Federica Fratini

Global proteomic analysis of the oocyst/ sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle

BackgroundToxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii and can be acquired either congenitally or via the oral route. In the latter case, transmission is mediated by two distinct invasive stages, i.e., bradyzoites residing in tissue cysts or sporozoites contained in environmentally resistant oocysts shed by felids in their feces. The oocyst plays a central epidemiological role, yet this stage has been scarcely investigated at the molecular level and the knowledge of its expressed proteome is very limited.ResultsUsing one-dimensional gel electrophoresis coupled to liquid chromatography-linked tandem mass spectrometry, we analysed total or fractionated protein extracts of partially sporulated T. gondii oocysts, producing a dataset of 1304 non reduntant proteins (~18% of the total predicted proteome), ~59% of which were classified according to the MIPS functional catalogue database. Notably, the comparison of the oocyst dataset with the extensively covered proteome of T. gondii tachyzoite, the invasive stage responsible for the clinical signs of toxoplasmosis, identified 154 putative oocyst/sporozoite-specific proteins, some of which were validated by Western blot. The analysis of this protein subset showed that, compared to tachyzoites, oocysts have a greater capability of de novo amino acid biosynthesis and are well equipped to fuel the Krebs cycle with the acetyl-CoA generated through fatty acid β-oxidation and the degradation of branched amino acids.ConclusionsThe study reported herein significantly expanded our knowledge of the proteome expressed by the oocyst/sporozoite of T. gondii, shedding light on a stage-specifc subset of proteins whose functional profile is consistent with the adaptation of T. gondii oocysts to the nutrient-poor and stressing extracellular environment.

Involvement of 14-3-3 protein post-translational modifications in Giardia duodenalis encystation

14-3-3s are a family of phosphoserine/phosphothreonine binding proteins directly affecting many protein functions by regulating enzyme activity, intracellular localisation or mediating protein-protein interaction. The single 14-3-3 (g14-3-3) of the flagellated parasite Giardia duodenalis is phosphorylated at residue threonine 214 (T214) and polyglycylated at the extreme C-terminus in a stage-specific manner. To define the role of each post-translational modification, Giardia transgenic lines expressing a N-terminally FLAG-tagged g14-3-3, or the single point mutant T214A, or the E246A and the E247A mutants of the putative polyglycylation sites, were generated in this study. By affinity chromatography and MALDI-MS analysis, Glu246 was identified as the only site of polyglycylation. The absence of a polyglycine chain results in the nuclear localisation of the protein at any parasite life-stage, suggesting a role for polyglycylation in 14-3-3 nucleo/cytoplasm shuttling. Moreover, cyst formation was strongly induced in parasites expressing the E246A mutant and delayed in those harbouring the T214A mutant. Finally, in vitro overlay assays with a GST_T214E mutant indicated that phosphorylation can alter in vitro the binding properties of 14-3-3. The present data suggest that g14-3-3 post-translational modifications act in combination to affect encystation efficiency in Giardia.

Distinct properties of the egress-related osmiophilic bodies in male and female gametocytes of the rodent malaria parasite Plasmodium berghei

Gametogenesis is the earliest event after uptake of malaria parasites by the mosquito vector, with a decisive impact on colonization of the mosquito midgut. This process is triggered by a drop in temperature and contact with mosquito molecules. In a few minutes, male and female gametocytes escape from the host erythrocyte by rupturing the parasitophorous vacuole and the erythrocyte membranes. Electron‐dense, oval‐shaped organelles, the osmiophilic bodies (OB), have been implicated in the egress of female gametocytes. By comparative electron microscopy and electron tomography analyses combined with immunolocalization experiments, we here define the morphological features distinctive of male secretory organelles, hereafter named MOB (male osmiophilic bodies). These organelles appear as club‐shaped, electron‐dense vesicles, smaller than female OB. We found that a drop in temperature triggers MOB clustering, independently of exposure to other stimuli. MDV1/PEG3, a protein associated with OB in Plasmodium berghei females, localizes to both non‐clustered and clustered MOB, suggesting that clustering precedes vesicle discharge. A P. berghei mutant lacking the OB‐resident female‐specific protein Pbg377 displays a dramatic reduction in size of the OB, accompanied by a delay in female gamete egress efficiency, while female gamete fertility is not affected. Immunolocalization experiments indicated that MDV1/PEG3 is still recruited to OB‐remnant structures.

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurers clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.

