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Walter Stöcker - One of the best experts on this subject based on the ideXlab platform.
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The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of Astacin metallopeptidases
Scientific Reports, 2019Co-Authors: Tibisay Guevara, Carlo Schmitz, Walter Stöcker, Irene Yiallouros, Michael Kuske, Hagen Körschgen, Anna Cuppari, Julia Floehr, Willi Jahnen-dechent, F. Xavier Gomis-rüthAbstract:Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovAstacin, a member of the Astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a “CPDCP-trunk” and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal Astacin from crayfish, which is a useful model of human ovAstacin. Two hairpins from CY2, the linker, and the tip of the “legumain-binding loop” of CY1 inhibit crayfish Astacin following the “raised-elephant-trunk mechanism” recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish Astacin and revealed that the cleaved human inhibitor blocks crayfish Astacin and human meprin α and β only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.
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functional and structural insights into Astacin metallopeptidases
Biological Chemistry, 2012Co-Authors: Xavier F Gomisruth, Sergio Trillomuyo, Walter StöckerAbstract:The Astacins are a family of multi-domain metallopeptidases with manifold functions in metabolism. They are either secreted or membrane-anchored and are regulated by being synthesized as inactive zymogens and also by colocalizing protein inhibitors. The distinct family members consist of N-terminal signal peptides and pro-segments, zincdependent catalytic domains, further downstream extracellular domains, transmembrane anchors, and cytosolic domains. The catalytic domains of four Astacins and the zymogen of one of these have been structurally characterized and shown to comprise compact ∼ 200-residue zinc-dependent moieties divided into an N-terminal and a C-terminal sub-domain by an active-site cleft. Astacins include an extended zinc-binding motif (HEXXHXXGXXH) which includes three metal ligands and groups them into the metzincin clan of metallopeptidases. In mature, unbound Astacins, a conserved tyrosine acts as an additional zinc ligand, which is swung out upon substrate or inhibitor binding in a ‘ tyrosine switch ’ motion. Other characteristic structural elements of Astacin catalytic domains are three large α -helices and a fi ve-stranded β -sheet, as well as two or three disulfi de bonds. The N-terminal pro-segments are variable in length and rather unstructured. They inhibit the catalytic zinc following an ‘ aspartate-switch ’ mechanism mediated by an aspartate embedded in a conserved motif (FXGD). Removal of the pro-segment uncovers a deep and extended active-site cleft, which in general shows preference for aspartate residues in the specifi city pocket (S 1 ′ ). Furthermore, Astacins undergo major rearrangement upon activation within an ‘ activation domain, ’ and show a slight hinge movement when binding substrates or inhibitors. In this review, we discuss the overall architecture of Astacin catalytic domains and their involvement in function and zymogenic activation.
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the metzincins topological and sequential relations between the Astacins adamalysins serralysins and matrixins collagenases define a superfamily of zinc peptidases
Protein Science, 2008Co-Authors: Walter Stöcker, F X Gomisruth, Ulrich Baumann, Peter Reinemer, Frank Grams, David B Mckay, Wolfram BodeAbstract:The three-dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin II from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and Astacin from crayfish are topologically similar, with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable "Met-turn." This structural relationship is supported by a sequence alignment performed on the basis of topological equivalence showing faint but distinct sequential similarity. The alkaline proteinase is about equally distant (26% sequence identity) to both human neutrophil collagenase and Astacin and a little further away from adamalysin II (17% identity). The pairs Astacin/adamalysin II, Astacin/human neutrophil collagenase, and adamalysin II/human neutrophil collagenase exhibit sequence identities of 16%, 14%, and 13%, respectively. Therefore, the corresponding four distinct families of zinc peptidases, the Astacins, the matrix metalloproteinases (matrixins, collagenases), the adamalysins/reprolysins (snake venom proteinases/reproductive tract proteins), and the serralysins (large bacterial proteases from Serratia, Erwinia, and Pseudomonas) appear to have originated by divergent evolution from a common ancestor and form a superfamily of proteolytic enzymes for which the designation "metzincins" has been proposed. There is also a faint but significant structural relationship of the metzincins to the thermolysin-like enzymes, which share the truncated zinc-binding motif HEXXH and, moreover, similar topologies in their N-terminal domains.
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News from an ancient world: two novel Astacin metalloproteases from the horseshoe crab.
