Urinary endogenous peptides as biomarkers for prostate cancer.

Prostate cancer (PCa) is one of the most prevalent types of cancer in men worldwide; however, the main diagnostic tests available for PCa have limitations and a biopsy is required for histopathological confirmation of the disease. Prostate-specific antigen (PSA) is the main biomarker used for the early detection of PCa, but an elevated serum concentration is not cancer-specific. Therefore, there is a need for the discovery of new non-invasive biomarkers that can accurately diagnose PCa. The present study used trichloroacetic acid-induced protein precipitation and liquid chromatography-mass spectrometry to profile endogenous peptides in urine samples from patients with PCa (n=33), benign prostatic hyperplasia (n=25) and healthy individuals (n=28). Receiver operating characteristic curve analysis was performed to evaluate the diagnostic performance of urinary peptides. In addition, Proteasix tool was used for in silico prediction of protease cleavage sites. Five urinary peptides derived from uromodulin were revealed to be significantly altered between the study groups, all of which were less abundant in the PCa group. This peptide panel showed a high potential to discriminate between the study groups, resulting in area under the curve (AUC) values between 0.788 and 0.951. In addition, urinary peptides outperformed PSA in discriminating between malignant and benign prostate conditions (AUC=0.847), showing high sensitivity (81.82%) and specificity (88%). From in silico analyses, the proteases HTRA2, KLK3, KLK4, KLK14 and MMP25 were identified as potentially involved in the degradation of uromodulin peptides in the urine of patients with PCa. In conclusion, the present study allowed the identification of urinary peptides with potential for use as non-invasive biomarkers in PCa diagnosis.

p53 mutants G245S and R337H associated with the Li-Fraumeni syndrome regulate distinct metabolic pathways.

Li-Fraumeni and Li-Fraumeni-like syndromes (LFS/LFL) are hereditary cancer predisposition disorders associated with germline mutations in the TP53 tumor suppressor gene. Here, we stably expressed LFS/LFL-associated p53 mutants R337H and G245S in p53-null H1299 cells to study their cellular and molecular effects. Mutant proteins showed distinct oligomerization states and opposing effects on cell proliferation and viability. Stable expression of p53G245S enhanced cell proliferation and spheroid formation, while cells stably expressing p53R337H showed reduced proliferation and clonogenicity, along with increased cell death. Mass spectrometry analysis revealed that proteins whose expression was induced by p53R337H or p53G245S expression were related to distinct metabolic profiles. Proteins upregulated by p53G245S expression were associated with a Warburg phenotype, while proteins upregulated by p53R337H expression were related to oxidative phosphorylation and fatty acid oxidation. Differences in mitochondrial mass and activity between cells stably expressing p53R337H or p53G245S were further corroborated by High Resolution Respirometry, flow cytometry and qPCR assays. The implications of the different oncogenic properties of p53R337H and p53G245S on the clinical manifestation and treatment of LFS/LFL patients carrying these mutations are discussed.

Proteomic profiling of hydatid fluid from pulmonary cystic echinococcosis.

BACKGROUND: Most cystic echinococcosis cases in Southern Brazil are caused by Echinococcus granulosus and Echinococcus ortleppi. Proteomic studies of helminths have increased our knowledge about the molecular survival strategies that are used by parasites. Here, we surveyed the protein content of the hydatid fluid compartment in E. granulosus and E. ortleppi pulmonary bovine cysts to better describe and compare their molecular arsenal at the host-parasite interface. METHODS: Hydatid fluid samples from three isolates of each species were analyzed using mass spectrometry-based proteomics (LC-MS/MS). In silico functional analyses of the identified proteins were performed to examine parasite survival strategies. RESULTS: The identified hydatid fluid protein profiles showed a predominance of parasite proteins compared to host proteins that infiltrate the cysts. We identified 280 parasitic proteins from E. granulosus and 251 from E. ortleppi, including 52 parasitic proteins that were common to all hydatid fluid samples. The in silico functional analysis revealed important molecular functions and processes that are active in pulmonary cystic echinococcosis, such as adhesion, extracellular structures organization, development regulation, signaling transduction, and enzyme activity. CONCLUSIONS: The protein profiles described here provide evidence of important mechanisms related to basic cellular processes and functions that act at the host-parasite interface in cystic echinococcosis. The molecular tools used by E. granulosus and E. ortleppi for survival within the host are potential targets for new therapeutic approaches to treat cystic echinococcosis and other larval cestodiases.

