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.
- Klíčová slova
- cathepsin, cysteine protease, parasite, protease inhibitor, protein structure, saliva, thyropin, tick,
- MeSH
- cystein MeSH
- glykosaminoglykany MeSH
- kathepsiny metabolismus MeSH
- klíště * metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- sliny * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cystein MeSH
- glykosaminoglykany MeSH
- kathepsiny MeSH
Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary glands, we demonstrated that saliva-producing type II and III acini are innervated by neuropeptidergic axons which release different classes of neuropeptides via their terminals (Šimo et al., 2009b, 2013). Among these, the neuropeptide SIFamide-along with its cognate receptor-were postulated to control the basally located acinar valve via basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionally characterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genome and proved that it senses a low nanomolar level of its corresponding ligand. Insect SIFamide paralogs, SMYamides, also activated the receptor but less effectively compared to SIFamide. Bioinformatic and molecular dynamic analyses suggested that I. scapularis SIFamide receptors are class A GPCRs where the peptide amidated carboxy-terminus is oriented within the receptor binding cavity. The receptor was found to be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, and ovaries, but not in Malpighian tubules. Investigation of the temporal expression patterns suggests that the receptor transcript is highly expressed in unfed I. ricinus female salivary glands and then decreases during feeding. In synganglia, a significant transcript increase was detected in replete ticks. In salivary gland acini, an antibody targeting the SIFa_R2 recognized basal epithelial cells, myoepithelial cells, and basal granular cells in close proximity to the SIFamide-releasing axon terminals. Immunoreactivity was also detected in specific neurons distributed throughout various I. ricinus synganglion locations. The current findings, alongside previous reports from our group, indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in I. ricinus salivary glands. Our study investigates the peptidergic regulation of the I. ricinus salivary gland in detail, emphasizing the complexity of this system.
- Klíčová slova
- SIFamide, SIFamide receptors, Salivary gland acini, Synganglion, Ticks,
- MeSH
- klíště * genetika metabolismus MeSH
- neurony metabolismus MeSH
- neuropeptidy * genetika metabolismus MeSH
- slinné žlázy metabolismus MeSH
- sliny MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- neuropeptidy * MeSH
Decorin binding proteins (Dbps) mediate attachment of spirochetes in host organisms during the early stages of Lyme disease infection. Previously, different binding mechanisms of Dbps to glycosaminoglycans have been elucidated for the pathogenic species Borrelia burgdorferi sensu stricto and B. afzelii. We are investigating various European Borrelia spirochetes and their interactions at the atomic level using NMR. We report preparative scale recombinant expression of uniformly stable isotope enriched B. afzelii DbpA in Escherichia coli, its chromatographic purification, and solution NMR assignments of its backbone and sidechain 1H, 13C, and 15N atoms. This data was used to predict secondary structure propensity, which we compared to the North American B. burgdorferi sensu stricto and European B. garinii DbpA for which solution NMR structures had been determined previously. Backbone dynamics of DbpA from B. afzelii were elucidated from spin relaxation and heteronuclear NOE experiments. NMR-based secondary structure analysis together with the backbone dynamics characterization provided a first look into structural differences of B. afzelii DbpA compared to the North American species and will serve as the basis for further investigation of how these changes affect interactions with host components.
- Klíčová slova
- Borrelia afzelii, Decorin-binding proteins, NMR resonance assignment,
- MeSH
- Borrelia burgdorferi komplex * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).
- MeSH
- adeninfosforibosyltransferasa metabolismus MeSH
- adeninnukleotidy metabolismus MeSH
- buněčné linie MeSH
- HeLa buňky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nukleosidy metabolismus MeSH
- organofosfonáty metabolismus MeSH
- puriny metabolismus MeSH
- Trypanosoma brucei brucei metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adeninfosforibosyltransferasa MeSH
- adeninnukleotidy MeSH
- nukleosidy MeSH
- organofosfonáty MeSH
- purine MeSH Prohlížeč
- puriny MeSH
BACKGROUND: The systematic of several marine diphyllobothriid tapeworms of pinnipeds has been revised in recent years. However, 20 species of Diphyllobothrium from phocids and otariids are still recognized as incertae sedis. We describe a new species of Diphyllobothrium from the intestine of California sea lions Zalophus californianus (Lesson) (type-host) and South American sea lions Otaria flavescens (Shaw). METHODS: Zalophus californianus from the Pacific coast of the USA and O. flavescens from Peru and Argentina were screened for parasites. Partial fragments of the large ribosomal subunit gene (lsrDNA) and the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene were amplified for 22 isolates. Properly fixed material from California sea lions was examined using light and scanning electron microscopy. RESULTS: A total of four lsrDNA and 21 cox1 sequences were generated and aligned with published sequences of other diphyllobothriid taxa. Based on cox1 sequences, four diphyllobothriid tapeworms from O. flavescens in Peru were found to be conspecific with Adenocephalus pacificus Nybelin, 1931. The other newly generated sequences fall into a well-supported clade with sequences of a putative new species previously identified as Diphyllobothrium sp. 1. from Z. californianus and O. flavescens. A new species, Diphyllobothrium sprakeri n. sp., is proposed for tapeworms of this clade. CONCLUSIONS: Diphyllobothrium sprakeri n. sp. is the first diphyllobothriid species described from Z. californianus from the Pacific coast of North America, but O. flavescens from Argentina, Chile and Peru was confirmed as an additional host. The present study molecularly confirmed the first coinfection of two diphyllobothriid species in sea lions from the Southern Hemisphere.
