RESUMO
The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNA(Gln) and a glutaminyl-tRNA amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln). Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyl-tRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNA(Gln) to Gln-tRNA(Gln) in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria.
Assuntos
Apicoplastos/metabolismo , Fatores de Transcrição GATA/metabolismo , Transferases de Grupos Nitrogenados/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Aminoacil-RNA de Transferência/biossíntese , Antimaláricos/química , Núcleo Celular/metabolismo , Biologia Computacional , Eritrócitos/parasitologia , Deleção de Genes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Malária/metabolismo , Malária/parasitologia , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína , RNA de Transferência de Glutamina/genética , Proteínas Recombinantes/metabolismoRESUMO
Malaria remains one of the most prevalent and lethal human infectious diseases worldwide. A comprehensive characterization of antibody responses to blood stage malaria is essential to support the development of future vaccines, sero-diagnostic tests, and sero-surveillance methods. We constructed a proteome array containing 4441 recombinant proteins expressed by the blood stages of the two most common human malaria parasites, P. falciparum (Pf) and P. vivax (Pv), and used this array to screen sera of Papua New Guinea children infected with Pf, Pv, or both (Pf/Pv) that were either symptomatic (febrile), or asymptomatic but had parasitemia detectable via microscopy or PCR. We hypothesized that asymptomatic children would develop antigen-specific antibody profiles associated with antidisease immunity, as compared with symptomatic children. The sera from these children recognized hundreds of the arrayed recombinant Pf and Pv proteins. In general, responses in asymptomatic children were highest in those with high parasitemia, suggesting that antibody levels are associated with parasite burden. In contrast, symptomatic children carried fewer antibodies than asymptomatic children with infections detectable by microscopy, particularly in Pv and Pf/Pv groups, suggesting that antibody production may be impaired during symptomatic infections. We used machine-learning algorithms to investigate the relationship between antibody responses and symptoms, and we identified antibody responses to sets of Plasmodium proteins that could predict clinical status of the donors. Several of these antibody responses were identified by multiple comparisons, including those against members of the serine enriched repeat antigen family and merozoite protein 4. Interestingly, both P. falciparum serine enriched repeat antigen-5 and merozoite protein 4 have been previously investigated for use in vaccines. This machine learning approach, never previously applied to proteome arrays, can be used to generate a list of potential seroprotective and/or diagnostic antigens candidates that can be further evaluated in longitudinal studies.
Assuntos
Malária Falciparum/imunologia , Malária Vivax/imunologia , Análise Serial de Proteínas/métodos , Proteínas de Protozoários/análise , Inteligência Artificial , Criança , Pré-Escolar , Humanos , Lactente , Malária Falciparum/parasitologia , Malária Falciparum/patologia , Malária Vivax/parasitologia , Malária Vivax/patologia , Nova Guiné , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Plasmodium vivax/imunologia , Plasmodium vivax/metabolismo , Proteínas de Protozoários/imunologiaRESUMO
After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection.
Assuntos
Suscetibilidade a Doenças , Endocitose , Hepatócitos/parasitologia , Malária/imunologia , Malária/parasitologia , Plasmodium yoelii/fisiologia , Animais , Feminino , Perfilação da Expressão Gênica , Camundongos Endogâmicos BALB CRESUMO
The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA(Gln) biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA(Glu) and tRNA(Gln), determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA(Gln) biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.
Assuntos
Apicoplastos/enzimologia , Glutamato-tRNA Ligase/metabolismo , Plasmodium berghei/enzimologia , Plasmodium falciparum/enzimologia , Biossíntese de Proteínas/fisiologia , Proteínas de Protozoários/metabolismo , Aminoacilação de RNA de Transferência/fisiologia , Apicoplastos/genética , Glutamato-tRNA Ligase/genética , Humanos , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , RNA de Transferência de Glutamina/genética , RNA de Transferência de Glutamina/metabolismo , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência de Ácido Glutâmico/metabolismoRESUMO
The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.
