Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Proteases and their inhibitors involved in Schistosoma mansoni egg-host interaction revealed by comparative transcriptomics with Fasciola hepatica eggs.

Schistosoma mansoni eggs are the main causative agents of the pathological manifestations of schistosomiasis. The eggs are laid in the host bloodstream, then they migrate through the intestinal wall into the lumen. However, a significant proportion of the eggs become lodged in the liver, where they cause inflammation and fibrosis. In this study, we focus on a specific group of proteins expressed by the egg, namely proteases and their inhibitors. These molecules are often involved in schistosome-host interactions, but are still unexplored in the egg stage. Using RNA-seq and comparative transcriptomics of immature and mature S. mansoni eggs, we mapped the portfolio of proteases and their inhibitors, and determined their gene expression levels. In addition, we compared these data with gene expression of proteases and their inhibitors in Fasciola hepatica eggs. Fasciola hepatica eggs served as a useful comparative model, as they do not migrate through tissues and inflict pathology. We detected transcription of 135 and 117 proteases in S. mansoni and F. hepatica eggs, respectively, with 87 identified as orthologous between the two species. In contrast, we observed only four orthologous inhibitors out of 21 and 16 identified in S. mansoni and F. hepatica eggs, respectively. Among others, we measured high and developmentally regulated levels of expression of metalloproteases in S. mansoni eggs, specifically aminopeptidase N1, endothelin-converting enzyme 1, and several leishmanolysin-like peptidases. We identified highly transcribed protease inhibitors serpin and alpha-2-macroglobulin that are unique to S. mansoni eggs, and antistasin-like inhibitor in F. hepatica eggs. This study provides new insights into the portfolio of proteases and inhibitors expressed by S. mansoni with potential roles in egg tissue migration, stimulation of angiogenesis, and interaction with host blood and immunity.

A literature review of schistosomiasis in Ghana: a reference for bridging the research and control gap.

Schistosomiasis is endemic in most sub-Saharan African countries, including Ghana, where the need for effective control involving preventive chemotherapy was indicated by the WHO. Mass drug administration commenced in 2008 and has continued since then in Ghana, but the country remains highly endemic. Here, we review the literature on schistosomiasis to identify research and knowledge gaps potentially affecting disease control. A total of 100 Ghana-related schistosomiasis literature sources were reviewed, showing that most studies were conducted on epidemiology, control of transmission and diagnosis. By contrast, many aspects of this disease remain neglected, including livestock schistosomiasis and its zoonotic potential, recent distribution of disease vectors or widely overlooked genital schistosomiasis. Stratified by region, the highest number of studies focus on Greater Accra, while studies are limited or absent for several other regions. Although this review shows apparent progress in terms of schistosomiasis research and control, a considerable amount of work remains to achieve at least a reduction in the prevalence of the disease, which affects a significant proportion of the population. National epidemiological data based on a nationwide survey, integrated control and improved monitoring and evaluation must be ensured.

Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization.

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.

Azanitrile Inhibitors of the SmCB1 Protease Target Are Lethal to Schistosoma mansoni: Structural and Mechanistic Insights into Chemotype Reactivity.

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.

Inactivation of Schistosoma Using Low-Temperature Plasma.

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.

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

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.

Dirofilaria repens: emergence of autochthonous human infections in the Czech Republic (case reports).

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.

Trichobilharzia regenti (Digenea: Schistosomatidae): changes of body wall musculature during the development from miracidium to adult worm.

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.

In vitro cultivation of early schistosomula of nasal and visceral bird schistosomes (Trichobilharzia spp., Schistosomatidae).

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.

Terminal phase of bird schistosomiasis caused by Trichobilharzia regenti (Schistosomatidae) in ducks (Anas platyrhynchos f. domestica).

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.