Genomics and functional genomics in Leishmania and Trypanosoma cruzi: statuses, challenges and perspectives

The availability of Trypanosomatid genomic data in public databases has opened myriad experimental possibilities that have contributed to a more comprehensive understanding of the biology of these parasites and their interactions with hosts. In this review, after brief remarks on the history of the Trypanosoma cruzi and Leishmania genome initiatives, we present an overview of the relevant contributions of genomics, transcriptomics and functional genomics, discussing the primary obstacles, challenges, relevant achievements and future perspectives of these technologies.

Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis

BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Symbiont modulates expression of specific gene categories in Angomonas deanei

Trypanosomatids are parasites that cause disease in humans, animals, and plants. Most are non-pathogenic and some harbor a symbiotic bacterium. Endosymbiosis is part of the evolutionary process of vital cell functions such as respiration and photosynthesis. Angomonas deanei is an example of a symbiont-containing trypanosomatid. In this paper, we sought to investigate how symbionts influence host cells by characterising and comparing the transcriptomes of the symbiont-containing A. deanei (wild type) and the symbiont-free aposymbiotic strains. The comparison revealed that the presence of the symbiont modulates several differentially expressed genes. Empirical analysis of differential gene expression showed that 216 of the 7625 modulated genes were significantly changed. Finally, gene set enrichment analysis revealed that the largest categories of genes that downregulated in the absence of the symbiont were those involved in oxidation-reduction process, ATP hydrolysis coupled proton transport and glycolysis. In contrast, among the upregulated gene categories were those involved in proteolysis, microtubule-based movement, and cellular metabolic process. Our results provide valuable information for dissecting the mechanism of endosymbiosis in A. deanei.

Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodium vivaxmalaria cases in China that are imported across its Southeast Asia border, especially in the China-Myanmar border area (CMB). To date, little is known about the genetic diversity of P. vivaxin this region. In this paper, we report the first genome sequencing of a P. vivaxisolate (CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto 96.43% of the P. vivaxSalvador I reference strain (Sal I) genome with 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Using the de novoassembly approach, we generated 8,541 scaffolds and assembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, we identified all 295 known virgenes, which is the largest subtelomeric multigene family in malaria parasites. These results provide an important foundation for further research onP. vivaxpopulation genetics.

Genômica comparativa em ambiente computacional distribuído: aplicabilidade e potencial no estudo de homologia entre protozoários / Comparative genomics in a distributed computing scenario: applicability and potential on protozoa homology study

A inferência de homologia entre organismos é uma atividade da genômicacomparativa que possibilita compreender melhor a relação entre os mesmos e, porconseguinte, sua distância evolutiva. Especificamente, a identificação de genesortólogos, ou seja, aqueles que têm sua origem em um ancestral comum, permiteoferecer melhorias na anotação funcional de genes, uma vez que genes ortólogostendem a ter sua função conservada.Com a crescente disponibilidade de genomas através de técnicas de NGS, aconstrução e atualização de bases de dados de ortólogos representam um desafioconstante, pois demandam o estudo e identificação das relações entre os genes detais organismos, em um volume de dados cada vez mais extenso e a um custocomputacional cada vez mais elevado.Nesta tese propomos a solução para nuvem computacional elastic-OrthoSearch, umworkflow científico de genômica comparativa inspirado no OrthoSearch, responsávelpela inferência de homologia entre organismos com o uso de abordagem baseadaem melhores hits recíprocos e perfis de Markov.Também propomos uma metodologia para criação de bases de ortólogos construídaatravés do reuso do OrthoSearch. Esta metodologia mostrou-se capaz de alavancara oferta de grupos ortólogos e assim auxiliar, por exemplo, na identificação de alvosde protozoários...

Homology inference among organisms is a comparative genomics tasks which allowsfor a better understanding on how such organisms are related to each other and ontheir evolutionary distance. Specifically, the identification of orthologous genes –those who share a common ancestor – allows for functional gene annotationimprovements, as orthologous genes tend to preserve their functions.The increasing amount of genomic data provided by the NGS techniques makes theorthologous databases’ building and update processes a challenging task. It requiresthe identification and study of the organisms’ genes relationships, in an extensivedata volume and at an increasing computational cost.In this thesis we propose elastic-OrthoSearch, a cloud-enabled comparativegenomics scientific workflow, derived from OrthoSearch. It aims at providinghomology inference among organisms, in a reciprocal best hits and Markov profilesapproach.We also propose an improved orthologous database creation methodology built ontop of OrthoSearch. Such methodology has shown means to offer a broaderorthologous groups dataset, which could in turn aid on Protozoa target identification...