Assuntos
Genoma de Protozoário/genética , Genômica , Malária Vivax/parasitologia , Plasmodium vivax/genética , Motivos de Aminoácidos , Animais , Artemisininas/metabolismo , Artemisininas/farmacologia , Atovaquona/metabolismo , Atovaquona/farmacologia , Núcleo Celular/genética , Cromossomos/genética , Sequência Conservada/genética , Eritrócitos/parasitologia , Evolução Molecular , Haplorrinos/parasitologia , Humanos , Isocoros/genética , Ligantes , Malária Vivax/metabolismo , Família Multigênica , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/patogenicidade , Plasmodium vivax/fisiologia , Análise de Sequência de DNA , Especificidade da Espécie , Sintenia/genéticaRESUMO
Vaccination with a single dose of genetically attenuated malaria parasites can induce sterile protection against sporozoite challenge in the rodent Plasmodium yoelii model. Protection is dependent on CD8(+) T cells, involves perforin and gamma interferon (IFN-γ), and is correlated with the expansion of effector memory CD8(+) T cells in the liver. Here, we have further characterized vaccine-induced changes in the CD8(+) T cell phenotype and demonstrated significant upregulation of CD11c on CD3(+) CD8b(+) T cells in the liver, spleen, and peripheral blood. CD11c(+) CD8(+) T cells are predominantly CD11a(hi) CD44(hi) CD62L(-), indicative of antigen-experienced effector cells. Following in vitro restimulation with malaria-infected hepatocytes, CD11c(+) CD8(+) T cells expressed inflammatory cytokines and cytotoxicity markers, including IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), perforin, and CD107a. CD11c(-) CD8(+) T cells, on the other hand, expressed negligible amounts of all inflammatory cytokines and cytotoxicity markers tested, indicating that CD11c marks multifunctional effector CD8(+) T cells. Coculture of CD11c(+), but not CD11c(-), CD8(+) T cells with sporozoite-infected primary hepatocytes significantly inhibited liver-stage parasite development. Tetramer staining for the immunodominant circumsporozoite protein (CSP)-specific CD8(+) T cell epitope demonstrated that approximately two-thirds of CSP-specific cells expressed CD11c at the peak of the CD11c(+) CD8(+) T cell response, but CD11c expression was lost as the CD8(+) T cells entered the memory phase. Further analyses showed that CD11c(+) CD8(+) T cells are primarily KLRG1(+) CD127(-) terminal effectors, whereas all KLRG1(-) CD127(+) memory precursor effector cells are CD11c(-) CD8(+) T cells. Together, these results suggest that CD11c marks a subset of highly inflammatory, short-lived, antigen-specific effector cells, which may play an important role in eliminating infected hepatocytes.
Assuntos
Antígeno CD11c/biossíntese , Linfócitos T CD8-Positivos/imunologia , Vacinas Antimaláricas/imunologia , Plasmodium yoelii/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Sangue/imunologia , Linfócitos T CD8-Positivos/química , Feminino , Imunofenotipagem , Fígado/imunologia , Vacinas Antimaláricas/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Subpopulações de Linfócitos T/química , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologiaRESUMO
While most Ascomycetes tend to associate principally with plants, the dimorphic fungi Coccidioides immitis and Coccidioides posadasii are primary pathogens of immunocompetent mammals, including humans. Infection results from environmental exposure to Coccidiodies, which is believed to grow as a soil saprophyte in arid deserts. To investigate hypotheses about the life history and evolution of Coccidioides, the genomes of several Onygenales, including C. immitis and C. posadasii; a close, nonpathogenic relative, Uncinocarpus reesii; and a more diverged pathogenic fungus, Histoplasma capsulatum, were sequenced and compared with those of 13 more distantly related Ascomycetes. This analysis identified increases and decreases in gene family size associated with a host/substrate shift from plants to animals in the Onygenales. In addition, comparison among Onygenales genomes revealed evolutionary changes in Coccidioides that may underlie its infectious phenotype, the identification of which may facilitate improved treatment and prevention of coccidioidomycosis. Overall, the results suggest that Coccidioides species are not soil saprophytes, but that they have evolved to remain associated with their dead animal hosts in soil, and that Coccidioides metabolism genes, membrane-related proteins, and putatively antigenic compounds have evolved in response to interaction with an animal host.
Assuntos
Coccidioides/genética , Genoma Fúngico , Fungos Mitospóricos/genética , Animais , Especiação Genética , Genômica/métodos , Histoplasma/genética , Humanos , Dados de Sequência Molecular , Onygenales/genética , Filogenia , Seleção Genética , Análise de Sequência de DNA , SinteniaRESUMO
Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary. Plasmodium falciparum has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an in vivo luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5' and 3' splicing requirements and experimentally determine the intron branch point in P. falciparum. This analysis indicates that P. falciparum mRNAs have canonical 5' and 3' splice sites. However, the 5' consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3' splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different P. falciparum pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that P. falciparum retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack.
