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1.
Mol Microbiol ; 84(5): 816-31, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22537295

RESUMO

Plasmodium vivax and P. cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81(95) and PcyPHIST/CVC-81(95) , analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81(95) is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-81(95) localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-81(95) gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81(95) is essential for survival of these malaria parasites.


Assuntos
Cavéolas/química , Eritrócitos/parasitologia , Plasmodium cynomolgi/química , Plasmodium vivax/química , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , Cromatografia Líquida , DNA de Protozoário/química , DNA de Protozoário/genética , Tomografia com Microscopia Eletrônica , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Genes Essenciais , Humanos , Imageamento Tridimensional , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Peso Molecular , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Espectrometria de Massas em Tandem
2.
BMC Microbiol ; 11: 75, 2011 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-21492426

RESUMO

BACKGROUND: Different models for biofilm in Streptococcus pneumoniae have been described in literature. To permit comparison of experimental data, we characterised the impact of the pneumococcal quorum-sensing competence system on biofilm formation in three models. For this scope, we used two microtiter and one continuous culture biofilm system. RESULTS: In both microtiter models the competence system influences stability and structure of biofilm in the late attachment phase and synthetic competence stimulating peptide (CSP) restored wild type phenotypes in the comC mutants unable to produce the peptide. Early attachment of single cells to well bottoms was found for both systems to be competence independent, while later phases, including microcolony formation correlated to an intact competence system. The continuous culture biofilm model was not affected by mutations in the competence locus, but deletion of capsule had a significant impact in this model. CONCLUSIONS: Since biofilm remains a largely uncharacterised multi-parameter phenotype it appears to be advisable to exploit more than one model in order to draw conclusion of possible relevance of specific genotypes on pneumococcal physiology.


Assuntos
Biofilmes/crescimento & desenvolvimento , Percepção de Quorum , Streptococcus pneumoniae/fisiologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Técnicas de Inativação de Genes , Variação Genética , Genótipo , Modelos Teóricos , Fenótipo , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/metabolismo
3.
Malar J ; 8: 181, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19646264

RESUMO

BACKGROUND: The SICAvar gene family, expressed at the surface of infected erythrocytes, is critical for antigenic variation in Plasmodium knowlesi. When this family was discovered, a prototypic SICAvar gene was characterized and defined by a 10-exon structure. The predicted 205-kDa protein lacked a convincing signal peptide, but included a series of variable cysteine-rich modules, a transmembrane domain encoded by the penultimate exon, and a cytoplasmic domain encoded by the final highly conserved exon. The 205 SICAvar gene and its family with up to 108 possible family members, was identified prior to the sequencing of the P. knowlesi genome. However, in the published P. knowlesi database this gene remains disjointed in five fragments. This study addresses a number of structural and functional questions that are critical for understanding SICAvar gene expression. METHODS: Database mining, bioinformatics, and traditional genomic and post-genomic experimental methods including proteomic technologies are used here to confirm the genomic context and expressed structure of the prototype 205 SICAvar gene. RESULTS: This study reveals that the 205 SICAvar gene reported previously to have a 10-exon expressed gene structure has, in fact, 12 exons, with an unusually large and repeat-laden intron separating two newly defined upstream exons and the bona fide 5'UTR from the remainder of the gene sequence. The initial exon encodes a PEXEL motif, which may function to localize the SICA protein in the infected erythrocyte membrane. This newly defined start of the 205 SICAvar sequence is positioned on chromosome 5, over 340 kb upstream from the rest of the telomerically positioned SICAvar gene sequence in the published genome assembly. This study, however, verifies the continuity of these sequences, a 9.5 kb transcript, and provides evidence that the 205 SICAvar gene is located centrally on chromosome 5. CONCLUSION: The prototype 205 SICAvar gene has been redefined to have a 12-exon structure. These data are important because they 1) address questions raised in the P. knowlesi genome database regarding SICAvar gene fragments, numbers and structures, 2) show that this prototype gene encodes a PEXEL motif, 3) emphasize the need for further refinement of the P. knowlesi genome data, and 4) retrospectively, provide evidence for recombination within centrally located SICAvar sequences.


Assuntos
Variação Antigênica , Antígenos de Protozoários/genética , Genes de Protozoários , Plasmodium knowlesi/genética , Regiões 5' não Traduzidas , Animais , Éxons , Íntrons
4.
Infect Genet Evol ; 6(1): 75-9, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16376842

RESUMO

Malaria variant antigens are encoded by large multigene families and expressed on the surface of infected erythrocytes. The Plasmodium knowlesi Schizont-infected cell agglutination (SICA) antigens are encoded by the SICAvar multigene family, and the P. falciparum erythrocyte membrane protein-1 (PfEMP1) antigens are encoded by the var gene family. Although these variant antigens share many fundamental features, P. knowlesi and P. falciparum are phylogenetically distantly related, and so far a significant level of sequence identity has not been observed in alignments of either the SICAvar and var gene families or their encoded proteins. In support of their orthologous relationship, however, here we demonstrate through proteomic analysis that the P. knowlesi SICA variant antigens share significant common sequences with P. falciparum EMP1 molecules. As many as forty P. knowlesi SICA peptides show identity with a particular P. falciparum EMP1, mapping throughout all characterized domains, including the externally exposed cysteine-rich domains that are characteristic of both proteins. These findings provide further validation of the classical in vivo P. knowlesi-rhesus monkey model system for advancing our understanding of the immunobiology of antigenic variation and variant antigen gene expression in Plasmodium.


Assuntos
Variação Antigênica , Antígenos de Protozoários/análise , Antígenos de Superfície/análise , Eritropoetina/imunologia , Peptídeos Cíclicos/imunologia , Plasmodium falciparum/metabolismo , Plasmodium knowlesi/metabolismo , Proteômica , Animais , Antígenos de Protozoários/metabolismo , Plasmodium falciparum/imunologia , Plasmodium knowlesi/imunologia , Análise Serial de Proteínas
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