Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

• Klíčová slova
• Arabidopsis thaliana, EVs, NTA, PTI, Pto DC3000, extracellular vesicles, nanoparticle tracking analysis, pattern-triggered immunity, proteomics,
• MeSH
• bakteriální proteiny metabolismus   MeSH
• extracelulární vezikuly * metabolismus   MeSH
• flagelin metabolismus   MeSH
• lidé MeSH
• nemoci rostlin mikrobiologie   MeSH
• proteomika MeSH
• Pseudomonas syringae genetika   metabolismus   MeSH
• Solanum lycopersicum * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• flagelin MeSH

Proinflammatory cytokines play important roles in the pathogenesis of diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157, but the spectrum of bacterial components involved in the proinflammatory responses is not fully understood. Here, we investigated the abilities of outer membrane vesicles (OMVs), nanoparticles released by EHEC O157 during growth, to induce production of proinflammatory cytokines in human intestinal epithelial cells. OMVs from both EHEC O157:H7 and sorbitol-fermenting (SF) EHEC O157:H- induced production of interleukin-8 (IL-8) in Caco-2, HCT-8, and HT-29 intestinal epithelial cell lines. H7 flagellin was the key IL-8-inducing component of EHEC O157:H7 OMVs, whereas cytolethal distending toxin V and O157 lipopolysaccharide (LPS) largely contributed to IL-8 production elicited by flagellin-lacking OMVs from SF EHEC O157:H-. The H7 flagellin-mediated signaling via Toll-like receptor (TLR) 5, and O157 LPS-mediated signaling via TLR4/MD-2 complex, which were followed by activation of the nuclear factor NF-κB were major pathways underlying IL-8 production induced by EHEC O157 OMVs. The proinflammatory and immunomodulatory capacities of EHEC O157 OMVs have pathogenetic implications and support the OMVs as suitable vaccine candidates.

• Klíčová slova
• Enterohemorrhagic Escherichia coli O157, Flagellin, Immunomodulation, Lipopolysaccharide, Outer membrane vesicles, Proinflammatory cytokines,
• MeSH
• buněčná membrána metabolismus   MeSH
• buňky HT-29 MeSH
• Caco-2 buňky MeSH
• epitelové buňky metabolismus   MeSH
• Escherichia coli O157 patogenita   MeSH
• faktory virulence metabolismus   MeSH
• flagelin metabolismus   MeSH
• infekce vyvolané Escherichia coli mikrobiologie   patologie   MeSH
• interleukin-8 biosyntéza   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• NF-kappa B metabolismus   MeSH
• proteiny vnější bakteriální membrány metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• signální transdukce MeSH
• střevní sliznice cytologie   mikrobiologie   patologie   MeSH
• toll-like receptor 4 metabolismus   MeSH
• toll-like receptor 5 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CXCL8 protein, human MeSH Prohlížeč
• faktory virulence MeSH
• flagelin MeSH
• interleukin-8 MeSH
• NF-kappa B MeSH
• proteiny vnější bakteriální membrány MeSH
• proteiny z Escherichia coli MeSH
• TLR4 protein, human MeSH Prohlížeč
• TLR5 protein, human MeSH Prohlížeč
• toll-like receptor 4 MeSH
• toll-like receptor 5 MeSH

BACKGROUND: One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. METHODS AND RESULTS: We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. CONCLUSION: We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

