PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.
- MeSH
- atrofie komplikace patologie MeSH
- běžná variabilní imunodeficience * komplikace imunologie MeSH
- CD8-pozitivní T-lymfocyty MeSH
- imunoglobulin A MeSH
- infekce viry z čeledi Caliciviridae * imunologie MeSH
- interferony MeSH
- lidé MeSH
- Norovirus * fyziologie MeSH
- zánět komplikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5'-untranslated region (5'-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways.IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.
- Publikační typ
- časopisecké články MeSH
EFLamide (EFLa) is a neuropeptide known for a long time from crustaceans, chelicerates and myriapods. Recently, EFLa-encoding genes were identified in the genomes of apterygote hexapods including basal insect species. In pterygote insects, however, evidence of EFLa was limited to partial sequences in the bed bug (Cimex), migratory locust and a few phasmid species. Here we present identification of a full length EFLa-encoding transcript in the linden bug, Pyrrhocoris apterus (Heteroptera). We created complete null mutants allowing unambiguous anatomical location of this peptide in the central nervous system. Only 2-3 EFLa-expressing cells are located very close to each other near to the surface of the lateral protocerebrum with dense neuronal arborization. Homozygous null EFLa mutants are fully viable and do not have any visible defect in development, reproduction, lifespan, diapause induction or circadian rhythmicity. Phylogenetic analysis revealed that EFLa-encoding transcripts are produced by alternative splicing of a gene that also produces Prohormone-4. However, this Proh-4/EFLa connection is found only in Hemiptera and Locusta, whereas EFLa-encoding transcripts in apterygote hexapods, chelicerates and crustaceans are clearly distinct from Proh-4 genes. The exact mechanism leading to the fused Proh-4/EFLa transcript is not yet determined, and might be a result of canonical cis-splicing, cis-splicing of adjacent genes (cis-SAG), or trans-splicing.
- MeSH
- fylogeneze MeSH
- Heteroptera genetika metabolismus MeSH
- hmyzí proteiny chemie genetika metabolismus MeSH
- hormon uvolňující thyreotropin genetika metabolismus MeSH
- neuropeptidy chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A novel actinobacterial strain, designated 15TR583T, was isolated from a waterlogged acidic soil collected near the town of Trebon, Czech Republic, and was subjected to a polyphasic taxonomic characterization. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that the organism forms an individual line of descent related to the order Streptosporangiales, class Actinomycetia. The strain shared highest 16S rRNA gene sequence similarity, yet of only 92.8%, with Actinocorallia aurea IFO 14752T. The strain grew in white colonies of aerobic, Gram-stain-positive, unbranching substrate mycelium bearing single spores at hyphae tips. The major fatty acids (>10%) were iso-C16 : 0, C16 : 0, iso-C17 : 1ω9 and 10-methyl-C17 : 0. The fatty acid pattern differed from all patterns currently described for actinobacterial genera. The organism contained as major menaquinones MK9(H6) and MK9(H8), which differentiated it from other actinobacterial families. Polar lipids were composed of six unidentified glycolipids, an unidentified phosphoglycolipid, two unidentified phospholipids and two unidentified aminolipids. Whole-cell sugars contained galactose, xylose and arabinose as major components. The peptidoglycan type was A1γ meso-diaminopimelic acid. The genomic DNA G+C content was 69.7 mol%. The distinct phylogenetic position and unusual combination of chemotaxonomic characteristics justify the proposal of Trebonia gen. nov., with the type species Trebonia kvetii sp. nov. (type strain 15TR583T=CCM 8942T=DSM 109105T), within Treboniaceae fam. nov.
- MeSH
- Actinobacteria klasifikace izolace a purifikace MeSH
- buněčná stěna chemie MeSH
- DNA bakterií genetika MeSH
- fosfolipidy chemie MeSH
- fylogeneze * MeSH
- glykolipidy chemie MeSH
- kyselina diaminopimelová chemie MeSH
- mastné kyseliny chemie MeSH
- peptidoglykan chemie MeSH
- půdní mikrobiologie * MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- vitamin K 2 analogy a deriváty chemie MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.
