The presence of rotenone-sensitive NADH dehydrogenase in the long slender bloodstream and the procyclic forms of Trypanosoma brucei brucei

The mitochondrial electron-transport chain present in the procyclic and long slender bloodstream forms of Trypanosoma brucei brucei was investigated by means of several experimental approaches. The oxidation of proline, glycerol and glucose in procyclic cells was inhibited 80-90% by antimycin A or cyanide, 15-19% by salicylhydroxamic acid, and 30-35% by rotenone. Cytochrom-c-reductase activity, with proline or glycerol 3-phosphate as substrate, in a mitochondrial fraction isolated from these cells was inhibited by antimycin and rotenone, but not by malonate, while cytochrome-c-reductase activity with succinate as substrate was inhibited by antimycin A and malonate, but not by rotenone. In addition, the reduction of dichloroindophenol by NADH was inhibited by rotenone but not by malonate, which suggests that rotenone-sensitive NADH dehydrogenase (complex I) is present in these mitochondria. The presence of three subunits of NADH dehydrogenase was observed in immunoblots of mitochondrial proteins with specific antibodies raised against peptides corresponding to predicted antigenic regions of these proteins, which provides further evidence for the presence of NADH dehydrogenase. In long slender bloodstream forms, the oxidation of glucose or glycerol was inhibited 100% by salicyhydroxamic acid, unaffected by cyanide or antimycin A, and inhibited 40% or 75%, respectively, by rotenone, which suggests that NADH dehydrogenase is present in these cells. In a mitochondrial fraction isolated from the bloodstream forms, oxygen uptake with glycerol 3-phosphate as substrate was inhibited 65% by rotenone. Low levels of rotenone-sensitive NADH-dependent reduction of dichloroindophenol and the presence of subunits 7 and 8 of NADH dehydrogenase provided additional evidence for the presence of NADH dehydrogenase in bloodstream forms of T. brucei.     

Interferon-gamma activation of a mitogen-activated protein kinase, KFR1, in the bloodstream form of Trypanosoma brucei

KFR1, a mitogen-activated protein (MAP) kinase identified in the African trypanosome, Trypanosoma brucei, is a serine protein kinase capable of phosphorylating the serine residues in histone H-1, myelin basic protein, and beta-casein. It phosphorylates four proteins with estimated molecular masses of 22, 34, 46, and 90 kDa from the T. brucei bloodstream-form lysate in vitro. KFR1 bears significant sequence similarity to the yeast MAP kinases KSS1 and FUS3 but cannot functionally complement the kss1/fus3 yeast mutant. It is encoded by a single-copy gene in the diploid T. brucei, and only one of the two alleles can be successfully disrupted, suggesting an essential function of KFR1 in T. brucei. KFR1 activity is present at a much enhanced level in the bloodstream form of T. brucei when compared with that in the insect (procyclic) form. This enhanced activity can be eliminated in vitro by the treatment with protein phosphatase HVH2 known to act specifically on MAP kinases. It can also be decreased in the bloodstream form of T. brucei by serum starvation but induced specifically by interferon-gamma. The production of interferon-gamma in the mammalian host is known to be triggered by T. brucei infection, and this cytokine, as has been reported, promotes the proliferation of T. brucei in the mammalian blood. Since none of these phenomena can be observed in the procyclic form of T. brucei, activation of KFR1 is most likely involved in mediating the interferon-gamma-induced proliferation of T. brucei in the mammalian host.     

A Trypanosoma brucei bloodstream form mutant deficient in ornithine decarboxylase can protect against wild-type infection in mice

