Judith Helena Prieto

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

BackgroundAlthough 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.ResultsThe 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.ConclusionOur 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.

The malarial parasite Plasmodium falciparum imports the human protein peroxiredoxin 2 for peroxide detoxification

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurers clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.

Large-Scale Differential Proteome Analysis in Plasmodium falciparum under Drug Treatment

Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using 14N-isoleucine and 13C6,15N1-isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (>1.5) whereas 14 and 8 proteins were down-regulated (<0.5). Apart from specifically regulated proteins we also identified sets of proteins which were regulated as a general response to drug treatment. The proteomic data was confirmed by Western blotting. The methodology described here allows for the efficient large-scale differential proteome analysis of P. falciparum to study the response to drug treatment or environmental changes. Only 100 µg of protein is required for the analysis suggesting that the method can also be transferred to other apicomplexan parasites.

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.

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

SUMMARY Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studies.

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility

BackgroundGametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown.MethodsPlasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163.Results615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway.ConclusionsThis study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.

Protein S-nitrosylation in Plasmodium falciparum.

AIMS Due to its life in different hosts and environments, the human malaria parasite Plasmodium falciparum is exposed to oxidative and nitrosative challenges. Nitric oxide (NO) and NO-derived reactive nitrogen species can constitute nitrosative stress and play a major role in NO-related signaling. However, the mode of action of NO and its targets in P. falciparum have hardly been characterized. Protein S-nitrosylation (SNO), a posttranslational modification of protein cysteine thiols, has emerged as a principal mechanism by which NO exerts diverse biological effects. Despite its potential importance, SNO has hardly been studied in human malaria parasites. Using a biotin-switch approach coupled to mass spectrometry, we systemically studied SNO in P. falciparum cell extracts. RESULTS We identified 319 potential targets of SNO that are widely distributed throughout various cellular pathways. Glycolysis in the parasite was found to be a major target, with glyceraldehyde-3-phosphate dehydrogenase being strongly inhibited by S-nitrosylation of its active site cysteine. Furthermore, we show that P. falciparum thioredoxin 1 (PfTrx1) can be S-nitrosylated at its nonactive site cysteine (Cys43). Mechanistic studies indicate that PfTrx1 possesses both denitrosylating and transnitrosylating activities mediated by its active site cysteines and Cys43, respectively. INNOVATION This work provides first insights into the S-nitrosoproteome of P. falciparum and suggests that the malaria parasite employs the thioredoxin system to deal with nitrosative challenges. CONCLUSION Our results indicate that SNO may influence a variety of metabolic processes in P. falciparum and contribute to our understanding of NO-related signaling processes and cytotoxicity in the parasites.

Insight into the selenoproteome of the malaria parasite Plasmodium falciparum.

AIMS The malaria parasite Plasmodium falciparum possesses four unique selenoproteins (PfSel1-PfSel4) which are likely to represent important components of the redox-regulatory network of this infectious agent. So far these proteins have only been characterized in silico. The aim of the present study was to gain further insight into the structural, biochemical, and functional properties of P. falciparum selenoproteins. RESULTS Using (75)Se labeling in P. falciparum cell culture, the presence of selenoproteins in the parasite could be verified for the first time. Bioinformatic analyses indicated distant relatedness between the Plasmodium proteins and selenoproteins described in other organisms, namely between PfSel1 and SelK, PfSel2 and SelT, and between PfSel4 and SelS. For PfSel3 no remarkable similarities with proteins from other organisms were identified. All four proteins were recombinantly produced in Escherichia coli as UGA→UGU (selenocysteine→cysteine) mutants. Using green fluorescent protein (GFP)-fusion proteins and immunofluorescence, the subcellular localization of the four selenoprotein mutants was studied. PfSel1, PfSel2, and PfSel4 localized to the endoplasmic reticulum whereas PfSel3 was visualized in the nucleus and/or the apicoplast. Functional assays support the roles of PfSel1 and PfSel4 in cellular redox reactions. Transcriptional profiles of the four selenoproteins, and proteins involved in selenoprotein biosynthesis, indicate that their expression is regulated via the availability of selenium and via oxidative and nitrosative stress. INNOVATION In this study the presence of selenoproteins in Plasmodium has been proven for the first time; the subcellular localization of the proteins and their relatedness to known selenoproteins have been systematically studied, and recombinant proteins as well as information on regulation of transcript levels have been obtained. CONCLUSION Taken together, our data enhance our understanding of the functional role of selenoproteins in Plasmodium.

Characterization of the 26S proteasome network in Plasmodium falciparum

In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system.