Escherichia coli Common Pilus (ECP) Targets Arabinosyl Residues in Plant Cell Walls to Mediate Adhesion to Fresh Produce Plants

International audienceBackground: Bacterial fimbriae mediate binding to host tissue through specific interactions. Results: ECP interacts with arabinosyl residues in pectin and other plant cell wall components. Conclusion: ECP-arabinan interactions facilitate binding of E. coli to plant hosts. Significance: The prevalence of arabinan targets in produce plants together with ECP expression may explain the association of pathogenic bacteria in edible plants

The role of l-arabinose metabolism for <i>Escherichia coli</i> O157:H7 in edible plants

International audienceArabinose is a major plant aldopentose in the form of arabinans complexed in cell wall polysaccharides or glycoproteins (AGP), but comparatively rare as a monosaccharide. l -arabinose is an important bacterial metabolite, accessed by pectolytic micro-organisms such as Pectobacterium atrosepticum via pectin and hemicellulose degrading enzymes. However, not all plant-associated microbes encode cell-wall-degrading enzymes, yet can metabolize l -arabinose, raising questions about their use of and access to the glycan in plants. Therefore, we examined l -arabinose metabolism in the food-borne pathogen Escherichia coli O157:H7 (isolate Sakai) during its colonization of plants. l -arabinose metabolism ( araBA ) and transport ( araF ) genes were activated at 18 °C in vitro by l -arabinose and expressed over prolonged periods in planta . Although deletion of araBAD did not impact the colonization ability of E. coli O157:H7 (Sakai) on spinach and lettuce plants (both associated with STEC outbreaks), araA was induced on exposure to spinach cell-wall polysaccharides. Furthermore, debranched and arabinan oligosaccharides induced ara metabolism gene expression in vitro , and stimulated modest proliferation, while immobilized pectin did not. Thus, E. coli O157:H7 (Sakai) can utilize pectin/AGP-derived l -arabinose as a metabolite. Furthermore, it differs fundamentally in ara gene organization, transport and regulation from the related pectinolytic species P. atrosepticum , reflective of distinct plant-associated lifestyles

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively

The impact of plasma membrane lipid composition on flagella-mediated adhesion of enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions

Bacterial Flagella: Twist and Stick, or Dodge across the Kingdoms

The flagellum organelle is an intricate multiprotein assembly best known for its rotational propulsion of bacteria. However, recent studies have expanded our knowledge of other functions in pathogenic contexts, particularly adherence and immune modulation, e.g., for Salmonella enterica, Campylobacter jejuni, Pseudomonas aeruginosa, and Escherichia coli. Flagella-mediated adherence is important in host colonisation for several plant and animal pathogens, but the specific interactions that promote flagella binding to such diverse host tissues has remained elusive. Recent work has shown that the organelles act like probes that find favourable surface topologies to initiate binding. An emerging theme is that more general properties, such as ionic charge of repetitive binding epitopes and rotational force, allow interactions with plasma membrane components. At the same time, flagellin monomers are important inducers of plant and animal innate immunity: variation in their recognition impacts the course and outcome of infections in hosts from both kingdoms. Bacteria have evolved different strategies to evade or even promote this specific recognition, with some important differences shown for phytopathogens. These studies have provided a wider appreciation of the functions of bacterial flagella in the context of both plant and animal reservoirs

Biological and pathological implications of MUC5AC mucin in gastro-intestinal tract

