Christian Müller

Leeches of the genus Placobdella Blanchard, 1893 (Annelida: Rhynchobdellida: Glossiphoniidae) are mainly distributed across North and Central America, but at least two species are also present in the palearctic region: the European turtle leech Placobdella costata Müller, 1846, and the North African turtle leech Placobdella nabeulensis Ben Ahmed et al., 2023. All species of the genus are hematophagous and feed on vertebrates, but are known to preferentially target aquatic and semi-aquatic reptiles like turtles and snakes. Placobdella ornata Verrill, 1872, is the original source of ornatins, a class of potent platelet aggregation inhibitors. However, the whole repertoire of bioactive peptides, including anticoagulation factors remains largely underexplored for Placobdella , and functional characterizations of putative antithrombotics are scarce. Here we describe the genes and cDNAs that encode putative hirudins and ornatins in both of the palearctic and three American species of the genus Placobdella . A selection of putative hirudins and ornatins was recombinantly expressed and functionally characterized using appropriate coagulation and platelet aggregation assays, and our data confirm the expression of functional hirudins and ornatins in the studied leeches. In addition, our genetic analyses strongly support the hypothesis that blood feeding evolved only once in the evolutionary history of leeches.

Medicinal leeches express a broad variety of anticoagulants and other bioactive factors that are involved in the blood feeding process. For most of the anticoagulants, several genes exist that may encode isoforms of the respective proteins, including hirudins and decorsins. Decorsins negatively affect platelet aggregation, whereas hirudins are potent thrombin inhibitors. Both factors belong to the hirudin superfamily that also includes the group of hirudin-like factors, and all hematophagous leeches analyzed so far contain several gene copies that encode representatives of at least two of the three groups of these factors. Members of the hirudin superfamily may contain only one central globular domain, but others may contain two or more copies. Here we describe the molecular identification and partial functional characterization of a broad variety of putative mono-, bi and multimeric hirudins, decorsins and hirudin-like factors in two Asian medicinal leech species, namely Hirudo nipponia and H. tianjinensis . Some of the monomeric hirudins and decorsins have already been described before, but they represent only a small part of the overall diversity. For the first time, putative monomeric and one oligomeric decorsins of H. tianjinensis were expressed as recombinant proteins, functionally characterized and successfully verified as platelet aggregation inhibitors. In addition we have conducted phylogenetic analyses based on genomic and mitochondrial markers and found convincing evidence that H. nipponia and H. tianjinensis together with members of the genus Whitmania form a monophyletic clade that is clearly distinct from clades that are formed either by European members of the genus Hirudo or by members of the genus Hirudinaria .

Hematophagous leeches rely on a broad diversity of bioactive factors that interfere with the host’s defense systems to secure a successful blood meal. The most prominent examples of such factors are the antithrombotics that address either the primary hemostasis (the platelet aggregation) or the secondary hemostasis (the blood coagulation). Whereas the inhibitors of platelet aggregation mainly cause continuous blood flow, coagulation inhibitors mainly keep the blood fluid within the stomach of the leech. The critical dependency of hematophagous leeches on the accurate action of all antithrombotic factors may explain the presence of multiple genes for each type of these factors that is regularly observed in leech genomes. The genome of Hirudinaria manillensis Lesson, 1842, the Asian buffalo leech, contains five individual genes that encode variants of the coagulation inhibitor hirudin. However, no genes that encode putative decorsins, the archetype of leech-derived inhibitors of platelet aggregation, have been described to date. Here, we report the identification of one monomeric and four multimeric decorsin encoding genes in the genome of H. manillensis. A selection of the putative decorsins was expressed as recombinant proteins, purified and functionally characterized. All but one of these putative decorsins displayed platelet aggregation-inhibitory potencies. Furthermore, we provide a mathematical calculation based on the mechanism of alternative pre-mRNA splicing that illustrates the potential to generate an enormous variety of different factors from one single multimeric ornatin gene.

