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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.

Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphical Abstract

The inactivation of Schistosoma mansoni cercariae and miracidia was achieved by exposure to plasma produced by the positive, negative, and axial negative corona discharges. The positive discharge appeared as the most effective, causing the death of cercariae and miracidia within 2–3 min of exposure. The negative discharge was less effective, and the axial discharge was ineffective. The water pre-activated (PAW) by the discharges showed similar efficiency, with the exception of the significantly effective PAW activated with axial discharge. These facts, together with the observation of various reactions among plasma-damaged schistosomes, suggest that the mechanisms of inactivation by different types of discharges are different.

Background Human 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 presentation The 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. Conclusions In 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.

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.

Trichobilharzia regenti (Schistosomatidae, Digenea), a parasite of birds, exhibits a unique strategy among schistosomes, having affinity to the nervous system of vertebrate hosts. Migration of parasitic stages within hosts and/or swimming of non-parasitic larvae in water environment depend on the action of body wall muscles which were studied with confocal and electron microscopy. In all stages, body wall musculature is comprised of differently organized circular and longitudinal muscles. During the development, an extensive change of musculature characteristics and/or formation of new muscle structures were recorded; cercariae, schistosomula and adult worms produce additional underlying diagonal muscle fibers and inner plexus of radial musculature. Substantial changes of the outer environment during penetration of a host (osmotic values of water vs. host tissues) are accompanied by surface transformation of miracidia/mother sporocysts and cercariae/schistosomula. Contrary to that, changes of body musculature in these stages are characterized only by growth and re-organization of existing structures, and never by formation of new components of body musculature. Future studies in this field may contribute to a better knowledge of morphology and function of trematode muscles, including those of schistosomes that are important pathogens of humans and animals.

Background Serine 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 findings SmSP2 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/Significance The 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., Author summary Schistosomiasis (bilharziasis) is a global parasitic infection with more than 240 million people infected. It is caused by Schistosoma flatworms that live in the bloodstream. Current treatment relies on one drug, and no effective vaccine has yet been developed. Proteolytic enzymes (proteases) help the parasite survive in the mammalian host, allowing the schistosome to invade, feed, grow, reproduce and, manipulate the immune system. Thus, proteases are considered potential drug and vaccine targets. We previously described, and herein, investigate at the protein level, SmSP2, the major serine protease produced by the blood-dwelling stages of S. mansoni. We show that SmSP2 is secreted by the parasite and effectively processes host blood bioactive peptides and proteins that are involved in fibrinolysis and regulation of vascular tone. We propose, therefore, that SmSP2 modulates host hemostasis to promote parasite infection and survival. Thus, SmSP2 and similar proteins in other parasitic flatworms represent potential targets for novel drug or vaccine interventions.

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.

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.

The terminal phase of the migration of Trichobilharzia regenti Horák, Kolárová et Dvorák, 1998 in the definitive host (Anas platyrhynchos f. domestica) was studied 12-27 days post infection (p.i.). Brain meninges were the last part of the nervous system where the worms were detected before their occurrence in the nasal cavity. In meninges, the parasites started to feed on red blood cells. Then the worms occurred in the nasal mucosa 14-25 days p.i. and the first immature eggs appeared 15 days p.i. The fully developed miracidia were recorded in the eggs from 17 days p.i. and freely in the nasal mucosa 19 days p.i. Infiltrates of lymphocytes, later also eosinophils and heterophils around the eggs and free miracidia, were observed from 15 and 19 days p.i., respectively. The haemorrhages occurring from 17 days p.i., and the granulomas with lymphocytes, eosinophils and heterophils forming around the eggs from 22 days p.i. were the most apparent pathological changes of nasal tissue.