• MeSH
• alanindehydrogenasa genetika   metabolismus   MeSH
• aspartátsemialdehyddehydrogenasa genetika   metabolismus   MeSH
• Bacillus subtilis účinky léků   enzymologie   genetika   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• delece genu MeSH
• Escherichia coli genetika   metabolismus   MeSH
• ethanol farmakologie   MeSH
• flagelin genetika   metabolismus   MeSH
• fosfoglycerátkinasa genetika   metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem nedostatek   genetika   MeSH
• fyziologická adaptace genetika   MeSH
• glutaminsynthetasa genetika   metabolismus   MeSH
• isocitrátdehydrogenasa genetika   metabolismus   MeSH
• malátdehydrogenasa genetika   metabolismus   MeSH
• operon MeSH
• osmolární koncentrace MeSH
• reakce na tepelný šok genetika   MeSH
• reduktoizomeráza pro obousměrnou transformaci ketokyselin genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanindehydrogenasa MeSH
• aspartátsemialdehyddehydrogenasa MeSH
• bakteriální proteiny MeSH
• ethanol MeSH
• flagelin MeSH
• fosfoglycerátkinasa MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• glutaminsynthetasa MeSH
• isocitrátdehydrogenasa MeSH
• malátdehydrogenasa MeSH
• reduktoizomeráza pro obousměrnou transformaci ketokyselin MeSH

Flagellin potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Recently, we were able to show that stimulation of bovine TLR5 resulted in neither NFκB signalling nor CXCL8 production. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Analysis of available TLR5 sequences revealed substitutions in all artiodactyl sequences at amo acid (AA) position 798 and 799. Interestingly, a putative binding site for PI3K was identified at tyrosine 798 in the human TLR5 TIR domain, analogous to the PI3K recruitment domain in the IL-1 receptor. Mutation of the artiodactyl residues at position 798, 799 or both with their corresponding human counterparts partially restored the response of bovine (bo)TLR5 to flagellin as well as phosphorylation of PI3K. Together, our results suggest a potential lack of phosphorylation of F798 and H799 in boTLR5 partially explains the lack in observed response.

• Klíčová slova
• Cattle, Flagellin, Human, TLR5,
• MeSH
• Bacteria chemie   MeSH
• flagelin metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• lidé MeSH
• signální transdukce MeSH
• skot MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• toll-like receptor 5 chemie   metabolismus   MeSH
• zánět imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• flagelin MeSH
• fosfatidylinositol-3-kinasy MeSH
• toll-like receptor 5 MeSH

Molecular cloning, nucleotide sequencing, and characterization of the flaA gene from additional isolates of urease-positive thermophilic Campylobacter (UPTC) were performed. These isolates were obtained from the natural environment in Northern Ireland (n = 9 from mussels) and in England (n = 1 from sea water). All isolates carried the shorter flaA gene, [open reading frames (ORFs), 1,461 to 1,503 base pairs], without any internal termination codons, and did not carry any flaA pseudogenes. The UPTC isolates were well discriminated by the neighbor joining (NJ) phylogenetic tree constructed based on the putative flaA genes ORFs nucleotide sequence information. In addition, the NJ tree constructed based on the flaA-short variable region sequence information discriminated the Campylobacter lari isolates with a similar degree of discrimination power.

• MeSH
• Campylobacter lari klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• Campylobacter klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• flagelin chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• klonování DNA MeSH
• mlži mikrobiologie   MeSH
• molekulární sekvence - údaje MeSH
• mořská voda mikrobiologie   MeSH
• otevřené čtecí rámce MeSH
• polymerázová řetězová reakce MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA metody   MeSH
• sekvenční seřazení MeSH
• ureasa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Anglie MeSH
• Severní Irsko MeSH
• Názvy látek
• flaA protein, bacteria MeSH Prohlížeč
• flagelin MeSH
• rekombinantní proteiny MeSH
• ureasa MeSH