African hematophagous leeches are largely underinvestigated both in terms of their biodiversity and the characterization of the repertoire of bioactive components of their salivary gland secretions. Asiaticobdella (formerly Aliolimnatis ) fenestrata Moore, 1939, also referred to as the African medicinal leech, is hence a promising candidate for the identification and functional characterization of putative anticoagulants that may serve as inspirations for the development of new or the enhancement of existing drugs for medical purposes. A. fenestrata inhabits flooded plains of southern African regions and mainly feeds on mammals like black lechwe ( Kobus leche Gray, 1850). Whereas several anticoagulants, including two putative hirudins of A. fenestrata , have already been identified in previous investigations, functional characterizations of these components are yet missing. We conducted an in-depth re-analysis of the salivary gland transcriptomic data set of A. fenestrata and identified two additional hirudin-like factors that exhibit a remarkable structural feature—the presence of an RGD motif at or near the N-terminal end of the molecules. The presence of such an RGD motif is a key feature of leech-derived platelet aggregation inhibitors such as ornatins and decorsins. However, both in ornatins and in decorsins, the respective RGD motif is located within the molecule between the 5th and the 6th cysteine residues, but not at or near the N-terminus. We named the two putative platelet aggregation inhibitors “fenestrins.” Both the two putative hirudins and the two fenestrins of A. fenestrata were expressed as recombinant proteins, purified, and functionally characterized in either coagulation or platelet aggregation assays.

The river nerite Theodoxus fluviatilis (Linneaus, 1758) is an euryhaline osmoconformer found in freshwater (FW) and brackish water (BW) habitats across Europe and western Asia. In northern Germany, T. fluviatilis forms regional subgroups, the FW and the BW ecotypes. Members of these ecotypes differ in shell morphology and in shell size as well as in their metabolic pathways of accumulating organic osmolytes under hyperosmotic stress. Oxygen consumption rates were measured, as a noninvasive fitness‐indicator, of animals exposed to external media with different salinities. The BW water snails were exposed to hyper‐ and hyposaline conditions, while the FW snails were naturally only exposed to hyperosmotic conditions. Respiration rates in specimens of the BW ecotype stayed more or less constant overall in all salinities tested. While the BW snails' respiration rates were not affected by the salinity treatment per se, they were affected by the sequence of salinity treatments that they were exposed to (hyperosmotic conditions first vs. hypoosmotic conditions first). Respiration rates strongly declined in the FW ecotype individuals with increasing medium salinity. We suggest that the hypersaline conditions pose greater stress for the animals than the hyposaline conditions because of the higher metabolic activities required for tissue volume regulation.

The expression of recombinant proteins in heterologous hosts is a common strategy to obtain larger quantities of the “protein of interest” (POI) for scientific, therapeutic or commercial purposes. However, the experimental success of such an approach critically depends on the choice of an appropriate host system to obtain biologically active forms of the POI. The correct folding of the molecule, mediated by disulfide bond formation, is one of the most critical steps in that process. Here we describe the recombinant expression of hirudin, a leech-derived anticoagulant and thrombin inhibitor, in the yeast Komagataella phaffii (formerly known and mentioned throughout this publication as Pichia pastoris) and in two different strains of Escherichia coli, one of them being especially designed for improved disulfide bond formation through expression of a protein disulfide isomerase. Cultivation of the heterologous hosts and expression of hirudin were performed at different temperatures, ranging from 22 to 42 °C for the bacterial strains and from 20 to 30 °C for the yeast strain, respectively. The thrombin-inhibitory potencies of all hirudin preparations were determined using the thrombin time coagulation assay. To our surprise, the hirudin preparations of P. pastoris were considerably less potent as thrombin inhibitors than the respective preparations of both E. coli strains, indicating that a eukaryotic background is not per se a better choice for the expression of a biologically active eukaryotic protein. The hirudin preparations of both E. coli strains exhibited comparable high thrombin-inhibitory potencies when the strains were cultivated at their respective optimal temperatures, whereas lower or higher cultivation temperatures reduced the inhibitory potencies.