Proteomic profiling of hydatid fluid from pulmonary cystic echinococcosis

Background Most cystic echinococcosis cases in Southern Brazil are caused by Echinococcus granulosus and Echinococcus ortleppi. Proteomic studies of helminths have increased our knowledge about the molecular survival strategies that are used by parasites. Here, we surveyed the protein content of the hydatid fluid compartment in E. granulosus and E. ortleppi pulmonary bovine cysts to better describe and compare their molecular arsenal at the host-parasite interface. Methods Hydatid fluid samples from three isolates of each species were analyzed using mass spectrometry-based proteomics (LC-MS/MS). In silico functional analyses of the identified proteins were performed to examine parasite survival strategies. Results The identified hydatid fluid protein profiles showed a predominance of parasite proteins compared to host proteins that infiltrate the cysts. We identified 280 parasitic proteins from E. granulosus and 251 from E. ortleppi, including 52 parasitic proteins that were common to all hydatid fluid samples. The in silico functional analysis revealed important molecular functions and processes that are active in pulmonary cystic echinococcosis, such as adhesion, extracellular structures organization, development regulation, signaling transduction, and enzyme activity. Conclusions The protein profiles described here provide evidence of important mechanisms related to basic cellular processes and functions that act at the host-parasite interface in cystic echinococcosis. The molecular tools used by E. granulosus and E. ortleppi for survival within the host are potential targets for new therapeutic approaches to treat cystic echinococcosis and other larval cestodiases.

Expression and distribution of glutathione transferases in protoscoleces of Echinococcus granulosus sensu lato.

Glutathione transferases (GSTs) belong to a diverse superfamily of multifunctional proteins involved in metabolic detoxification. In helminth parasite, GSTs are particularly relevant since they are also involved in host immunomodulation. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least three phylogenetically distant cytosolic GSTs: EgGST1 and EgGST2 previously grouped within Mu and Sigma classes, respectively; and EgGST3 related to both Omega and Sigma classes. To better characterize E. granulosus s.l. GSTs, herein their expression and distribution were assessed in the pre-adult protoscolex (PSC) parasite stage. Potential transcriptional regulatory mechanisms of the corresponding EgGST genes were also explored. Firstly, the transcription of the three EgGSTs was significantly induced during the early stages of the murine model of infection, suggesting a potential role during parasite establishment. EgGST1 was detected in the parenchyma of PSCs and its expression increased after H2O2 exposure, supporting its role in detoxification. EgGST2 was mainly detected on the PSCs tegument, strategically localized for potential immunoregulation functions due to its Sigma-class characteristics. In addition, its expression increased after anthelmintic treatment, suggesting a role in chemotherapy resistance. Finally, the Omega-related EgGST3 was localized throughout the entire PSC body, including suckers and tegument, and since its expression also increased after H2O2 treatment, a potential role in oxidative stress response could also be ascribed. On the other hand, known cis-acting regulatory motifs were detected in EgGST genes, suggesting similar transcription processes to other eukaryotes. The results herein reported provide additional data regarding the roles of EgGSTs in E. granulosus s.l. biology, contributing to a better understanding of its host-parasite interaction.

Proteomic Analysis of the Non-genetic Response to Cisplatin in Lung Cancer Cells.

Background: Drug resistance is the main cause of therapy failure in advanced lung cancer. Although non-genetic mechanisms play important roles in tumor chemoresistance, drug-induced epigenetic reprogramming is still poorly understood. Materials and Methods: The A549 cell line was used to generate cells with non-genetic resistance to cisplatin (CDDP), namely A549/CDDP cells. Bioorthogonal non-canonical amino acid tagging (BONCAT) and mass spectrometry were used to identify proteins modulated by CDDP in A549 and A549/CDDP cells. Results: Proteins related to proteostasis, telomere maintenance, cell adhesion, cytoskeletal remodeling, and cell redox homeostasis were found enriched in both cell lines upon CDDP exposure. On the other hand, proteins involved in drug response, metabolic pathways and mRNA processing and splicing were up-regulated by CDDP only in A549/CDDP cells. Conclusion: Our study revealed proteome dynamics involved in the non-genetic response to CDDP, pointing out potential targets to monitor and overcome epigenetic resistance in lung cancer.

Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations.

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

Protein Profiling of Extracellular Vesicles Associated With Cisplatin Resistance in Lung Cancer.

BACKGROUND/AIM: Extracellular vesicles (EVs) can mediate drug resistance within the tumor microenvironment by delivering bioactive molecules, including proteins. Here, we performed a comparative proteomic analysis of EVs secreted by A549 lung cancer cells and their cisplatin-resistant counterparts in order to identify proteins involved in drug resistance. MATERIALS AND METHODS: Cells were co-cultivated using a transwell system to evaluate EV exchange. EVs were isolated by ultracentrifugation and analyzed using microscopy and nanoparticle tracking. EV proteome was analyzed by mass spectrometry. RESULTS: EV-mediated communication was observed between co-cultured A549 and A549/CDDP cells. EVs isolated from both cells were mainly exosome-like structures. Extracellular matrix components, cell adhesion proteins, complement factors, histones, proteasome subunits and membrane transporters were found enriched in the EVs released by cisplatin-resistant cells. CONCLUSION: Proteins identified in this work may have a relevant role in modulating the chemosensitivity of the recipient cells and could represent useful biomarkers to monitor cisplatin response in lung cancer.

Dynamics of protein synthesis in the initial steps of strobilation in the model cestode parasite Mesocestoides corti (syn. vogae).

Mesocestoides corti (syn. vogae) is a useful model for developmental studies of platyhelminth parasites of the Cestoda class, such as Taenia spp. or Echinococcus spp. It has been used in studies to characterize cestode strobilation, i.e. the development of larvae into adult worms. So far, little is known about the initial molecular events involved in cestode strobilation and, therefore, we carried out a study to characterize newly synthesized (NS) proteins upon strobilation induction. An approach based on bioorthogonal noncanonical amino acid tagging and mass spectrometry was used to label, isolate, identify, and quantify NS proteins in the initial steps of M. corti strobilation. Overall, 121 NS proteins were detected exclusively after induction of strobilation, including proteins related to development pathways, such as insulin and notch signaling. Metabolic changes that take place in the transition from the larval stage to adult worm were noted in special NS protein subsets related to developmental processes, such as focal adhesion, cell leading edge, and maintenance of location. The data shed light on mechanisms underlying early steps of cestode strobilation and enabled identification of possible developmental markers. We also consider the use of developmental responsive proteins as potential drug targets for developing novel anthelmintics. BIOLOGICAL SIGNIFICANCE: Larval cestodiases are life-threatening parasitic diseases that affect both man and domestic animals worldwide. Cestode parasites present complex life cycles, in which they undergo major morphological and physiological changes in the transition from one life-stage to the next. One of these transitions occurs during cestode strobilation, when the mostly undifferentiated and non-segmented larval or pre-adult form develops into a fully segmented and sexually differentiated (strobilated) adult worm. Although the proteomes of bona fide larvae and strobialted adults have been previously characterized for a few cestode species, little is still known about the dynamic of protein synthesis during the early steps of cestode strobilation. Now, the assessment of newly synthesized (NS) proteins within the first 48 h of strobilation the model cestode M. corti allowed to shed light on molecular mechanisms that are triggered by strobilation induction. The functional analyses of this repertoire of over a hundred NS proteins pointed out to changes in metabolism and activation of classical developmental signaling pathways in early strobilation. Many of the identified NS proteins may become valuable cestode developmental markers and their involvement in vital processes make them also good candidate targets for novel anthelmintic drugs.

Transcriptomic Analysis of the Early Strobilar Development of Echinococcus granulosus.

Echinococcus granulosus has a complex life cycle involving two mammalian hosts. The transition from one host to another is accompanied by changes in gene expression, and the transcriptional events that underlie this transition have not yet been fully characterized. In this study, RNA-seq was used to compare the transcription profiles of samples from E. granulosus protoscoleces induced in vitro to strobilar development at three time points. We identified 818 differentially expressed genes, which were divided into eight expression clusters formed over the entire 24 h period. An enrichment of gene transcripts with molecular functions of signal transduction, enzymes, and protein modifications was observed upon induction and developmental progression. This transcriptomic study provides insights for understanding the complex life cycle of E. granulosus and contributes for searching for the key genes correlating with the strobilar development, which can be used to identify potential candidates for the development of anthelmintic drugs.

