A single-nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor alters the development of host immunity.

The acquisition of malaria immunity is both remarkably slow and unpredictable. At present, we know little about the malaria parasite genes that influence the host's ability to mount a protective immune response. Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasite Plasmodium berghei (Pb) NK65 allowed infected mice to mount a T helper cell 1 (TH1)-type immune response that controlled subsequent infections. As compared to PbNK65S, PbNK65F parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. PbNK65F infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell-specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies. Thus, the Pb ApiAP2 transcription factor functions as a critical parasite virulence factor in malaria infections.

Exploring Plasmodium falciparum Var Gene Expression to Assess Host Selection Pressure on Parasites During Infancy.

In sub-Saharan Africa, children below 5 years bear the greatest burden of severe malaria because they lack naturally acquired immunity that develops following repeated exposure to infections by Plasmodium falciparum. Antibodies to the surface of P. falciparum infected erythrocytes (IE) play an important role in this immunity. In children under the age of 6 months, relative protection from severe malaria is observed and this is thought to be partly due to trans-placental acquired protective maternal antibodies. However, the protective effect of maternal antibodies has not been fully established, especially the role of antibodies to variant surface antigens (VSA) expressed on IE. Here, we assessed the immune pressure on parasites infecting infants using markers associated with the acquisition of naturally acquired immunity to surface antigens. We hypothesized that, if maternal antibodies to VSA imposed a selection pressure on parasites, then the expression of a relatively conserved subset of var genes called group A var genes in infants should change with waning maternal antibodies. To test this, we compared their expression in parasites from children between 0 and 12 months and above 12 months of age. The transcript quantity and the proportional expression of group A var subgroup, including those containing domain cassette 13, were positively associated with age during the first year of life, which contrasts with above 12 months. This was accompanied by a decline in infected erythrocyte surface antibodies and an increase in parasitemia during this period. The observed increase in group A var gene expression with age in the first year of life, when the maternal antibodies are waning and before acquisition of naturally acquired antibodies with repeated exposure, is consistent with the idea that maternally acquired antibodies impose a selection pressure on parasites that infect infants and may play a role in protecting these infants against severe malaria.

An improved Plasmodium cynomolgi genome assembly reveals an unexpected methyltransferase gene expansion.

BACKGROUND: Plasmodium cynomolgi, a non-human primate malaria parasite species, has been an important model parasite since its discovery in 1907. Similarities in the biology of P. cynomolgi to the closely related, but less tractable, human malaria parasite P. vivax make it the model parasite of choice for liver biology and vaccine studies pertinent to P. vivax malaria. Molecular and genome-scale studies of P. cynomolgi have relied on the current reference genome sequence, which remains highly fragmented with 1,649 unassigned scaffolds and little representation of the subtelomeres.  Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated a new reference genome sequence, PcyM, sourced from an Indian rhesus monkey. We compare the newly assembled genome sequence with those of several other Plasmodium species, including a re-annotated P. coatneyi assembly. RESULTS: The new PcyM genome assembly is of significantly higher quality than the existing reference, comprising only 56 pieces, no gaps and an improved average gene length. Detailed manual curation has ensured a comprehensive annotation of the genome with 6,632 genes, nearly 1,000 more than previously attributed to P. cynomolgi. The new assembly also has an improved representation of the subtelomeric regions, which account for nearly 40% of the sequence. Within the subtelomeres, we identified more than 1300 Plasmodium interspersed repeat ( pir) genes, as well as a striking expansion of 36 methyltransferase pseudogenes that originated from a single copy on chromosome 9. CONCLUSIONS: The manually curated PcyM reference genome sequence is an important new resource for the malaria research community. The high quality and contiguity of the data have enabled the discovery of a novel expansion of methyltransferase in the subtelomeres, and illustrates the new comparative genomics capabilities that are being unlocked by complete reference genomes.

Characterization and gene expression analysis of the cir multi-gene family of Plasmodium chabaudi chabaudi (AS).

