Translocation of outer membrane vesicles from enterohemorrhagic Escherichia coli O157 across the intestinal epithelial barrier

Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS

Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov., two multidrug-resistant psychrotrophic species isolated from Antarctica

Despite unfavourable Antarctic conditions, such as cold temperatures, freeze-thaw cycles, high ultraviolet radiation, dryness and lack of nutrients, microorganisms were able to adapt and surprisingly thrive in this environment. In this study, eight cold-adapted Flavobacterium strains isolated from a remote Antarctic island, James Ross Island, were studied using polyphasic taxonomic approach to determine their taxonomic position. Phylogenomic analyses based on 16S rRNA gene and 92 core genes clearly showed that these strains formed two distinct phylogenetic clusters comprising three and five strains, with average nucleotide identities significantly below 90 % in between both proposed species as well as between their closest phylogenetic relatives. Phenotyping revealed a unique pattern of biochemical and physiological characteristics enabling differentiation from the closest phylogenetically related Flavobacterium spp. Chemotaxonomic analyses showed that type strains P4023T and P7388T were characterized by the major polyamine sym-homospermidine and a quinone system containing predominantly menaquinone MK-6. In the polar lipid profile phosphatidylethanolamine, an ornithine lipid and two unidentified lipids lacking a functional group were detected as major lipids. These characteristics along with fatty acid profiles confirmed, that these species belong to the genus Flavobacterium. Thorough genomic analysis revealed presence of numerous cold-inducible or cold-adaptation associated genes, such as cold-shock proteins, proteorhodopsin, carotenoid biosynthetic genes or oxidative-stress response genes. Genomes of type strains surprisingly harboured multiple prophages, with many of them predicted to be active. Genome-mining identified biosynthetic clusters in type strain genomes with majority not matching any known biosynthetic genes which indicates further research possibilities involving these psychrotrophic bacteria. Antibiotic susceptibility testing revealed multidrug-resistant phenotype that was correlated with in silico antibiotic resistance prediction. Interestingly, while typical resistance finder tools failed to detect genes responsible for antibiotic resistance, genomic prediction confirmed multidrug-resistant profile and suggested even broader resistance than tested. Results of this study confirmed and thoroughly characterized two novel psychrotrophic Flavobacterium species, for which names Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov. are proposed

The role of carbohydrates in abiotic stresses

Department of Experimental Plant BiologyKatedra experimentální biologie rostlinPřírodovědecká fakultaFaculty of Scienc

Abiotic stress resposes in apple (Malus domestica Borkh.) tissue cultures

Abiotic stress factors, primarily those which disturb plant water balance, cause extensive crop losses. With regard to sessile lifestyle, many mechanisms how to cope with these unfavourable conditions were developed during evolution of plants. As common impact of many of these stresses like drought, salinity or low temperatures is of osmotic nature, the osmotic adjustment represents important part of plant stress response. Mostly, this component of stress reaction is provided by organic compounds, which are referred to as compatible solutes, including polyhydroxyl compounds (sucrose, sugar alcohols, cyclitols, and oligosacharides) and nitrogen-containing compounds (mainly proline). The effect of these solutes is not only osmotic, but also osmoprotective as they are able to preserve integrity of membranes and macromolecules by mimicking their water envelope. The compatible solutes also contribute to quenching of reactive oxygen species overproduced under these stress conditions. This study is focused on apple (Malus domestica) producing, beside sucrose, sugar alcohol sorbitol as primary photosynthetic product and transporting these carbohydrates along with raffinose family oligosaccharides (RFO) for a long distance. In vitro tissue cultures derived from leaves of two apple cultivars differing in..

Abiotic stress resposes in apple (Malus domestica Borkh.) tissue cultures

Abiotic stress factors, primarily those which disturb plant water balance, cause extensive crop losses. With regard to sessile lifestyle, many mechanisms how to cope with these unfavourable conditions were developed during evolution of plants. As common impact of many of these stresses like drought, salinity or low temperatures is of osmotic nature, the osmotic adjustment represents important part of plant stress response. Mostly, this component of stress reaction is provided by organic compounds, which are referred to as compatible solutes, including polyhydroxyl compounds (sucrose, sugar alcohols, cyclitols, and oligosacharides) and nitrogen-containing compounds (mainly proline). The effect of these solutes is not only osmotic, but also osmoprotective as they are able to preserve integrity of membranes and macromolecules by mimicking their water envelope. The compatible solutes also contribute to quenching of reactive oxygen species overproduced under these stress conditions. This study is focused on apple (Malus domestica) producing, beside sucrose, sugar alcohol sorbitol as primary photosynthetic product and transporting these carbohydrates along with raffinose family oligosaccharides (RFO) for a long distance. In vitro tissue cultures derived from leaves of two apple cultivars differing in..

Behaviour of silver nanoparticles in simulated saliva and gastrointestinal fluids

Continuously increasing application of silver nanoparticles (AgNPs) requires information on their safety and performance under biological conditions. Assessment of AgNPs in biological systems is also related to availability of robust toxicological methods for evaluation of toxic potential of AgNPs and information on their physicochemical state. Silver nanoparticles were subjected to action of simulated saliva, gastric and intestinal fluids, appropriately supplemented with digestive enzymes pepsin or pancreatin. The behaviour of AgNPs was determined using dynamic light scattering and transmission electron microscopy, and their toxicity as well as capability to induce inflammatory reactions were assessed using reconstructed human tissue models (EpiOral, EpiGingival, EpiIntestinal). The study revealed that during exposure to the fluids, AgNPs size and morphology changed and depended on composition and pH of the respective fluid. If present, the change in terms of growth of AgNPs size occurred immediately after contact of AgNPs with the respective fluid and continued with prolonged time of contact. A pilot study on reconstituted human tissue models revealed low toxicity and inflammatory effects of AgNPs and confirmed the suitability of 3-D models for toxicological studies including bioavailability. © 201717-05095S, GACR, Grantová Agentura České RepublikyCzech Science Foundation [17-05095S]; Ministry of Health, Czech Republic conceptual development of research organization (National Institute of Public Health - NIPH) [75010330]; TBU in Zlin; funds of specific academic research [IGA/CPS/2017/001

Characteristics of silver nanoparticles in vehicles for biological applications

Silver nanoparticles (AgNPs) have been used for decades as anti-bacterial agents in various industrial fields such as cosmetics, health industry, food storage, textile coatings and environmental applications, although their toxicity is not fully recognized yet. Antimicrobial and catalytic activity of AgNPs depends on their size as well as structure, shape, size distribution, and physico-chemical environment. The unique properties of AgNPs require novel or modified toxicological methods for evaluation of their toxic potential combined with robust analytical methods for characterization of nanoparticles applied in relevant vehicles, e.g., culture medium with/without serum and phosphate buffered saline. © 2015 Elsevier B.V. All rights reserved.Internal Grant Agency of the Ministry of Health of the Czech Republic [NT 14060-3/2013]; TBU in Zlin [IGA/FT/2014/004