- Publikační typ
- časopisecké články MeSH
Adult females of the genus Ixodes imbibe blood meals exceeding about 100 times their own weight within 7‒9 days. During this period, ticks internalise components of host blood by endocytic digest cells that line the tick midgut epithelium. Using RNA-seq, we aimed to characterise the midgut transcriptome composition in adult Ixodes ricinus females during early and late phase of engorgement. To address specific adaptations to the haemoglobin-rich diet, we compared the midgut transcriptomes of genetically homogenous female siblings fed either bovine blood or haemoglobin-depleted serum. We noted that tick gut transcriptomes are subject to substantial temporal-dependent expression changes between day 3 and day 8 of feeding. In contrast, the number of transcripts significantly affected by the presence or absence of host red blood cells was low. Transcripts relevant to the processes associated with blood-meal digestion were analysed and involvement of selected encoded proteins in the tick midgut physiology discussed. A total of 7215 novel sequences from I. ricinus were deposited in public databases as an additional outcome of this study. Our results broaden the current knowledge of tick digestive system and may lead to the discovery of potential molecular targets for efficient tick control.
- MeSH
- klíště genetika metabolismus MeSH
- sekvenční analýza RNA * MeSH
- skot MeSH
- stanovení celkové genové exprese * MeSH
- střeva metabolismus patologie MeSH
- transkriptom fyziologie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
Circadian clocks keep organisms in synchrony with external day-night cycles. The free running period (FRP) of the clock, however, is usually only close to-not exactly-24 h. Here, we explored the geographical variation in the FRP of the linden bug, Pyrrhocoris apterus, in 59 field-lines originating from a wide variety of localities representing geographically different environments. We have identified a remarkable range in the FRPs between field-lines, with the fastest clock at ~21 h and the slowest close to 28 h, a range comparable to the collections of clock mutants in model organisms. Similarly, field-lines differed in the percentage of rhythmic individuals, with a minimum of 13.8% and a maximum of 86.8%. Although the FRP correlates with the latitude and perhaps with the altitude of the locality, the actual function of this FRP diversity is currently unclear. With the recent technological progress of massive parallel sequencing and genome editing, we can expect remarkable progress in elucidating the genetic basis of similar geographic variants in P. apterus or in similar emerging model species of chronobiology.
- MeSH
- časové faktory MeSH
- cirkadiánní hodiny fyziologie MeSH
- cirkadiánní rytmus fyziologie MeSH
- fylogeneze MeSH
- Heteroptera klasifikace genetika fyziologie MeSH
- pohybová aktivita fyziologie MeSH
- Tilia parazitologie MeSH
- zeměpis MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Geografické názvy
- Evropa MeSH
- Izrael MeSH
- východní Evropa MeSH
The ability to perceive geomagnetic fields (GMFs) represents a fascinating biological phenomenon. Studies on transgenic flies have provided evidence that photosensitive Cryptochromes (Cry) are involved in the response to magnetic fields (MFs). However, none of the studies tackled the problem of whether the Cry-dependent magnetosensitivity is coupled to the sole MF presence or to the direction of MF vector. In this study, we used gene silencing and a directional MF to show that mammalian-like Cry2 is necessary for a genuine directional response to periodic rotations of the GMF vector in two insect species. Longer wavelengths of light required higher photon fluxes for a detectable behavioral response, and a sharp detection border was present in the cyan/green spectral region. Both observations are consistent with involvement of the FADox, FAD(•-) and FADH(-) redox forms of flavin. The response was lost upon covering the eyes, demonstrating that the signal is perceived in the eye region. Immunohistochemical staining detected Cry2 in the hemispherical layer of laminal glia cells underneath the retina. Together, these findings identified the eye-localized Cry2 as an indispensable component and a likely photoreceptor of the directional GMF response. Our study is thus a clear step forward in deciphering the in vivo effects of GMF and supports the interaction of underlying mechanism with the visual system.