Isolation and characterization of exosomes derived from fertile sheep hydatid cysts

Cystic echinococcosis (CE) is a chronic and complex zoonotic disease. Information on the mechanisms involved in parasite establishment, growth and persistence remain limited. These may be modulated by a crosstalk between extracellular vesicles (EVs). EVs including exosomes and microvesicles are able to carry developmental signaling proteins which coordinate growth and establishment of several parasites. Here, an exosome enriched EV fraction was isolated from hydatid fluid (HF) of fertile sheep cysts. A proteomic analysis of this fraction identified a number of parasite-derived vesicle-membrane associated proteins as well as cytosolic proteins. Additionally, the exosomal enriched fraction contained proteins of host origin. Specific proteins -antigen B2 and TSPAN14- in the exosomal fraction were further assayed by immunoblot and transmission electron microscopy. To the best of our knowledge, this is the first report on the presence of parasite exosomes in fertile hydatid cyst fluid. Further characterization of the exosome cargo will allow the discovery of new markers for the detection of CE in humans and animals, and the treatment of CE patients, and provide new insights regarding the role of these EVs in the establishment and persistence of hydatid cysts.

The telomeric protein AKTIP interacts with A- and B-type lamins and is involved in regulation of cellular senescence

AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence.

Increased levels of acute-phase inflammatory proteins in plasma of patients with sporadic CJD

Objective: Screening plasma samples from patients with sporadic Creutzfeldt-Jakob disease (CJD) to discover diagnostic biomarkers. Methods: Plasma samples were collected from 17 patients with sporadic CJD, 17 patients with Alzheimer disease (AD), and 20 healthy subjects. A 2-phase screening was carried out using quantitative protein mass spectrometry. The putative sporadic CJD biomarkers were then validated independently by immunoturbidimetry. Results: Mass spectrometry uncovered 7 candidate sporadic CJD protein biomarkers, all belonging to the acute-phase response. Highly significant increases of these markers in patients with sporadic CJD, compared with healthy subjects and patients with AD, was confirmed by immunoturbidimetry. Conclusions: The increase in plasma levels of a related set of acute-phase reactants in patients with sporadic CJD is a novel finding that suggests new pathogenetic hypotheses. The possible value of this set of proteins as biomarkers in the diagnosis of sporadic CJD or for blood/tissue donor screening remains to be further explored and validated in larger studies.

Phosphorylation on threonine 11 of β-dystrobrevin alters its interaction with kinesin heavy chain.

Dystrobrevin family members (α and β) are cytoplasmic components of the dystrophin‐associated glycoprotein complex, a multimeric protein complex first isolated from skeletal muscle, which links the extracellular matrix to the actin cytoskeleton. Dystrobrevin shares high homology with the cysteine‐rich and C‐terminal domains of dystrophin and a common domain organization. The β‐dystrobrevin isoform is restricted to nonmuscle tissues, serves as a scaffold for signaling complexes, and may participate in intracellular transport through its interaction with kinesin heavy chain. We have previously characterized the molecular determinants affecting the β‐dystrobrevin–kinesin heavy chain interaction, among which is cAMP‐dependent protein kinase [protein kinase A (PKA)] phosphorylation of β‐dystrobrevin. In this study, we have identified β‐dystrobrevin residues phosphorylated in vitro by PKA with pull‐down assays, surface plasmon resonance measurements, and MS analysis. Among the identified phosphorylated residues, we demonstrated, by site‐directed mutagenesis, that Thr11 is the regulatory site for the β‐dystrobrevin–kinesin interaction. As dystrobrevin may function as a signaling scaffold for kinases/phosphatases, we also investigated whether β‐dystrobrevin is phosphorylated in vitro by kinases other than PKA. Thr11 was phosphorylated by protein kinase C, suggesting that this represents a key residue modified by the activation of different signaling pathways.

An Integrated Approach to Explore Composition and Dynamics of Cholesterol-rich Membrane Microdomains in Sexual Stages of Malaria Parasite

Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions.

Phylo_dCor: distance correlation as a novel metric for phylogenetic profiling

BackgroundElaboration of powerful methods to predict functional and/or physical protein-protein interactions from genome sequence is one of the main tasks in the post-genomic era. Phylogenetic profiling allows the prediction of protein-protein interactions at a whole genome level in both Prokaryotes and Eukaryotes. For this reason it is considered one of the most promising methods.ResultsHere, we propose an improvement of phylogenetic profiling that enables handling of large genomic datasets and infer global protein-protein interactions. This method uses the distance correlation as a new measure of phylogenetic profile similarity. We constructed robust reference sets and developed Phylo-dCor, a parallelized version of the algorithm for calculating the distance correlation that makes it applicable to large genomic data. Using Saccharomyces cerevisiae and Escherichia coli genome datasets, we showed that Phylo-dCor outperforms phylogenetic profiling methods previously described based on the mutual information and Pearson’s correlation as measures of profile similarity.ConclusionsIn this work, we constructed and assessed robust reference sets and propose the distance correlation as a measure for comparing phylogenetic profiles. To make it applicable to large genomic data, we developed Phylo-dCor, a parallelized version of the algorithm for calculating the distance correlation. Two R scripts that can be run on a wide range of machines are available upon request.