Structure of trypanosome coat protein VSGsur and function in suramin resistance.

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

The Alkaloid-Enriched Fraction of Pachysandra terminalis (Buxaceae) Shows Prominent Activity against Trypanosoma brucei rhodesiense.

In the course of our studies on antiprotozoal natural products and following our recent discovery that certain aminosteroids and aminocycloartanoid compounds from Holarrhena africana A. DC. (Apocynaceae) and Buxus sempervirens L. (Buxaceae), respectively, are strong and selective antitrypanosomal agents, we have extended these studies to another plant, related to the latter-namely, Pachysandra terminalis Sieb. and Zucc. (Buxaceae). This species is known to contain aminosteroids similar to those of Holarrhena and structurally related to the aminocycloartanoids of Buxus. The dicholoromethane extract obtained from aerial parts of P. terminalis and, in particular, its alkaloid fraction obtained by acid-base partitioning showed prominent activity against Trypanosoma brucei rhodesiense (Tbr). Activity-guided fractionation along with extended UHPLC-(+)ESI QTOF MS analyses coupled with partial least squares (PLS) regression modelling relating the analytical profiles of various fractions with their bioactivity against Tbr highlighted eighteen constituents likely responsible for the antitrypanosomal activity. Detailed analysis of their (+)ESI mass spectral fragmentation allowed identification of four known constituents of P. terminalis as well as structural characterization of ten further amino-/amidosteroids not previously reported from this plant.

The structure of serum resistance-associated protein and its implications for human African trypanosomiasis.

Only two trypanosome subspecies are able to cause human African trypanosomiasis. To establish an infection in human blood, they must overcome the innate immune system by resisting the toxic effects of trypanolytic factor 1 and trypanolytic factor 2 (refs. 1,2). These lipoprotein complexes contain an active, pore-forming component, apolipoprotein L1 (ApoL1), that causes trypanosome cell death 3 . One of the two human-infective subspecies, Trypanosoma brucei rhodesiense, differs from non-infective trypanosomes solely by the presence of the serum resistance-associated protein, which binds directly to ApoL1 and blocks its pore-forming capacity3-5. Since this interaction is the single critical event that renders T. b. rhodesiense human- infective, detailed structural information that allows identification of binding determinants is crucial to understand immune escape by the parasite. Here, we present the structure of serum resistance-associated protein and reveal the adaptations that occurred as it diverged from other trypanosome surface molecules to neutralize ApoL1. We also present our mapping of residues important for ApoL1 binding, giving molecular insight into this interaction at the heart of human sleeping sickness.

Localization of serum resistance-associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei.

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism.

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Crystal structures of T. b. rhodesiense adenosine kinase complexed with inhibitor and activator: implications for catalysis and hyperactivation.

BACKGROUND: The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP) utilizing adenosine triphosphate (ATP) as the preferred phosphoryl donor. METHODS AND FINDINGS: Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK): the structure of TbrAK in complex with the bisubstrate inhibitor P(1),P(5)-di(adenosine-5')-pentaphosphate (AP5A) at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) at 2.8 Å resolution. CONCLUSIONS: The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.

A search for Trypanosoma brucei rhodesiense diagnostic antigens by proteomic screening and targeted cloning.

BACKGROUND: The only available diagnostic method for East African trypanosomiasis is light microscopy of blood samples. A simple immunodiagnostic would greatly aid trypanosomiasis control. METHODOLOGY AND PRINCIPAL FINDINGS: To find trypanosome proteins that are specifically recognised by sera from human sleeping sickness patients, we have screened the Trypanosoma brucei brucei proteome by Western blotting. Using cytosolic, cytoskeletal and glycosomal fractions, we found that the vast majority of abundant trypanosome proteins is not specifically recognised by patient sera. We identified phosphoglycerate kinase (PGKC), heat shock protein (HSP70), and histones H2B and H3 as possible candidate diagnostic antigens. These proteins, plus paraflagellar rod protein 1, rhodesain (a cysteine protease), and an extracellular fragment of the Trypanosoma brucei nucleoside transporter TbNT10, were expressed in E. coli and tested for reactivity with patient and control sera. Only TbHSP70 was preferentially recognized by patient sera, but the sensitivity and specificity were insufficient for use of TbHSP70 alone as a diagnostic. Immunoprecipitation using a native protein extract revealed no specifically reacting proteins. CONCLUSIONS: No abundant T. brucei soluble, glycosomal or cytoskeletal protein is likely to be useful in diagnosis. To find useful diagnostic antigens it will therefore be necessary to use more sophisticated proteomic methods, or to test a very large panel of candidate proteins.

Expression and localization of serum resistance associated protein in Trypanosoma brucei rhodesiense.