- Klíčová slova
- Helminths, Otaria flavescens, Otariidae, Parasites, Pinnipedia, Zalophus californianus, cox1, lsrDNA,
- MeSH
- difylobotriáza veterinární MeSH
- Diphyllobothrium anatomie a histologie klasifikace genetika izolace a purifikace MeSH
- fylogeneze MeSH
- lachtani parazitologie MeSH
- mitochondriální geny MeSH
- střeva parazitologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Jižní Amerika MeSH
- Severní Amerika MeSH
The northern fur seal (NFS), Callorhinus ursinus (Mammalia: Otariidae), is a marine mammal species included into the IUCN Red List as the vulnerable species which population is dramatically declining. A significant amount of parasitological data collected previously and our recent data allowed us to clarify the list of NFS metazoan parasites and to perform a comprehensive analysis of the gastrointestinal helminth community. Gastrointestinal tracts from 756 NFSs (3- to 4-year-old males) were collected during the annual Aleut subsistence harvests in July-August of 2011-2014 from five separate rookeries on St. Paul Island, Alaska. Totally, 27,625 specimens of helminths and approximately 1000 nasal mites were collected and identified. Detailed analysis of the previously published and newly obtained data revealed 32 species of metazoan parasites, including trematodes (6 species), cestodes (4), nematodes (9), acanthocephalans (9) and arthropods (4). The gastrointestinal helminth community of newly studied NFSs comprised 19 species including trematodes (4), cestodes (3), nematodes (5) and acanthocephalans (7). Temporal changes in the helminth community structure were small but statistically significant. Gastrointestinal helminth infracommunities comprised from 1 to 10 species (average of 4). Small but significant correlation was found between the abundances of acanthocephalans (Corynosoma similis and C. strumosum), nematodes (Contracaecum osculatum, Pseudoterranova spp.) and cestode Diphyllobothrium tetrapterum.
- Klíčová slova
- Acanthocephala, Cestoda, Marine mammals, Nematoda, Parasite community, Trematoda,
- MeSH
- cizopasní červi klasifikace cytologie izolace a purifikace MeSH
- gastrointestinální trakt parazitologie MeSH
- helmintózy zvířat epidemiologie parazitologie MeSH
- lachtani rodu Arctocephalus a Callorhinus parazitologie MeSH
- ostrovy epidemiologie MeSH
- paraziti klasifikace izolace a purifikace MeSH
- prevalence MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Aljaška epidemiologie MeSH
- ostrovy epidemiologie MeSH
The cercarial emergence patterns of three species of Diplostomum (Diplostomum 'mergi', Diplostomum spathaceum and Diplostomum parviventosum) parasitizing freshwater first intermediate host Radix lagotis sampled in Most Lake, Czech Republic, were studied under various experimental conditions, i.e. field, laboratory and incubator, and seasons, i.e. spring, summer and autumn. We discovered unexpected daily periodicity-dependent species-specific emergence patterns among the three Diplostomum spp. depending on experimental conditions. At the same time, the intraspecific variation of D. spathaceum cercarial release in response to seasonal conditions was observed. We found that a complex array of mechanisms can affect Diplostomum species-specific patterns in cercarial emergence, of which behavioural characteristics of fish related to reproduction and feeding processes are considered the most important factors. This might represent a specific adaptive evolutionary mechanism to maximise transmission success while avoiding competition for host resources. Our results contribute to a better understanding of ecological and epidemiological aspects with respect to specific adaptive strategies compartmentalised among species of Diplostomum and consequences for infection risk in fish hosts.
- Klíčová slova
- Cercariae, Diplostomum, Emergence, Freshwater, Radix lagotis, Transmission, Trematoda,
- MeSH
- cerkárie MeSH
- druhová specificita MeSH
- hlemýždi parazitologie MeSH
- periodicita MeSH
- roční období MeSH
- Trematoda * fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Under drought stress, Phytoseiulus persimilis females are able to lay drought-resistant eggs through an adaptive maternal effect. The mechanisms making these eggs drought resistant still remain to be investigated. For this purpose, we studied the physiological differences between drought-resistant and drought-sensitive eggs. We compared the volume and the surface-area-to-volume ratio (SA:V) of the eggs, their sex ratio, their chemical composition (by gas chromatography-mass spectrometry), their internal and external structure [by scanning electron microscope (SEM) and transmission electron microscope (TEM) images], and their developmental time. Our results show that drought-resistant and drought-sensitive eggs have a different chemical composition: drought-resistant eggs contain more compatible solutes (free amino acids and sugar alcohols) and saturated hydrocarbons than drought-sensitive eggs. This difference may contribute to reducing water loss in drought-resistant eggs. Moreover, drought-resistant eggs are on average 8.4% larger in volume, and have a 2.4% smaller SA:V than drought-sensitive eggs. This larger volume and smaller SA:V, probably the result of a higher water content, may make drought-resistant eggs less vulnerable to water loss. We did not find any difference in sex ratio, internal or external structure nor developmental time between drought-resistant and drought-sensitive eggs. These results mark the first step in the understanding of the strategies and the energetic costs involved in the production of drought-resistant eggs in P. persimilis females.
- Klíčová slova
- Desiccation, Egg, Maternal effect, Metabolomic profiling, Phytoseiidae,
- MeSH
- období sucha * MeSH
- ovum fyziologie MeSH
- roztoči * MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
