Characterisation of Plasmodium invasive organelles; an ookinete microneme proteome.

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.

Proteomic comparison of four Eimeria tenella life-cycle stages: unsporulated oocyst, sporulated oocyst, sporozoite and second-generation merozoite.

We report the proteomes of four life-cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second-generation merozoites, respectively. A multidimensional protein identification technology shotgun approach identified 812 sporozoites, 1528 merozoites and all of the oocyst proteins, whereas 2-D gel proteomics identified 230 sporozoites and 98 merozoite proteins. Comparing the invasive stages, we find moving junction components RON2 in both, whereas AMA-1 and RON4 are found only in merozoites and AMA-2 and RON5 are only found in sporozoites, suggesting stage-specific moving junction proteins. During early oocyst to sporozoite development, refractile body and most "glideosome" proteins are found throughout, whereas microneme and most rhoptry proteins are only found after sporulation. Quantitative analysis indicates glycolysis and gluconeogenesis are the most abundant metabolic groups detected in all stages. The mannitol cycle "off shoot" of glycolysis was not detected in merozoites but was well represented in the other stages. However, in merozoites we find more protein associated with oxidative phosphorylation, suggesting a metabolic shift mobilising greater energy production. We find a greater abundance of protein linked to transcription, protein synthesis and cell cycle in merozoites than in sporozoites, which may be residual protein from the preceding massive replication during schizogony.

Determining the protein repertoire of Cryptosporidium parvum sporozoites.

The genome of the intracellular parasite Cryptosporidium parvum has recently been sequenced, but protein expression data for the invasive stages of this important zoonotic gastrointestinal pathogen are limited. In this paper a comprehensive analysis of the expressed protein repertoire of an excysted oocyst/sporozoite preparation of C. parvum is presented. Three independent proteome platforms were employed which yielded more than 4800 individual protein identifications representing 1237 nonredundant proteins, corresponding to approximately 30% of the predicted proteome. Peptide data were mapped to the corresponding locations on the C. parvum genome and a publicly accessible interface for proteome data was developed for data-mining and visualisation at CryptoDB (http://cryptodb.org). These data provide a timely and valuable resource for improved annotation of the genome, verification of predicted hypothetical proteins and identification of proteins not predicted by current gene models. The data indicated the expression of proteins likely to be important to the invasion and intracellular establishment of the parasite, including surface proteins, constituents of the remnant mitochondrion and apical organelles. Comparison of the expressed proteome with existing transcriptional data indicated only a weak correlation. For approximately half the proteome there was limited functional and structural information, highlighting the limitations in the current understanding of Cryptosporidium biology.

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin.

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

Solid-phase library synthesis, screening, and selection of tight-binding reduced peptide bond inhibitors of a recombinant Leishmania mexicana cysteine protease B.

A one-bead-two-compound inhibitor library was synthesized by the split-mix method for the identification of inhibitors of a recombinant cysteine protease from Leishmania mexicana, CPB2.8DeltaCTE. The inhibitor library was composed of octapeptides with a centrally located reduced bond introduced by reductive amination of the resin-bound amines with Fmoc amino aldehydes. The library was screened on solid phase, and less than 1% of the library contained active compounds. The inhibitors displayed great specificity in the subsites flanking the enzyme catalytic triad with Cha and Ile/Leu preferred in P(2), Phe in P(1), Cha and Ile/Leu in P(1)', and Ile/Leu in P(2)'. Some of the inhibitors were resynthesized, and the kinetics of inhibition were determined in solution-phase assays. Most of the inhibitors had micromolar K(i) values, and a few inhibited the enzyme at nanomolar concentrations. One inhibitor, DKHF(CH(2)NH)LLVK (K(i) = 1 microm), was tested for antiparasite efficacy and shown to affect parasite survival with an IC(50) of approximately 50 microm.

Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

BACKGROUND: Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity. METHODOLOGY/PRINCIPAL FINDINGS: THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i) it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold), and also decrease in k 3 (3.5-fold). The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25))-S(-)/(His(163))-Im(+) H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme. CONCLUSIONS/SIGNIFICANCE: Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

The proteome of Toxoplasma gondii: integration with the genome provides novel insights into gene expression and annotation.

BACKGROUND: Although the genomes of many of the most important human and animal pathogens have now been sequenced, our understanding of the actual proteins expressed by these genomes and how well they predict protein sequence and expression is still deficient. We have used three complementary approaches (two-dimensional electrophoresis, gel-liquid chromatography linked tandem mass spectrometry and MudPIT) to analyze the proteome of Toxoplasma gondii, a parasite of medical and veterinary significance, and have developed a public repository for these data within ToxoDB, making for the first time proteomics data an integral part of this key genome resource. RESULTS: The draft genome for Toxoplasma predicts around 8,000 genes with varying degrees of confidence. Our data demonstrate how proteomics can inform these predictions and help discover new genes. We have identified nearly one-third (2,252) of all the predicted proteins, with 2,477 intron-spanning peptides providing supporting evidence for correct splice site annotation. Functional predictions for each protein and key pathways were determined from the proteome. Importantly, we show evidence for many proteins that match alternative gene models, or previously unpredicted genes. For example, approximately 15% of peptides matched more convincingly to alternative gene models. We also compared our data with existing transcriptional data in which we highlight apparent discrepancies between gene transcription and protein expression. CONCLUSION: Our data demonstrate the importance of protein data in expression profiling experiments and highlight the necessity of integrating proteomic with genomic data so that iterative refinements of both annotation and expression models are possible.

Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii.

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.

Combinatorial library of peptidotriazoles: identification of [1,2,3]-triazole inhibitors against a recombinant Leishmania mexicana cysteine protease.

A library consisting of about half of 800 000 possible peptidotriazoles on 450 000 beads was prepared by solid-phase peptide synthesis combined with a regiospecific copper(I)-catalyzed 1,3-dipolar cycloaddition between a resin-bound alkyne and a protected amino azide. The central [1,2,3]-triazole was flanked on each side by two randomized amino acids introduced in a combinatorial approach. Importantly, the formation of the triazole could be performed quantitatively in a randomized fashion. The library was screened on solid phase for inhibitory effect against a recombinant cysteine protease, Leishmania mexicana CPB2.8DeltaCTE and sorted by a high-throughput instrument, COPAS beadsorter (up to 200 000 beads/h). Forty-eight hits were analyzed by MALDI-TOF MS providing structural information about the protease specificity, and 23 peptidotriazoles were resynthesized and evaluated in solution, with the best inhibitor displaying a K(i) value of 76 nM. A one-pot procedure was used to convert Fmoc-amino azides into their corresponding Boc derivatives. The crucial influence of weak interactions with a spacer used for detection by MALDI-TOF MS on screening results was observed.

Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes.

The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.