Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro.

In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations greater than 24 mg/liter (2 X 10(-4) M), cysteine was acutely toxic to trypanosome concentrations of 3 X 10(7)/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H2O2 produced by cysteine autoxidation. In uptake studies using [35S]cysteine and [35S]cystine, T. brucei efficiently incorporated only cysteine. The Km for cysteine uptake was 4 X 10(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.

Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii.

A series of conditional mutants of the algal, biflagellate Chlamydomonas reinhardtii with temperature-sensitive defects in flagellar assembly and function were isolated. The genetics and phenotypes of 21 mutants displaying a rapid alteration in flagellar function upon shift from the permissive (20 degrees C) to the restrictive (32 degrees C) temperatures are described. These mutants designated as "drop-down" or dd-mutants have been placed in four categories on the basis of their defective phenotypes: (a) dd-assembly mutants - the preformed flagella are resorbed at 32 degrees C and reassembly of flagella is inhibited; (b) dd-fragile flagella mutants - the flagella are lost by detachment at 32 degrees C, but can be reassembled; (c) dd-motility mutants - the flagella are retained at 32 degrees C, but are functionally defective; (d) dd-lethal mutants - display combined defects in flagellar function and cell growth. Tetrad analysis of the mutants back-crossed to wild-type, recombination analysis of intermutant crosses, and complementation tests in the construction of heterozygous diploid strains indicate that at least 14 nuclear genetic loci are represented among 21 mutants. The availability of temperature-sensitive mutations affecting the assembly and function of the flagellum suggests that the morphogenesis of this complex eukaryotic organelle is amenable to genetic dissection.

Transport of ethanolamine and its incorporation into the variant surface glycoprotein of bloodstream forms of Trypanosoma brucei.

The membrane-attached form of the variant surface glycoprotein (mf-VSG) of bloodstream forms of Trypanosoma brucei is anchored to the plasma membrane by a hydrophobic C-terminal lipo-oligosaccharide containing ethanolamine. Analysis by polyacrylamide gel electrophoresis showed that several different cloned T. brucei strains (strain EATRO 110 and variants 117 and 118 of strain 427) incorporated [3H]ethanolamine into both mf-VSG and the soluble VSG derived from it, but not into other proteins. Other trypanosomatids, e.g. Leishmania mexicana promastigotes, T. cruzi epimastigotes, and T. brucei procyclic forms, did not incorporate ethanolamine into cellular proteins. Thus, [3H]ethanolamine can be used as a specific biosynthetic label for T. brucei VSG polypeptides. The time course of incorporation of [3H]ethanolamine into VSG showed a lag period of about 15 min. Double-labelling experiments using [3H]ethanolamine and H3[32P]O4 demonstrated that ethanolamine labelled only the C-terminal moiety and was not incorporated into other portions of the VSG molecule. Cellular uptake of ethanolamine occurred via a specific carrier-mediated transport system having a Vmax of 132 pmol min-1 mg-1 protein and a Km of 3.7 microM. The properties of this transport system are consistent with the possibility that ethanolamine is derived entirely from the host.

Human serum-sensitive Trypanosoma brucei rhodesiense: a comparison with serologically identical human serum-resistant clones.

Trypanosoma brucei rhodesiense clones, which are susceptible to lysis by normal human serum, were isolated from 3 different human serum-resistant clones originally derived from strain ETat 1.10. Serologically, these pairs of serum-sensitive and serum-resistant clones displayed the same variant surface glycoprotein (VSG) on their surface. Acquisition of human serum sensitivity correlated with susceptibility to lysis by human high density lipoprotein, a trypanocidal factor in normal human serum. Analysis of these paired populations by two-dimensional gel electrophoresis of whole trypanosomes and various subcellular fractions failed to reveal any differences in mobility of VSG and other proteins. Northern blot analysis of mRNAs from serum-sensitive and serum-resistant clones showed no differences when probed with a previously described resistance-specific probe. In addition, the ethanolamine membrane transport system and the overall membrane lipid fluidity did not reveal any detectable biochemical or biophysical differences in membrane properties. If resistance to lysis is indeed mediated by membrane changes at the enzymatic or structural level, the data presented suggest that the gene product(s) responsible for this change in human serum sensitivity may be present in very small quantities.

High-density-lipoprotein-independent killing of Trypanosoma brucei by human serum.

The cattle pathogen Trypanosoma brucei brucei is morphologically indistinguishable from the human pathogens T.b. rhodesiense and T.b. gambiense. However, unlike the human pathogens, T.b. brucei is lysed by normal human serum (NHS). The trypanolytic factor in NHS co-purifies with high-density lipoproteins (HDL), but its precise nature is unknown. Using a new fluorescence-based viability assay to assess T.b. brucei killing, we find that the HDL-deficient sera from two patients with Tangier disease are as trypanolytic as NHS. Fractionation of the Tangier sera by density ultracentrifugation revealed that the activity resides only in lipoprotein-depleted fractions. Tangier and NHS were also subjected to molecular sieving chromatography, and the activity profiles were identical. Lytic fractions to T. brucei (but not to T. rhodesiense) appeared under two distinct peaks of 100-600 kDa and > 1000 kDa. Neither peak coincided with the position of the major serum lipoproteins, as determined by cholesterol titrations. The high-molecular-mass peak did not contain the HDL-associated apolipoprotein-A1. Further, we did not find that purified apolipoproteins A1 or J are lytic for the trypanosomes. We conclude that the killing of T. brucei by human serum can be independent of HDL.