The nodulin/glutamine synthetase-like protein (NodGS) that we identified proteomically in Arabidopsis thaliana is a fusion protein composed of an N-terminal amidohydrolase domain that shares homology with nodulins and a C-terminal domain of prokaryotic glutamine synthetase type I. The protein is homologous to the FluG protein, a morphogenetic factor in fungi. Although genes encoding NodGS homologues are present in many plant genomes, their products have not yet been characterized. The Arabidopsis NodGS was present in an oligomeric form of ~700-kDa, mainly in the cytosol, and to a lesser extent in the microsomal membrane fraction. The oligomeric NodGS was incorporated into large heterogeneous protein complexes >700 kDa and partially co-immunoprecipitated with γ-tubulin. In situ and in vivo microscopic analyses revealed a NodGS signal in the cytoplasm, with endomembranes, particularly in the perinuclear area. NodGS had no detectable glutamine synthetase activity. Downregulation of NodGS by RNAi resulted in plants with a short main root, reduced meristematic activity and disrupted development of the root cap. Y2H analysis and publicly available microarray data indicated a role for NodGS in biotic stress signalling. We found that flagellin enhanced the expression of the NodGS protein, which was then preferentially localized in the nuclear periphery. Our results point to a role for NodGS in root morphogenesis and microbial elicitation. These data might help in understanding the family of NodGS/FluG-like fusion genes that are widespread in prokaryotes, fungi and plants.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• flagelin genetika   metabolismus   MeSH
• glutaminsynthetasa genetika   metabolismus   fyziologie   MeSH
• kořeny rostlin genetika   růst a vývoj   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   fyziologie   MeSH
• morfogeneze fyziologie   MeSH
• proteiny huseníčku genetika   metabolismus   fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   metabolismus   fyziologie   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• flagelin MeSH
• glutaminsynthetasa MeSH
• membránové proteiny MeSH
• nodulin MeSH Prohlížeč
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny MeSH

In this study we have compared protein secretion in the wild type of S. Typhimurium and the rfaC mutant. We found out that the rfaC mutant was defective in protein secretion. In addition, the rfaC mutant was defective in its invasion into an IPEC-J2 porcine epithelial cell line and also in motility in semisolid agar. Consistent with this, reduced flagella numbers were observed in the rfaC mutant. In the rfaC mutant, there were no defects in flagellin expression as detected by western blot and immune electron microscopy which demonstrated equal amounts of flagellin in the cytoplasm of both the rfaC mutant and the wild-type S. Typhimurium. However, in the wild-type strain only, the flagellin was assembled to spatially restricted areas on the inner side of cytoplasmic membrane. The oligosaccharide core of LPS is therefore required for the assembly of flagella and T3SS secretion machinery followed by protein secretion.

• MeSH
• bakteriální sekreční systémy * MeSH
• buněčné linie MeSH
• cytoplazma chemie   MeSH
• epitelové buňky mikrobiologie   MeSH
• flagelin biosyntéza   metabolismus   MeSH
• flagella metabolismus   MeSH
• imunoelektronová mikroskopie MeSH
• lipopolysacharidy chemie   MeSH
• mutace MeSH
• prasata MeSH
• Salmonella enterica genetika   metabolismus   ultrastruktura   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální sekreční systémy * MeSH
• flagelin MeSH
• lipopolysacharidy MeSH

We investigated the presence of glycoproteins in Borrelia burgdorferi. We did not find any evidence for glycosylation of the major outer membrane proteins OspA and OspB or the structural flagellar proteins FlaB and FlaA. We suggest that glycoproteins present on the surface of B. burgdorferi may be tightly bound culture medium glycoproteins.

• MeSH
• antigeny bakteriální genetika   metabolismus   MeSH
• antigeny povrchové genetika   metabolismus   MeSH
• bakteriální vakcíny genetika   metabolismus   MeSH
• Borrelia burgdorferi genetika   metabolismus   ultrastruktura   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• elektronová kryomikroskopie MeSH
• flagelin genetika   metabolismus   MeSH
• glykosylace MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• lipoproteiny genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• proteiny vnější bakteriální membrány genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• antigeny povrchové MeSH
• bakteriální vakcíny MeSH
• flaA protein, bacteria MeSH Prohlížeč
• flaB flagellin MeSH Prohlížeč
• flagelin MeSH
• lipoproteiny MeSH
• OspA protein MeSH Prohlížeč
• OspB protein, Borrelia burgdorferi MeSH Prohlížeč
• proteiny vnější bakteriální membrány MeSH