Ticks are important ectoparasites and vectors of a variety of pathogens in both animals and humans, and their increasing global distribution poses a growing health risk. Unlike other blood-feeding vectors, ticks feed for an extended period at each life stage and rely exclusively on blood for development and reproduction. Blood digestion in ticks is mediated by a complex multienzyme network within the endolysosomal system of the midgut (MG) epithelial cells. Previous studies have focused largely on protein digestion during the slow feeding phase. However, the processing of the blood meal after the mating-induced rapid engorgement ("big sip") remains unclear, although the rapid turnover of proteins from host blood proteins into yolk proteins in fully fed females is a crucial step for tick reproduction. In this study, we performed a label-free quantitative proteomic analysis of MG tissue extracts and MG contents of the hard tick Ixodes ricinus to characterize proteases and protease inhibitors expressed during selected timepoints of female feeding and off-host digestion. In addition, we analyzed the distribution of digestive enzymes by activity profiling in MG extracts and contents with specific diagnostic substrates. Our results show that the multienzyme network, mainly based on aspartic acid and cysteine cathepsins and complemented by specific types of serine proteases and metalloproteases, is involved in the intracellular and probably also in the luminal digestion of blood meal proteins in fully engorged female ticks. We also detected different types of protease inhibitors and proposed their regulatory role in controlling both endogenous (tick-derived) and host protease activities in the MG tissue and luminal contents storing ingested blood. These results provide comprehensive insights into the physiology of the tick MG and offer new opportunities for the development of future control strategies against ticks and tick-borne diseases.

• Keywords
• adult Ixodes ricinus, label-free proteomics, midgut proteome, proteolytic system, tick physiology,
• MeSH
• Ixodes * metabolism   physiology   enzymology   MeSH
• Peptide Hydrolases metabolism   MeSH
• Arthropod Proteins * metabolism   MeSH
• Proteome * metabolism   MeSH
• Proteomics * methods   MeSH
• Feeding Behavior MeSH
• Digestion * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptide Hydrolases MeSH
• Arthropod Proteins * MeSH
• Proteome * MeSH

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.

• Keywords
• Schistosoma mansoni, acrylamide inhibitor, cathepsin B, cysteine protease, drug target, parasite,
• MeSH
• Cathepsin B * antagonists & inhibitors   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Schistosoma mansoni * enzymology   drug effects   MeSH
• Schistosomicides pharmacology   chemistry   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cathepsin B * MeSH
• Schistosomicides MeSH

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

• Keywords
• Borrelia, Ixodes, antimicrobial peptides, label-free quantification, lipid metabolism, midgut, protease inhibitors, proteases, proteome, ticks,
• MeSH
• Ixodes * parasitology   MeSH
• Proteome MeSH
• Proteomics MeSH
• Digestive System MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH

Dermanyssus gallinae is a blood-feeding mite that parasitises wild birds and farmed poultry. Its remarkably swift processing of blood, together with the capacity to blood-feed during most developmental stages, makes this mite a highly debilitating pest. To identify specific adaptations to digestion of a haemoglobin-rich diet, we constructed and compared transcriptomes from starved and blood-fed stages of the parasite and identified midgut-enriched transcripts. We noted that midgut transcripts encoding cysteine proteases were upregulated with a blood meal. Mapping the full proteolytic apparatus, we noted a reduction in the suite of cysteine proteases, missing homologues for Cathepsin B and C. We have further identified and phylogenetically analysed three distinct transcripts encoding vitellogenins that facilitate the reproductive capacity of the mites. We also fully mapped transcripts for haem biosynthesis and the ferritin-based system of iron storage and inter-tissue trafficking. Additionally, we identified transcripts encoding proteins implicated in immune signalling (Toll and IMD pathways) and activity (defensins and thioester-containing proteins), RNAi, and ion channelling (with targets for commercial acaricides such as Fluralaner, Fipronil, and Ivermectin). Viral sequences were filtered from the Illumina reads and we described, in part, the RNA-virome of D. gallinae with identification of a novel virus, Red mite quaranjavirus 1.

• MeSH
• Poultry MeSH
• Mite Infestations * veterinary   parasitology   MeSH
• Chickens MeSH
• Poultry Diseases * MeSH
• Mites * genetics   MeSH
• RNA-Seq MeSH
• Virome MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

• Keywords
• Cathepsin K, azadipeptide nitrile, cyanohydrazide warhead, protease inhibitor, structure,
• MeSH
• Hydrazines chemistry   pharmacology   MeSH
• Protease Inhibitors chemistry   pharmacology   MeSH
• Cathepsin K antagonists & inhibitors   metabolism   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Tumor Cells, Cultured MeSH
• Nitriles chemistry   pharmacology   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CTSK protein, human MeSH Browser
• Hydrazines MeSH
• Protease Inhibitors MeSH
• Cathepsin K MeSH
• Nitriles MeSH

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

• Keywords
• Ixodes ricinus, cathepsin, crystal structure, cysteine protease, digestion, midgut, parasite,
• MeSH
• Cystatins metabolism   MeSH
• Phylogeny MeSH
• Cathepsin L metabolism   MeSH
• Ticks metabolism   MeSH
• Ixodes metabolism   MeSH
• Blood Proteins metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Proteolysis MeSH
• Amino Acid Sequence MeSH
• Digestive System metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cystatins MeSH
• Cathepsin L MeSH
• Blood Proteins MeSH