Role of phospholipids in the cytotoxic action of high density lipoprotein on trypanosomes.

Host range among the African trypanosomes, protozoa that cause fatal diseases both in humans and livestock, may be, in part, regulated by toxic properties associated with host high density lipoproteins (HDL). High density lipoproteins from hosts resistant (baboon, human) or susceptible (rabbit, rat) to Trypanosoma brucei infection were isolated and their trypanocidal activity was determined in in vitro cell lysis assays. Rabbit and rat HDL were not cytotoxic while baboon and human HDL rapidly lysed trypanosomes within 2 h at 37 degrees C. Analysis of the phospholipid composition of HDL preparations from these species suggested a correlation between trypanocidal activity and low phosphatidylinositol content. Phospholipase digestion of HDL resulted in a loss of trypanocidal activity, indicating the importance of native phospholipids in maintaining this biological activity of HDL. Cell lysis and loss of trypanosome infectivity induced by baboon HDL could be inhibited either by addition of rabbit or rat HDL to the incubation medium or by addition of purified phospholipids, phosphatidylinositol being the most effective inhibitor. Although the mechanism by which HDL lyses trypanosomes remains to be elucidated, these results suggest an important role for phospholipids in determining the specificity of this cytotoxic property of HDL.

Identification of the trypanocidal factor in normal human serum: high density lipoprotein.

The differentiation of Trypanosoma brucei from T. rhodesiense, the causative agent of human sleeping sickness, depends on their relative sensitivities to the cytotoxic effects of normal human serum. The molecule responsible for the specific lysis of T. brucei has now been isolated. Serum lipoproteins were fractionated and purified by ultracentrifugal flotation and chromatography on Bio-Gel A-5m. Trypanocidal activity was recovered in the high density lipoprotein fraction (density, 1.063-1.216 g/ml). Contamination by other serum proteins was checked by crossed immunoelectrophoresis and sodium dodecyl sulfate/acrylamide gel electrophoresis. Only a trace of beta-lipoprotein was found. The trypanocidal activity of pure human high density lipoprotein was identical to that of unfractionated serum when the following were tested: (i) time course of in vitro lysis of T. bruceli; (ii) in vivo destruction of T. brucei; (iii) relative resistance of T. rhodesiense to lysis. Rat or rabbit high density lipoprotein had no trypanocidal activity. Identification of the trypanocidal factor as high density lipoprotein was confirmed by the finding that serum from patients with Tangier disease, an autosomal recessive disorder characterized by a severe deficiency of high density lipoprotein, had no trypanocidal activity.

Interaction of high-density lipoprotein with Trypanosoma brucei: effect of membrane stabilizers.

The specific lysis of bloodstream trypanosomes by serum from a nonpermissive mammalian host is the result of interaction between the serum trypanocidal factor (high-density lipoprotein) and the trypanosome surface. The studies described in this paper attempt to define further the mode of action of this cytotoxic lipoprotein. The binding of high-density lipoprotein to Trypanosoma brucei was instantaneous at 4 degrees C and readily reversible. Binding was not mediated by the surface glycoprotein as removal of the surface coat enhanced binding at 4 degrees C, and no stable glycoprotein-lipoprotein complex could be detected. Pretreatment of trypanosomes with the cross linker dimethylsuberimidate rendered cells resistant to lysis. Addition of membrane-stabilizing drugs, such as cytochalasins C, D, and E, and local anesthetics (dibucaine, tetracaine, and procaine), also inhibited high-density lipoprotein-induced cell lysis. The data presented support the idea that at 37 degrees C lateral diffusion of the variant surface glycoprotein, an integral membrane protein, allows maximal high-density lipoprotein-cell interaction in serum-sensitive cells, and that altered properties of the plasma membrane induced by low temperature or the addition of cytochalasins, local anesthetics, or zinc inhibit this interaction, possibly by increasing the shielding of the plasma membrane by more rigidly anchored surface glycoprotein molecules.

Trypanosoma brucei: biochemical and morphological studies of cytotoxicity caused by normal human serum.

The biochemical and morphological events which accompany lysis of Trypanosoma brucei by normal human serum have been described. The prelytic events include loss of infectivity and rapid cation shifts across the cell membrane. This is followed by cell swelling, fraying of the surface coat of the cell, loss of intracellular organelles, and eventually cell lysis. The data presented are consistent with a colloid osmotic mechanism of lysis induced by irreversible acute damage to the normal permeability properties of the trypanosome plasma membrane.

