Summary
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‐8195 and PcyPHIST/CVC‐8195, analysed their structural features, including a PEXEL motif, repeated sequences and a C‐terminal PHIST domain, and show that PHIST/CVC‐8195 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‐8195 localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc‐8195 gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC‐8195 is essential for survival of these malaria parasites.
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.
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.
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.
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.
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.
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.
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.
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.
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 95kDa 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 81kDa. We named the orthologous proteins PvPHIST/CVC-8195 and PcyPHIST/CVC-8195, analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-8195 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-8195 localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-8195 gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-8195 is essential for survival of these malaria parasites.
Plasmodium vivax
and
P. cynomolgi
produce numerous caveolae-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
P. falciparum
infected erythrocytes. Here we investigate the 3-dimensional 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 sub-telomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81
95
and PcyPHIST/CVC-81
95
, analyzed 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, and used immuno-Electron Tomography (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.
Abstract 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.
Plasmodium vivax is the causative infectious agent of 80–300 million annual cases of malaria. Many aspects of this parasite's biology remain unknown. To further elucidate the interaction of P. vivax ...with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax's known reticulocyte host–cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host–parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function.
Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host–cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte membranes. Studies of stage-specific P. vivax expressed proteomes have been limited in scope and focused mainly on pathogen proteins, thus limiting understanding of the biology of this pathogen and its host interactions. Here three P. vivax proteomes are reported from biological replicates based on purified trophozoite-infected reticulocytes from different Saimiri boliviensis infections (the main non-human primate experimental model for P. vivax biology and pathogenesis). An in-depth analysis of two of the proteomes using 2D LC/MS/MS and multiple search engines identified 1375 pathogen proteins and 3209 host proteins. Numerous functional categories of both host and pathogen proteins were identified, including several known P. vivax protein family members (e.g., PHIST, eTRAMP and VIR), and 33% of protein identifications were classified as hypothetical. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with this parasite species' known reticulocyte host–cell specificity. In two biological replicates analyzed for post-translational modifications, hemoglobin was extensively oxidized, and various other proteins were also oxidized or nitrated in one of the two replicates. The cause of such protein modification remains to be determined but could include oxidized heme and oxygen radicals released from the infected red blood cell's parasite-induced acidic digestive vacuoles. In any case, the data suggests the presence of distinct infection-specific conditions whereby both the pathogen and host infected red blood cell proteins may be subject to significant oxidative stress.
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•We use 2D LC/MS/MS to obtain Plasmodium vivax trophozoite-enriched proteomes.•In two biological replicates, 5 search engines identify 1375 P. vivax proteins.•3209 host S. boliviensis proteins are also identified.•In both replicates extensive hemoglobin oxidation was observed.•Oxidation/nitration of other proteins in 1 replicate could alter protein function.
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