Blood of African Hedgehog Atelerix albiventris Contains 115-kDa Trypanolytic Protein that Kills Trypanosoma congolense.

INTRODUCTION: Protozoan parasites of the Order Trypanosomatida infect a wide range of multicellular plants and animals, causing devastating and potentially fatal diseases. Trypanosomes are the most relevant members of the order in sub-Saharan Africa because of mortalities and morbidities caused to humans and livestock. PURPOSE: There are growing concerns that trypanosomes are expanding their reservoirs among wild animals, which habours the parasites, withstand the infection, and from which tsetse flies transmit the parasites back to humans and livestock. This study was designed to investigate the potentials of the African hedgehog serving as reservoir for African animal trypanosomes. METHODS: Five adult hedgehogs alongside five laboratory mice were intraperitoneally inoculated with 106 and 104 of Trypanosoma congolense cells, respectively, and monitored for parasitemia and survival. Serum from twenty hedgehogs was subjected to trypanocidal activity-guided fractionation by successive ion-exchange and gel-filtration chromatographies, followed by characterization with Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). RESULTS: Hedgehogs were resistant to the infection as no parasite was detected and none died even after 60 days, while all the mice died within 12 days. Both the serum and plasma prepared from hedgehogs demonstrated trypanocidal activity- rapidly killed trypanosomes even when diluted 1000 times. The trypanolytic factor was identified to be proteinaceous with an estimated molecular weight of 115-kDa. CONCLUSION: For the first time, it is here demonstrated that hedgehog blood has significant trypanolytic activity against T. congolense. The potential application of the hedgehog protein for the breeding of trypanosomosis-resistant livestock in tsetse fly belt is discussed.

Crystal structure of TbEsa1 presumed Tudor domain from Trypanosoma brucei.

The essential SAS2-related acetyltransferase 1 (Esa1), as a acetyltransferase of MYST family, is indispensable for the cell cycle and transcriptional regulation. The Tudor domain consists of 60 amino acids and belongs to the Royal family, which serves as a module interacting with methylated histone and/or DNA. Although Tudor domain has been widely studied in higher eukaryotes, its structure and function remain unclear in Trypanosoma brucei (T. brucei), a protozoan unicellular parasite causing sleeping sickness in human and nagana in cattle in sub-Saharan Africa. Here, we determined a high-resolution structure of TbEsa1 presumed Tudor domain from T. brucei by X-ray crystallography. TbEsa1 Tudor domain adopts a conserved Tudor-like fold, which is comprised of a five-stranded ß-barrel surrounded by two short α-helices. Furthermore, we revealed a non-specific DNA binding pattern of TbEsa1 Tudor domain. However, TbEsa1 Tudor domain showed no methyl-histone binding ability, due to the absence of key aromatic residues forming a conserved aromatic cage.

Innate immunity in the tsetse fly (Glossina), vector of African trypanosomes.

Tsetse flies (Glossina sp.) are medically and veterinary important vectors of African trypanosomes, protozoan parasites that cause devastating diseases in humans and livestock in sub-Saharan Africa. These flies feed exclusively on vertebrate blood and harbor a limited diversity of obligate and facultative bacterial commensals. They have a well-developed innate immune system that plays a key role in protecting the fly against invading pathogens and in modulating the fly's ability to transmit African trypanosomes. In this review, we briefly summarize our current knowledge on the tsetse fly innate immune system and its interaction with the bacterial commensals and the trypanosome parasite.

Colonization of the tsetse fly midgut with commensal Kosakonia cowanii Zambiae inhibits trypanosome infection establishment.

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco_Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco_Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.

A Molecular Hybridization Approach for the Design of Potent, Highly Selective, and Brain-Penetrant N-Myristoyltransferase Inhibitors.

Crystallography has guided the hybridization of two series of Trypanosoma brucei N-myristoyltransferase (NMT) inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of crossing the blood-brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for human African trypanosomiasis in both the peripheral and central nervous system stages of disease.

Phe71 in Type III Trypanosomal Protein Arginine Methyltransferase 7 (TbPRMT7) Restricts the Enzyme to Monomethylation.

Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal SN2 methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.

The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion.

The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic forms. We used an in vitro system based on expressing the RNA binding protein 6 to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNA-Seq) in comparison to non-infectious procyclic trypanosomes. We showed that metacyclics are quiescent cells, and propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.

Structure Elucidation and Activity of Kolossin†A, the D-/L-Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase.

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness.