Defective production of mitochondrial ribosomes in the poky mutant of Neurospora crassa.

A strain of Neurospora crassa containing a cytoplasmic mutation (poky) affecting mitochondrial function is shown to be deficient in small ribosomal subunits in the mitochondrion during the exponential growth phase. In the stationary growth phase, small subunits are more abundant and are present in mitochondrial ribosomal monomers. This change can be correlated with the return of mitochondrial cytochrome content to amounts approaching those of wild type mitochondria. The ribosomal defect shows an extrachromosomal pattern of inheritance in crosses of poky with wild type.

Trypanosome variant surface glycoprotein transfer to target membranes: a model for the pathogenesis of trypanosomiasis.

The variant surface glycoprotein (VSG) of trypanosomes is attached to the cell surface by means of a phosphatidylinositol-containing glycolipid membrane anchor. The studies presented in this paper support the hypothesis that the transfer of VSG from trypanosomes to erythrocytes could lead to one of the pathological features associated with trypanosome infection--i.e., anemia. Migration of trypanosome VSG from live trypanosomes to target cells (sheep erythrocytes) could be shown by preincubating erythrocytes with trypanosomes and subsequently testing the washed erythrocytes for insertion of VSG by their susceptibility to lysis by complement in the presence of an anti-VSG antibody. Complement-mediated lysis was found to depend on the strain-specific anti-VSG antibody used. Extent of erythrocyte lysis increased with time of cell exposure to trypanosomes and with trypanosome concentration. No erythrocyte lysis was observed when trypanosomes were preincubated with anti-VSG antibody before adding erythrocytes. Purified membrane-form VSG (which retains the glycolipid anchor), but not soluble VSG (which no longer has the terminal diacylglycerol moiety), could sensitize erythrocytes to anti-VSG antibody-mediated complement lysis. The intermembrane transfer of VSG from trypanosomes to cells of the infected host could provide a molecular mechanism for the pathogenesis of trypanosomiasis.

Specificity of ethanolamine transport and its further metabolism in Trypanosoma brucei.

Ethanolamine is found in trypanosomes as an integral component of the variant surface glycoprotein (VSG) and the membrane phospholipid phosphatidylethanolamine (PE). Steps in the utilization of ethanolamine could represent novel targets for the development of chemotherapeutic drugs and were therefore investigated in detail. Transport of [3H]ethanolamine was studied using structural analogs of ethanolamine. Compounds with substitutions in the amino group or of one of the methylene hydrogens of ethanolamine were the most effective inhibitors. Those analogs studied in detail with respect to their kinetic properties were all found to be competitive inhibitors of ethanolamine transport. Following uptake, ethanolamine is rapidly phosphorylated by an ethanolamine-specific kinase to form phosphoethanolamine. Other acid-soluble intermediates identified by thin layer chromatography were CDP-ethanolamine, dCDP-ethanolamine, and glycerophosphorylethanolamine. The relative amounts of these metabolites varied between slender (dividing) and stumpy (non-dividing) trypanosomes and may reflect special biosynthetic needs of the different morphological forms. Pulse-chase experiments indicated that the acid-soluble metabolites served as precursors for chloroform/methanol-soluble lipids. Radioactive lipids included PE, mono-methyl and dimethyl PE, and lysoPE. Further methylation of dimethylPE to phosphatidylcholine was not observed under the experimental conditions described. These results are consistent with the conclusion that trypanosomes are able to synthesize phospholipids via the Kennedy pathway.

Isolation and partial characterization of mutants of the trypanosomatid Crithidia fasciculata and their use in detecting genetic recombination.

Crithidia fasciculata was used as a model trypanosomatid to study the possible existence of genetic recombination in this group of protozoa. The approach was based on the ability to select a variety of mutants on agar plates. Following mutagenesis of wild type cells by nitrosoguanidine or ethylmethanesulfonate, stable mutant phenotypes were obtained. These included mutants resistant to the drugs actinomycin D, 6-azauracil, 6-azauridine, and 5-fluorouracil, auxotrophs and colony morphology mutants. Following mixed growth of pairs of drug-resistant mutants on selective media, isolates exhibiting stable recombinant phenotypes were obtained. The data presented suggest that 1) Crithidia undergoes some type of genetic recombination and 2) Crithidia must be diploid at some time during this process.

In utero analysis of heterozygous achondroplasia: variable time of onset as detected by femur length measurements.

Seven cases of heterozygous achondroplasia were examined in utero. Although the head shape and growth were normal, the initially normal femur length showed a decrease in growth and fell below the lower 99 per cent confidence limit in all cases. The time of presentation of achondroplasia varied between 21 and 27 gestational weeks. This study suggests that the diagnosis of achondroplasia can be reasonably made when the femur is abnormally short. When the femoral length is appropriate prior to 30 weeks, more caution should be taken in interpretation. Because of the variability in presentation, the fetus could be normal or still could be affected.