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Background Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. Results We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Conclusions Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. We annotated theHirudo medicinalisgenome and performed RNA-seq on salivary cells isolated from three closely related leech species,H. medicinalis, Hirudo orientalis,andHirudo verbana.Differential expression analysis verified by proteomics identified salivary cell-specific genes, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants were not differentially expressed in the salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Thus, our study provides one of the most comprehensive knowledge of the genetic fundamentals of the blood-sucking lifestyle in leeches.

The procedure for obtaining an active recombinant destabilase from the medicinal leech in Escherichia coli cells was developed. The plasmids encoding an analogue of native destabilase, as well as the protein forms carrying polyhistidine sequence at the Cand/or N-terminus of the polypeptide were obtained during the work. The producing strains of different forms of the protein were constructed, the cultivation process was optimized. The conditions of renaturation of destabilase recombinant forms by dialysis and using chromatographic absorbent were selected. The muramidase activity towards cell walls of Micrococcus lysodeikticus bacferia and lytic activity towards E. coli were investigated. The dependence of pH and ionic strength of the solution on the activities was determined. The total antibacterial activity of destabilase towards E. coli was shown.

Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.

Here we present two incomplete mitochondrial genome sequences of Hirudo medicinalis and Hirudo verbana (Annelida, Hirudinea). The corresponding sequences are 14,729 and 14,604 base pairs in length. They contain all mitochondrial genes (13 protein-coding genes, 22 tRNAs and two rRNAs) but lack the non-coding region. Nevertheless, the robust reconstruction of their phylogenetic relationships presented here reveals distinct separation of both leeches from other annelids and at the same time relatively high dissimilarity between each other.

Background Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. Results In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. Conclusions The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics. Electronic supplementary material The online version of this article (doi:10.1186/s12858-015-0056-3) contains supplementary material, which is available to authorized users.