The bacterial flora of tsetse fly midgut and its effect on trypanosome transmission.

The tsetse fly, Glossina palpalis is a vector of the trypanosome that causes sleeping sickness in humans and nagana in cattle along with associated human health problems and massive economic losses. The insect is also known to carry a number of symbionts such as Sodalis, Wigglesworthia, Wolbachia whose effects on the physiology of the insect have been studied in depth. However, effects of other bacterial flora on the physiology of the host and vector competence have received little attention. Epidemiological studies on tsetse fly populations from different geographic sites revealed the presence of a variety of bacteria in the midgut. The most common of the flora belong to the genera Entrobacter (most common), Enterococcus, and Acinetobacter. It was a little surprising to find such diversity in the tsetse midgut since the insect is monophagous consuming vertebrate blood only. Diversity of bacteria is normally associated with polyphagous insects. In contrast to the symbionts, the role of resident midgut bacterial flora on the physiology of the fly and vector competence remains to be elucidated. With regard, Sodalis glossinidius, our data showed that flies harbouring this symbiont have three times greater probability of being infected by trypanosomes than flies without the symbiont. The data delineated in these studies under score the need to carry out detailed investigations on the role of resident bacteria on the physiology of the fly and vector competence.

Trypanosoma brucei rhodesiense infection in a German traveller returning from the Masai Mara area, Kenya, January 2012.

In January 2012, a case of Human African Trypanosomiasis (HAT) has been identified in Germany in a traveller returning from the Masai Mara area in Kenya. The 62-year-old man had travelled to the Masai Mara game park from 18 to 19 January 2012 and developed fever on 28 January. The infection with Trypanosoma brucei rhodesiense was confirmed by laboratory testing three days hereafter.

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei.

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC(50)) of PCF was 1.0 ± 0.2 µM for Isoxyl and 5 ± 2 µM for 10-TS, whereas BSF appeared more susceptible with EC(50) values 0.10 ± 0.03 µM (Isoxyl) and 1.0 ± 0.6 µM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

Elongator protein 3b negatively regulates ribosomal DNA transcription in african trypanosomes.

Eukaryotic cells limit ribosomal DNA (rDNA) transcription by RNA polymerase I (RNAP-I) to maintain genome integrity. African trypanosomes present an excellent model for studies on RNAP-I regulation because they possess a bifunctional RNAP-I and because RNAP-II transcription appears unregulated. Since Elp3, the catalytic component of Elongator, controls RNAP-II transcription in yeast and human cells, we predicted a role for a trypanosome Elp3-related protein, ELP3a or ELP3b, in RNAP-I regulation. elp3b null and conditional strains specifically exhibited resistance to a transcription elongation inhibitor, suggesting that ELP3b negatively impacts elongation. Nascent RNA analysis and expression of integrated reporter cassettes supported this interpretation and revealed negative control of rDNA transcription. ELP3b specifically localized to the nucleolus, and ELP3b loss rendered cells hypersensitive to DNA damage and to translation inhibition, suggesting that anti-Elongator function was important to maintain genome integrity rather than to modulate ribosome production. Finally, ELP3b displayed discrimination between RNAP-I compartments in the same cell. Our results establish ELP3b as a major negative regulator of rDNA transcription and extend the roles of the Elp3-related proteins to RNAP-I transcription units. ELP3b is also the first trypanosome protein shown to distinguish between rDNA and variant surface glycoprotein transcription within different RNAP-I compartments.

The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II) into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5' and 3' ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq) was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp.) and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

In vitro activities of plant extracts from Saudi Arabia against malaria, leishmaniasis, sleeping sickness and Chagas disease.

The in vitro activity of the methanol extracts of 51 plants randomly collected from the Kingdom of Saudi Arabia and some of their fractions (petroleum ether, chloroform, ethyl acetate and aqueous) were evaluated against Plasmodium falciparum, Trypanosoma brucei brucei, T. cruzi and Leishmania infantum, as well as toxicity against MRC-5 fibroblast cells. Ten crude methanolic extracts that demonstrated potent and adequately selective antiprotozoal activity were subjected to solvent fractionation using petroleum ether, ethyl acetate and chloroform. Only three samples showed promising antiprotozoal activity. Argemone ochroleuca (CHCl(3) fraction) showed pronounced activity against P. falciparum(GHA) (IC(50) 0.32 microg/mL) and T. cruzi (IC(50) 0.30 microg/mL) with low cytotoxicity against MRC-5 cells (CC(50) 11.6 microg/mL). Capparis spinosa (EtOAc fraction) showed pronounced activity against P. falciparum(GHA) with an IC(50) 0.50 microg/mL in the absence of toxicity against MRC-5 cell line (CC(50) > 30 microg/mL). Heliotropium curassavicum (CHCl(3) fraction) showed similar activity against P. falciparum (IC(50) 0.65 microg/mL; MRC-5 CC(50) > 30 microg /mL). These three extracts will be subjected for further extensive studies to isolate and identify their active constituents.