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

Malaria parasite carbonic anhydrase: inhibition of aromatic/heterocyclic sulfonamides and its therapeutic potential

Plasmodium falciparum (P. falciparum) is responsible for the majority of life-threatening cases of human malaria, causing 1.5-2.7 million annual deaths. The global emergence of drug-resistant malaria parasites necessitates identification and characterization of novel drug targets and their potential inhibitors. We identified the carbonic anhydrase (CA) genes in P. falciparum. The pfCA gene encodes anα-carbonic anhydrase, a Zn(2+)-metalloenzme, possessing catalytic properties distinct from that of the human host CA enzyme. The amino acid sequence of the pfCA enzyme is different from the analogous protozoan and human enzymes. A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds were found to be very good inhibitors for the malarial enzyme at moderate-low micromolar and submicromolar inhibitions. The structure of the groups substituting the aromatic-ureido- or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide were critical parameters for the inhibitory properties of the sulfonamides. One derivative, that is, 4- (3, 4-dichlorophenylureido)thioureido-benzenesulfonamide (compound 10) was the most effective in vitro Plasmodium falciparum CA inhibitor, and was also the most effective antimalarial compound on the in vitro P. falciparum growth inhibition. The compound 10 was also effective in vivo antimalarial agent in mice infected with Plasmodium berghei, an animal model of drug testing for human malaria infection. It is therefore concluded that the sulphonamide inhibitors targeting the parasite CA may have potential for the development of novel therapies against human malaria.

Spliceosomal genes in the D. discoideum genome: a comparison with those in H. sapiens, D. melanogaster, A. thaliana and S. cerevisiae

Little is known about pre-mRNA splicing in Dictyostelium discoideum although its genome has been completely sequenced. Our analysis suggests that pre-mRNA splicing plays an important role in D. discoideum gene expression as two thirds of its genes contain at least one intron. Ongoing curation of the genome to date has revealed 40 genes in D. discoideum with clear evidence of alternative splicing, supporting the existence of alternative splicing in this unicellular organism. We identified 160 candidate U2-type spliceosomal proteins and related factors in D. discoideum based on 264 known human genes involved in splicing. Spliceosomal small ribonucleoproteins (snRNPs), PRP19 complex proteins and late-acting proteins are highly conserved in D. discoideum and throughout the metazoa. In non-snRNP and hnRNP families, D. discoideum orthologs are closer to those in A. thaliana, D. melanogaster and H. sapiens than to their counterparts in S. cerevisiae. Several splicing regulators, including SR proteins and CUG-binding proteins, were found in D. discoideum, but not in yeast. Our comprehensive catalog of spliceosomal proteins provides useful information for future studies of splicing in D. discoideum where the efficient genetic and biochemical manipulation will also further our general understanding of pre-mRNA splicing.

Roles of histologic examination and polymerase chain reaction in diagnosis of toxoplasmic lymphadenitis / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the roles of histologic examination and polymerase chain reaction in diagnosis of toxoplasmic lymphadenitis (TL).</p><p><b>METHODS</b>Forty-six archival cases of histologically diagnosed TL, encountered during the period from April, 1999 to September, 2009 and with the paraffin-embedded lymph node tissue blocks available, were enrolled into the study. The presence of genome fragments of Toxoplasma gondii (T. gondii) was analyzed using semi-nested polymerase chain reaction (PCR). Thirty cases of one or two histopathologic triad of TL as the controls.</p><p><b>RESULTS</b>The positive rate of PCR in TL group was 76.1% (35/46), as compared to 10.0% (3/30) in the control group. The difference was of statistical significance. The sensitivity and specificity of the histologic triad in diagnosing TL was 92.1% (35/38) and 71.1% (27/38), respectively. The predictive value of positive and negative PCR results was 76.1% (35/46) and 90.0% (27/30). respectively.</p><p><b>CONCLUSIONS</b>The high specificity but low sensitivity of applying the histologic triad in diagnosing TL cases may be due to the occurrence of atypical histologic pattern. The sensitivity is improved with the use of semi-nested PCR in detecting T. gondii DNA.</p>

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

The current drug options for the treatment of chronic Chagas disease have not been sufficient and high hopes have been placed on the use of genomic data from the human parasite Trypanosoma cruzi to identify new drug targets and develop appropriate treatments for both acute and chronic Chagas disease. However, the lack of a complete assembly of the genomic sequence and the presence of many predicted proteins with unknown or unsure functions has hampered our complete view of the parasite's metabolic pathways. Moreover, pinpointing new drug targets has proven to be more complex than anticipated and has revealed large holes in our understanding of metabolic pathways and their integrated regulation, not only for this parasite, but for many other similar pathogens. Using an in silicocomparative study on pathway annotation and searching for analogous and specific enzymes, we have been able to predict a considerable number of additional enzymatic functions in T. cruzi. Here we focus on the energetic pathways, such as glycolysis, the pentose phosphate shunt, the Krebs cycle and lipid metabolism. We point out many enzymes that are analogous to those of the human host, which could be potential new therapeutic targets.

