A genome-wide expression analysis was undertaken to identify novel genes specifically activated from early stages of gametocytogenesis in Plasmodium falciparum. A comparative analysis was conducted on sexually induced cultures of reference parasite clone 3D7 and its gametocyteless derivative clone F12. Competitive hybridisations on long-oligomer microarrays representing 4488 P. falciparum genes identified a remarkably small number of transcripts differentially produced in the two clones. Upregulation of the mRNAs for the early gametocyte markers Pfs16 and Pfg27 was however readily detected in 3D7, and such genes were used as reference transcripts in a comparative time course analysis of 3D7 and F12 parasites between 30 and 40 h post-invasion in cultures induced to enter gametocytogenesis. One hundred and seventeen genes had expression profiles which correlated to those of pfs16 and pfg27, and Northern blot analysis and published proteomic data identified those whose expression was gametocyte-specific. Immunofluorescence analysis with antibodies against two of these gene products identified two novel parasite membrane associated, sexual stage-specific proteins. One was produced from stage I gametocytes and the second showed peak production in stage II gametocytes. The two proteins were named Pfpeg-3 and Pfpeg-4, for P. falciparum proteins of early gametocytes.
BackgroundMicroenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described.MethodsWe studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis.Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression.ResultsWe found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive.We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis.A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases.ConclusionsA crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0915-z) contains supplementary material, which is available to authorized users.
BackgroundPlasmodium parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism.ResultsUsing various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i) fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii) 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv) several PfaaRSs posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs.ConclusionWe have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.
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.
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