Journal of molecular biology, 2008Co-Authors: Christoph Becker-pauly, Bernd Cem Bruns, Olga Damm, André Schütte, Kada Hammouti, Thorsten Burmester, Walter StöckerAbstract:In this work, we report the cloning, heterologous expression, and characterization of two novel Astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus Astacin) and LAST_MAM (Limulus Astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and Astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphatase micro) domain, which so far only has been found in few Astacins such as the vertebrate meprin Hydra and squid enzymes, and in a number of other extracellular proteins such as A5 protein and tyrosine phosphatase micro. These gave rise to the designation MAM for this protein module. MAM domains have been shown to be responsible for protein oligomerization in meprin proteases and tyrosine phosphatase micro. Since the horseshoe crab has kept its body plan for almost half a billion years, it is therefore a privileged organism for the study of protease evolution. In this context, we could show by phylogenetic analysis that this protease is not related to the other MAM-domain-containing Astacins indicating different evolutionary origins of these proteins. Moreover, we clearly demonstrated the divergent evolvement of the MAM module itself, and not only with regard to proteases. However, there are some unique functional features that are not shared by other members of this protein family. For example, LAST_MAM is the only Astacin protease known so far that is active in its zymogen form, indicating that the presence of the N-terminal propeptide does not prevent proteolytic activity.
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meprins membrane bound and secreted Astacin metalloproteinases
Molecular Aspects of Medicine, 2008Co-Authors: Erwin E. Sterchi, Walter Stöcker, Judith S BondAbstract:The Astacins are a subfamily of the metzincin superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme Astacin. To date more than 200 members of this family have been identified in species ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus Astacin), the 'hatching' subfamily comprising alveolin, ovAstacin, LCE, HCE ('low' and 'high' choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins. In the human and mouse genomes, there are six Astacin family genes (two meprins, three BMP1/tolloid-like, one ovAstacin), but in Caenorhabditis elegans there are 40. Meprins are the only Astacin proteinases that function on the membrane and extracellularly by virtue of the fact that they can be membrane-bound or secreted. They are unique in their domain structure and covalent subunit dimerization, oligomerization propensities, and expression patterns. They are normally highly regulated at the transcriptional and post-translational levels, localize to specific membranes or extracellular spaces, and can hydrolyse biologically active peptides, cytokines, extracellular matrix (ECM) proteins and cell-surface proteins. The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions.
Adam Petrusek - One of the best experts on this subject based on the ideXlab platform.
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aphanomyces astaci genotypes involved in recent crayfish plague outbreaks in central italy
Diseases of Aquatic Organisms, 2018Co-Authors: Riccardo Caprioli, Agata Mrugala, Cesare Camma, Marco Di Domenico, Valentina Curini, Carla Giansante, Adam PetrusekAbstract:The oomycete Aphanomyces astaci is the causative agent of crayfish plague in native European freshwater crayfish. Molecular analyses showed that several distinct genotype groups of this pathogen, apparently associated with different original host taxa, are present in Europe. Tracking their distribution may contribute to understanding the introduction pathways of A. astaci. We used microsatellite markers to genotype the pathogen strains involved in 7 mass mortalities of the endangered indigenous crayfish Austropotamobius pallipes that occurred between 2009 and 2016 in the Abruzzi and Molise regions, central Italy. Three A. astaci genotype groups (A, B, and D, with the latter represented by 2 distinct multilocus genotypes) were identified, suggesting the existence of multiple infection sources even in a relatively small area. Most crayfish plague episodes were due to genotype groups associated with the North American host species Pacifastacus leniusculus and Procambarus clarkii, although these crayfish are not widespread in the study area. A. astaci genotype group A was detected not only in crayfish plague outbreaks but also in apparently healthy Astacus leptodactylus imported for human consumption from Armenia and kept alive in an aquaculture facility. Imports of chronically infected A. leptodactylus from Armenia, Turkey, and possibly Eastern Europe are an underestimated introduction pathway for A. astaci. Although we cannot exclude the presence of latently infected native populations of A. pallipes in the region, A. astaci infections in legally imported crayfish species considered vulnerable to crayfish plague may represent further reservoirs of A. astaci; this should be reflected in the policies regulating the trade of live crayfish.
