Martin Kašný

Peptidases of trematodes.

Among human and veterinary parasitic diseases the trematodiases (e.g. schistosomiasis, fascioliasis) represent a problem of global importance with vast social, economic and public health impacts, especially in developing countries. Therefore, host-parasite (host-trematode) interactions represent a key topic in many research laboratories, and modern approaches and technologies allow us to study the molecular basis of these interactions. As a consequence, key molecules produced by trematodes in order to ensure parasite invasion and survival within a hosts can be characterized. Trematode peptidases certainly belong to such molecules; as they are indispensable biocatalysts in a number of basal biological processes in trematodes (e.g. tissue invasion/migration, nutrition, immune evasion or other host-parasite interactions). Schistosoma mansoni cercarial elastase (CE) (penetration enzyme), cathepsin B (CB) (mainly nutrition enzyme) and Fasciola hepatica cathepsin L (CL) (nutrition, immune evasion enzyme) are probably the most studied trematode peptidases with well-characterized critical functions. Due to the importance of peptidases in host-parasite interactions they are considered to be promising targets for the development of novel chemotherapeutic drugs and vaccines against a number of trematodiases, including schistosomiasis, fascioliasis, paragonimiasis and opisthorchiasis. The present chapter summarizes the data on the biochemical and molecular features of the major trematode peptidases, and describes their role in trematode biology and host-parasite interactions based on proteolysis (peptidolysis).

Comparison of cysteine peptidase activities in Trichobilharzia regenti and Schistosoma mansoni cercariae.

Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin.

The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S2 subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

Radix spp.: Identification of trematode intermediate hosts in the Czech Republic

Lymnaeid snails of the genus Radix serve as intermediate hosts of some schistosomes and fasciolids. In Europe, delineation of species within the genus Radix is unresolved and, therefore, spectrum of snail hosts susceptible to trematode infections is under discussion. We used and compared three criteria for species delineation using snails collected at 43 localities. (a) Sequence analysis of ITS-2 rDNA disclosed that the collected snails belong to four species — R. auricularia (Linnaeus, 1758), R. peregra (Müller, 1774), R. lagotis (Schrank, 1803) and R. labiata (Rossmaessler, 1835) (criteria and names are based on the work of Bargues et al. 2001). Occurrence of R. peregra in the Czech Republic was confirmed by molecular data for the first time. (b) Characterization of reproductive system disclosed differences in location, size and shape of bursa copulatrix and its ductus. Unfortunately, some R. labiata specimens shared morphological features of reproductive organs with R. lagotis. (c) Statistical analysis of shell morphology proved that significant differences exist among particular species. One prediction model showed that correct classification of species may be achieved in 82-84% of cases. However, identification of individual snails in the field (without knowledge of respective snail population and use of statistical tools) still remains a complicated issue due to overlaps of shell characteristics. Concerning the role in trematode transmission, R. lagotis, R. labiata and R. peregra are susceptible to Trichobilharzia regenti. Also, successful experimental infections of R. lagotis and R. labiata by Fascioloides magna were accomplished.

Antibody responses induced by Trichobilharzia regenti antigens in murine and human hosts exhibiting cercarial dermatitis.

Cercariae of bird schistosomes (genus Trichobilharzia) are able to penetrate the skin of mammals (noncompatible hosts), including humans, and cause a Th2‐associated inflammatory cutaneous reaction termed cercarial dermatitis. The present study measured the antibody reactivity and antigen specificity of sera obtained after experimental infection of mice and natural infection of humans. Sera from mice re‐infected with T. regenti showed a bias towards the development of antigen‐specific IgM and IgG1 antibodies and elevated levels of total serum IgE, indicative of a Th2 polarized immune response. We also demonstrate that cercariae are a source of antigens triggering IL‐4 release from basophils collected from healthy human volunteers. Analysis of sera from patients with a history of cercarial dermatitis revealed elevated levels of cercarial‐specific IgG, particularly for samples collected from adults (> 14 years old) comparedwith children (8–14 years old), although elevated levels of antigen‐specific IgE were not detected. In terms of antigen recognition, IgG and IgE antibodies in the sera of both mice and humans preferentially bound an antigen of 34 kDa. The 34 kDa molecule was present in both homogenate of cercariae, as well as cercarial excretory/secretory products, and we speculate it may represent a major immunogen initiating the Th2‐immune response associated with cercarial dermatitis.

Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna

Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rDNA, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna.

Secretory glands in cercaria of the neuropathogenic schistosome Trichobilharzia regenti - ultrastructural characterization, 3-D modelling, volume and pH estimations.

BackgroundCercariae of schistosomes employ bioactive molecules for penetration into their hosts. These are released from specialized unicellular glands upon stimuli from host skin. The glands were previously well-described in the human pathogen Schistosoma mansoni. As bird schistosomes can also penetrate human skin and cause cercarial dermatitis, our aim was to characterize the architecture and ultrastructure of glands in the neurotropic bird schistosome Trichobilharzia regenti and compare it with S. mansoni. In the context of different histolytic enzymes used by these two species, we focused also on the estimations of gland volumes and pH in T. regenti.ResultsThe architecture and 3-D models of two types of acetabular penetration glands, their ducts and of the head gland are shown here. We characterized secretory vesicles in all three gland types by means of TEM and confirmed accuracy of the models obtained by confocal microscopy. The results of two independent approaches showed that the glands occupy ca. one third of cercarial body volume (postacetabular glands ca. 15%, circumacetabular 12% and head gland 6%). The inner environment within the two types of acetabular glands differed significantly as evidenced by dissimilar ability to bind fluorescent markers and by pH value which was higher in circumacetabular (7.44) than in postacetabular (7.08) glands.ConclusionsAs far as we know, this is the first presentation of a 3-D model of cercarial glands and the first exact estimation of the volumes of the three gland types in schistosomes. Our comparisons between T. regenti and S. mansoni implied that the architecture and ultrastructure of the glands is most likely conserved within the family. Only minor variations were found between the two species. It seems that the differences in molecular composition have no effect on general appearance of the secretory cells in TEM. Fluorescent markers employed in this study, distinguishing between secretory vesicles and gland types, can be useful in further studies of mechanisms used by cercariae for host invasion. Results of the first attempts to estimate pH within schistosome glands may help further understanding of regulation of enzymatic activities present within the glands.

