Characterization Of Invariant Membrane Proteins Of Trypanosoma (duttonella) Vivax

Monoclonal antibodies (mAbs), raised against whole, fixed, uncoated, culture forms of Trypanosoma (Duttonella) vivax, were used to identify two invariant membrane proteins of this protozoan parasite. Since non-variant membrane proteins of the cell surface, flagellar pocket and endocytic pathway are potential targets for the control of trypanosomiasis of livestock by immunization, the identification and characterization of invariant membrane proteins is a necessary preliminary step.;A 65 kDA invariant membrane glycoprotein (gp65), identified using mAb 4E1, was the main focus of this study. Immunolocalization studies using the monoclonal antibody (mAb 4E1) for immunofluorescence staining and immunoelectron microscopy, demonstrated that the 65 kDa antigen was associated with tubulo-vesicular profiles in the posterior region of the bloodstream form parasite. Endocytosis and co-localization experiments revealed that gp65 was associated with an endocytic compartment of T. vivax which is morphologically and temporally similar to the endosomal system of mammalian cells. Double labelling experiments using the mAb and a polyclonal anti-variant surface glycoprotein antibody (R{dollar}{lcub}\alpha{rcub}{dollar}VSG) to simultaneously localize both gp65 and intracellular VSG, demonstrated that there was little overlap in the distribution of these antigens. Thus, gp65 is associated with tubules and vesicles that are involved in endocytosis but which appear to be distinct from VSG processing pathways in the cell.;A 35 kDa T. vivax antigen was shown in immunolocalization studies to be associated primarily with the surface of bloodstream forms of the parasite. Although T. vivax 3{dollar}\sp{lcub}\prime{rcub}{dollar}-nucleotidase activity, a surface membrane enzyme in other trypanosomatids, migrated at 35 kDa on SDS-PAGE gels, it is doubtful that the 35 kDa antigen identified with the monoclonal antibody (mAb 4B11) is specific for the T. vivax 3{dollar}\sp\prime{dollar}-nucleotidase since the two proteins exhibited different capacities to bind to immobilized Concanavalin A.;Both T. vivax invariant antigens have potential as targets for disease control based on their location in the cell and thus merit further study to this end. In addition, gp65 is the first putative marker for an endosomal compartment of trypanosomes and has potential for use in the further study of endocytosis in African trypanosomes, a process upon which these parasites are dependent upon for survival

Trypanosome Lytic Factor, an Antimicrobial High-Density Lipoprotein, Ameliorates Leishmania Infection

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system

Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening.

Novel technologies that include recombinant pathogens and rapid detection methods are contributing to the development of drugs for neglected diseases. Recently, the results from the first high throughput screening (HTS) to test compounds for activity against Trypanosoma cruzi trypomastigote infection of host cells were reported. We have selected 23 compounds from the hits of this HTS, which were reported to have high anti-trypanosomal activity and low toxicity to host cells. These compounds were highly purified and their structures confirmed by HPLC/mass spectrometry. The compounds were tested in vitro, where about half of them confirmed the anti-T. cruzi activity reported in the HTS, with IC50 values lower than 5 µM. We have also adapted a rapid assay to test anti-T. cruzi compounds in vivo using mice infected with transgenic T. cruzi expressing luciferase as a model for acute infection. The compounds that were active in vitro were also tested in vivo using this assay, where we found two related compounds with a similar structure and low in vitro IC50 values (0.11 and 0.07 µM) that reduce T. cruzi infection in the mouse model more than 90% after five days of treatment. Our findings evidence the benefits of novel technologies, such as HTS, for the drug discovery pathway of neglected diseases, but also caution about the need to confirm the results in vitro. We also show how rapid methods of in vivo screening based in luciferase-expressing parasites can be very useful to prioritize compounds early in the chain of development

A novel IFN regulatory factor 3-dependent pathway activated by trypanosomes triggers IFN-beta in macrophages and fibroblasts

Innate immune recognition of intracellular pathogens involves both extracellular and cytosolic surveillance mechanisms. The intracellular protozoan parasite Trypanosoma cruzi triggers a robust type I IFN response in both immune and nonimmune cell types. In this study, we report that signaling through TBK1 and IFN regulatory factor 3 is required for T. cruzi-mediated expression of IFN-beta. The TLR adaptors MyD88 and TRIF, as well as TLR4 and TLR3, were found to be dispensable, demonstrating that T. cruzi induces IFN-beta expression in a TLR-independent manner. The potential role for cytosolic dsRNA sensing pathways acting through RIG-I and MDA5 was ruled out because T. cruzi was shown to trigger robust expression of IFN-beta in macrophages lacking the MAVS/IPS1/VISA/CARDif adaptor protein. The failure of T. cruzi to activate HEK293-IFN-beta-luciferase cells, which are highly sensitive to cytosolic triggers of IFN-beta expression including Listeria, Sendai virus, and transfected dsRNA and dsDNA, further indicates that the parasite does not engage currently recognized cytosolic surveillance pathways. Together, these findings identify the existence of a novel TLR-independent pathogen-sensing mechanism in immune and nonimmune cells that converges on TBK1 and IFN regulatory factor 3 for activation of IFN-beta gene expression

Stable expression of luciferase after <i>in vitro</i> or <i>in vivo</i> passaging of <i>T. cruzi</i> trypomastigotes.

<p>Luciferase activity in 1×10⁵ epimastigotes (Epi) under continued drug selection (G418 200 µg/ml) (Epi); 1×10⁵ trypomastigotes (Tryp); 1×10⁵ trypomastigotes 2 weeks after differentiation into metacyclics, removal of drug selection and <i>in vitro</i> passage through LLCMK2 cells and subsequent <i>in vivo</i> passage in mice where luciferase activity was measured in 10⁵ trypomastigotes acquired from the blood of an infected 6 week-old Balb/c mouse 1 week post infection (<i>in vivo</i> trypo).</p

Test for activity <i>in vivo</i> of compounds active <i>in vitro</i>.

<p>Groups of five mice were infected with <i>T. cruzi</i> and treated with different compounds following the protocol shown in (A). (B) Quantification of parasite infection levels in the groups of mice treated with the different compounds is expressed as <i>T. cruzi</i> index. Compounds are identified by their CID. Results are expressed as average ± standard deviation (*, <i>P</i><0.05). (C) One representative mouse of each group treated with compounds CID-12402750 and CID-24892493.</p

Compound CID-12402750 shows trypanostatic activity <i>in vitro</i>.

<p>NIH-3T3 fibroblasts were incubated with <i>T. cruzi</i> trypomastigotes for 2 h before washing of extracellular <i>T. cruzi</i> and addition of drugs. Cells were incubated for 3 days, stained with an anti-<i>T. cruzi</i> antibody and DAPI to visualize DNA. Control infection (A) or infection in the presence of compound CID-12402750 at IC5 (B) and IC100 (C) concentration. These are representative images from a total of 50 fields observed in each condition. Scale bar: 10 µm.</p