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apparent interspecific transmission of aphanomyces astaci from invasive signal to virile crayfish in a sympatric wild population
Journal of Invertebrate Pathology, 2017Co-Authors: Joanna James, Agata Mrugala, Birgit Oidtmann, Adam Petrusek, Joanne CableAbstract:The crayfish plague pathogen (Aphanomyces astaci) causes mass mortalities of European crayfish when transmitted from its original North American crayfish hosts. Little is known, however, about interspecific transmission of the pathogen between different American crayfish species, although evidence from trade of ornamental crayfish suggests this may happen in captivity. We screened signal and virile crayfish for A. astaci at allopatric and sympatric sites in a UK river. Whilst the pathogen was detected in signal crayfish from both sites, infected virile crayfish were only found in sympatry. Genotyping of A. astaci from virile crayfish suggested the presence of a strain related to one infecting British signal crayfish. We conclude that virile crayfish likely contracted A. astaci interspecifically from infected signal crayfish. Interspecific transmission of A. astaci strains differing in virulence between American carrier species may influence the spread of this pathogen in open waters with potential exacerbated effects on native European crayfish.
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hosts and transmission of the crayfish plague pathogen aphanomyces astaci a review
Journal of Fish Diseases, 2017Co-Authors: Jiři Svoboda, Agata Mrugala, Eva Kozubikovabalcarova, Adam PetrusekAbstract:The crayfish plague pathogen, Aphanomyces astaci Schikora, has become one of the most well-studied pathogens of invertebrates. Since its introduction to Europe in the mid-19th century, it has caused mass crayfish mortalities, resulting in drastic declines of local populations. In contrast, North American crayfish usually serve as latent carriers, although they may also be negatively affected by A. astaci infections under some circumstances. Recent research benefiting from molecular tools has improved our knowledge about various aspects of A. astaci biology. In this review, we summarize these advances, particularly with respect to the host range and transmission. We highlight several aspects that have recently received particular attention, in particular newly confirmed or suspected A. astaci hosts, latent A. astaci infections in populations of European crayfish, and the relationship between A. astaci genotype groups and host taxa.
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resistance to the crayfish plague pathogen aphanomyces astaci in two freshwater shrimps
Journal of Invertebrate Pathology, 2014Co-Authors: Jiři Svoboda, Javier Dieguezuribeondo, Agata Mrugala, Eva Kozubikovabalcarova, Antonin Kouba, Adam PetrusekAbstract:Aphanomyces astaci, the causal agent of the crayfish plague, has recently been confirmed to infect also freshwater-inhabiting crabs. We experimentally tested the resistance of freshwater shrimps, another important decapod group inhabiting freshwaters, to this pathogen. We exposed individuals of two Asian shrimp species, Macrobrachium dayanum and Neocaridina davidi, to zoospores of the pathogen strain isolated from Procambarus clarkii, a known A. astaci carrier likely to get into contact with shrimps. The shrimps were kept in separate vessels up to seven weeks; exuviae and randomly chosen individuals were sampled throughout the experiment. Shrimp bodies and exuviae were tested for A. astaci presence by a species-specific quantitative PCR. The results were compared with amounts of A. astaci DNA in an inert substrate to distinguish potential pathogen growth in live specimens from persisting spores or environmental DNA attached to their surface. In contrast to susceptible crayfish Astacus astacus, we did not observe mortality of shrimps. The amount of detected pathogen DNA was decreasing steadily in the inert substrate, but it was still detectable several weeks after zoospore addition, which should be considered in studies relying on molecular detection of A. astaci. Probably due to moulting, the amount of A. astaci DNA was decreasing in N. davidi even faster than in the inert substrate. In contrast, high pathogen DNA levels were detected in some non-moulting individuals of M. dayanum, suggesting that A. astaci growth may be possible in tissues of this species. Further experiments are needed to test for the potential of long-term A. astaci persistence in freshwater shrimp populations.
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the crayfish plague pathogen can infect freshwater inhabiting crabs
Freshwater Biology, 2014Co-Authors: Jiři Svoboda, Trude Vrålstad, Frederic Grandjean, Antonin Kouba, David A Strand, Lennart Edsman, Pavel Kozak, Rosa F Fristad, Seval Bahadir Koca, Adam PetrusekAbstract:SUMMARY 1. The oomycete Aphanomyces astaci is generally considered a parasite specific to freshwater crayfish, and it has become known as the crayfish plague pathogen. Old experimental work that reported transmission of crayfish plague to the Chinese mitten crab Eriocheir sinensis, and the ability of A. astaci to grow in non-decapod crustaceans, has never been tested properly. 2. We re-evaluated the host range of A. astaci by screening for the presence of A. astaci in two crab species cohabiting with infected crayfish in fresh waters, as well as in other higher crustaceans from such localities. The animals were tested with species-specific quantitative PCR, and the pathogen determination was confirmed by sequencing of an amplified fragment of the nuclear internal transcribed spacer. Furthermore, we examined microscopically cuticle samples from presumably infected crab individuals for the presence of A. astaci-like hyphae and checked for the presence of pathogen DNA in such samples. 3. Screenings of benthopelagic mysids, amphipods and benthic isopods did not suggest infection by A. astaci in non-decapod crustaceans. In contrast, both studied lake populations of crabs (a native semiterrestrial species Potamon potamios in Turkey, and an invasive catadromous E. sinensis in Sweden) were infected with this parasite according to both molecular and microscopic evidence. 4. Analyses of polymorphic microsatellite loci demonstrated that A. astaci strains in the crabs and in cohabiting crayfish belonged to the same genotype group, suggesting crayfish as the source for crab infection. 5. The potential for A. astaci transmission in the opposite direction, from crabs to crayfish, and potential impact of this pathogen on populations of freshwater crabs requires further investigations, because of possible consequences for crayfish and freshwater crab conservation and aquaculture.