Trypanosoma cruzi: ancestral genomes and population structure

Although the genome of Trypanosoma cruzi has been completely sequenced, little is known about its population structure and evolution. Since 1999, two major evolutionary lineages presenting distinct epidemiological characteristics have been recognised: T. cruzi I and T. cruzi II. We describe new and important aspects of the population structure of the parasite, and unequivocally characterise a third ancestral lineage that we propose to name T. cruzi III. Through a careful analysis of haplotypes (blocks of genes that are stably transmitted from generation to generation of the parasite), we inferred at least two hybridisation events between the parental lineages T. cruzi II and T. cruzi III. The strain CL Brener, whose genome was sequenced, is one such hybrid. Based on these results, we propose a simple evolutionary model based on three ancestral genomes, T. cruzi I, T. cruzi II and T. cruzi III. At least two hybridisation events produced evolutionarily viable progeny, and T. cruzi III was the cytoplasmic donor for the resulting offspring (as identified by the mitochondrial clade of the hybrid strains) in both events. This model should be useful to inform evolutionary and pathogenetic hypotheses regarding T. cruzi.

Caracterização de proteínas MAPKs e MKPs no genoma de Schistosoma mansoni: uma abordagem in silico e experimental / Characterization of proteins and MAPKs MKPS in genome of Schistosoma mansoni: An experimental approach in silico

Pouco se sabe a respeito dos mecanismos de transdução de sinal no parasito Schistosoma mansoni. A identificação e caracterização dos mecanismos e das moléculas envolvidas em vias de transdução de sinal são essenciais para se entender a biologia do parasito e interações hospedeiro-parasito. As proteínas MAPK (Mitogen-activated protein kinases) desempenham um importante papel na transdução de sinais extracelulares, controlando vários mecanismos celulares essências. As MKPs são o principal grupo de proteína fosfatases que controlam a magnitude e a duração da ativação das proteínas MAPK. Até o presente momento, nenhuma proteína da fàmília das MAPKs ou proteínas MKPs havia sido detalhadamente caracterizada no parasito S. mansoni. Foi realizada uma análise in silico abragente da versão 3.1 do genoma de S. mansoni utilizando diferentes abordagens de bioinformática, incluindo buscas por similaridade de seqüências realizadas com o algoritmo blast ou com modelos ocultos de Markov (HMM) especificamente elaborados e construídos para identificação de MAPKs e MKPs. Complementarmente foi realizada a anotação manual dos genes que apresentaram hits positivos para proteínas MAPK e MKP utilizando o programa Artemis para integrar as anotações. Com base nesses resultados quatro proteínas foram selecionadas para posterior validação experimental: uma proteína ERK (SmMAPKl), uma proteína JNK (SmMAPK2) e duas proteínas fosfatase de dupla especificidade (SmMKPl e SmMKP2). Análises in silico revelaram ainda que os aminoácidos importantes para a funcionalidade das proteínas estão conservados. Nas MAPKs os resíduos dos domínios CD e ED, essenciais para o reconhecimento dos ativadores, substratos e desativadores, estão conservados. Nas fosfatases, o sítio de ligação as MAPKs está presente e apresenta os aminoácidos essenciais para a interação entre as proteínas. O cDNA de SmMAPK 1, SmMAPK2, SmMKPl e SmMKP2 está presente em todas as fases de desenvolvimento do parasito...

At present, little is known about signal transduction mechanisms in Schistosomes. The identification and characterization of signal transduction mechanisms and molecules are essential to elucidate Schistosoma mansoni host-parasite interactions and parasite biology.Mitogen-activated protein kinases (MAPKs) are important signal transducing enzymes that are involved in many aspects of cellular regulation. Specifically MAP kinase phosphatases (MKPs) that catalyze dephosphorylation of activated MAPK. Therefore, MKPs play an important role in determining the magnitude and duration of MAPK activities. In silicoanalysis using blast or HMMs builted from known MAPK and MKP characterized in related species resulted in the identification of 7 MAPK’s and 6 MKP’s with strong similarity with Schistosoma mansoni predicted proteome (ab initio gene prediction using Augustus). A highly curated manual annotation of S. mansoni genome supercontigs presenting positive hits for MAPK and MKP was produced using Artemis and in house developed Perl scripts. Based on the quality and confidence of the in silico predictions and annotation we select four targets: one ERK (SmMAPK1), one JNK (SmMAPK2), and 2 MKP (SmMKP1 and SmMKP2).Preliminary results of experimental validation will be presented. In addition, our in silico analysis reveled that all amino acids of the CD and ED domains that are essential for activators, regulators and substrate recognition are conserved in MAPKs and that the Kinaseinteractingmotif are conserved for the 2 MKP identified.

