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.

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.