Harri Kokko - One of the best experts on this subject based on the ideXlab platform.
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controlled infection experiment with aphanomyces astaci provides additional evidence for latent infections and resistance in freshwater crayfish
Frontiers in Ecology and Evolution, 2021Co-Authors: Caterina Francesconi, Harri Kokko, Japo Jussila, Jenny Makkonen, Anne Schrimpf, Kathrin TheissingerAbstract:For 150 years the crayfish plague disease agent Aphanomyces astaci has been the cause of mass mortalities among native European crayfish populations. However, recently several studies have highlighted the great variability of A. astaci virulence and crayfish resistance towards the disease. The main aim of this study was to compare the response of two crayfish species, the European native noble crayfish (Astacus astacus) and the invasive alien marbled crayfish (Procambarus virginalis), to an A. astaci challenge with a highly virulent strain from haplogroup B and a lowly virulent strain from haplogroup A. In a controlled infection experiment we showed a high resistance of marbled crayfish against an A. astaci infection, with zoospores from the highly virulent haplogroup B strain being able to infect the crayfish, but unable to cause signs of disease. Furthermore, we demonstrated a reduced virulence in the A. astaci strain belonging to haplogroup A, as shown by the light symptoms and the lack of mortality in the generally susceptible noble crayfish. Interestingly, in both marbled crayfish and noble crayfish challenged with this strain, we observed a significant decrease of the detected amount of pathogen’s DNA during the experiment, suggesting that this A. astaci haplogroup A strain has a decreased ability of penetrating into the cuticle of the crayfish. Our results provide additional evidence of how drastically strains belonging to A. astaci haplogroup B and haplogroup A differ in their virulence. This study confirmed the adaptation of one specific A. astaci haplogroup A strain to their novel European hosts, supposedly due to reduced virulence. This feature might be the consequence of A. astaci´s reduced ability to penetrate into the crayfish. Finally, we experimentally showed that marbled crayfish are remarkably resistant against the crayfish plague disease and could potentially be latently infected, acting as carriers of highly virulent A. astaci strains.
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aphanomyces astaci isolate from latently infected stone crayfish austropotamobius torrentium population is virulent
Journal of Invertebrate Pathology, 2017Co-Authors: Japo Jussila, Jenny Makkonen, Al Vrezec, Tina Jaklic, Hobo Kukkonen, Harri KokkoAbstract:Aphanomyces astaci infection is the cause of crayfish plague in European crayfish. Here the virulence of an A. astaci As strain isolated from apparently healthy stone crayfish (Austropotamobius torrentium) from Slovenia was compared to that of the Psl-Puujarvi A. astaci isolate in 3 crayfish species: noble crayfish (Astacus astacus), signal crayfish (Pacifastacus leniusculus) from Finland and stone crayfish from Slovenia. All 3 crayfish species were challenged with PsI-Puujarvi A. astaci and succumbed to crayfish plague, with both noble crayfish and stone crayfish showing 100% mortality, while 25% of the signal crayfish died during the challenge. In comparison, the As-Slovenia A. astaci isolate was pathogenic for noble crayfish but not for signal crayfish or stone crayfish. This finding suggests that A. astaci virulence could be species specific and a strain from latent A. astaci infection in one native European crayfish species could be detrimental to other native European crayfish species.
