BackgroundSerine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis.Methodology/Principal findingsSmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting.Conclusions/SignificanceThe data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.

BackgroundHuman dirofilariasis is a zoonotic infection that continues to spread to previously unaffected areas of Europe. In the South Moravian Region of the Czech Republic (CR), imported as well as autochthonous canine infections were recorded in the last decade, and parasite DNA was detected in mosquitoes of Aedes vexans. In the present paper, human Dirofilaria infections are reported from the country for the first time.Case presentationThe samples from five patients with suspected tissue helminthiases were investigated. In particular cases, nematodes were isolated from various tissues including skin of lower leg, soft tissues of finger, subcutaneous tissue of hypogastrium, lymph node and peritoneum. The diagnosis was based on light microscopic morphology and/or DNA analysis of the worms. In addition, ELISA examination of patients’ sera for anti-filaria IgG antibodies was performed.ConclusionsIn the CR, five cases of human dirofilariasis caused by Dirofilaria repens were recorded during 2010–2014 (species determination for three of them was confirmed besides morphological also by DNA analysis). At least, three of the cases were of autochthonous origin (the patients are Czech citizens residing in South Moravian Region who have never travelled abroad). The findings confirm the natural setting of D. repens in South Moravian Region of the CR. Dirofilariasis should be therefore considered as endemic in this area where it may represent a significant risk factor for public health.

Abstract. The terminal phase

The passage of Trichobilharzia szidati schistosomula through the vertebrate lungs was examined in natural and abnormal hosts--birds (ducks Anas platyrhynchos f. domestica) and mammals (mice Mus musculus Bagg albino/c [BALB/c]), respectively. Using the methods of classical histology, the migratory route of worms was characterized, and the impact of migration on host tissues and the host cell reactions were evaluated. Living schistosomula were recorded in the lungs of ducks 2-10 days post infection (p.i.) and in the lungs of mice 2-4 days p.i. In ducks, the schistosomula migrated from the blood vessels through the blood capillaries to the lung tissue; then, they entered free air space of the lungs. The infection resulted in inflammatory reaction with nodules composed of infiltrated lymphocytes, heterophils, eosinophils and macrophages. These structures were formed around the blood vessels and in the gas-exchange tissues of the parabronchial walls and, consequently, in the walls of secondary bronchi. An extensive inflammation of secondary bronchi and parabronchi was observed. In the lungs of mice, the parasites were localized extravascularly in the alveolar walls. No migratory pattern similar to that in the lungs of ducks was recorded. No specific inflammatory reaction occurred. However, alveolar wall congestion, edema and lymphocyte infiltrates appeared and, therefore, pathogenicity of T. szidati was also confirmed in the murine host.

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E-to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

Cercariae of bird schistosomes (Trichobilharzia szidati and Trichobilharzia regenti) were mechanically stimulated to transform to schistosomula and kept in different cultivation media supplemented with duck red blood cells and/or homogenized nervous tissue. The development under in vitro conditions was compared with that in vivo, using the following characters: emptying of penetration glands, surface changes, food uptake, and growth of early schistosomula. The results show that the cultivation medium routinely used for human schistosomes is also suitable for mass production of early schistosomula of bird schistosomes, including the unique nasal species-T. regenti. The changes observed resemble those present in worms developing in vivo; therefore, the in vitro produced early schistosomula might be used for further studies of host-parasite interactions.

No abstract

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.