Backround/Objectives: Mobile genetic elements (MGEs), in general, and transposable elements (TEs), in particular, constitute a major part of almost every eukaryotic genome, and several types of such elements have been classified based on size, genetic structure and transposition intermediate. Methods: The fast-growing availability of whole genome sequences of species across the living world provides almost unlimited possibilities for in-depth molecular analyses of all kinds, including the search for TEs. The aim of the present study was to perform the first molecular description and characterization of selected MGEs in leeches, namely, short interspersed nuclear element (SINE), long interspersed nuclear element (LINE) and long terminal repeat (LTR) retrotransposons. Results: Several representatives of all three groups of TEs could be identified, and some of the newly described elements display unique structural features compared to the archetype elements of the respective groups. Conclusions: Non-model organisms like leeches are an excellent source for new information on long-term studied objects like TEs and may provide new insights into the diversity and the putative biological impact of these MGEs.

The expression of recombinant proteins in heterologous hosts is a common strategy to obtain larger quantities of the "protein of interest" (POI) for scientific, therapeutic or commercial purposes. However, the experimental success of such an approach critically depends on the choice of an appropriate host system to obtain biologically active forms of the POI. The correct folding of the molecule, mediated by disulfide bond formation, is one of the most critical steps in that process. Here we describe the recombinant expression of hirudin, a leech-derived anticoagulant and thrombin inhibitor, in the yeast Komagataella phaffii (formerly known and mentioned throughout the manuscript as Pichia pastoris) and in two different strains of Escherichia coli, one of them being especially designed for improved disulfide bond formation. Cultivation of the heterologous hosts and expression of hirudin were performed at different temperatures, ranging from 22 to 42 °C for the bacterial strains and from 20 to 30 °C for the yeast strain, respectively. The thrombin inhibitory potencies of all hirudin preparations were determined using the thrombin time coagulation assay. To our surprise, the hirudin preparations of P. pastoris were considerably less potent as thrombin inhibitors compared to the respective preparations of both E. coli strains, indicating that an eukaryotic background is not per se a better choice for the expression of a biologically active eukaryotic protein. The hirudin preparations of both E. coli strains exhibited comparable high thrombin-inhibitory potencies when the strains were cultivated at their respective optimal temperatures, whereas lower or higher cultivation temperatures reduced the inhibitory potencies.

Haematophagous leeches rely on a broad variety of bioactive compounds to secure a sufficient blood meal from their vertebrate prey. Both the primary (platelet aggregation) and secondary (blood coagulation) haemostasis are the main targets of action. The platelet aggregation inhibitor decorsin was first described in the North American leech, Macrobdella decora Say, 1824, whereas the bivalent thrombin inhibitor hirudin was originally identified in the European medicinal leech, Hirudo medicinalis Linnaeus, 1758. Hirudin blocks both the catalytic site and the fibrinogen-binding site (exosite I) of thrombin. Haemadin of the Indian land leech Haemadipsa sylvestris Blanchard, 1894, is also a highly efficient bivalent thrombin inhibitor but blocks exosite II of thrombin. So far, only the archetypal form of haemadin from H. sylvestris has been purified and functionally characterized, and two putative haemadins have been identified in the salivary transcriptome of Haemadipsa interrupta Moore, 1835, a terrestrial leech inhabiting mainly the Malayan peninsula. Here, we describe the identification of ten additional putative haemadins in the transcriptomic data set of H. interrupta, first generated by another study. Furthermore, we identified a putative oligomeric decorsin, which represents the first finding of this anticoagulant in a haemadipsid leech. Both the putative decorsin and a selection of haemadins were expressed, purified, and functionally characterized. The putative haemadins displayed a broad spectrum of thrombin-inhibitory potencies, whereas the putative oligomeric decorsin was indeed a weak inhibitor of platelet aggregation.