Histone as future drug target for malaria.

Malaria continues to be a major cause of mortality and morbidity in tropical countries and affecting around 100 countries of the world. As per WHO estimates, 300-500 million are being infected and 1-3 million deaths annually due to malaria. With the emerging knowledge about genome sequence of all the three counterparts involved in the disease of malaria, the parasite Plasmodium, vector Anopheles and host Homo sapien have helped the scientists to understand interactions between them. Simultaneous advancement in technology further improves the prospects to discover new targets for vaccines and drugs. Though the malaria vaccine is still far away in this situation there is need to develop a potent and affordable drug(s). Histones are the key protein of chromatin and play an important role in DNA packaging, replication and gene expression. They also show frequent post-translation modifications. The specific combinations of these posttranslational modifications are thought to alter chromatin structure by forming epigenetic bar codes that specify either transient or heritable patterns of genome function. Chromatin regulators and upstream pathways are therefore seen as promising targets for development of therapeutic drugs.

Cloning and molecular characterization of Trypanosoma cruzi U2, U4, U5, and U6 small nuclear RNAs

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.

Intraflagellar transport complex in Leishmania spp. In silico genome-wide screening and annotation of gene function

Flagella are constructed and maintained through the highly conserved process of intraflagellar transport (IFT), which is a rapid movement of particles along the axonemal microtubules of cilia/flagella. Particles that are transported by IFT are composed of several protein subunits comprising two complexes (A and B), which are conserved among green algae, nematodes, and vertebrates. To determine whether or not homologues to members of the IFT complex proteins are conserved in Leishmania spp, we scanned genomes, transcriptomes and proteomes of Leishmania species in a search for putative IFT factors, which were then identified in silico, compared, cataloged, and characterized. Since a large proportion of newly identified genes in L. major remain unclassified, with many of these being potentially Leishmania- (or kinetoplastid-) specific, there is a need for detailed analyses of homologs/orthologs that could help us understand the functional assignment of these gene products. We used a combination of integrated bioinformatics tools in a pathogenomics approach to contribute to the annotation of Leishmania genomes, particularly regarding flagellar genes and their roles in pathogenesis. This resulted in the formal in silico identification of eight of these homologs in Leishmania (IFT subunits, 20, 27, 46, 52, 57, 88, 140, and 172), along with others (IFTs 71, 74/72, and 81), as well as sequence comparisons and structural predictions. IFT, an important flagellar pathway in Leishmania, begins to be revealed through screening of trypanosomatid genomes; this information could also be used to better understand fundamental processes in Leishmania, such as motility and pathogenesis.

Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death mediators

Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family), about 250 metallopeptidases (most leishmanolysin homologues), 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2), probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins), which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

Rapid turnover of Plasmodium falciparum var gene transcripts and genotypes during natural non-symptomatic infections

Os genes var de Plasmodium falciparum codificam as proteínas variantes da superfície do eritrócito infectado (PfEMP1). Neste estudo examinamos a mudança de transcritos destes genes var em duas infecções assintomáticas durante um curto prazo e estimamos simultaneamente o número de genomas circulantes nas mesmas amostras por análise de microssatélites. Nas duas infecções observamos uma rápida mudança de genótipos e transcritos de genes var. A mudança acelerada do repertório de transcritos possivelmente foi causada pela rápida eliminação de parasitas circulantes transcrevendo genes var a partir de genomas iguais ou diferentes, ou pela mudança acelerada da própria transcrição (switching) de genes var.

Statistical properties of nucleotide clusters in DNA sequences / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Using the complete genome of Plasmodium falciparum 3D7 which has 14 chromosomes as an example, we have examined the distribution functions for the amount of C or G and A or T consecutively and non-overlapping blocks of m bases in this system. The function P(S) about the number of the consecutive C-G or A-T content cluster conforms to the relation P(S) proportional, variante(-alphas); values of the scaling exponent alpha(CG) are much larger than alpha(AT); and alpha(AT) of 14 chromosomes are hardly changed, whereas alpha(CG) of 14 chromosomes have a number of fluctuations. We found maximum value of A-T cluster size is much larger than C-G, which implies the existence of large A-T cluster. Our study of the width function xi(m) of cluster C-G content showed that follows good power law xi(m) proportional, variantm(-gamma). The average gamma for 14 chromosomes is 0.931. These investigations provide some insight into the nucleotide clusters of DNA sequences, and help us understand other properties of DNA sequences.