Overproduction, purification and characterisation of Tbj1, a novel Type III Hsp40 from Trypanosoma brucei, the African sleeping sickness parasite.

The heat shock protein 40 (Hsp40) family of proteins act as co-chaperones of the heat shock protein 70 (Hsp70) chaperone family, and together they play a vital role in the maintenance of cellular homeostasis. The Type III class of Hsp40s are diverse in terms of both sequence identity and function and have not been extensively characterised. The Trypanosoma brucei parasite is the causative agent of Human African Trypanosomiasis, and possesses an unusually large Hsp40 complement, consisting mostly of Type III Hsp40s. A novel T. brucei Type III Hsp40, Tbj1, was heterologously expressed, purified, and found to exist as a compact monomer in solution. Using polyclonal antibodies to the full-length recombinant protein, Tbj1 was found by Western analysis to be expressed in the T. brucei bloodstream-form. Tbj1 was found to be able to assist two different Hsp70 proteins in the suppression of protein aggregation in vitro, despite being unable to stimulate their ATPase activity. This indicated that while Tbj1 did not possess independent chaperone activity, it potentially functioned as a novel co-chaperone of Hsp70 in T. brucei.

Propulsion of African trypanosomes is driven by bihelical waves with alternating chirality separated by kinks.

Trypanosoma brucei, a parasitic protist with a single flagellum, is the causative agent of African sleeping sickness. Propulsion of T. brucei was long believed to be by a drill-like, helical motion. Using millisecond differential interference-contrast microscopy and analyzing image sequences of cultured procyclic-form and bloodstream-form parasites, as well as bloodstream-form cells in infected mouse blood, we find that, instead, motility of T. brucei is by the propagation of kinks, separating left-handed and right-handed helical waves. Kink-driven motility, previously encountered in prokaryotes, permits T. brucei a helical propagation mechanism while avoiding the large viscous drag associated with a net rotation of the broad end of its tapering body. Our study demonstrates that millisecond differential interference-contrast microscopy can be a useful tool for uncovering important short-time features of microorganism locomotion.

Trypanocidal activity of 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]-(methylamino)}adenosine (Genz-644131), an adenosylmethionine decarboxylase inhibitor.

Genzyme 644131, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.

The effect of aqueous extracts of Hibiscus sabdariffa (Sorrel) calyces on heamatological profile and organ pathological changes in Trypanasoma congolense - infected rats.

The effects of aqueous extract of Hibiscus sabdariffa calyces on haematology and pathological changes in some selected organs during experimental Trypanosoma congolense infection of rats were investigated. Three groups of rats were intraperitoneally infected with T. congolense (Karu stock). One group was administered with the aqueous extract and another given a solution of vitamin C in drinking water; the remaining infected group was left untreated. Data from these groups were compared with those of two groups of healthy rats, one of which was similarly treated with the aqueous extract. The experiment was terminated three weeks, post-infection (pi). The uninfected and infected rats administered the extract consumed the equivalent of 9.94 mg - and 9.61 mg ascorbic acid / 100g / day during the experiment. Consumption of the extract significantly (p<0.01) retarded the rate of weight gain in both healthy and infected rats; even though the feed-intake was not significantly affected. After two weeks of infection the extract and vitamin C kept the parasitaemia significantly (p<0.01) lower than the untreated infected group. The anaemia in the untreated infected group was significantly (p<0.01) more severe than that of the corresponding extract- or vitamin-treated groups. Trypanosoma congolense infection caused significant (p<0.01) decreases in serum total proteins and albumin; serum and organ ascorbic acid as well as significant (p<0.01) elevation of serum alanine amino transferase levels in untreated rats. Consumption of the extract or vitamin C, however, prevented these disease-induced anomalies in the treated infected rats. Serum creatinine and urea levels were not affected by infection but the extract elevated these parameters significantly (p<0.01) above infection levels. It was concluded that consumption of the extract ameliorated the pathological changes in blood and organs of T. congolense-infected rats.

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