Les mucines sont impliquées dans de nombreux mécanismes biologiques comme la cancérogenèse et les interactions hôte/pathogènes, faisant de ces glycoprotéines de potentielles cibles thérapeutiques ou diagnostiques. Il a, par exemple, été démontré une néoexpression de MUC5AC dans les tumeurs coliques et dans les foyers de cryptes aberrantes (FCA) qui apparaissent dès les premières étapes de développement du cancer du côlon. MUC5AC constitue ainsi un biomarqueur de choix pour le diagnostic et le pronostic du cancer du côlon. La première partie de notre travail de thèse a consisté à développer un produit de contraste adapté à l’imagerie du cancer du côlon. Pour cela, nous avons tout d’abord sélectionné, par la technique du phage display, un peptide affin vis-à-vis de MUC5AC qui a été préalablement purifiée à partir d’adénomes coliques humains. Ce peptide a ensuite été conjugué à la biotine et greffé à un produit de contraste pour l’imagerie par résonance magnétique (IRM) (USPIO : Ultrasmall Particles of Iron Oxide). Cela nous a permis d’apporter la preuve de l’efficacité de détection par IRM des polypes pré-cancéreux dans des modèles animaux et cellulaires qui surexpriment MUC5AC et dans des tissus coliques humains. Dans l’estomac, MUC5AC est exprimée de façon physiologique et est impliquée dans l’adhésion d’Helicobacter pylori à la muqueuse gastrique. Cette adhésion est un prérequis aux pathologies induites par H. pylori. Dans la deuxième partie de notre travail de thèse, nous avons démontré que parmi les différents oligosaccharides portés par les mucines gastriques humaines, un nouveau type de O-glycane portant un motif LacdiNAc (LDN) était impliqué dans l’adhésion de la bactérie à la muqueuse gastrique. Le LDN est exclusivement porté par la mucine MUC5AC gastrique et son expression est corrélée à la localisation de H. pylori. Toutes les souches de H. pylori testées adhèrent fortement au LDN. L’analyse protéomique et la construction de mutants ont permis d’identifier une nouvelle adhésine bactérienne, LabA (LacdiNAc antigen binding adhesin) qui reconnait spécifiquement le LDN. Cette découverte permet de mieux comprendre les mécanismes d’adhésion d’H. pylori et son tropisme d’organe. Ces résultats sont le point de départ d’études visant à étudier l’implication du LDN dans la physiologie gastrique et l’homéostasie, mais aussi pour le développement de stratégies alternatives pour le traitement de l’infection à H. pylori.Mucins are implicated in different biological phenomena such as cancer development and host/pathogen interactions. Mucins have thus been identified as promising therapeutic targets and have been proposed as potential prognostic or diagnostic markers.Improved detection sensitivity of early colorectal cancer would have important clinical applications. In this PhD thesis work, we report the development of new peptides against MUC5AC, a good marker of aberrant crypt foci (ACF), for the early diagnostic of colorectal cancer. Peptides have been identified by screening phage display peptide libraries against MUC5AC purified from fresh human colonic adenomas. One heptapeptide has been selected and further conjugated with biotin for immunohistochemistry studies and USPIO (ultrasmall superparamagnetic iron oxides) for nuclear magnetic resonance imagery (MRI). Its efficiency to detect MUC5AC has been evaluated on cellular and animal models which overexpressed this mucin, as well as on human colonic tissues. In stomach, MUC5AC is physiologically expressed and is implicated in adhesion of Helicobacter pylori to the gastric mucosa. This adhesion is a necessary prerequisite for the pathogenesis of H. pylori related diseases. Here we demonstrated that, among all the oligosaccharides expressed on human gastric mucins, a new type of O-glycan carrying a LacdiNAc motif is implicated in the binding of the bacteria to the gastric mucosa. LacdiNAc was exclusively carried by gastric MUC5AC mucin and its expression correlated with H. pylori localization. All strains tested adhere strongly to LacdiNAc. Proteomic analysis and construction of mutants allowed us to identify a novel bacterial adhesin, LabA (LacdiNAc antigen binding adhesin), which specifically recognizes LacdiNAc. These findings give new insights into the mechanisms of adhesion of H. pylori and its specific tissue tropism. We anticipate our results to be a starting point to study implication of LDN in gastric physiology and homeostasis and for development of alternative strategies for treatment of H. pylori infection

Flagellar Phenotypes Impact on Bacterial Transport and Deposition Behavior in Porous Media: Case of <i>Salmonella enterica</i> Serovar Typhimurium

Bacterial contamination of groundwater has always been an ecological problem worthy of attention. In this study, Salmonella enterica serovar Typhimurium with different flagellar phenotypes mainly characterized during host-pathogen interaction were analyzed for their transport and deposition behavior in porous media. Column transport experiments and a modified mobile-immobile model were applicated on different strains with flagellar motility (wild-type) or without motility (ΔmotAB), without flagella (ΔflgKL), methylated and unmethylated flagellin (ΔfliB), and different flagella phases (fliCON, fljBON). Results showed that flagella motility could promote bacterial transport and deposition due to their biological advantages of moving and attaching to surfaces. We also found that the presence of non-motile flagella improved bacterial adhesion according to a higher retention rate of the ΔmotAB strain compared to the ΔflgKL strain. This indicated that bacteria flagella and motility both had promoting effects on bacterial deposition in sandy porous media. Flagella phases influenced the bacterial movement; the fliCON strain went faster through the column than the fljBON strain. Moreover, flagella methylation was found to favor bacterial transport and deposition. Overall, flagellar modifications affect Salmonella enterica serovar Typhimurium transport and deposition behavior in different ways in environmental conditions

Cloning and molecular characterization of three lysophosphatidic acid acyltransferases expressed in flax seeds

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Simultaneous Quantification of Trehalose and Trehalose 6-Phosphate by Hydrophilic Interaction Chromatography/Electrospray Accurate Mass Spectrometry with Application in Non-Targeted Metabolomics

High-resolution mass spectrometry (HRMS) was coupled with ultra-high-performance liquid chromatography (UHPLC) to simultaneously quantify trehalose and trehalose 6-phosphate without derivatization or sample preparation. The use of full scan mode and exact mass analysis also makes it possible to carry out metabolomic analyses as well as semi-quantification. In addition, the use of different clusters in negative mode makes it possible to compensate for deficiencies in linearity and inerrant saturation at time-of-flight detectors. The method has been approved and validated for different matrices, yeasts, and bacteria, and has shown differentiation between bacteria as a function of growth temperatures