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dose dependent mortality of the noble crayfish astacus astacus to different strains of the crayfish plague aphanomyces astaci
Journal of Invertebrate Pathology, 2014Co-Authors: Jenny Makkonen, Harri Kokko, Raine Kortet, Anssi Vainikka, Japo JussilaAbstract:Several reports of the European crayfish species carrying a latent infection of the crayfish plague (Aphanomyces astaci) have emerged and the discussion has focused especially on the lowered virulence of As-genotypes behind decreased mortality. The aim of this study was to compare the killing rate of different A. astaci strains in controlled infection experiments. Two separate infection experiments with three A. astaci strains (UEFT2B (As), Evira6462/06 (As) and UEF8866-2 (PsI)) were made to compare the noble crayfish populations from the Lake Viitajarvi, Tervo, (Expt I) and the Lake Mikitanjarvi, Hyrynsalmi (Expt II). In the Expt III, the Lake Koivujarvi population noble crayfish were infected with A. astaci strains UEF8866-2 (PsI) and Evira6462/06 (As) using different dosages (1, 10, 100 and 1000sporesml(-1)) of A. astaci zoospores. The results confirmed that PsI-genotype strain is highly virulent and kills all the crayfish within a few days. The tested two As-genotype strains caused the mortalities more slowly, and part of the challenged crayfish survived until the end of the follow-up period. Our results also confirmed the variance of virulence among A. astaci strains within the As-genotype and demonstrated that the mortality is dependent on the number of zoospores used in the infections. It also appeared, that some noble crayfish populations show increased resistance towards the crayfish plague, especially against the As-genotype of A. astaci.
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differing virulence of aphanomyces astaci isolates and elevated resistance of noble crayfish astacus astacus against crayfish plague
Diseases of Aquatic Organisms, 2012Co-Authors: Jenny Makkonen, Japo Jussila, Raine Kortet, Anssi Vainikka, Harri KokkoAbstract:Crayfish plague epidemics (caused by Aphanomyces astaci) have been causing population collapses among native European crayfish stocks since the late 1800s. Recent indirect and direct evidence has shown that its virulence has been variable, with native European crayfish even acting as carriers. We tested the differences in A. astaci virulence under experimental conditions using both PsI- and As-genotypes with 3 Finnish noble crayfish Astacus astacus populations. We infected crayfish with adjusted quantities of A. astaci zoospores and monitored the symptoms and mortality of the crayfish. The PsI-genotype isolate caused rapid and total mortality among the tested populations, while the As-genotype isolates expressed more variable virulence. In some cases, mortality among the As-genotype-infected crayfish did not exceed the mortality level of the control group. All of the tested noble crayfish stocks showed lower mortality towards the As-genotype of A. astaci isolated from the River Kemijoki epidemic. We conclude that there are clear differences in virulence between different A. astaci genotypes and also differences in virulence within As-genotypes. Furthermore, we observed clear signs of increased resistance in different populations of noble crayfish towards some of the tested strains belonging to the As-genotype of A. astaci.
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the diversity of the pathogenic oomycete aphanomyces astaci chitinase genes within the genotypes indicate adaptation to its hosts
Fungal Genetics and Biology, 2012Co-Authors: Jenny Makkonen, Japo Jussila, Harri KokkoAbstract:The aim of this work was to evaluate the genetic diversity of the crayfish plague pathogen Aphanomyces astaci (Oomycete) among different isolates and genotypes. Partial chitinase genes were cloned and sequenced from 28 A. astaci isolates including four of the five previously identified RAPD (random amplification of polymorphic DNA)-genotypes. The cloned chitinase sequences (n=176) formed three main groups, CHI1, CHI2 and CHI3, with the CHI2 group then further divided into three subgroups, CHI2A, CHI2B and CHI2C. Some of these chitinases were specific for certain genotypes of A. astaci, as CHI2B and CHI2C were only found from the As-genotype and CHI3 from the Ps-genotypes of A. astaci. Highest diversity rate was observed in the CHI2 group, while the CHI3 group specific for Ps-genotypes was highly homologous. Based on our chitinase data, As- and Pc-genotypes seem to be related, while the two Ps-genotypes were identical to each other, but considerably different from the genotypes As and Pc. These are the first genotype specific differences that are located in the coding region of the chitinase gene of A. astaci and the differences observed here also enable the genotyping of A. astaci. The diversity observed here can also reflect differences in the epidemiological properties of the different genotypes.
Jenny Makkonen - One of the best experts on this subject based on the ideXlab platform.