Urinary peptidomics and bioinformatics for the detection of diabetic kidney disease.

The aim of this study was to establish a peptidomic profile based on LC-MS/MS and random forest (RF) algorithm to distinguish the urinary peptidomic scenario of type 2 diabetes mellitus (T2DM) patients with different stages of diabetic kidney disease (DKD). Urine from 60 T2DM patients was collected: 22 normal (stage A1), 18 moderately increased (stage A2) and 20 severely increased (stage A3) albuminuria. A total of 1080 naturally occurring peptides were detected, which resulted in the identification of a total of 100 proteins, irrespective of the patients' renal status. The classification accuracy showed that the most severe DKD (A3) presented a distinct urinary peptidomic pattern. Estimates for peptide importance assessed during RF model training included multiple fragments of collagen and alpha-1 antitrypsin, previously associated to DKD. Proteasix tool predicted 48 proteases potentially involved in the generation of the 60 most important peptides identified in the urine of DM patients, including metallopeptidases, cathepsins, and calpains. Collectively, our study lightened some biomarkers possibly involved in the pathogenic mechanisms of DKD, suggesting that peptidomics is a valuable tool for identifying the molecular mechanisms underpinning the disease and thus novel therapeutic targets.

Functional characterization of the translation initiation factor eIF4E of Echinococcus granulosus.

The eukaryotic initiation factor 4E (eIF4E) specifically recognizes the 5' mRNA cap, a rate-limiting step in the translation initiation process. Although the 7-methylguanosine cap (MMGcap) is the most common 5' cap structure in eukaryotes, the trans-splicing process that occurs in several organism groups, including nematodes and flatworms, leads to the addition of a trimethylguanosine cap (TMGcap) to some RNA transcripts. In some helminths, eIF4E can have a dual capacity to bind both MMGcap and TMGcap. In the present work, we evaluated the distribution of eIF4E protein sequences in platyhelminths and we showed that only one gene coding for eIF4E is present in most parasitic flatworms. Based on this result, we cloned the Echinococcus granulosus cDNA sequence encoding eIF4E in Escherichia coli, expressed the recombinant eIF4E as a fusion protein to GST, and tested its ability to capture mRNAs through the 5' cap using pull-down assay and qPCR. Our results indicate that the recombinant eIF4E was able to bind preferentially 5'-capped mRNAs compared with rRNAs from total RNA preparations of E. granulosus. By qPCR, we observed an enrichment in MMG-capped mRNA compared with TMG-capped mRNAs among Eg-eIF4E-GST pull-down RNAs. Eg-eIF4E structural model using the Schistosoma mansoni eIF4E as template showed to be well preserved with only a few differences between chemically similar amino acid residues at the binding sites. These data showed that E. granulosus eIF4E can be used as a potential tool to study full-length 5'-capped mRNA, besides being a potential drug target against parasitic flatworms.

Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways.

BACKGROUND: Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid) of Echinococcus granulosus (Cestoda, Taeniidae). The hydatid develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. The lipoprotein Antigen B (AgB) is the major component of E. granulosus metacestode hydatid fluid. Functionally, AgB has been implicated in immunomodulation and lipid transport. However, the mechanisms underlying AgB functions are not completely known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated AgB interactions with different mammalian cell types and the pathways involved in its internalization. AgB uptake was observed in four different cell lines, NIH-3T3, A549, J774 and RH. Inhibition of caveolae/raft-mediated endocytosis causes about 50 and 69% decrease in AgB internalization by RH and A549 cells, respectively. Interestingly, AgB colocalized with the raft endocytic marker, but also showed a partial colocalization with the clathrin endocytic marker. Finally, AgB colocalized with an endolysosomal tracker, providing evidence for a possible AgB destination after endocytosis. CONCLUSIONS/SIGNIFICANCE: The results indicate that caveolae/raft-mediated endocytosis is the main route to AgB internalization, and that a clathrin-mediated entry may also occur at a lower frequency. A possible fate for AgB after endocytosis seems to be the endolysosomal system. Cellular internalization and further access to subcellular compartments could be a requirement for AgB functions as a lipid carrier and/or immunomodulatory molecule, contributing to create a more permissive microenvironment to metacestode development and survival.