A Trypanosoma brucei bloodstream mutant in which both copies of the ornithine decarboxylase (ODC) gene were knocked out (ODC mutant) was used to determine the biological functions of ODC in T. brucei. Growth of the mutant cells ceased within 12-24 h in regular culture medium deficient in polyamines, but could be rescued by supplementation with 1 mM putrescine. A mouse model of T. brucei infection was used to determine whether the mutant was still infective and was found to develop either extremely low or undetectable levels of parasitemia, suggesting that in T. brucei, ODC activity is essential for establishing an infection. Furthermore, when these mice were subsequently challenged with wild-type T. brucei cells expressing the same variant surface glycoprotein (VSG), they did not develop any parasitemia, indicating that inoculating the mice with the attenuated ODC mutant had conferred protection against challenge by wild-type cells. These results were reproduced in C57BL/6J mice deficient in alpha-beta and gamma-delta T-cell receptors. However, no protection was observed in rag-2 knockout mice deficient in both B and T lymphocytes or in C57BL/10J mice deficient only in B lymphocytes. The results thus suggest that the ODC mutant could induce a T-lymphocyte-independent but B-lymphocyte-dependent immunity against wild-type cells of the same VSG. Such a mechanism of immunity has been elicited only by live T. brucei cells, but not by isolated VSGs or radiation-killed trypanosomes. This ODC mutant may thus represent a genuinely attenuated T. brucei bloodstream form capable of immunizing mammals against infections by African trypanosomes of the same VSG subtype without causing detectable infection by itself. The observation also raises the interesting likelihood that the in vivo treatment of T. brucei bloodstream forms with alpha-DL-difluoromethylornithine is a de facto attenuation of the parasitic organisms, which may very well result in B-lymphocyte-dependent host immune responses to subsequent infections by parasites of the same VSG subtypes.     

Trypanosoma brucei rhodesiense: membrane glycoproteins localized primarily in endosomes and lysosomes of bloodstream forms

Bloodstream forms of Trypanosoma brucei rhodesiense take up macromolecules in endocytic vesicles that form in a large coated pit called the flagellar pocket. Glycoproteins that bind to ricin are concentrated in the flagellar pocket and in intracellular vesicles. We purified Triton X-100-soluble ricin-binding glycoproteins by lectin affinity chromatography and immunized mice to generate hybridomas. Monoclonal antibody produced by the CB1 hybridoma recognized heterodisperse trypanosome components migrating with M(r) 84-140 kDa in immunoblots. CB1 binding was specifically inhibited by lactose. The CB1-reactive material was purified by sequential affinity chromatography on ricin- and CB1-Sepharose. N-Glycosidase F, but not endoglycosidase H, digestion destroyed CB1-reactivity of purified material. This suggests that N-linked oligosaccharides contribute to the CB1 epitope. Glycosidase digestion of biosynthetically radiomethionine-labeled, affinity purified, CB1-reactive material yielded two radiolabeled polypeptides, p57 and p42. Thirteen methionyl peptides were resolved in one-dimensional peptide maps of V8 protease digests of p57; p42 had 10 methionyl peptides with mobilities indistinguishable from those of peptides of p57. This suggests that p57 and p42 are closely related. In cryoimmunoelectron microscopy studies CB1 specifically labeled the interior surface of tubular and vesicular membranes located between the nucleus and the flagellar pocket. These membranes were morphologically identical to structures that have been previously identified as trans Golgi, lysosomal, and endosomal elements. In double-labeling studies endocytosed serum albumen-gold complexes were found in the lumen of vesicles that had CB1-reactive material in their membranes. This provides direct evidence that vesicles containing high levels of CB1-reactive material are part of the lysosome/endosomal system. Some CB1-reactive material was also detected in the flagellar pocket by cryoimmunoelectron microscopy. Corrolated flow cytofluorimetry and immunofluorescence analysis showed that 85-96% of the total CB1-reactive material was intracellular and inaccessible to antibody in living cells. The 4-15% of the total CB1-reactive material accessible to antibody in living cells was localized in the flagellar pocket. Bloodstream forms of Trypanosoma brucei brucei, Trypanosoma brucei gambiense, and T.b. rhodesiense all expressed the CB1 epitope. However, expression of this epitope is developmentally regulated during the parasite life cycle, for no CB1-reactive material was detected in procyclic forms. The trypanosome proteins detected by CB1 show some similarities to vertebrate lysosomal and endosomal membrane proteins.     