BACKGROUND: The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due to antigenic variation. In this model, pir genes are called cirs and may be involved in this mechanism, allowing evasion of host immune responses. In order to fully understand the role(s) of CIR proteins during P. chabaudi infection, a detailed characterization of the cir gene family was required. RESULTS: The cir repertoire was annotated and a detailed bioinformatic characterization of the encoded CIR proteins was performed. Two major sub-families were identified, which have been named A and B. Members of each sub-family displayed different amino acid motifs, and were thus predicted to have undergone functional divergence. In addition, the expression of the entire cir repertoire was analyzed via RNA sequencing and microarray. Up to 40% of the cir gene repertoire was expressed in the parasite population during infection, and dominant cir transcripts could be identified. In addition, some differences were observed in the pattern of expression between the cir subgroups at the peak of P. chabaudi infection. Finally, specific cir genes were expressed at different time points during asexual blood stages. CONCLUSIONS: In conclusion, the large number of cir genes and their expression throughout the intraerythrocytic cycle of development indicates that CIR proteins are likely to be important for parasite survival. In particular, the detection of dominant cir transcripts at the peak of P. chabaudi infection supports the idea that CIR proteins are expressed, and could perform important functions in the biology of this parasite. Further application of the methodologies described here may allow the elucidation of CIR sub-family A and B protein functions, including their contribution to antigenic variation and immune evasion.

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Leishmania (Viannia) braziliensis: influence of successive in vitro cultivation on the expression of promastigote proteinases.

Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes.

Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally.

BACKGROUND: Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS: Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS: Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS: We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

ProteinWorldDB: querying radical pairwise alignments among protein sets from complete genomes.

MOTIVATION: Many analyses in modern biological research are based on comparisons between biological sequences, resulting in functional, evolutionary and structural inferences. When large numbers of sequences are compared, heuristics are often used resulting in a certain lack of accuracy. In order to improve and validate results of such comparisons, we have performed radical all-against-all comparisons of 4 million protein sequences belonging to the RefSeq database, using an implementation of the Smith-Waterman algorithm. This extremely intensive computational approach was made possible with the help of World Community Grid, through the Genome Comparison Project. The resulting database, ProteinWorldDB, which contains coordinates of pairwise protein alignments and their respective scores, is now made available. Users can download, compare and analyze the results, filtered by genomes, protein functions or clusters. ProteinWorldDB is integrated with annotations derived from Swiss-Prot, Pfam, KEGG, NCBI Taxonomy database and gene ontology. The database is a unique and valuable asset, representing a major effort to create a reliable and consistent dataset of cross-comparisons of the whole protein content encoded in hundreds of completely sequenced genomes using a rigorous dynamic programming approach. AVAILABILITY: The database can be accessed through http://proteinworlddb.org

Abordagens computacionais aplicadas aos genomas de micobactérias e Trypanossomatídeos: da montagem à anotação / Computational methods applied to the genomes of the mycobacteria and trypanosomatids: of the assembly to the notation

Esta dissertação trata do desenvolvimento de novas abordagens e ferramentas computacionais focadas nas seguintes aplicações: aperfeiçoamento do fluxo de informação de Plataformas Tecnológicas de Seqüenciamento e de Bioinformática; montagem de seqüências em projetos genomas; detecção de seqüências repetitivas; análise comparativa e anotação de genomas. A maioria das ferramentas foram desenvolvidas e/ou aplicadas no projeto genoma do Mycobacterium bovis cepa BCG Moreau-RJ, que foi seqüenciado, montado e anotado em nosso laboratório. Uma parte dos resultados deste projeto genoma foi utilizada no desenvolvimento de ferramentas de identificação e controle de qualidade que possam ser utilizadas na manutenção e produção da vacina BCG Moreau-RJ, e foram objetos de um depósito de patente, no âmbito nacional e internacional. Além disto, vários sistemas independentes, porém complementares, foram desenvolvidos para a análise de genomas em geral, os quais permitem uma série de aplicações na interface in silico- in vitro. (1) ReRep (Read Repeat Finder) é um sistema que detecta seqüências repetitivas em um conjunto de dados oriundos de seqüenciamento aleatório (GSS), visando a caracterização de repetições e permitindo um auxílio na montagem de seqüências genômicas, tendo sido aplicado em um conjunto de GSS com cobertura de 1,4por cento do genoma de Leishmania braziliensis. (2) AnEnPi (Analogous Enzyme Pipeline), composto por um banco de dados de enzimas para a anotação de vias metabólicas e detecção de possíveis alvos terapêuticos, com várias possibilidades de comparação e geração de mapas metabólicas, aplicado em L. major e Trypanosoma cruzi. (3) Conjunto de ferramentas para assistir a montagem e a anotação do genoma BCG Moreau-RJ: páginas web do projeto, incluindo monitoramento de estatísticas de montagem e visualização do mapeamento das leituras contra o genoma do Mycobacterium bovis; fluxo...ao Programa de Desenvolvimento Tecnológico em Insumos para Saúde (PDTIS).