A Family of Diverse Kunitz Inhibitors from Echinococcus granulosus Potentially Involved in Host-Parasite Cross-Talk (original) (raw)
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We previously reported a multigene family of monodomain Kunitz proteins from Echinococ-cus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close para-logs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (K v); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.
Microbes and infection, 2018
Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases.
Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family
Biochemical Journal, 2003
Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38kDa subunits. Whereas the 22kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residu...
2012
Background: The cestode Echinococcus granulosus-the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide-is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages. Methodology/Principal Findings: We generated ,10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H +-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol-and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissuedwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development. Conclusions/Significance: This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths.
Proteomics, 2003
We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.
A gene family from Echinococcus granulosus differentially expressed in mature adult worms
Molecular and Biochemical Parasitology, 2003
Differences in mRNA expression between immature adult worms (IAW) and mature adult worms (MAW) of Echinococcus granulosus were determined using polymerase chain reaction-based differential display (DDRT-PCR). Twenty-eight putative differential cDNA fragments were isolated, cloned and sequenced. mRNAs from IAW and MAW were probed with the labelled fragments. Six cDNA fragments (coded as egM12, egM13, egM22, egM26, egM30 and egM34) were putatively determined to be specific to MAW by Northern hybridisation. The stage-specificity of egM12, egM13 and egM34 was confirmed by RT-PCR. RNAs of IAW, MAW, protoscoleces and oncospheres, probed with egM13 and egM30, showed that the mRNAs were expressed exclusively in MAW, which implied involvement in the regulation of egg development. Using the labelled fragments to screen a cDNA library of MAW, 99 clones were identified and analysed. An alignment of selected clones showed that the MAW-specific mRNAs belonged to a family. Examination of the deduced amino acid sequence of three of the corresponding cDNAs (egM4, egM9 and egM123) indicated they were cysteine-rich and contained a 24 amino acid repeat sequence, repeated four to six times. The repeat regions were predominantly alpha helical in nature with interspersed turns, forming alternating zones of positive and negative charge. The functional significance of each of the cDNAs identified is unclear as none had significant sequence similarity to genes of known function. However, polypeptides encoded by egM4 and egM123 were recognised by antibodies in a serum pool from dogs experimentally infected with E. granulosus, suggesting they could prove of value in serodiagnosis of definitive hosts.
Parasites & Vectors, 2016
Background: Echinostoma caproni is an intestinal trematode extensively used as experimental model for the study of factors that determine the course of intestinal helminth infections, since this markedly depends on the host species. Although the host-dependent mechanisms for either chronic establishment or early parasite rejection have been broadly studied, little is known regarding the parasite response against different host environments. Methods: To identify host-dependent differentially expressed proteins, a comparative proteomic analysis of the excretory/secretory products released from E. caproni adults, isolated from hosts displaying different compatibility with this trematode, was performed. Results: A total of 19 differential protein spots were identified (14 overexpressed in mice and 5 overexpressed in rats). The establishment of chronic infections in mice is mainly associated with the overexpression by adult worms of antioxidant and detoxifying enzymes (e.g. glutathione S-transferase, hydroxyacylglutathione hydrolase, thiopurine S-transferase, etc.) and metabolic enzymes like enolase, leucine aminopeptidase or malate dehydrogenase. However, the overexpression of cathepsin L and the structural protein actin observed in worms isolated from rats seems not to be effective for the colonization of the intestinal mucosa of this host. Conclusions: The observed differences suggest that protein expression and/or release is modulated by the local environment generated inside the host and provide useful insights in regards to the resistance mechanisms developed by parasites to ensure their long-term survival.
Journal of proteome research, 2015
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...
Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage
Journal of proteome research, 2015
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 an...