Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

Expression of the histone chaperone SET/TAF-Iß during the strobilation process of Mesocestoides corti (Platyhelminthes, Cestoda).

The histone chaperone SET/TAF-Iß is implicated in processes of chromatin remodelling and gene expression regulation. It has been associated with the control of developmental processes, but little is known about its function in helminth parasites. In Mesocestoides corti, a partial cDNA sequence related to SET/TAF-Iß was isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Here, the full-length coding sequence of the M. corti SET/TAF-Iß gene was analysed and the encoded protein (McSET/TAF) was compared with orthologous sequences, showing that McSET/TAF can be regarded as a SET/TAF-Iß family member, with a typical nucleosome-assembly protein (NAP) domain and an acidic tail. The expression patterns of the McSET/TAF gene and protein were investigated during the strobilation process by RT-qPCR, using a set of five reference genes, and by immunoblot and immunofluorescence, using monospecific polyclonal antibodies. A gradual increase in McSET/TAF transcripts and McSET/TAF protein was observed upon development induction by trypsin, demonstrating McSET/TAF differential expression during strobilation. These results provided the first evidence for the involvement of a protein from the NAP family of epigenetic effectors in the regulation of cestode development.

Proteomic survey of the cestode Mesocestoides corti during the first 24 hours of strobilar development.

Despite the fact that cestodes represent major etiological agents of both human and domestic animal diseases, little is known about the molecular aspects of cestode development. In this work, Mesocestoides corti, a model cestode species, was studied from the early development of its larval form (tetrathyridium) into adult worms (strobilation) using different proteomic approaches. The protein profiles of M. corti tetrathyridia induced or not induced to undergo strobilation were compared. Proteomic mapping by two-dimensional gel electrophoresis showed the resolution of 248 and 154 spots from tetrathyridia that were subjected or not subjected to strobilation induction, respectively, allowing for the detection of at least nine spots exclusive to each group. Spot analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) or MALDI-TOF MS/MS identified four reference proteins (six spots). LC-MS/MS analyses of protein extracts identified 66 proteins, eight of which were found exclusively in non-induced tetrathyridia, while 13 were found exclusively in strobilation-induced tetrathyridia. Among the proteins exclusively identified in strobilation-induced worms, there was a predominance of proteins with functions relating to chaperone activity and protein synthesis and turnover. Quantitative differential expression analysis between M. corti tetrathyridia prior to and after strobilation induction revealed six proteins upregulated in strobilation-induced worms; these proteins were involved in metabolic pathways, cell proliferation, and cytoskeletal rearrangement. Overall, despite the absence of a sequenced M. corti genome, using sequences from other platyhelminthes, we were able to establish comprehensive protein profiles for tetrathyridia prior to and after strobilation induction and identify several proteins potentially involved in the early events leading to strobilation.

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 Echinococcus granulosus metacestode during infection of its intermediate host.

Cystic hydatid disease (CHD) is caused by infection with the Echinococcus granulosus metacestode and affects both humans and livestock. In this work, we performed a proteomic analysis of the E. granulosus metacestode during infection of its intermediate bovine host. Parasite proteins were identified in different metacestode components (94 from protoscolex, 25 from germinal layer and 20 from hydatid cyst fluid), along with host proteins (58) that permeate into the hydatid cyst, providing new insights into host-parasite interplay. E. granulosus and platyhelminth EST data allowed successful identification of proteins potentially involved in downregulation of host defenses, highlighting possible evasion mechanisms adopted by the parasite to establish infection. Several intracellular proteins were found in hydatid cyst fluid, revealing a set of newly identified proteins that were previously thought to be inaccessible for inducing or modulating the host immune response. Host proteins identified in association with the hydatid cyst suggest that the parasite may bind/adsorb host molecules with nutritional and/or immune evasion purposes, masking surface antigens or inhibiting important effector molecules of host immunity, such as complement components and calgranulin. Overall, our results provide valuable information on parasite survival strategies in the adverse host environment and on the molecular mechanisms underpinning CHD immunopathology.

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.

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers.

Echinococcus granulosus antigen B is an oligomeric protein of 120-160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed approximately 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.

Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B.

Antigen B (AgB) is a major protein component of the Echinococcus granulosus metacestode. It is oligomeric and this raises several questions regarding the subunit structure and composition of AgB. Several genes that encode different AgB subunits have been identified, and some of these have been cloned and expressed to produce recombinant subunits. The study of these recombinant subunits may provide new insights into the structure, physical-chemical properties, and functional aspects of AgB. Like native AgB, the AgB8/1, AgB8/2, and AgB8/3 recombinant subunits produced in our laboratory form 120-160 kDa oligomers that have stable secondary structures, are strongly antigenic and immunogenic, and selectively bind hydrophobic compounds. Here, we review these results and discuss their implications for the elucidation of the structure and function of AgB. This includes a possible role for AgB in host-parasite interactions.

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.

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.

Excretory/secretory products from in vitro-cultured Echinococcus granulosus protoscoleces.

Cystic hydatid disease (CHD) is caused by infection with Echinococcus granulosus metacestodes and affects humans and livestock. Proteins secreted or excreted by protoscoleces, pre-adult worms found in the metacestode, are thought to play fundamental roles in the host-parasite relationship. In this work, we performed an LC-MS/MS proteomic analysis of the excretory-secretory products obtained from the first 48 h of an in vitro culture of the protoscoleces. We identified 32 proteins, including 18 that were never detected previously in metacestode proteomic studies. Among the novel identified excretory-secretory products are antigenic proteins, such as EG19 and P-29 and a calpain protease. We also identified other important protoscolex excretory-secretory products, such as thioredoxin peroxidase and 14-3-3 proteins, which are potentially involved in evasion mechanisms adopted by parasites to establish infection. Several intracellular proteins were found in the excretory-secretory products, revealing a set of identified proteins not previously thought to be exposed at the host-parasite interface. Additionally, immunological analyses established the antigenic profiles of the newly identified excretory-secretory products and revealed, for the first time, the in vitro secretion of the B antigen by protoscoleces. Considering that the excretory-secretory products obtained in vitro might reflect the products released and exposed to the host in vivo, our results provide valuable information on parasite survival strategies in adverse host environments and on the molecular mechanisms underpinning CHD immunopathology.

Echinococcus granulosus antigen B structure: subunit composition and oligomeric states.

BACKGROUND: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. METHODOLOGY/PRINCIPAL FINDINGS: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. CONCLUSIONS/SIGNIFICANCE: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

Comparative analysis of the diagnostic performance of six major Echinococcus granulosus antigens assessed in a double-blind, randomized multicenter study.

The serodiagnosis of hydatid disease is a valuable instrument for clinical diagnosis and epidemiological surveillance of high-risk populations. In the past decade a wealth of reports on the diagnostic performance of numerous antigens have been produced. However, their diagnostic value has been estimated under different conditions, using different serum collection, therefore precluding their direct comparison. Here we report an unbiased comparison of the same batch of six major E. granulosus antigens, namely, hydatid cyst fluid (HCF), native antigen B (AgB), two recombinant AgB subunits, an AgB-derived synthetic peptide, and recombinant cytosolic malate dehydrogenase from E. granulosus (EgMDH), against the same serum collection. The double-blind analysis was performed using a standardized protocol and receiver operating characteristic (ROC) data analysis by a network of six South American laboratories. High intercenter reproducibility was attained, and the intralaboratory analysis allowed the comparative ranking of the antigen panel. HCF, AgB, and its AgB8/1 subunit exhibited equivalent diagnostic efficiencies, 81.4% +/- 0.5%, 81.3% +/- 0.6%, and 81.9% +/- 2.0%, respectively; with a more favorable balance toward specificity in the case of the last antigen. The diagnostic efficiencies for the other three antigens were 76.8% +/- 6.8%, 69.1% +/- 2.7%, and 66.8% +/- 2.1%, for the peptide, the AgB8/2 subunit, and the EgMDH, respectively. The study also included an analysis of batch-to-batch variation in the diagnostic performance of different HCF regional preparations. Based on these results, a suggested recommendation on the use of these antigens was drawn.