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controlled infection experiment with aphanomyces astaci provides additional evidence for latent infections and resistance in freshwater crayfish
Frontiers in Ecology and Evolution, 2021Co-Authors: Caterina Francesconi, Harri Kokko, Japo Jussila, Jenny Makkonen, Anne Schrimpf, Kathrin TheissingerAbstract:For 150 years the crayfish plague disease agent Aphanomyces astaci has been the cause of mass mortalities among native European crayfish populations. However, recently several studies have highlighted the great variability of A. astaci virulence and crayfish resistance towards the disease. The main aim of this study was to compare the response of two crayfish species, the European native noble crayfish (Astacus astacus) and the invasive alien marbled crayfish (Procambarus virginalis), to an A. astaci challenge with a highly virulent strain from haplogroup B and a lowly virulent strain from haplogroup A. In a controlled infection experiment we showed a high resistance of marbled crayfish against an A. astaci infection, with zoospores from the highly virulent haplogroup B strain being able to infect the crayfish, but unable to cause signs of disease. Furthermore, we demonstrated a reduced virulence in the A. astaci strain belonging to haplogroup A, as shown by the light symptoms and the lack of mortality in the generally susceptible noble crayfish. Interestingly, in both marbled crayfish and noble crayfish challenged with this strain, we observed a significant decrease of the detected amount of pathogen’s DNA during the experiment, suggesting that this A. astaci haplogroup A strain has a decreased ability of penetrating into the cuticle of the crayfish. Our results provide additional evidence of how drastically strains belonging to A. astaci haplogroup B and haplogroup A differ in their virulence. This study confirmed the adaptation of one specific A. astaci haplogroup A strain to their novel European hosts, supposedly due to reduced virulence. This feature might be the consequence of A. astaci´s reduced ability to penetrate into the crayfish. Finally, we experimentally showed that marbled crayfish are remarkably resistant against the crayfish plague disease and could potentially be latently infected, acting as carriers of highly virulent A. astaci strains.
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non destructive method for detecting aphanomyces astaci the causative agent of crayfish plague on the individual level
Journal of Invertebrate Pathology, 2020Co-Authors: Dora Pavic, Jenny Makkonen, Milan Cankovic, Ines Petric, Sandra Hudina, Ivana Maguire, Tomislav Vladusic, Lidija Sver, Reno Hrascan, Karla OrlicAbstract:Abstract The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a non-destructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.
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hidden sites in the distribution of the crayfish plague pathogen aphanomyces astaci in eastern europe relicts of genetic groups from older outbreaks
Journal of Invertebrate Pathology, 2018Co-Authors: Jorn Panteleit, Javier Dieguezuribeondo, Jenny Makkonen, Anne Schrimpf, Laura Martintorrijos, Nina Sophie Keller, Viorica Patrulea, Mălina Pirvu, Cristina Preda, Lucian PârvulescuAbstract:The crayfish plague agent Aphanomyces astaci is one of the world's most threatening invasive species. Originally from North America, the pathogen is being imported alongside American crayfish species, which are used for various purposes. In this study, we investigated the marginal, currently known distribution area of the pathogen in Eastern Europe by sampling narrow-clawed crayfish (Astacus leptodactylus) and spiny-cheek crayfish (Orconectes limosus) populations. In addition, using specific real-time PCR, we tested several marine decapod species, which also occur in brackish waters of the Danube at the West coast of the Black Sea and the Dniester River basin. By sequencing the nuclear chitinase gene, mitochondrial rnnS/rnnL DNA and by genotyping using microsatellite markers, we identified the A. astaci haplogroups of highly infected specimens. The A. astaci DNA was detected in 9% of the investigated A. leptodactylus samples, both in invaded and non-invaded sectors, and in 8% of the studied O. limosus samples. None of the marine decapods tested positive for A. astaci. The results revealed that narrow-clawed crayfish from the Dniester River carried the A. astaci B-haplogroup, while A. astaci from the Danube Delta belonged to the A- and B-haplogroups. In the invaded sector of the Danube, we also identified the A-haplogroup. Microsatellite analysis revealed a genotype identical to the genotype Up. It might be that some of the detected A. astaci haplogroups are relics from older outbreaks in the late 19th century, which may have persisted as a chronic infection for several decades in crayfish populations.
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aphanomyces astaci isolate from latently infected stone crayfish austropotamobius torrentium population is virulent
Journal of Invertebrate Pathology, 2017Co-Authors: Japo Jussila, Jenny Makkonen, Al Vrezec, Tina Jaklic, Hobo Kukkonen, Harri KokkoAbstract:Aphanomyces astaci infection is the cause of crayfish plague in European crayfish. Here the virulence of an A. astaci As strain isolated from apparently healthy stone crayfish (Austropotamobius torrentium) from Slovenia was compared to that of the Psl-Puujarvi A. astaci isolate in 3 crayfish species: noble crayfish (Astacus astacus), signal crayfish (Pacifastacus leniusculus) from Finland and stone crayfish from Slovenia. All 3 crayfish species were challenged with PsI-Puujarvi A. astaci and succumbed to crayfish plague, with both noble crayfish and stone crayfish showing 100% mortality, while 25% of the signal crayfish died during the challenge. In comparison, the As-Slovenia A. astaci isolate was pathogenic for noble crayfish but not for signal crayfish or stone crayfish. This finding suggests that A. astaci virulence could be species specific and a strain from latent A. astaci infection in one native European crayfish species could be detrimental to other native European crayfish species.