Assuntos
Íntrons/genética , Plasmodium falciparum/genética , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Sequência de Bases , Plasmodium falciparum/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Análise de Sequência de DNARESUMO
TriTrypDB (http://tritrypdb.org) is an integrated database providing access to genome-scale datasets for kinetoplastid parasites, and supporting a variety of complex queries driven by research and development needs. TriTrypDB is a collaborative project, utilizing the GUS/WDK computational infrastructure developed by the Eukaryotic Pathogen Bioinformatics Resource Center (EuPathDB.org) to integrate genome annotation and analyses from GeneDB and elsewhere with a wide variety of functional genomics datasets made available by members of the global research community, often pre-publication. Currently, TriTrypDB integrates datasets from Leishmania braziliensis, L. infantum, L. major, L. tarentolae, Trypanosoma brucei and T. cruzi. Users may examine individual genes or chromosomal spans in their genomic context, including syntenic alignments with other kinetoplastid organisms. Data within TriTrypDB can be interrogated utilizing a sophisticated search strategy system that enables a user to construct complex queries combining multiple data types. All search strategies are stored, allowing future access and integrated searches. 'User Comments' may be added to any gene page, enhancing available annotation; such comments become immediately searchable via the text search, and are forwarded to curators for incorporation into the reference annotation when appropriate.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Leishmania/genética , Trypanosoma/genética , Animais , Biologia Computacional/tendências , Bases de Dados de Proteínas , Genoma de Protozoário , Armazenamento e Recuperação da Informação/métodos , Internet , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Software , Interface Usuário-ComputadorRESUMO
BACKGROUND: Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (e.g. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date. RESULTS: To gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated>31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two P. marinus strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-valueAssuntos
Alveolados/genética
, Etiquetas de Sequências Expressas
, Alveolados/classificação
, Animais
, Ostreidae/parasitologia
, Filogenia
RESUMO
The RTS,S/AS01 vaccine provides partial protection against Plasmodium falciparum infection but determinants of protection and/or disease are unclear. Previously, anti-circumsporozoite protein (CSP) antibody titers and blood RNA signatures were associated with RTS,S/AS01 efficacy against controlled human malaria infection (CHMI). By analyzing host blood transcriptomes from five RTS,S vaccination CHMI studies, we demonstrate that the transcript ratio MX2/GPR183, measured 1 day after third immunization, discriminates protected from non-protected individuals. This ratiometric signature provides information that is complementary to anti-CSP titer levels for identifying RTS,S/AS01 immunized people who developed protective immunity and suggests a role for interferon and oxysterol signaling in the RTS,S mode of action.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/genética , Malária Falciparum/prevenção & controle , Proteínas de Resistência a Myxovirus/genética , Plasmodium falciparum/imunologia , Receptores Acoplados a Proteínas G/genética , Transcriptoma , Vacinação , Vacinas Sintéticas/imunologia , Anticorpos Antiprotozoários/imunologia , Estudos de Coortes , Humanos , Imunogenicidade da Vacina/genética , Controle de Infecções/métodos , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Proteínas de Protozoários/imunologia , RNA-Seq , Análise de Célula ÚnicaRESUMO
MOTIVATION: The sequencing of the Plasmodium yoelii genome, a model rodent malaria parasite, has greatly facilitated research for the development of new drug and vaccine candidates against malaria. Unfortunately, only preliminary gene models were annotated on the partially sequenced genome, mostly by in silico gene prediction, and there has been no major improvement of the annotation since 2002. RESULTS: Here we report on a systematic assessment of the accuracy of the genome annotation based on a detailed analysis of a comprehensive set of cDNA sequences and proteomics data. We found that the coverage of the current annotation tends to be biased toward genes expressed in the blood stages of the parasite life cycle. Based on our proteomic analysis, we estimate that about 15% of the liver stage proteome data we have generated is absent from the current annotation. Through comparative analysis we identified and manually curated a further 510 P. yoelii genes which have clear orthologs in the P. falciparum genome, but were not present or incorrectly annotated in the current annotation. This study suggests that improvements of the current P. yoelii genome annotation should focus on genes expressed in stages other than blood stages. Comparative analysis will be critically helpful for this re-annotation. The addition of newly annotated genes will facilitate the use of P. yoelii as a model system for studying human malaria. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Mapeamento Cromossômico/métodos , Genoma de Protozoário/genética , Plasmodium yoelii/genética , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Animais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Massively parallel signature sequencing (MPSS) was used to analyze the transcriptome of the intracellular protozoan Theileria parva. In total 1,095,000, 20 bp sequences representing 4371 different signatures were generated from T.parva schizonts. Reproducible signatures were identified within 73% of potentially detectable predicted genes and 83% had signatures in at least one MPSS cycle. A predicted leader peptide was detected on 405 expressed genes. The quantitative range of signatures was 4-52,256 transcripts per million (t.p.m.). Rare transcripts (<50 t.p.m.) were detected from 36% of genes. Sequence signatures approximated a lognormal distribution, as in microarray. Transcripts were widely distributed throughout the genome, although only 47% of 138 telomere-associated open reading frames exhibited signatures. Antisense signatures comprised 13.8% of the total, comparable with Plasmodium. Eighty five predicted genes with antisense signatures lacked a sense signature. Antisense transcripts were independently amplified from schizont cDNA and verified by sequencing. The MPSS transcripts per million for seven genes encoding schizont antigens recognized by bovine CD8 T cells varied 1000-fold. There was concordance between transcription and protein expression for heat shock proteins that were very highly expressed according to MPSS and proteomics. The data suggests a low level of baseline transcription from the majority of protein-coding genes.