BACKGROUND: Ectoparasites from the family Diplozoidae (Platyhelminthes, Monogenea) belong to obligate haematophagous helminths of cyprinid fish. Current knowledge of these worms is for the most part limited to their morphological, phylogenetic, and population features. Information concerning the biochemical and molecular nature of physiological processes involved in host-parasite interaction, such as evasion of the immune system and its regulation, digestion of macromolecules, suppression of blood coagulation and inflammation, and effect on host tissue and physiology, is lacking. In this study, we report for the first time a comprehensive transcriptomic/secretome description of expressed genes and proteins secreted by the adult stage of Eudiplozoon nipponicum (Goto, 1891) Khotenovsky, 1985, an obligate sanguivorous monogenean which parasitises the gills of the common carp (Cyprinus carpio). RESULTS: RNA-seq raw reads (324,941 Roche 454 and 149,697,864 Illumina) were generated, de novo assembled, and filtered into 37,062 protein-coding transcripts. For 19,644 (53.0%) of them, we determined their sequential homologues. In silico functional analysis of E. nipponicum RNA-seq data revealed numerous transcripts, pathways, and GO terms responsible for immunomodulation (inhibitors of proteolytic enzymes, CD59-like proteins, fatty acid binding proteins), feeding (proteolytic enzymes cathepsins B, D, L1, and L3), and development (fructose 1,6-bisphosphatase, ferritin, and annexin). LC-MS/MS spectrometry analysis identified 721 proteins secreted by E. nipponicum with predominantly immunomodulatory and anti-inflammatory functions (peptidyl-prolyl cis-trans isomerase, homolog to SmKK7, tetraspanin) and ability to digest host macromolecules (cathepsins B, D, L1). CONCLUSIONS: In this study, we integrated two high-throughput sequencing techniques, mass spectrometry analysis, and comprehensive bioinformatics approach in order to arrive at the first comprehensive description of monogenean transcriptome and secretome. Exploration of E. nipponicum transcriptome-related nucleotide sequences and translated and secreted proteins offer a better understanding of molecular biology and biochemistry of these, often neglected, organisms. It enabled us to report the essential physiological pathways and protein molecules involved in their interactions with the fish hosts.

• Keywords
• Annotation, Assembly, Eudiplozoon nipponicum, Mass spectrometry, Monogenea, NGS, Secretome, Transcriptome,
• MeSH
• Molecular Sequence Annotation MeSH
• Chromatography, Liquid MeSH
• Phylogeny MeSH
• Carps * genetics   MeSH
• Gene Expression Profiling MeSH
• Tandem Mass Spectrometry MeSH
• Transcriptome MeSH
• Trematoda * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

• MeSH
• Carps parasitology   MeSH
• Cathepsins analysis   metabolism   MeSH
• Laser Capture Microdissection MeSH
• Parenchymal Tissue metabolism   MeSH
• Platyhelminths metabolism   MeSH
• Peptide Hydrolases analysis   metabolism   MeSH
• Proteome analysis   MeSH
• Proteomics methods   MeSH
• Intestinal Mucosa metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Chromatography, High Pressure Liquid MeSH
• Gills parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cathepsins MeSH
• Peptide Hydrolases MeSH
• Proteome MeSH

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.

• MeSH
• Fibrinolysis drug effects   MeSH
• Blood Coagulation drug effects   MeSH
• Hemostatics antagonists & inhibitors   MeSH
• Blood Pressure drug effects   MeSH
• Models, Molecular MeSH
• Plasminogen drug effects   MeSH
• Protein Domains MeSH
• Helminth Proteins chemistry   genetics   pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Recombinant Proteins MeSH
• Schistosoma mansoni enzymology   MeSH
• Schistosomiasis mansoni parasitology   MeSH
• Amino Acid Sequence MeSH
• Serine Endopeptidases chemistry   genetics   pharmacology   MeSH
• Tissue Plasminogen Activator drug effects   MeSH
• Vasodilation drug effects   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hemostatics MeSH
• Plasminogen MeSH
• Helminth Proteins MeSH
• Recombinant Proteins MeSH
• Serine Endopeptidases MeSH
• Tissue Plasminogen Activator MeSH
• trypsin-like serine protease MeSH Browser

The publication of the first tick sialome (salivary gland transcriptome) heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

• Keywords
• cystatins, immunomodulation, protease inhibitors, serpins, tick-host interaction,
• MeSH
• Cystatins physiology   therapeutic use   MeSH
• Immunomodulation MeSH
• Protease Inhibitors classification   metabolism   therapeutic use   MeSH
• Serine Proteinase Inhibitors physiology   therapeutic use   MeSH
• Host-Parasite Interactions MeSH
• Ticks metabolism   MeSH
• Humans MeSH
• Serpins physiology   therapeutic use   MeSH
• Saliva enzymology   metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Cystatins MeSH
• Protease Inhibitors MeSH
• Serine Proteinase Inhibitors MeSH
• Serpins MeSH