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.

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.

Secretomes of Mycoplasma hyopneumoniae and Mycoplasma flocculare reveal differences associated to pathogenesis.

Mycoplasma hyopneumoniae and Mycoplasma flocculare cohabit the porcine respiratory tract. However, M. hyopneumoniae causes the porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. Comparative analyses demonstrated high similarity between these species, which includes the sharing of all predicted virulence factors. Nevertheless, studies related to soluble secretomes of mycoplasmas were little known, although they are important for bacterial-host interactions. The aim of this study was to perform a comparative analysis between the soluble secreted proteins repertoires of the pathogenic Mycoplasma hyopneumoniae and its closely related commensal Mycoplasma flocculare. For that, bacteria were cultured in medium with reduced serum concentration and secreted proteins were identified by a LC-MS/MS proteomics approach. Altogether, 62 and 26 proteins were identified as secreted by M. hyopneumoniae and M. flocculare, respectively, being just seven proteins shared between these bacteria. In M. hyopneumoniae secretome, 15 proteins described as virulence factors were found; while four putative virulence factors were identified in M. flocculare secretome. For the first time, clear differences related to virulence were found between these species, helping to elucidate the pathogenic nature of M. hyopneumoniae to swine hosts. BIOLOGICAL SIGNIFICANCE: For the first time, the secretomes of two porcine respiratory mycoplasmas, namely the pathogenic M. hyopneumoniae and the commensal M. flocculare were compared. The presented results revealed previously unknown differences between these two genetically related species, some of which are associated to the M. hyopneumoniae ability to cause porcine enzootic pneumonia.

Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

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.

Protective effect of high alkalinity against the deleterious effects of chronic waterborne cadmium exposure on the detection of alarm cues by juvenile silver catfish (Rhamdia quelen).

The objective of the present study was to analyze the effect of chronic cadmium (Cd) exposure at two alkalinity levels (63 and 92 mg l(-1) CaCO(3)) on the antipredatory behavior of juvenile silver catfish (Rhamdia quelen) exposed to conspecific skin extract and predator odor. At an alkalinity of 63 mg l(-1) CaCO(3), 30 days of exposure to either 4.5 or 8.0 microg l(-1) Cd impaired the catfish's antipredatory response to alarm cues. However, silver catfish exposed to 4.5 microg l(-1) Cd at an alkalinity of 92 mg l(-1) CaCO(3) responded to skin extract and predator odor. In catfish exposed to 8.0 microg l(-1) Cd at the same alkalinity, only the number of feeding bites decreased, and this occurred only for specimens exposed to predator odor. Our results show that higher alkalinity protected against the deleterious effects of Cd on alarm cue detection but only in the larvae exposed to the lowest waterborne Cd level.