Trypanocidal and leishmanicidal activity of six limonoids

Six limonoids [kotschyienone A and B (1, 2), 7-deacetylgedunin (3), 7-deacetyl-7-oxogedunin (4), andirobin (5) and methyl angolensate (6)] were investigated for their trypanocidal and leishmanicidal activities using bloodstream forms of Trypanosoma brucei and promastigotes of Leishmania major. Whereas all compounds showed anti-trypanosomal activity, only compounds 1–4 displayed anti-leishmanial activity. The 50% growth inhibition (GI 50) values for the trypanocidal and leishmanicidal activity of the compounds ranged between 2.5 and 14.9 μM. Kotschyienone A (1) was found to be the most active compound with a minimal inhibition concentration (MIC) value of 10 μM and GI 50 values between 2.5 and 2.9 μM. Only compounds 1 and 3 showed moderate cytotoxicity against HL-60 cells with MIC and GI 50 values of 100 μM and 31.5–46.2 μM, respectively. Compound 1 was also found to show activity against intracellular amastigotes of L. major with a GI 50 value of 1.5 μM. The results suggest that limonoids have potential as drug candidates for the development of new treatments against trypanosomiasis and leishmaniasis

Cymantrene–Triazole "Click" Products: Structural Characterization and Electrochemical Properties

We report the first known examples of triazole-derivatized cymantrene complexes (η5-[4-substituted triazol-1-yl]cyclopentadienyl)tricarbonylmanganese(I), obtained via a “click” chemical synthesis, bearing a phenyl, 3-aminophenyl, or 4-aminophenyl moiety at the 4-position of the triazole ring. Structural characterization data using multinuclear NMR, UV–vis, ATR-IR, and mass spectrometric methods are provided, as well as crystallographic data for (η5-[4-phenyltriazol-1-yl]cyclopentadienyl)tricarbonylmanganese(I) and (η5-[4-(3-aminophenyl)triazol-1-yl]cyclopentadienyl)tricarbonylmanganese(I). Cyclic voltammetric characterization of the redox behavior of each of the three cymantrene–triazole complexes is presented together with digital simulations, in situ infrared spectroelectrochemistry, and DFT calculations to extract the associated kinetic and thermodynamic parameters. The trypanocidal activity of each cymantrene–triazole complex is also examined, and these complexes are found to be more active than cymantrene alone

Differences between <i>Trypanosoma brucei gambiense</i> groups 1 and 2 in their resistance to killing by Trypanolytic factor 1

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; The three sub-species of &lt;i&gt;Trypanosoma brucei&lt;/i&gt; are important pathogens of sub-Saharan Africa. &lt;i&gt;T. b. brucei&lt;/i&gt; is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. &lt;i&gt;T. b. rhodesiense&lt;/i&gt; and &lt;i&gt;T. b. gambiense&lt;/i&gt; are able to resist lysis by TLF. There are two distinct sub-groups of &lt;i&gt;T. b. gambiense&lt;/i&gt; that differ genetically and by human serum resistance phenotypes. Group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (&lt;i&gt;HpHbR&lt;/i&gt;)) gene. Here we investigate if this is also true in group 2 parasites.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methodology:&lt;/b&gt; Isogenic resistant and sensitive group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the &lt;i&gt;HpHbR&lt;/i&gt; gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to &lt;i&gt;T. b. brucei&lt;/i&gt;. Both resistant and sensitive group 2, as well as group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt;, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Our data indicate that, despite group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of &lt;i&gt;HpHbR&lt;/i&gt;. Thus there are differences in the mechanism of human serum resistance between &lt;i&gt;T. b. gambiense&lt;/i&gt; groups 1 and 2.&lt;/p&gt

T. brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

Aims: African trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response. Methods and results: Using confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events. Conclusion: These data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function

Modulation of the surface proteome through multiple ubiquitylation pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes

How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

Variations on the statement "the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier" appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.The authors’ lab is funded by the Wellcome Trust (093008/Z10/Z) and the Medical Research Council (MR/L008246/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It was first available from PLOS via http://dx.doi.org/10.1371/journal.ppat.100525

A new initiative for the development of new diagnostic tests for human African trypanosomiasis

Human African trypanosomiasis is a threat to millions of people living in sub-Saharan countries and is fatal unless treated. At present, the serological and parasitological tests used in the field for diagnosis of sleeping sickness have low specificity and sensitivity. There is clearly an urgent need for accurate tools for both diagnosis and staging of the disease. The Foundation for Innovative New Diagnostics and the World Health Organization have announced that they will collaborate to develop and evaluate new diagnostic tests for human African trypanosomiasis

A Deep Insight into the Sialome of Rhodnius neglectus, a vector of chagas disease

Background Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immuneresponses. Methods/Principal Findings Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva.We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant. Conclusions/Significance The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts

In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC(50)) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development

The genome landscape of indigenous African cattle

Background: The history of African indigenous cattle and their adaptation to environmental and human selection pressure is at the root of their remarkable diversity. Characterization of this diversity is an essential step towards understanding the genomic basis of productivity and adaptation to survival under African farming systems. Results: We analyze patterns of African cattle genetic variation by sequencing 48 genomes from five indigenous populations and comparing them to the genomes of 53 commercial taurine breeds. We find the highest genetic diversity among African zebu and sanga cattle. Our search for genomic regions under selection reveals signatures of selection for environmental adaptive traits. In particular, we identify signatures of selection including genes and/ or pathways controlling anemia and feeding behavior in the trypanotolerant N’Dama, coat color and horn development in Ankole, and heat tolerance and tick resistance across African cattle especially in zebu breeds. Conclusions: Our findings unravel at the genome-wide level, the unique adaptive diversity of African cattle while emphasizing the opportunities for sustainable improvement of livestock productivity on the continent