Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure.

Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused a significant reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol-a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

Unique Aspects of rRNA Biogenesis in Trypanosomatids.

Trypanosomatids are protozoan parasites that cycle between an insect and a mammalian host. The large-subunit rRNA of these organisms undergoes unique processing events absent in other eukaryotes. Recently, small nucleolar RNAs (snoRNAs) that mediate these specific cleavages were identified. Trypanosomatid rRNA is rich in RNA modifications such as 2'-O-methylation (Nm) and pseudouridylation (Ψ) that are also guided by these snoRNAs. A subset of these modifications is developmentally regulated and increased in the parasite form that propagates in the mammalian host. Such hypermodification contributes the temperature adaptation and hence infectivity during cycling of the parasite. rRNA processing and modification should be considered promising drug targets for fighting the diseases caused by these parasites.

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum.

Micro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, comparably little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts, generally preceded by a T-rich putative promoter motif, accumulate in a drnB knock-out strain. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. The generation of the 3'-ends of these pri-miRNAs differs, with pri-mir-1177 reading into the downstream gene, and pri-mir-1176 displaying a distinct end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development.

Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum.

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite.

New insights into the Plasmodium vivax transcriptome using RNA-Seq.

Historically seen as a benign disease, it is now becoming clear that Plasmodium vivax can cause significant morbidity. Effective control strategies targeting P. vivax malaria is hindered by our limited understanding of vivax biology. Here we established the P. vivax transcriptome of the Intraerythrocytic Developmental Cycle (IDC) of two clinical isolates in high resolution by Illumina HiSeq platform. The detailed map of transcriptome generates new insights into regulatory mechanisms of individual genes and reveals their intimate relationship with specific biological functions. A transcriptional hotspot of vir genes observed on chromosome 2 suggests a potential active site modulating immune evasion of the Plasmodium parasite across patients. Compared to other eukaryotes, P. vivax genes tend to have unusually long 5' untranslated regions and also present multiple transcription start sites. In contrast, alternative splicing is rare in P. vivax but its association with the late schizont stage suggests some of its significance for gene function. The newly identified transcripts, including up to 179 vir like genes and 3018 noncoding RNAs suggest an important role of these gene/transcript classes in strain specific transcriptional regulation.

Antisense Transcripts Delimit Exonucleolytic Activity of the Mitochondrial 3' Processome to Generate Guide RNAs.

Small, noncoding RNA biogenesis typically involves cleavage of structured precursor by RNase III-like endonucleases. However, guide RNAs (gRNAs) that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5' triphosphate characteristic of the transcription initiation and possess a U-tail indicative of 3' processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase, DSS1 3'-5' exonuclease, and three additional subunits. This complex, termed mitochondrial 3' processome (MPsome), is responsible for primary uridylation of ∼800 nt gRNA precursors, their processive degradation to a mature size of 40-60 nt, and secondary U-tail addition. Both strands of the gRNA gene are transcribed into sense and antisense precursors of similar lengths. Head-to-head hybridization of these transcripts blocks symmetrical 3'-5' degradation at a fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5' extremity of a primary molecule by uridylation-induced, antisense transcription-controlled 3'-5' exonucleolytic degradation.

Deciphering RNA Regulatory Elements Involved in the Developmental and Environmental Gene Regulation of Trypanosoma brucei.

Trypanosoma brucei is a vector-borne parasite with intricate life cycle that can cause serious diseases in humans and animals. This pathogen relies on fine regulation of gene expression to respond and adapt to variable environments, with implications in transmission and infectivity. However, the involved regulatory elements and their mechanisms of actions are largely unknown. Here, benefiting from a new graph-based approach for finding functional regulatory elements in RNA (GRAFFER), we have predicted 88 new RNA regulatory elements that are potentially involved in the gene regulatory network of T. brucei. We show that many of these newly predicted elements are responsive to both transcriptomic and proteomic changes during the life cycle of the parasite. Moreover, we found that 11 of predicted elements strikingly resemble previously identified regulatory elements for the parasite. Additionally, comparison with previously predicted motifs on T. brucei suggested the superior performance of our approach based on the current limited knowledge of regulatory elements in T. brucei.

Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

Life without post-transcriptional addition of G-1: two alternatives for tRNAHis identity in Eukarya.

