Leishmania donovani: physicochemical, immunological, and biological characterization of excreted factor from promastigotes.

Leishmanial excreted factor (EF) from promastigote cultures was enriched from the crude product by differential precipitation with ammonium sulfate and perchloric acid, followed by column chromatography; and by boiling EF-antibody complex. Boiling destroyed the antibody, releasing the EF, which retained its ability to precipitate antibody. Enriched EF from Leishmania donovani promastigotes was found to be a highly negatively charged, carbohydrate-like material with a molecular weight approximating to 33,000, when monitored against a series of protein markers by gel filtration. Its ability to precipitate with antibody was unimpaired by boiling, lyophilization, pH changes from 1 to 11, treatment with high concentrations of NaCl, 10% phosphotungstic acid in 10% HCl, 0.6 M perchloric acid, 5% H₂SO₄, acetone, or dioxan. It did not absorb at wavelengths between 220 and 750 nm. Treatment with trypsin, Pronase, neuraminidase, and hyaluronidase did not affect its activity. Biochemical analysis showed that enriched EF contains carbohydrates but, at our level of detection, no protein, lipid, triglycerides, fatty acids, DNA, RNA, pentoses, amino sugars, sialic or uronic acid. Precipitation of EF by antibody was studied and the optimal molecular proportions for complete precipitation determined. EF-antibody complex, prepared at optimal proportions, and EF complexed with methylated bovine serum albumin, like EF alone, did not elicit antibody production in rabbits. EF in 0.5% phenol-saline elicited a delayed skin response of induration and erythema in guinea pigs cured of L. enriettii. Elevated temperature increased the release of EF from promastigotes, while the presence of trypsin acting at 37 C seemed to inhibit this effect slightly. Fractionation of mechanically broken promastigotes, by differential centrifugation and stepwise sucrose gradients, revealed a factor that precipitated rabbit antibody against whole promastigotes. This factor was associated with the soluble, organelle-free fraction and resembled EF when monitored by gel diffusion. This factor did not migrate when the complete extract from the broken promastigotes was run in immunoelectrophoresis. Boiling the extract for 5 min released a factor, which migrated to the anode. This factor appeared to be associated with another component in the promastigote, from which it dissociated on boiling. Boiling hamster tissues infected with leishmanial amastigotes, i.e., spleens containing L. donovani and epididymides containing L. tropica, also released factors similar to EF. These precipitated antibody in the same way, producing precipitation arcs that were continuous with those formed by EF from the homologous promastigotes. EF acted as a conditioner for culture promastigotes. Conditioned cultures showed maximal growth before similar, unconditioned cultures. However, both types of culture produced equal numbers of promastigotes per unit volume by the end of exponential growth.     

Phenotypic diversity of cloned lines of Leishmania major promastigotes

In vitro cultured promastigotes of virulent (V) and avirulent (A) cloned lines of Leishmania major, and the parental isolate LRC-L137, were examined with respect to morphology, cell size, growth rate, and apparent DNA content. Growth rates of all lines were comparable and both virulent (V121, LRC-L137) and avirulent parasites (A12, A52, A59) exhibited a progressive decrease in apparent DNA content with time in culture, as measured by incorporation of Hoechst Dye 33342. The four cloned lines and the parental isolate showed differences in the content of morphological variants and in the mean body length. Morphologically, there were similarities between A12 and A52 and between A59 and V121. Promastigote populations were also examined for the expression of the target antigen of a previously characterized monoclonal antibody, WIC-79.3. This antibody binds to a membrane antigen that is also present in culture supernatants of Leishmania of A1 serotype. Three different assays with culture supernatants all showed that V121, A59, and A12 were high producers with LRC-L137 and A52, low producers. Similar variation in expression of the 79.3 target antigen was detected in intact organisms of the various lines by immunofluorescence with flow cytometry. No simple correlation was found between the expression or release of the WIC-79.3 target antigen and virulence. The virulence or avirulence of all cloned lines for BALB/c mice remained stable. The data are discussed in terms of differentiation stages of L. major promastigotes and the continuing search for morphological and biochemical markers of virulence.     

Passive transfer of Leishmania lipopolysaccharide confers parasite survival in macrophages

Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. We have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study we have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here we show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, we show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.     

Leishmania spp.: effect of inhibitors on growth and on polyamine and macromolecular syntheses

Ethidium bromide, pentamidine isethionate, and MGBG [methylglyoxal-bis (guanylhydrazone)] inhibited the uptake of radioactive putrescine by leishmanial (Leishmania spp.; Leishmania tropica major; Leishmania mexicana; Leishmania donovani) promastigotes and interfered with their polyamine synthesis. Inhibition was apparent as early as 1 hr after adding these drugs to the parasites at growth-inhibiting concentrations. Ethidium bromide also inhibited the incorporation of radioactive uracil into leishmanial RNA at growth-inhibiting concentrations, while DNA synthesis was inhibited by ethidium bromide at high concentrations after a lag period. MGBG inhibited the synthesis of leishmanial DNA and RNA at growth-inhibiting concentrations.     

Ablepharon macrostomia syndrome with associated cutis laxa: Possible localization to 18q

The ablepharon-macrostomia (AMS) and Barber-Say syndromes (BSS) are rare disorders characterized by absence of the eyelids or ectropion, macrostomia, ambiguous genitalia, abnormal ears, rudimentary nipples, and dry, redundant skin. Patients with Barber-Say syndrome also have hypertrichosis. We present a patient with a phenotype similar to AMS who has a complex rearrangement of chromosome 18, involving both an inversion and interstitial deletion. Our patient lacks the typical features of the 18q deletion syndrome. We review AMS and BSS as compared with our patient, and recognize cutis laxa as a feature shared by all. We propose that the gene(s) for this phenotype may lie on chromosome 18 in the region of the deletion or inversion breakpoints. Received: 1 March 1995 / Revised: 20 May 1995