Comparative proteomics of the larval and adult stages of the model cestode parasite Mesocestoides corti.

Mesocestoides corti is a widely used model for the study of cestode biology, and its transition from the larval tetrathyridium (TT) stage to the strobilated, adult worm (ST) stage can be induced and followed in vitro. Here, a proteomic approach was used to describe and compare M. corti TT and ST protein repertories. Overall, 571 proteins were identified, 238 proteins in TT samples and 333 proteins in ST samples. Among the identified proteins, 207 proteins were shared by TTs and STs, while 157 were stage-specific, being 31 exclusive from TTs, and 126 from STs. Functional annotation revealed fundamental metabolic differences between the TT and the ST stages. TTs perform functions related mainly to basic metabolism, responsible for growth and vegetative development by asexual reproduction. STs, in contrast, perform a wider range of functions, including macromolecule biosynthetic processes, gene expression and control pathways, which may be associated to its proglottization/segmentation, sexual differentiation and more complex physiology. Furthermore, the generated results provided an extensive list of cestode proteins of interest for functional studies in M. corti. Many of these proteins are novel candidate diagnostic antigens, and/or potential targets for the development of new and more effective antihelminthic drugs. BIOLOGICAL SIGNIFICANCE: Cestodiases are parasitic diseases with serious impact on human and animal health. Efforts to develop more effective strategies for diagnosis, treatment or control of cestodiases are impaired by the still limited knowledge on many aspects of cestode biology, including the complex developmental processes that occur in the life cycles of these parasites. Mesocestoides corti is a good experimental model to study the transition from the larval to the adult stage, called strobilation, which occur in typical cestode life-cycles. The performed proteomics approach provided large-scale identification and quantification of M. corti proteins. Many stage-specific or differentially expressed proteins were detected in the larval tetrathyridium (TT) stage and in the strobilated, adult worm (ST) stage. Functional comparative analyses of the described protein repertoires shed light on function and processes associated to specific features of both stages, such as less differentiation and asexual reproduction in TTs, and proglottization/segmentation and sexual differentiation in ST. Moreover, many of the identified stage-specific proteins are useful as cestode developmental markers, and are potential targets for development of novel diagnostic methods and therapeutic drugs for cestodiases.

Comparative proteomics of hydatid fluids from two Echinococcus multilocularis isolates.

The hydatid fluid (HF) that fills Echinococcus multilocularis metacestode vesicles is a complex mixture of proteins from both parasite and host origin. Here, a LC-MS/MS approach was used to compare the HF composition of E. multilocularis H95 and G8065 isolates (EmH95 and EmG8065, respectively), which present differences in terms of growth and fertility. Overall, 446 unique proteins were identified, 392 of which (88%) were from parasite origin and 54 from culture medium. At least 256 of parasite proteins were sample exclusive, and 82 of the 136 shared proteins presented differential abundance between E. multilocularis isolates. The parasite's protein repertoires in EmH95 and EmG8065 HF samples presented qualitative and quantitative differences involving antigens, signaling proteins, proteolytic enzymes, protease inhibitors and chaperones, highlighting intraspecific singularities that could be correlated to biological features of each isolate. The repertoire of medium proteins found in the HF was also differential between isolates, and the relevance of the HF exogenous protein content for the parasite's biology is discussed. The repertoires of identified proteins also provided potential molecular markers for important biological features, such as parasite growth rate and fertility, as well potential protein targets for the development of novel diagnostic and treatment strategies for alveolar echinococcosis. BIOLOGICAL SIGNIFICANCE: E. multilocularis metacestode infection of mammal hosts involve complex interactions mediated by excretory/secretory (ES) products. The hydatid fluid (HF) that fills the E. multilocularis metacestode vesicles contains complex repertoires of parasite ES products and host proteins that mediate important molecular interactions determinant for parasite survival and development, and, consequently, to the infection outcome. HF has been also extensively reported as the main source of proteins for the immunodiagnosis of echinococcosis. The performed proteomic analysis provided a comprehensive profiling of the HF protein composition of two E. multilocularis isolates. This allowed us to identify proteins of both parasite and exogenous (medium) origin, many of which present significant differential abundances between parasite isolates and may correlate to their differential biological features, including fertility and growth rate.