Comparative Transcriptomic Exploration Reveals Unique Molecular Adaptations of Neuropathogenic Trichobilharzia to Invade and Parasitize Its Avian Definitive Host

To date, most molecular investigations of schistosomatids have focused principally on blood flukes (schistosomes) of humans. Despite the clinical importance of cercarial dermatitis in humans caused by Trichobilharzia regenti and the serious neuropathologic disease that this parasite causes in its permissive avian hosts and accidental mammalian hosts, almost nothing is known about the molecular aspects of how this fluke invades its hosts, migrates in host tissues and how it interacts with its hosts’ immune system. Here, we explored selected aspects using a transcriptomic-bioinformatic approach. To do this, we sequenced, assembled and annotated the transcriptome representing two consecutive life stages (cercariae and schistosomula) of T. regenti involved in the first phases of infection of the avian host. We identified key biological and metabolic pathways specific to each of these two developmental stages and also undertook comparative analyses using data available for taxonomically related blood flukes of the genus Schistosoma. Detailed comparative analyses revealed the unique involvement of carbohydrate metabolism, translation and amino acid metabolism, and calcium in T. regenti cercariae during their invasion and in growth and development, as well as the roles of cell adhesion molecules, microaerobic metabolism (citrate cycle and oxidative phosphorylation), peptidases (cathepsins) and other histolytic and lysozomal proteins in schistosomula during their particular migration in neural tissues of the avian host. In conclusion, the present transcriptomic exploration provides new and significant insights into the molecular biology of T. regenti, which should underpin future genomic and proteomic investigations of T. regenti and, importantly, provides a useful starting point for a range of comparative studies of schistosomatids and other trematodes.

Ultrastructure of the digestive tract of Paradiplozoon homoion (Monogenea)

Paradiplozoon homoion is a representative of blood-feeding ectoparasites from the family Diplozoidae (Polyopisthocotylea, Monogenea). Although these worms have been the subject of numerous taxonomical, phylogenetic and ecological studies, the ultrastructure of the alimentary system and related structures, as well as the mechanisms of essential processes like fish blood digestion, remain mostly unknown. Our observation of P. homoion using a transmission electron microscopy (TEM) revealed two main types of digestive cells—U-shaped haematin cells and connecting syncytium. Particular structures such as mouth cavity with specialised receptors, two oval-shaped muscular buccal suckers, pharynx surrounded with the glandular cells, oesophagus, the intestinal caeca with intact erythrocytes in the lumen, the apical pinocytotic fibrous surface complex and haematin vesicles of U-shaped cells have been shown in detail. According to our results, the P. homoion is degrading the blood components predominantly intracellularly.

Non-native gobies facilitate the transmission of Bucephalus polymorphus (Trematoda)

BackgroundIntroduced species can modify local host-parasite dynamics by amplifying parasite infection which can ‘spill-back’ to the native fauna, whether they are competent hosts for local parasites, or by acting as parasite sinks with ‘dilution’ of infection decreasing the parasite burden of native hosts. Recently infection by the trematode Bucephalus polymorphus has increased in several European rivers, being attributed to the introduction of intermediate host species from the Ponto-Caspian region. Using a combination of field and experimental data, we evaluated the competence of non-native and native fish as intermediate hosts for B. polymorphus and its role for parasite development in a definitive host.MethodsThe density of 0+ juvenile fish (the second intermediate hosts for B. polymorphus) was measured in the River Morava, Czech Republic and fish were screened for natural metacercariae infection. The stomach contents of predatory fish that are definitive hosts of B. polymorphus were examined to assess the importance of non-native gobies for parasite transmission. In semi-natural conditions, parasite establishment, initial survival, and maturity rates in experimentally infected definitive hosts pikeperch Sander lucioperca were measured in flukes recovered from native white bream Abramis bjoerkna and non-native tubenose goby Proterorhinus semilunaris and round goby Neogobius melanostomus. Adult fluke size and egg production was also measured to evaluate the potential effect of intermediate host species on parasite fitness.ResultsWe detected high natural infection parameters of B. polymorphus in native cyprinids and non-native gobies compared to data from the period prior to goby establishment. Both fish groups are consumed by predatory fish and represent a major component of the littoral fish community. Parasite establishment and adult size in definitive hosts was equivalent among the second intermediate host species, despite a lower size of metacercariae recovered from round gobies. However, development in the definitive host of flukes recovered from gobies was reduced, showing higher mortality, delayed maturity and lower egg production, in comparison with parasites from native hosts.ConclusionsSubstantial ‘spill-back’ of B. polymorphus due to higher transmission rates after establishment of non-native gobies was partially buffered by decreased fitness of B. polymorphus that underwent development in gobies.