Both IgM and IgG anti-VSG antibodies initiate a cycle of aggregation-disaggregation of bloodstream forms of Trypanosoma brucei without damage to the parasite

Bloodstream forms of Trypanosoma brucei, when aggregated in the presence of either acute immune plasma, acute immune serum, purified IgM anti-VSG antibodies or purified IgG anti-VSG antibodies, subsequently disaggregated with a t1/2 for disaggregation of 15 min at 37 degrees C as long as the trypanosomes were metabolically active at the beginning of the experiment and maintained during the experiment in a suitable supporting medium. The t1/2 for disaggregation was found to be directly dependent upon temperature and inversely proportional to the antibody concentration. The trypanosomes were always motile and metabolically active during aggregation and after disaggregation and were fully infective for a mammalian host following disaggregation as well as able to grow and divide normally during axenic culture. The disaggregation was strictly energy dependent and was inhibited when intracellular ATP levels were reduced by salicylhydroxamic acid or following addition of oligomycin while respiring glucose. In addition the process of disaggregation was dependent upon normal endosomal activity as evidenced by its sensitivity to a wide variety of inhibitors of various endosomal functions. Disaggregation was not due to separation of immunoglobulin chains by either disulphide reduction or disulphide exchange reactions and gross proteolytic cleavage of the immunoglobulins attached to the surface of the parasite was not detected. In addition, gross cleavage or release of the VSG from the surface of the cell did not occur during disaggregation but proteolytic cleavage of a small proportion of either the VSG or the immunoglobulins could not be eliminated from consideration. Finally the mechanism of disaggregation was found to be a regulated process, independent of Ca2+ movements but dependent upon the activity of protein kinase C or related kinases and inhibited by the activity of protein kinase A as evidenced by the effects of a panel of inhibitors and cAMP analogues on the process of disaggregation. The mechanism of disaggregation displayed by trypanosomes aggregated by anti-VSG antibody is proposed to form part of the parasite's defence against the host immune system and functions to aid survival of trypanosomes in the presence of antibody in the host prior to the occurrence of a VSG switching event.     

Stable expression of mosaic coats of variant surface glycoproteins in Trypanosoma brucei

The paradigm of antigenic variation in parasites is the variant surface glycoprotein (VSG) of African trypanosomes. Only one VSG is expressed at any time, except for short periods during switching. The reasons for this pattern of expression and the consequences of expressing more than one VSG are unknown. Trypanosoma brucei was genetically manipulated to generate cell lines that expressed two VSGs simultaneously. These VSGs were produced in equal amounts and were homogeneously distributed on the trypanosome surface. The double-expressor cells had similar population doubling times and were as infective as wild-type cells. Thus, the simultaneous expression of two VSGs is not intrinsically harmful.     

High molecular mass agarose matrix supports growth of bloodstream forms of pleomorphic Trypanosoma brucei strains in axenic culture

Primary axenic culture of Trypanosoma brucei bloodstream forms almost invariably requires a period of culture adaptation with cell death and clonal selection. This has been particularly difficult and in many cases unsuccessful for natural pleomorphic strains, which are characterized by their ability to differentiate from replicating long slender bloodstream forms into short stumpy forms. Here we show that a representative set of pleomorphic T. brucei strains can be cultured in vitro on agarose plates without any previous adaptation period and selection. The slender morphology was retained and the growth rate was identical to the growth rate in vivo. Long term in vitro culture for 3 months with this method did not affect the ability of the AnTat 1.1 strain to give rise to pleomorphic infections in mice. Stumpy populations emanating from these infections transformed rapidly and synchronously into dividing procyclic forms when triggered with cis-aconitate and a temperature shift. The growth supporting activity of agarose plates could be traced to high molecular mass polymeric agarose; beta-agarase digestion destroyed the activity. Membrane chamber experiments show that direct physical contact of trypanosomes with the agarose matrix is essential. In the absence of high molecular mass agarose, the cell division process is grossly impaired. We suggest that agarose mimics an interaction of trypanosomes with the host's extracellular matrix. Applications of the culture method are discussed.     

Host plasma low density lipoprotein particles as an essential source of lipids for the bloodstream forms of Trypanosoma brucei

In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.     