Proteomic analysis of Toxocara canis excretory and secretory (TES) proteins.

Toxocariasis is a neglected disease, and its main etiological agent is the nematode Toxocara canis. Serological diagnosis is performed by an enzyme-linked immunosorbent assay using T. canis excretory and secretory (TES) antigens produced by in vitro cultivation of larvae. Identification of TES proteins can be useful for the development of new diagnostic strategies since few TES components have been described so far. Herein, we report the results obtained by proteomic analysis of TES proteins using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. TES fractions were separated by one-dimensional SDS-PAGE and analyzed by LC-MS/MS. The MS/MS spectra were compared with a database of protein sequences deduced from the genome sequence of T. canis, and a total of 19 proteins were identified. Classification according to the signal peptide prediction using the SignalP server showed that seven of the identified proteins were extracellular, 10 had cytoplasmic or nuclear localization, while the subcellular localization of two proteins was unknown. Analysis of molecular functions by BLAST2GO showed that the majority of the gene ontology (GO) terms associated with the proteins present in the TES sample were associated with binding functions, including but not limited to protein binding (GO:0005515), inorganic ion binding (GO:0043167), and organic cyclic compound binding (GO:0097159). This study provides additional information about the exoproteome of T. canis, which can lead to the development of new strategies for diagnostics or vaccination.

Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions.

Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts.

Excretory/secretory products in the Echinococcus granulosus metacestode: is the intermediate host complacent with infection caused by the larval form of the parasite?

The genus Echinococcus consists of parasites that have a life cycle with two mammalian hosts. Their larval stage, called the hydatid cyst, develops predominantly in the liver and lungs of intermediate hosts. The hydatid cyst is the causative agent of cystic hydatid disease and the species Echinococcus granulosus, G1 haplotype, is responsible for the vast majority of cases in humans, cattle and sheep. Protein characterization in hydatid cysts is essential for better understanding of the host-parasite relationship and the fertility process of Echinococcus. The aims of this work were the identification and quantitative comparison of proteins found in hydatid fluid from fertile and infertile cysts from E. granulosus, in order to highlight possible mechanisms involved in cyst fertility or infertility. Hydatid fluid samples containing proteins from both E. granulosus and Bos taurus were analysed by LC-MS/MS. Our proteomic analysis of fertile and infertile cysts allowed identification of a total of 498 proteins, of which 153 proteins were exclusively identified in the fertile cyst, 271 in the infertile cyst, and 74 in both. Functional in silico analysis allowed us to highlight some important aspects: (i) clues about the possible existence of an "arms race" involving parasite and host responses in fertile and infertile cysts; (ii) a number of proteins in hydatid fluid without functional annotation or with possible alternative functions; (iii) the presence of extracellular vesicles such as exosomes, which was confirmed by transmission electron microscopy.

Proteomics analysis of Echinococcus granulosus protoscolex stage.

Echinococcus granulosus protoscolex proteins were separated using two-dimensional electrophoresis and then identified using mass spectrometry; we identified 61 proteins, 28 which are newly described of which 4 could be involved in hydatid cyst fertility molecular mechanisms.

Identification of Newly Synthesized Proteins by Echinococcus granulosus Protoscoleces upon Induction of Strobilation.

BACKGROUND: The proteins responsible for the key molecular events leading to the structural changes between the developmental stages of Echinococcus granulosus remain unknown. In this work, azidohomoalanine (AHA)-specific labeling was used to identify proteins expressed by E. granulosus protoscoleces (PSCs) upon the induction of strobilar development. METHODOLOGY/PRINCIPAL FINDINGS: The in vitro incorporation of AHA with different tags into newly synthesized proteins (NSPs) by PSCs was analyzed using SDS-PAGE and confocal microscopy. The LC-MS/MS analysis of AHA-labeled NSPs by PSCs undergoing strobilation allowed for the identification of 365 proteins, of which 75 were differentially expressed in comparison between the presence or absence of strobilation stimuli and 51 were expressed exclusively in either condition. These proteins were mainly involved in metabolic, regulatory and signaling processes. CONCLUSIONS/SIGNIFICANCE: After the controlled-labeling of proteins during the induction of strobilar development, we identified modifications in protein expression. The changes in the metabolism and the activation of control and signaling pathways may be important for the correct parasite development and be target for further studies.