calpain and Schistosomiasis

Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1).. Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host-parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion.. SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Topics: Animals; Antigens, Helminth; Calpain; Coumarins; Cysteine Proteases; Female; Immunization; Male; Mice; Mice, Inbred ICR; Oligopeptides; Schistosoma; Schistosomiasis; Sequence Analysis, DNA

Exosomes are small vesicles of endocytic origin, which are released into the extracellular environment and mediate a variety of physiological and pathological conditions. Here we show that Schistosoma mansoni releases exosome-like vesicles in vitro. Vesicles were purified from culture medium by sucrose gradient fractionation and fractions containing vesicles verified by western blot analyses and electron microscopy. Proteomic analyses of exosomal contents unveiled 130 schistosome proteins. Among these proteins are common exosomal markers such as heat shock proteins, energy-generating enzymes, cytoskeletal proteins, and others. In addition, the schistosome extracellular vesicles contain proteins of potential importance for host-parasite interaction, notably peptidases, signaling proteins, cell adhesion proteins (e.g., integrins) and previously described vaccine candidates, including glutathione-S-transferase (GST), tetraspanin (TSP-2) and calpain. S. mansoni exosomes also contain 143 microRNAs (miRNA), of which 25 are present at high levels, including miRNAs detected in sera of infected hosts. Quantitative PCR analysis confirmed the presence of schistosome-derived miRNAs in exosomes purified from infected mouse sera. The results provide evidence of vesicle-mediated secretion in these parasites and suggest that schistosome-derived exosomes could play important roles in host-parasite interactions and could be a useful tool in the development of vaccines and therapeutics.

Topics: Animals; Calpain; Exosomes; Female; Glutathione Transferase; Humans; Mice; Proteomics; Schistosoma mansoni; Schistosomiasis; Tetraspanins; Transport Vesicles; Vaccines

Schistosomes are blood dwelling parasitic worms that cause the debilitating disease schistosomiasis. Here we examined the influence of the parasites on their external environment by monitoring the impact of adult Schistosoma mansoni worms on the murine plasma proteome in vitro and, in particular, on how the worms affect the blood coagulation protein high molecular weight kininogen (HK).. Following the incubation of adult schistosomes in murine plasma, two-dimensional differential in-gel electrophoresis (2D-DIGE) was conducted to look for changes in the plasma proteome compared with control plasma. A major change to the blood protein kininogen (HK) was observed, and the interaction of Schistosoma mansoni parasite with this protein alone was then investigated by western blot analysis and activity assays. Finally, the generation of bradykinin from HK was monitored using a bradykinin detection kit.. The most striking change to the plasma proteome concerned HK; while the full-length protein was more abundant in control plasma, carboxyl-terminal truncated forms were more abundant in plasma that contained schistosomes. Incubating parasites in buffer with pure HK followed by Western blot analysis confirmed that human HK is degraded by the worms. The resulting digestion pattern differed from that brought about by kallikrein, a host serine protease that normally acts on HK to release the vasodilator bradykinin. We found that live schistosomes, while digesting HK, do not generate bradykinin nor do they cleave a chromogenic kallikrein substrate. Since the cleavage of HK by the worms is not impeded by the serine protease inhibitor PMSF but is blocked by the cysteine protease inhibitor E64c, we hypothesize that schistosome tegumental cysteine proteases are responsible for HK cleavage.. Since proteomic and biochemical studies have revealed that the schistosome tegument contains two cysteine proteases belonging to the calpain family (SmCalp1 and SmCalp2) we conclude that these are likely responsible for the HK cleavage reported here. Schistosome cleavage of HK should help impede blood clotting and inflammation around the worms in vivo and so promote their ease of movement within the vasculature of their hosts.

Topics: Animals; Blood Coagulation; Blotting, Western; Bradykinin; Calpain; Cysteine Proteases; Electrophoresis, Gel, Two-Dimensional; Humans; Kininogen, High-Molecular-Weight; Mice; Molecular Weight; Proteomics; Schistosoma mansoni; Schistosomiasis

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.

Topics: Amino Acid Sequence; Animals; Antibodies, Helminth; Base Sequence; Blotting, Northern; Calpain; Cloning, Molecular; DNA, Helminth; Electrophoresis, Agar Gel; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Humans; Immunization; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA, Helminth; Schistosoma japonicum; Schistosomiasis; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid

Schistosomiasis is a parasitic disease affecting over 200 million people every year in tropical regions of the world. Drug treatment and other existing control measures are costly and have failed to eliminate the incidence of infection, morbidity and mortality due to Schistosoma mansoni infection. Vaccination of susceptible individuals using recombinant vaccines encoding key S. mansoni antigens may be the most effective and least expensive means of controlling schistosomiasis. A candidate vaccine antigen is p80, the large subunit of the S. mansoni protease, calpain. In our vaccine studies, we have employed both the wild-type p80 and a mutant p80 (mut p80) in which an active site amino acid was genetically altered to create a less proteolytically-active enzyme. Two vaccine delivery approaches were implemented using p80 or mut p80 as vaccine antigen: recombinant vaccinia virus (RVV) inoculation and DNA immunization via the Accell gene gun (GG) delivery system. RVV's expressing p80 and mut p80 were generated and tested for recombinant protein expression in vitro. These RVV's were tested for protective capacity in mouse challenge studies. Neither subcutaneous nor intranasal vaccinations with RVV-p80 or RVV-mut p80 were capable of significantly reducing the mean worm burdens of vaccinated mice. A GG-RVV combination immunization regime using WRG-vectors encoding p80 and mut p80 for GG priming and the RVV's for boosting prior to S. mansoni challenge infection was performed and no significant protection was obtained over two repeated studies. However, duplicate challenge studies involving GG immunization of mice with WRG-vectors encoding p80 or mut p80 revealed that 3 inoculations of mice with WRG-full5' mut p80 (containing the full 5' untranslated region of mut p80) provided 60% protection which was statistically significant (p < 0.05). These preliminary in vivo studies demonstrate the potential for further study of the protection afforded by gene gun-delivered WRG-full5' mut p80 into subsequently-challenged mice. Such studies may pave the way to effective vaccination of humans using WRG DNA vectors expressing a schistosomal calcium-activated neutral protease.

Topics: Animals; Antigens, Helminth; Biolistics; Calpain; Cell Line; Genetic Vectors; Mice; Schistosoma mansoni; Schistosomiasis; Vaccines, DNA; Vaccinia virus