- MeSH
- fenotyp MeSH
- fotoreceptory bezobratlých metabolismus účinky záření MeSH
- kryptochromy metabolismus MeSH
- magnetické pole * MeSH
- složené oko členovců účinky záření MeSH
- švábi metabolismus účinky záření MeSH
- ultrafialové záření MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Diapause is a developmental alternative to direct ontogeny in many invertebrates. Its primary adaptive meaning is to secure survival over unfavourable seasons in a state of developmental arrest usually accompanied by metabolic suppression and enhanced tolerance to environmental stressors. During photoperiodically triggered diapause of insects, the ontogeny is centrally turned off under hormonal control, the molecular details of this transition being poorly understood. Using RNAseq technology, we characterized transcription profiles associated with photoperiodic diapause induction in the larvae of the drosophilid fly Chymomyza costata with the goal of identifying candidate genes and processes linked to upstream regulatory events that eventually lead to a complex phenotypic change. RESULTS: Short day photoperiod triggering diapause was associated to inhibition of 20-hydroxy ecdysone (20-HE) signalling during the photoperiod-sensitive stage of C. costata larval development. The mRNA levels of several key genes involved in 20-HE biosynthesis, perception, and signalling were significantly downregulated under short days. Hormonal change was translated into downregulation of a series of other transcripts with broad influence on gene expression, protein translation, alternative histone marking by methylation and alternative splicing. These changes probably resulted in blockade of direct development and deep restructuring of metabolic pathways indicated by differential expression of genes involved in cell cycle regulation, metabolism, detoxification, redox balance, protection against oxidative stress, cuticle formation and synthesis of larval storage proteins. This highly complex alteration of gene transcription was expressed already during first extended night, within the first four hours after the change of the photoperiodic signal from long days to short days. We validated our RNAseq differential gene expression results in an independent qRT-PCR experiment involving wild-type (photoperiodic) and NPD-mutant (non-photoperiodic) strains of C. costata. CONCLUSIONS: Our study revealed several strong candidate genes for follow-up functional studies. Candidate genes code for upstream regulators of a complex change of gene expression, which leads to phenotypic switch from direct ontogeny to larval diapause.
- MeSH
- Drosophilidae embryologie genetika MeSH
- genetická transkripce * MeSH
- larva genetika MeSH
- reprodukovatelnost výsledků MeSH
- sekvenční analýza RNA MeSH
- shluková analýza MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), prevents metamorphosis in larvae and stimulates vitellogenesis in adult females. Whether the same JH signaling pathway regulates both processes is presently unknown. Here, we employ the robust JH response during reproduction and development of the linden bug, Pyrrhocoris apterus, to compare the function of key JH-signaling genes encoding the JH receptor, Methoprene-tolerant (Met), its binding partner Taiman (Tai), and a JH-inducible protein, Krüppel-homolog 1 (Kr-h1). RNA interference (RNAi) with Met or Tai, but not Kr-h1, blocked ovarian development and suppressed vitellogenin gene expression in the fat body of females raised under reproduction-inducing conditions. Loss of Met and Tai matched the effects of CA ablation or the natural absence of JH during reproductive diapause. Stimulation of vitellogenesis by treatment of diapausing females with a JH mimic methoprene also required both Met and Tai in the fat body, whereas Kr-h1 RNAi had no effect. Therefore, the Met-Tai complex likely functions as a JH receptor during vitellogenesis. In contrast to Met and Kr-h1 that are both required for JH to prevent precocious metamorphosis in P. apterus larvae, removal of Tai disrupted larval ecdysis without causing premature adult development. Our results show that while Met operates during metamorphosis in larvae and reproduction in adult females, its partner Tai is only required for the latter. The diverse functions of JH thus likely rely on a common receptor whose actions are modulated by distinct components.
- MeSH
- diapauza hmyzu MeSH
- Heteroptera růst a vývoj metabolismus fyziologie MeSH
- juvenilní hormony metabolismus MeSH
- larva růst a vývoj metabolismus fyziologie MeSH
- rozmnožování MeSH
- sexuální chování zvířat MeSH
- shazování tělního pokryvu MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