Assuntos
Genoma de Protozoário , Genômica/métodos , RNA de Protozoário/biossíntese , Theileria parva/genética , Animais , Fases de Leitura Aberta , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , RNA Antissenso/biossíntese , RNA Antissenso/química , RNA de Protozoário/análise , RNA de Protozoário/química , Análise de Sequência de RNA , Telômero/química , Theileria parva/crescimento & desenvolvimento , Theileria parva/metabolismo , Ativação TranscricionalRESUMO
Transcriptome analysis can provide useful data for refining genome sequence annotation. Application of massively parallel signature sequencing (MPSS) revealed reproducible transcription, in multiple MPSS cycles, from 73% of computationally predicted genes in the Theileria parva schizont lifecycle stage. Signatures spanning consecutive exons confirmed 142 predicted introns. MPSS identified 83 putative genes, >100 codons overlooked by annotation software, and 139 potentially incorrect gene models (with either truncated ORFs or overlooked exons) by interfacing signature locations with stop codon maps. Twenty representative models were confirmed as likely to be incorrect using reverse transcription PCR amplification from independent schizont cDNA preparations. More than 50% of the 60 putative single copy genes in T. parva that were absent from the genome of the closely related T. annulata had MPSS signatures. This study illustrates the utility of MPSS for improving annotation of small, gene-rich microbial eukaryotic genomes.
Assuntos
Genoma de Protozoário/genética , Fases de Leitura Aberta/genética , Theileria parva/genética , Transcrição Gênica/genética , Animais , Análise de Sequência de DNA/métodosRESUMO
High throughput immunomics is a powerful platform to discover potential targets of host immunity and develop diagnostic tests for infectious diseases. We screened the sera of Plasmodium vivax-exposed individuals to profile the antibody response to blood-stage antigens of P. vivax using a P. vivax protein microarray. A total of 1936 genes encoding the P. vivax proteins were expressed, printed and screened with sera from P. vivax-exposed individuals and normal subjects. Total of 151 (7.8% of the 1936 targets) highly immunoreactive antigens were identified, including five well-characterized antigens of P. vivax (ETRAMP11.2, Pv34, SUB1, RAP2 and MSP4). Among the highly immunoreactive antigens, 5 antigens were predicted as adhesins by MAAP, and 11 antigens were predicted as merozoite invasion-related proteins based on homology with P. falciparum proteins. There are 40 proteins that have serodiagnostic potential for antibody surveillance. These novel Plasmodium antigens identified provide the clues for understanding host immune response to P. vivax infection and the development of antibody surveillance tools.
Assuntos
Antígenos HLA-D/sangue , Malária Vivax/sangue , Plasmodium vivax/imunologia , Proteínas de Protozoários/sangue , Antígenos HLA-D/imunologia , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Malária Vivax/imunologia , Malária Vivax/parasitologia , Plasmodium vivax/patogenicidade , Análise Serial de Proteínas , Proteínas de Protozoários/imunologiaRESUMO
Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX_003775, PVX_082650, PVX_097625, and PVX_114145); sexual-stage antigen s16 (PVX_000930); early transcribed membrane protein (PVX_090230); tryptophan-rich antigen (Pv-fam-a) (PVX_092995); apical merozoite antigen 1 (PVX_092275); and proteins of unknown function (PVX_081830, PVX_117680, PVX_118705, PVX_121935, PVX_097730, PVX_110935, PVX_115450, and PVX_082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions.
Assuntos
Anticorpos Antiprotozoários/imunologia , Malária Vivax/imunologia , Plasmodium vivax/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Formação de Anticorpos , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Feminino , Expressão Gênica/imunologia , Humanos , Masculino , Plasmodium vivax/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Análise Serial de Proteínas , Recidiva , Adulto JovemRESUMO
The malaria genome has proved invaluable to researchers worldwide in the continuing fight against malaria by stimulating and underpinning molecular approaches in gene expression studies, vaccine and drug discovery research, and by providing data to facilitate hypothesis-driven research. The combination of in silico and experimental investigations has already yielded dividends by strengthening our understanding of the many facets of the malaria parasite Plasmodium falciparum. The recently initiated curation of the genome resource is a vital investment for maintaining and enhancing the use of this genomic information in the post-genomic era.
