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

Ticks are obligate hematophagous arthropods that transmit a wide range of pathogens to humans as well as wild and domestic animals. They also harbor a non-pathogenic microbiota, although our previous study has shown that the diverse bacterial microbiome in the midgut of Ixodes ricinus is quantitatively poor and lacks a core. In artificial infections by capillary feeding of ticks with two model bacteria (Gram-positive Micrococcus luteus and Gram-negative Pantoea sp.), rapid clearance of these microbes from the midgut was observed, indicating the presence of active immune mechanisms in this organ. In the current study, RNA-seq analysis was performed on the midgut of I. ricinus females inoculated with either M. luteus or Pantoea sp. or with sterile water as a control. While no immune-related transcripts were upregulated by microbial inoculation compared to that of the sterile control, capillary feeding itself triggered dramatic transcriptional changes in the tick midgut. Manual curation of the transcriptome from the midgut of unfed I. ricinus females, complemented by the proteomic analysis, revealed the presence of several constitutively expressed putative antimicrobial peptides (AMPs) that are independent of microbial stimulation and are referred to here as 'guard' AMPs. These included two types of midgut-specific defensins, two different domesticated amidase effector 2 (Dae2), microplusin/ricinusin-related molecules, two lysozymes, and two gamma interferon-inducible lysosomal thiol reductases (GILTs). The in vitro antimicrobial activity assays of two synthetic mature defensins, defensin 1 and defensin 8, confirmed their specificity against Gram-positive bacteria showing exceptional potency to inhibit the growth of M. luteus at nanomolar concentrations. The antimicrobial activity of midgut defensins is likely part of a multicomponent system responsible for the rapid clearance of bacteria in the tick midgut. Further studies are needed to evaluate the role of other identified 'guard' AMPs in controlling microorganisms entering the tick midgut.

• Keywords
• Ixodes, Micrococcus luteus, antimicrobial peptide, defensin, immune system, midgut microbiome, tick,
• MeSH
• Antimicrobial Peptides metabolism   MeSH
• Gastrointestinal Tract microbiology   immunology   MeSH
• Ixodes * microbiology   immunology   MeSH
• Micrococcus luteus immunology   MeSH
• Proteomics MeSH
• Gene Expression Profiling MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antimicrobial Peptides MeSH

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

• Keywords
• cathepsin, cysteine protease, parasite, protease inhibitor, protein structure, saliva, thyropin, tick,
• MeSH
• Cysteine MeSH
• Glycosaminoglycans MeSH
• Cathepsins metabolism   MeSH
• Ixodes * metabolism   MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy MeSH
• Saliva * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cysteine MeSH
• Glycosaminoglycans MeSH
• Cathepsins MeSH