The mRNA-bound Proteome of Leishmania mexicana: Novel Genetic Insight into an Ancient Parasite.

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.

Conserved regions as markers of different patterns of expression and distribution of the mucin-associated surface proteins of Trypanosoma cruzi.

The MASP gene family is the second most widely represented gene family in the genome of Trypanosoma cruzi. One of its main characteristics is that its 5' and 3' regions are highly conserved. We assessed the expression of these conserved regions as a marker for T. cruzi and also analyzed the expression of the masp genes and MASP proteins. In parasite strains CL-Brener (DTUVI lineage) and PAN4 (DTUI lineage), masp genes were expressed at different levels both with regard to the two strains and between stages in the parasite's life cycle. We also studied the expression of the family during the intracellular cycle of T. cruzi, using antibodies against the conserved MASP signal peptide (SP). Fluorescence intensity showed an increase in expression from 24 h onwards, with a peak in intensity at 72 h postinfection. After 24 and 48 h, the MASP proteins were expressed in 33.33% and 57.14% of the amastigotes, respectively. Our data show that not only the extracellular forms of T. cruzi but also the intracellular phases express this type of protein, though to different extents in the various forms of the parasite.

Methods for the Isolation and Study of Exovesicle DNA from Trypanosomatid Parasites.

Extracellular vesicles (EVs) or exovesicles are a heterogeneous group of small cell-derived membranous structures that carry complex cargoes including lipids, proteins, RNA, and DNA. Emerging evidence suggest that EVs secreted by kinetoplastid parasites play a cardinal role in the pathogenesis of diseases they cause, becoming valuable structures for understanding parasite-host interactions. Moreover, the characterization of EVs molecular cargo may provide a new approach to develop alternative tools for diagnosis and therapy of infectious diseases. EVs have a potential use as biomarkers since it contains a repertoire of DNA species that could be detected at different stages of infection by PCR-based assays. Here, we provide a detailed protocol for the isolation of Trypanosoma cruzi-derived EVs and purification of its DNA cargo for subsequent characterization. The methods described here are transferrable to other medically important parasites that are well adapted to grow in vitro and, therefore, suitable volume of EVs-containing supernatants can be obtained.

Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi.

BACKGROUND: Chagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes. METHODOLOGY/PRINCIPAL FINDINGS: To obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication. CONCLUSION/SIGNIFICANCE: This research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action.

Action of a pentacyclic triterpenoid, maslinic acid, against Toxoplasma gondii.

The action of maslinic acid (2alpha,3beta-dihydroxyolean-12-en-28-oic acid) (1), a pentacyclic derivative present in the pressed fruits of the olive (Olea europaea), has been studied against the tachyzoites of Toxoplasma gondii. The capability of tachyzoites to infect Vero cells treated with 1 was affected. The LD(50) values were 58.2 muM for the isolated tachyzoites and 236 muM for the noninfected Vero cells. Zymograms of the T. gondii proteases incubated with 1 showed a dosage-dependent inhibition of some of the proteases. The parasites treated with 1 showed gliding motility and ultrastructural alterations. The present findings suggest that protease activity of the parasite required for cell invasion is the action target for maslinic acid (1).

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.