The trypanosome lytic factor (TLF) is a primate specific innate defense mechanism that restricts the host range of African trypanosomes. Trypanosoma brucei rhodesiense, the causative agent of the acute form of human sleeping sickness, is resistant to the cytolytic action of TLF. By differential display PCR we have identified a gene in T. b. rhodesiense that is preferentially expressed in cell lines resistant to TLF. The protein sequence predicted from the gene shows homology to the trypanosome variable surface glycoprotein (VSG) gene family and in particular, to the previously reported human serum resistance associated gene (SRA). The amount of SRA mRNA is over 1000-fold higher in TLF resistant cells relative to TLF sensitive trypanosomes. Treatment of TLF sensitive trypanosomes with increasing concentrations of TLF in mice results in the selection of parasites that have reverted back to the TLF resistant phenotype. These trypanosomes also showed high levels of SRA mRNA. Antibodies against recombinant SRA react with a 59 kDa protein on western blots of total cell protein from TLF resistant trypanosomes but not TLF sensitive cells. Indirect immunofluorescence revealed that SRA is a cell surface protein present only in TLF resistant trypanosomes. These results suggest that TLF resistance in human sleeping sickness trypanosomes is a consequence of the selective, high level expression of a cell surface molecule(s). In addition, these studies support the role of TLF as a major factor in human serum mediated killing of susceptible trypanosomes.

Genetic diversity among Trypanosoma brucei rhodesiense isolates from Tanzania.

We compared 19 stocks of Trypanosoma brucei rhodesiense collected in 1991 and 1994 from Tanzania with representative stocks from other foci of Rhodesian sleeping sickness in Zambia, Kenya and Uganda. Stocks were characterized by isoenzyme electrophoresis, restriction fragment length polymorphisms in variant surface glycoprotein genes and random amplification of polymorphic DNA; the banding patterns obtained were coded for numerical analysis. In addition, the Tanzanian stocks were compared by pulsed field gel electrophoresis. Overall the Tanzanian stocks formed a homogeneous group and the predominant genotype isolated in 1991 was still present in the 1994 sample, although at a reduced level. The Tanzanian stocks were distinct from representative stocks from other East African foci. This observation does not support the proposal that there are northern and southern strains of T. b. rhodesiense, but is consistent with the view that T. b. rhodesiense stocks form a mosaic of different genotypes varying from focus to focus in East Africa.

Crystallization and preliminary X-ray investigation of the recombinant Trypanosoma brucei rhodesiense calmodulin.

Bipyramidal crystals of the recombinant calmodulin from Trypanosoma brucei rhodesiense were obtained by vapor diffusion against 55% (v/v) 2-methyl-2,4-pentanediol in 0.05 M cacodylate buffer, pH 5.6. When few nucleation events occurred, crystals grew to 0.25 x 0.25 x 1.20 mm. The space group of the crystal is I4(1)22, with unit cell dimensions a = b = 56.88 A, c = 230.11 A, alpha = beta = gamma = 90 degrees, z = 16. The molecular mass and volume of the unit cell suggest that there is one molecule in the asymmetric unit. The I/sigma (I) ratio for data at 3.0 A resolution was 3.67, indicating that the final structure can be refined at higher resolution. Molecular replacement methods and the PC-refinement technique have not yet yielded the structure under a variety of search conditions. We are currently investigating the multiple isomorphous replacement approach to determine this crystal structure.

Trypanosoma brucei rhodesiense: the inhibition of HL-60 cell growth by the African trypanosomes in vitro.

Trypanosomiasis is of major public health importance in Africa where the disease affects man and livestock. In order to explore the underlying mechanisms of pathogenesis in African trypanosomiasis, we studied the inhibition of host cell (human promyelocytic HL-60 cells) growth by Trypanosoma brucei rhodesiense using an in vitro system. This inhibition was not due to changes in pH or nutritional depletion of the culture medium by the trypanosomes as inhibitory activity was still observed in cultures that had been supplemented with glucose or fresh culture medium. Our study suggests that the African trypanosomes produce a soluble factor which inhibits the growth of HL-60 cells. This growth inhibitor does not appear to kill the HL-60 cells as determined by the trypan blue dye exclusion test. The production of this factor does not require host cell contact nor does it require a host cell cofactor. The trypanosome growth inhibitor is strictly a trypanosome product. Estimation of the molecular weight of the trypanosome growth inhibitor with Amicon filters revealed that the factor is greater than 30,000 Da in size. Protease and heat treatment of the factor resulted in the depletion of inhibitory activity. These results indicate that the African trypanosomes produce a large-molecular-weight protein growth inhibitory factor which could play a role in the pathogenesis of the disease.

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.

Identification of basic fibroblast growth factor-like proteins in African trypanosomes and Leishmania.

Basic fibroblast growth factor (bFGF) is a multifunctional, heparin-binding, mitogenic polypeptide found in all tissues or cells of multicellular organisms so far examined. Here we report that Trypanosoma brucei rhodesiense procyclic culture forms (PCF) and Leishmania donovani promastigotes grown in serum-containing and serum-free medium, contained peptides of 15-34 kDa which bound heparin-sepharose with high affinity and which reacted in immunoblots with several preparations of antibodies specific for bovine brain bFGF. Similar peptides were not detectable in foetal bovine serum. Immunofluorescence studies showed bFGF-like molecules to have a cytoplasmic distribution in both species growing in serum-free media. A nuclear and/or perinuclear distribution of immunoreactivity was also observed in parasites which had been grown in the presence of serum. The data indicate that both species of parasites synthesize their own bFGF-like molecules. Association of an ubiquitous growth factor with parasitic protozoa may play an important role in parasite multiplication and in host-parasite interactions.