucose and L-proline (Krassner, 1969a) on growth was also followed. Preliminary accounts of portions of this study have appeared in earlier communications (Krassner, 1969a, b). MATERIALS AND METHODS Stock cultures of L. tarentolae, strain TAR II (obtained from Dr. L. Parrot of the Institut Pasteur d'Algerie) were maintained at 26 C in 25-ml Erlenmeyer flasks containing 3.0 ml of Trager's Received for publication 5 January 1971. * This work was supported by U. S. Public Health Service Grant AI06827. Defined Medium C (Trager, 1957). The concentration and sources of amino acids used in this study are listed in Table I. Promastigotes cultured in complete Medium C, in the log phase of growth, were used to inoculate flasks containing 3.0 ml of Medium C lacking: (1) one of the amino acids or (2) an amino acid and either L-proline or D-glucose. Inocula contained 0.2 ml (. 4 X 106 cells) of unwashed organisms so that a small residual amount of the deleted material was carried over into the first culture. Successful cultures were transferred every 5 to 7 days into fresh identical media and growth was considered continuous if cells grew for at least 6 successive subcultures. Culture studies were carried out in triplicate and each experiment was repeated at least 3 times. Control cultures containing complete media were run in parallel with test cultures. Each amino acid was tested for purity and homogeneity on Cellulose MN300 (Brinkman Instruments, Inc., Westbury, New York) thin-layer chromatogram plates according to the method devised by Jones and Heathcote (1966). In most cases the L-form of the amino acid was used. Promastigotes from each subculture were studied under phase contrast microscopy to determine if there were any gross morphological changes.