Partitioning of large and minichromosomes in Trypanosoma brucei

The Trypanosoma brucei nuclear genome contains about 100 minichromosomes of between 50 to 150 kilobases and about 20 chromosomes of 0.2 to 6 megabase pairs. Minichromosomes contain nontranscribed copies of variant surface glycoprotein (VSG) genes and are thought to expand the VSG gene pool. Varying VSG expression allows the parasite to avoid elimination by the host immune system. The mechanism of inheritance of T. brucei chromosomes was investigated by in situ hybridization in combination with immunofluorescence. The minichromosome population segregated with precision, by association with the central intranuclear mitotic spindle. However, their positional dynamics differed from that of the large chromosomes, which were partitioned by kinetochore microtubules.     

Localization of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei

African trypanosomes such as Trypanosoma brucei undergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing the variant surface glycoprotein (VSG) gene expressed. The transcribed VSG gene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change the VSG gene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously that VSG genes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base beta-D-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomeric VSG expression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomeric VSG genes in silenced expression sites and not in actively transcribed telomeric VSG genes. J was absent from inactive chromosome-internal VSG genes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of the VSG gene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites.     

Time-dose response of Trypanosoma congolense bloodstream forms to diminazene and isometamidium

Trypanosoma congolense bloodstream forms were propagated in vitro axenically in a simplified cultivation medium at 34 degrees C. Viability of a drug-sensitive and a drug-resistant clone were examined for 10 days following exposure to 0.1, 1.0 and 10.0 micrograms ml-1 of diminazene aceturate and 0.1, 1.0 and 10.0 ng ml-1 of isometamidium chloride for various time intervals. Drug-sensitive T. congolense were irreversibly damaged after incubation with 10 micrograms ml-1 or 1 microgram ml-1 diminazene aceturate for 30 min or 2 h, respectively, while drug-resistant trypanosomes were not affected. Exposure to 10 ng ml-1 isometamidium chloride eliminated drug-sensitive trypanosomes after 24 h and drug-resistant trypanosomes after 96 h. The data obtained on in vitro time-dose responses of T. congolense were related to pharmacokinetic data of diminazene and isometamidium in cattle plasma.     

Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli

A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.     

Changes in ion channel expression during in vitro differentiation of trout oligodendrocyte precursor cells

A prospective study of conduct disorder (CD) was conducted using 4 annual structured diagnostic interviews of 171 clinic-referred boys, their parents, and their teachers. Only about half of the 65 boys who met criteria for CD in Year 1 met criteria again during the next year, but 88% met criteria for CD again at least once during the next 3 years. For most boys with CD, the number of symptoms fluctuated above and below the diagnostic threshold from year to year but remained relatively high. Lower socioeconomic status, parental antisocial personality disorder (APD), and attention-deficit hyperactivity disorder were significant correlates of CD in Year 1, but the interaction of parental APD and the boy's verbal intelligence predicted the persistence of CD symptoms over time (i.e., only boys without a parent with APD and with above-average verbal intelligence clearly improved).     

Novel post-replicative DNA modification in Streptomyces: analysis of the preferred modification site of plasmid pIJ101

BACKGROUND & AIMS: Chronic alcoholic pancreatitis occurs in only a limited number of heavy drinkers. Other factors than alcohol are necessary for the occurrence of chronic alcoholic pancreatitis. The aim of this study was to examine whether pancreatic duct obstruction resulted in increased alcohol-induced parenchymal cell damage. METHODS: Four groups of adult mongrel dogs were used. In group A, 2. 0 g . kg-1 . day-1 of ethanol was administered via a gastric cannula. In group O, after ligation of the minor pancreatic duct, a polyethylene tube was inserted transduodenally into the major duct. In group AO, the protocols used in groups A and O were combined. Laparotomy was repeated after 3 months in each group. RESULTS: Three of the 9 dogs in group AO had pancreatic calculi in the main pancreatic duct. Moderate interlobular fibrosis, parenchymal cell loss, and inflammatory cell infiltration resembling human chronic alcoholic pancreatitis were observed in group AO. Little change was observed in groups A and O. Exocrine function assessed by secretin test in group AO was significantly reduced. Total protein, hexosamine, and calcium contents of the pancreatic juice in group AO were significantly increased. CONCLUSIONS: Pancreatic duct obstruction is an aggravating factor in chronic alcoholic pancreatitis.