The identity of tRNA(His) is strongly associated with the presence of an additional 5'-guanosine residue (G-1) in all three domains of life. The critical nature of the G-1 residue is underscored by the fact that two entirely distinct mechanisms for its acquisition are observed, with cotranscriptional incorporation observed in Bacteria, while post-transcriptional addition of G-1 occurs in Eukarya. Here, through our investigation of eukaryotes that lack obvious homologs of the post-transcriptional G-1-addition enzyme Thg1, we identify alternative pathways to tRNA(His) identity that controvert these well-established rules. We demonstrate that Trypanosoma brucei, like Acanthamoeba castellanii, lacks the G-1 identity element on tRNA(His) and utilizes a noncanonical G-1-independent histidyl-tRNA synthetase (HisRS). Purified HisRS enzymes from A. castellanii and T. brucei exhibit a mechanism of tRNA(His) recognition that is distinct from canonical G-1-dependent synthetases. Moreover, noncanonical HisRS enzymes genetically complement the loss of THG1 in Saccharomyces cerevisiae, demonstrating the biological relevance of the G-1-independent aminoacylation activity. In contrast, in Caenorhabditis elegans, which is another Thg1-independent eukaryote, the G-1 residue is maintained, but here its acquisition is noncanonical. In this case, the G-1 is encoded and apparently retained after 5' end processing, which has so far only been observed in Bacteria and organelles. Collectively, these observations unearth a widespread and previously unappreciated diversity in eukaryotic tRNA(His) identity mechanisms.

Activity of benzimidazoles against Dientamoeba fragilis (Trichomonadida, Monocercomonadidae) in vitro and correlation of beta-tubulin sequences as an indicator of resistance.

Recently, Dientamoeba fragilis has emerged as a significant and common enteropathogen. The majority of patients with dientamoebiasis present with gastrointestinal complaints and chronic symptoms are common. Numerous studies have successfully demonstrated parasite clearance, coupled with complete resolution of clinical symptoms following treatment with various antiparasitic compounds. Despite this, there is very little in vitro susceptibility data available for the organism. Benzimidazoles are a class of antiparasitic drugs that are commonly used for the treatment of protozoan and helminthic infections. Susceptibility testing was undertaken on four D. fragilis clinical isolates against the following benzimidazoles: albendazole, flubendazole, mebendazole, nocodazole, triclabendazole and thiabendazole. The activities of the antiprotozoal compounds at concentrations ranging from 2 µg/mL to 500 µg/mL were determined via cell counts of D. fragilis grown in xenic culture. All tested drugs showed no efficacy. The beta-tubulin transcript was sequenced from two of the D. fragilis isolates and amino acid sequences predicted a susceptibility to benzimidazoles. This is the first study to report susceptibility profiles for benzimidazoles against D. fragilis, all of which were not active against the organism. This study also found that beta-tubulin sequences cannot be used as a reliable marker for resistance of benzimidazoles in D. fragilis.

PmMGST3, a novel microsomal glutathione S-transferase gene in the dinoflagellate Prorocentrum minimum, is a potential biomarker of oxidative stress.

In this study, we evaluated a novel microsomal glutathione S-transferase3 (MGST3) gene from the dinoflagellate Prorocentrum minimum, and examined its expression pattern in response to copper-and nickel-induced stresses. The full length of PmMGST3 was 732 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the poly (A) tail, covering a 441-bp ORF, 97-bp 5'UTR, and 194-bp 3'UTR. The PmMGST3 was up-regulated by metals, including copper and nickel. The highest up-regulation levels of the PmMGST3 were found under 0.1 mg/L copper and 0.5 mg/L nickel treatment, respectively. In addition, the PmMGST3 was gradually up-regulated by 0.1 mg/L copper with increasing exposure time. Furthermore, ROS production and reduced GSH was measured in the copper treated cells. A significant increased ROS production and reduced GSH were found in the copper treated cells. These results suggest that PmMGST3 may be related to defense mechanisms associated with oxidative stress in dinoflagellates.

The var3 genes of Plasmodium falciparum 3D7 strain are differentially expressed in infected erythrocytes.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important virulence factor encoded by a family of 59 var genes, including 56 var genes plus 3 small var3 genes. The var genes are among the most diverse sequences in the P. falciparum genome, but the var3 genes are found conserved in most P. falciparum strains. Previous studies have been mainly focused on the typical var genes, while the biological characteristics of the var3 genes remain unknown. In this study, the three var3 genes, PF3D7_0100300, PF3D7_0600400, and PF3D7_0937600, were found to be transcribed in the erythrocytic stages of P. falciparum, with a peak in the transcription level at 16 h post-invasion, but terminated immediately after 16 h post-invasion. The encoded protein of PF3D7_0600400 could be detected in both the late trophozoite stage and schizont stage, while the encoded proteins of PF3D7_0100300 and PF3D7_0937600 could only be detected in the late trophozoite stage and schizont stage, respectively. Thus, the var3 genes of the P. falciparum 3D7 strain were differentially expressed during the erythrocytic development of the parasite.

Entamoeba histolytica: gene expression analysis of cells invading tissues.

Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis.

Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina / Differential expression of proteins in Leishmania (Viannia) panamensis associated with mechanisms of resistance to meglumine antimoniate.