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dose dependent mortality of the noble crayfish astacus astacus to different strains of the crayfish plague aphanomyces astaci
Journal of Invertebrate Pathology, 2014Co-Authors: Jenny Makkonen, Harri Kokko, Raine Kortet, Anssi Vainikka, Japo JussilaAbstract:Several reports of the European crayfish species carrying a latent infection of the crayfish plague (Aphanomyces astaci) have emerged and the discussion has focused especially on the lowered virulence of As-genotypes behind decreased mortality. The aim of this study was to compare the killing rate of different A. astaci strains in controlled infection experiments. Two separate infection experiments with three A. astaci strains (UEFT2B (As), Evira6462/06 (As) and UEF8866-2 (PsI)) were made to compare the noble crayfish populations from the Lake Viitajarvi, Tervo, (Expt I) and the Lake Mikitanjarvi, Hyrynsalmi (Expt II). In the Expt III, the Lake Koivujarvi population noble crayfish were infected with A. astaci strains UEF8866-2 (PsI) and Evira6462/06 (As) using different dosages (1, 10, 100 and 1000sporesml(-1)) of A. astaci zoospores. The results confirmed that PsI-genotype strain is highly virulent and kills all the crayfish within a few days. The tested two As-genotype strains caused the mortalities more slowly, and part of the challenged crayfish survived until the end of the follow-up period. Our results also confirmed the variance of virulence among A. astaci strains within the As-genotype and demonstrated that the mortality is dependent on the number of zoospores used in the infections. It also appeared, that some noble crayfish populations show increased resistance towards the crayfish plague, especially against the As-genotype of A. astaci.
Robert Zwilling - One of the best experts on this subject based on the ideXlab platform.
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The Astacin protein family in Caenorhabditis elegans
European journal of biochemistry, 2003Co-Authors: Frank Möhrlen, Harald Hutter, Robert ZwillingAbstract:In the nematode Caenorhabditis elegans, 40 genes code for Astacin-like proteins (nematode Astacins, NAS). The Astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans Astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans Astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.
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activation of pro Astacin immunological and model peptide studies on the processing of immature Astacin a zinc endopeptidase from the crayfish astacus astacus
FEBS Journal, 2001Co-Authors: Frank Möhrlen, Stefanie Baus, Anja Gruber, Hansrichard Rackwitz, Martina Schnolzer, Gunter Vogt, Robert ZwillingAbstract:To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease Astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of Astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions. According to its genomic structure, Astacin is produced as a zymogen [Geier, G., Jacob, E., Stocker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys.337, 300–307]. To localize and follow the processing of pro-Astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro-Astacin and raised antibodies against them. In addition, antiserum against the whole active Astacin was produced. Using immunohistochemical investigation, we detected pro-Astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active Astacin, and never pro-Astacin, present in the cardiac stomach. We conclude from these studies that Astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To investigate which of the two endopeptidases found in the digestive tract of crayfish, Astacin or trypsin, is responsible for cleaving the propeptide from pro-Astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of Astacin and trypsin showed that Astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position −1 of the activation site does not prevent Astacin activity. As most members of the Astacin protein family have basic amino-acid residues in this position, in these cases also Astacin-specific cleavage would be possible.
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cdna cloning bacterial expression in vitro renaturation and affinity purification of the zinc endopeptidase Astacin
Biochemical Journal, 1999Co-Authors: Sabine Reyda, Robert Zwilling, Elard Jacob, Walter StöckerAbstract:Astacin (EC 3.4.24.21) from the freshwater crayfish (Astacus astacus) is a prototype for the metzincin superfamily and for the Astacin family of zinc peptidases, enzymes which are involved in hatching processes, embryonic patterning and tissue remodelling. Here we report on the cloning and overexpression in Escherichia coli of an Astacin cDNA which was reverse-transcribed from crayfish midgut-gland mRNA. A cDNA construct based on this clone was generated which comprised the nucleotide sequence encoding mature Astacin devoid of the signal and propeptide. This construct was cloned into the pET3a vector and used to transform E. coli BL21(DE3) cells. Recombinant Astacin was purified from inclusion bodies and dissolved under reducing conditions. For folding, the protein was diluted into neutral buffer containing l-arginine, GSH and EDTA. Eventually, Zn(2+) was added by dialysis and the fraction of active enzyme was affinity-purified on immobilized Pro-Leu-Gly hydroxamate. As shown by superimposition of the corresponding three-dimensional structures, this inhibitor binds to a region of the active-site cleft that is conserved in most metzincins. Therefore this principle behind this affinity technique, originally introduced for fibroblast collagenase by Moore and Spilburg [Biochemistry (1986) 25, 5189-5195], is applicable throughout the metzincin superfamily of metalloproteases, despite their otherwise differing cleavage specificities. Recombinant Astacin is active on gelatine zymograms and in a quenched fluorescence assay, yielding kinetic parameters comparable with those of wild-type Astacin purified from crayfish stomach.
