INTRODUCTION: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. METHODS: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria - Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. RESULTS: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. DISCUSSION: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.

• Klíčová slova
• Bacillus thuringiensis, comparative immune response, gut immunity, hematopoietic organ, insect immunity, insect midgut, lysozyme,
• MeSH
• larva MeSH
• Manduca * genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• muramidasa genetika   metabolismus   MeSH
• přirozená imunita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• muramidasa MeSH

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography.

• Klíčová slova
• crystal structure, endolysin, histidine tag, lysozyme, phage T4, protein purification,
• MeSH
• bakteriofág T4 * enzymologie   genetika   MeSH
• chromatografie afinitní metody   MeSH
• krystalografie rentgenová metody   MeSH
• muramidasa * chemie   genetika   izolace a purifikace   MeSH
• mutace * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• muramidasa * MeSH

Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.

• MeSH
• Bacteria klasifikace   genetika   imunologie   MeSH
• biologické toxiny genetika   imunologie   MeSH
• cytotoxicita imunologická genetika   imunologie   MeSH
• druhová specificita MeSH
• ekosystém * MeSH
• exprese genu MeSH
• hemolýza genetika   imunologie   MeSH
• hnůj mikrobiologie   parazitologie   MeSH
• molekulární sekvence - údaje MeSH
• muramidasa genetika   imunologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• Oligochaeta klasifikace   genetika   imunologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• přirozená imunita genetika   imunologie   MeSH
• proteiny genetika   imunologie   MeSH
• půdní mikrobiologie MeSH
• respirační komplex IV genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie nukleových kyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické toxiny MeSH
• fetidin MeSH Prohlížeč
• hnůj MeSH
• lysenin MeSH Prohlížeč
• muramidasa MeSH
• proteiny MeSH
• respirační komplex IV MeSH

Caenorhabditis elegans has been increasingly used to study the innate immunity and for the screening of microbe/host-specific pathogenic factors. Staphylococcus aureus-mediated infections with live C. elegans were performed on solid (full-lawn) and liquid assays. S. aureus required 90 ± 10 h for the complete killing of C. elegans, but the infection was started only after 32 h of exposure with 20% inoculum of S. aureus. The short time exposure studies revealed that, in 20% of inoculum, continuous exposure to the pathogen was required for the killing of nematode. In 100% of inoculum, only 8 h of exposure was sufficient to kill the C. elegans. To evaluate kinetically at the innate immune level, the regulation of representative candidate antimicrobial genes was investigated. Both semi-quantitative reverse transcriptase polymerase chain reaction (PCR) and real-time PCR analyses indicated the regulation of candidate immune regulatory genes of lysozyme (lys-7), cysteine protease (cpr-2), and C-type lectin (clec-60 and clec-87) family members during the course of S. aureus infections, indicating the possible contribution of the above players during the host immune response against S. aureus exposures.

• MeSH
• bakteriální léková rezistence MeSH
• Caenorhabditis elegans genetika   imunologie   metabolismus   MeSH
• cysteinové proteasy genetika   imunologie   metabolismus   MeSH
• exprese genu MeSH
• interakce hostitele a patogenu MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lektiny typu C genetika   imunologie   metabolismus   MeSH
• muramidasa genetika   imunologie   metabolismus   MeSH
• počet mikrobiálních kolonií MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• přirozená imunita * MeSH
• proteiny Caenorhabditis elegans genetika   imunologie   metabolismus   MeSH
• stafylokokové infekce genetika   imunologie   mikrobiologie   MeSH
• Staphylococcus aureus fyziologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cysteinové proteasy MeSH
• lektiny typu C MeSH
• muramidasa MeSH
• proteiny Caenorhabditis elegans MeSH

Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria.

• MeSH
• Bacillus subtilis imunologie   patogenita   MeSH
• bakteriální adheze MeSH
• chitinasy metabolismus   MeSH
• Echinodermata genetika   MeSH
• endopeptidasy metabolismus   MeSH
• Escherichia coli genetika   imunologie   patogenita   MeSH
• glukosamin analogy a deriváty   imunologie   metabolismus   MeSH
• grampozitivní bakteriální infekce imunologie   MeSH
• hydrolýza MeSH
• infekce vyvolané Escherichia coli imunologie   MeSH
• interakce hostitele a patogenu MeSH
• klonování DNA MeSH
• kyseliny muramové imunologie   metabolismus   MeSH
• lyasy štěpící vazby C-N metabolismus   MeSH
• muramidasa genetika   imunologie   metabolismus   MeSH
• Oligochaeta enzymologie   genetika   imunologie   MeSH
• pijavka lékařská genetika   MeSH
• sekvenční homologie MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chitinasy MeSH
• endopeptidasy MeSH
• fibrin destabilase MeSH Prohlížeč
• glukosamin MeSH
• isopeptidase MeSH Prohlížeč
• kyseliny muramové MeSH
• lyasy štěpící vazby C-N MeSH
• muramidasa MeSH
• N-acetylglucopyranosylamine MeSH Prohlížeč
• N-acetylmuramic acid MeSH Prohlížeč

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.

• MeSH
• DNA bakterií analýza   MeSH
• fenotyp MeSH
• Lactococcus lactis klasifikace   genetika   MeSH
• muramidasa genetika   MeSH
• polymerázová řetězová reakce metody   MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• ribozomální DNA analýza   MeSH
• RNA ribozomální 16S genetika   MeSH
• technika náhodné amplifikace polymorfní DNA * MeSH
• techniky typizace bakterií MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AcmA protein, Lactococcus lactis MeSH Prohlížeč
• DNA bakterií MeSH
• muramidasa MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH

Sequence of a tick gut lysozyme (TGL) from the soft tick Ornithodoros moubata was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and RACE PCR products. It is the first lysozyme sequence representing the subphylum Chelicerata. The resulting open reading frame codes for a putative signal peptide of 22 amino-acid residues and a mature protein composed of 124 amino-acids. Calculated mass of the protein is 14037.75 Da and a theoretical isoelectric point is 8.16. The phylogenetic analysis revealed that the TGL belongs to the c-type lysozymes. It forms a distinct monophyletic group together with multiple lysozyme-like sequences found in the gene products agCP6542 from Anopheles gambiae strain PEST and CG8492-PA from Drosophila melanogaster. This group is referred to as an H-branch due to a unique histidine residue at position 52 which replaces the highly conserved tyrosine present in the vast majority of c-type lysozymes. TGL seems to be an interesting case in which the features of lysozymes with anti-bacterial and digestive function are combined. Semi-quantitative RT-PCR and Northern blotting analysis demonstrated that TGL is strongly up-regulated at the transcriptional level after a bloodmeal. The maximum lysozyme mRNA level was detected 16 h post bloodmeal and the message remained stable for 5 days and then it slowly dropped down to the level of non-fed ticks within 2 weeks.

• MeSH
• fylogeneze * MeSH
• molekulární sekvence - údaje MeSH
• muramidasa genetika   metabolismus   MeSH
• northern blotting MeSH
• Ornithodoros fyziologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční analýza proteinů MeSH
• sekvenční homologie aminokyselin MeSH
• trávicí systém enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• muramidasa MeSH