Introducción. Los mecanismos de resistencia al antimonio pentavalente conocidos hasta el momento, se han descrito ampliamente en cepas del subgénero Leishmania, pero poco se sabe sobre las proteínas involucradas en los mecanismos de resistencia presentes en cepas del subgénero Viannia, como Leishmania panamensis. Objetivo. Identificar proteínas diferencialmente expresadas entre las cepas de L. panamensis (UA140), sensible y resistente al antimonio pentavalente, y analizar el posible papel de estas proteínas en mecanismos de resistencia. Materiales y métodos. Las proteínas de las cepas, sensible y resistente al antimonio pentavalente, se compararon usando electroforesis bidimensional. Las proteínas con aumento de la expresión fueron aisladas e identificadas por espectrometría de masas mediante MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). La expresión del ARNm de cinco de estas proteínas se cuantificó mediante PCR en tiempo real. Resultados. Los geles bidimensionales de las cepas sensible y resistente detectaron 532±39 y 541±43 manchas proteicas. Se encontraron 10 manchas con aumento de la expresión en la cepa resistente, identificadas como proteínas de choque térmico (Hsp60 mitocondrial, Hsp70 mitocondrial y citosólica), isomerasa de disulfuro, proteasa de cisteína, enolasa, factor de elongación 5-α, la subunidad 5-α del proteasoma y dos proteínas hipotéticas nombradas como Sp(2) y Sp(25). Conclusión. Este es el primer estudio llevado a cabo con una cepa resistente al antimonio pentavalente en L. panamensis, en el cual se han identificado proteínas que están relacionadas con el mecanismo de resistencia del parásito frente al medicamento, abriendo el camino para futuros estudios de estas proteínas como blancos terapéuticos.

Introduction. The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. Objective. Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. Materials and methods. The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. Results. On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). Conclusion. This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.

[Differential expression of proteins in Leishmania (Viannia) panamensis associated with mechanisms of resistance to meglumine antimoniate]. / Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina.

INTRODUCTION: The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. OBJECTIVE: Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. MATERIALS AND METHODS: The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. RESULTS: On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). CONCLUSION: This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.

A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor.

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes--a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Characterization and tissue-specific expression patterns of the Plasmodium chabaudi cir multigene family.

BACKGROUND: Variant antigens expressed on the surface of parasitized red blood cells (pRBCs) are important virulence factors of malaria parasites. Whereas Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1) are responsible for sequestration of mature parasites, little is known about putative ligands mediating cytoadherence to host receptors in other Plasmodium species. Candidates include members of the pir superfamily found in the human parasite Plasmodium vivax (vir), in the simian pathogen Plasmodium knowlesi (kir) and in the rodent malarias Plasmodium yoelii (yir), Plasmodium berghei (bir) and Plasmodium chabaudi (cir). The aim of this study was to reveal a potential involvement of cir genes in P. chabaudi sequestration. METHODS: Subfamilies of cir genes were identified by bioinformatic analyses of annotated sequence data in the Plasmodium Genome Database. In order to examine tissue-specific differences in the expression of cir mRNAs, RT-PCR with subfamily-specific primers was used. In total, 432 cDNA clones derived from six different tissues were sequenced to characterize the transcribed cir gene repertoire. To confirm differences in transcription profiles of cir genes, restriction fragment length polymorphism (RFLP) analyses were performed to compare different host tissues and to identify changes during the course of P. chabaudi infections in immunocompetent mice. RESULTS: The phylogenetic analysis of annotated P. chabaudi putative CIR proteins identified two major subfamilies. Comparison of transcribed cir genes from six different tissues revealed significant differences in the frequency clones belonging to individual cir gene subgroups were obtained from different tissues. Further hints of difference in the transcription of cir genes in individual tissues were obtained by RFLP. Whereas only minimal changes in the transcription pattern of cir genes could be detected during the developmental cycle of the parasites, switching to expression of other cir genes during the course of an infection was observed around or after peak parasitemia. CONCLUSIONS: The tissue-specific expression of cir mRNAs found in this study indicates correlation between expression of CIR antigens and distribution of parasites in inner organs. Together with comparable results for other members of the pir superfamily this suggests a role of cir and other pir genes in antigenic variation and sequestration of malaria parasites.

A single zinc ion is sufficient for an active Trypanosoma brucei tRNA editing deaminase.

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.

A zinc finger protein, TbZC3H20, stabilizes two developmentally regulated mRNAs in trypanosomes.

CCCH zinc finger proteins (ZC3Hs) are a novel class of RNA-binding protein involved in post-transcriptional mechanisms controlling gene expression. We show TbZC3H20 from Trypanosoma brucei, the causative agent of sleeping sickness and other diseases, stabilizes two developmentally regulated transcripts encoding a mitochondrial carrier protein (MCP12) and trans-sialidase (TS-like E). TbZC3H20 is shown to be an RNA-binding protein that is enriched in insect procyclic form T. brucei and is the first ZC3H discovered controlling gene expression through modulating mRNA abundance in trypanosomes. Previous studies have demonstrated that RNA recognition motif-containing and PUF family RNA-binding proteins can control gene expression by stabilizing specific target mRNA levels. This work is the first to describe a ZC3H stabilizing rather than destabilizing target mRNAs as a regulatory mechanism and the first report of a ZC3H regulating a gene encoding a mitochondrial protein. This suggests a broader role for ZC3Hs in post-transcriptional regulation of gene expression than previously thought.