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cloning and characterization of a cdna coding for astacus embryonic Astacin a member of the Astacin family of metalloproteases from the crayfish astacus astacus
FEBS Journal, 1998Co-Authors: Gebhard Geier, Robert ZwillingAbstract:The Astacin family of zinc endopeptidases was named after the digestive enzyme Astacin isolated from the crayfish Astacus astacus. Employing a reverse transcription/PCR strategy with degenerate oligonucleotide primers specific for two signature seqences of the Astacin family, we have isolated a 1602-bp cDNA from embryos of developing A. astacus eggs, which was designated Astacus embryonic Astacin (AEA). This cDNA was found to code for an Astacin-like protease domain which accounts for the N-terminal half of the predicted protein. The C-terminal half mainly consists of two complement subcomponent C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein 1 (CUB) domains. The metalloprotease domain displays an amino acid sequence identity of 42% with Astacin. A higher sequence similarity was found to Astacin family members that act as hatching enzymes in different species, e.g. chorioallantoic membrane protein 1 (CAM-1; from quail) and Xenopus hatching enzyme (formerly UVS.2), both of which show 54% identity, and high and low choriolytic enzymes (HCE and LCE) from the teleost Oryzias latipes (52% and 48% identity, respectively). A relationship to Astacin-like hatching enzymes is further supported by a phylogenetic analysis of the protease domains. Expression of AEA mRNA in developing embryos was found to be restricted to unhatched juveniles (larvae) during the last 8 days before hatching. AEA transcripts could not be detected in various tissues of adult animals or in eggs and embryos from an earlier developmental stage. AEA expression starts about 8 days prior to hatching, followed by a strong (18-fold) induction with a maximum at day 4 before hatching. Newly hatched juveniles were found not to express the AEA mRNA.
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phosphinic peptides the first potent inhibitors of Astacin behave as extremely slow binding inhibitors
Biochemical Journal, 1998Co-Authors: Irene Yiallouros, Robert Zwilling, Walter Stöcker, Stamatia Vassiliou, Athanasios Yiotakis, Vincent DiveAbstract:A series of phosphinic pseudo-peptides varying in length and composition have been designed as inhibitors of the crayfish zinc endopeptidase Astacin, the prototype of the Astacin family and of the metzincin superfamily of metalloproteinases. The most efficient phosphinic peptide, fluorenylmethyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-P ro-Leu-Val, binds to Astacin with a Ki value of 42 nM, which is about three orders of magnitude below the corresponding values for previously used hydroxamic acid derivatives. However, the rate constants for association (kon = 96.8 M-1.s-1) and dissociation (koff = 4.1 x 10(-6) s-1) are evidence for the extremely slow binding behaviour of this compound. N-terminally or C-terminally truncated phosphinic analogues of this parent molecule are much less potent, indicating a critical role of the peptide size on the potency. In particular, omission of the N-terminal proline residue leads to a 40-fold increase in Ki which is mostly due to a 75-fold higher koff value. These findings are consistent with the previously solved crystal structure of Astacin complexed with one of the phosphinic peptides, benzyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-Pro-O-methyl, Ki = 14 microM [Grams, Dive, Yiotakis, Yiallouros, Vassiliou, Zwilling, Bode and Stocker (1996) Nature Struct. Biol. 3, 671-675]. This structure also reveals that the phosphinic group binds to the active site as a transition-state analogue. The extremely slow binding behaviour of the phosphinic peptides is discussed in the light of the conformational changes involving a unique 'tyrosine switch' in the structure of Astacin upon inhibitor binding. The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the Astacin family which, like bone morphogenetic protein 1 (BMP1; i.e. the procollagen C-proteinase), have become targets of pharmacological research.