Assuntos
Genoma de Protozoário , Genômica/métodos , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Animais , DNA de Protozoário/química , DNA de Protozoário/genética , Bases de Dados Genéticas , Humanos , Análise de Sequência de DNARESUMO
The internal defense mechanism of the snail Biomphalaria glabrata during a schistosome infection is activated and mediated via the immune effector cells known as hemocytes. Since resistance and susceptibility to schistosome infection is known to be genetically determined, our interest was to use the EST approach as a gene discovery tool to examine transcription profiles in hemocytes of resistant snails pre- and post-exposure to Schistosoma mansoni. Comparative analysis of the transcripts suggested that parasite exposure caused an active metabolic response in the hemocytes. The most abundant transcripts were those showing 23-74% similarity to known reverse transcriptases (RT). Further characterization by RT-PCR indicated the RT transcripts were expressed in normal snails, parasite exposed snails, and the embryonic cell line Bge. To determine whether the occurrence of RT transcripts correlates to the presence of functional enzyme activity in the snails, RT assays were performed from both resistant and susceptible snails, pre- and post-exposure to miracidia, using protein extracts from the head-foot and posterior region tissues. Results indicated that in the resistant snail, RT activity was greater in the posterior region than in the head-foot. After exposure, however, RT activity increased dramatically in the head-foot, with peak activity at 24 h post-exposure. The detection of RT activity in B. glabrata was unexpected and the role of this enzyme in the hemocyte-mediated killing of parasites is not yet known. However, identification of this and other transcripts from these cells by the EST approach provides a useful resource towards elucidating the molecular basis of resistance/susceptibility in this snail-host parasite relationship.
Assuntos
Biomphalaria/genética , Hemócitos/parasitologia , Schistosoma mansoni/patogenicidade , Sequência de Aminoácidos , Animais , Biomphalaria/enzimologia , Biomphalaria/parasitologia , Southern Blotting , Linhagem Celular , Ciona intestinalis/enzimologia , Ciona intestinalis/genética , Etiquetas de Sequências Expressas , Hemócitos/fisiologia , Dados de Sequência Molecular , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The genome of the malaria parasite, Plasmodium falciparum, appears to contain the proteins necessary for a Type II dissociated fatty acid biosynthetic system. Here we report the functional characterization of two proteins from this system. Purified recombinant acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase III (KASIII) from P. falciparum are soluble and active in a truncated form. Malarial ACP is activated by the addition of a 4'-phosphopantetheine prosthetic group derived from coenzyme A, generating holo-PfACP. Holo-PfACP is an effective substrate for the transacylase activity of PfKASIII, but substitution of a key active site cysteine in PfKASIII to alanine or serine abolishes enzymatic activity. During the schizont stage of parasite development, there is a significant up-regulation of the mRNAs corresponding to these proteins, indicating an important metabolic requirement for fatty acids during this stage.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteína de Transporte de Acila/metabolismo , Panteteína/análogos & derivados , Plasmodium falciparum/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Clonagem Molecular , Ácido Graxo Sintases/genética , Ácidos Graxos/biossíntese , Holoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Panteteína/metabolismo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Random clones from a cDNA library made from mRNA purified from dissected salivary glands of feeding female Amblyomma variegatum ticks were subjected to single pass sequence analysis. A total of 3992 sequences with an average read length of 580 nucleotides have been used to construct a gene index called AvGI that consists of 2109 non-redundant sequences. A provisional gene identity has been assigned to 39% of the database entries by sequence similarity searches against a non-redundant amino acid database and a protein database that has been assigned gene ontology terms. Homologs of genes encoding basic cellular functions including previously characterised enzyme activities, such as stearoyl CoA saturase and protein phosphatase, of ixodid tick salivary glands were found. Several families of abundant cDNA sequences that may code for protein components of tick cement and A. variegatum proteins which may contribute to anti-haemostatic and anti-inflammatory responses, and, one with potential immunosuppressive activity, were also identified. Interference with the function of such proteins might disrupt the life cycle of A. variegatum and help to control this ectoparasite or to reduce its ability to transmit disease causing organisms. AvGI represents an electronic knowledge base, which can be used to launch investigations of the biology of the salivary glands of this tick species. The database may be accessed via the World Wide Web at http://www.tigr.org/tdb/tgi.shtml.