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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.

Most Escherichia coli strains associated with neonatal meningitis express the K1 capsule, a sialic acid polysaccharide that is directly related to their pathogenicity. Metabolic oligosaccharide engineering (MOE) has mostly been developed in eukaryotes, but has also been successfully applied to the study of several oligosaccharides or polysaccharides constitutive of the bacterial cell wall. However, bacterial capsules are seldom targeted despite their important role as virulence factors, and the K1 polysialic acid (PSA) antigen that shields bacteria from the immune system still remains untackled. Herein, we report a fluorescence microplate assay that allows the fast and facile detection of K1 capsules with an approach that combines MOE and bioorthogonal chemistry. We exploit the incorporation of synthetic analogues of N-acetylmannosamine or N-acetylneuraminic acid, metabolic precursors of PSA, and copper-catalysed azide-alkyne cycloaddition (CuAAC) as the click chemistry reaction to specifically label the modified K1 antigen with a fluorophore. The method was optimized, validated by capsule purification and fluorescence microscopy, and applied to the detection of whole encapsulated bacteria in a miniaturized assay. We observe that analogues of ManNAc are readily incorporated into the capsule while those of Neu5Ac are less efficiently metabolized, which provides useful information regarding the capsule biosynthetic pathways and the promiscuity of the enzymes involved. Moreover, this microplate assay is transferable to screening approaches and may provide a platform to identify novel capsule-targeted antibiotics that would circumvent resistance issues.

Arabinose 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.

Molecularly imprinted polymers (MIPs), often dubbed "synthetic antibodies", can recognize and bind their target molecule with high affinity and selectivity, making them serious competitors with regard to biological antibodies. MIPs have gained popularity in various clinical applications and have even been applied

Fresh produce is often a source of enterohaemorrhagic Escherichia coli (EHEC) outbreaks. Fimbriae are extracellular structures involved in cell-to-cell attachment and surface colonisation. F9 (Fml) fimbriae have been shown to be expressed at temperatures lower than 37 &deg, C, implying a function beyond the mammalian host. We demonstrate that F9 fimbriae recognize plant cell wall hemicellulose, specifically galactosylated side chains of xyloglucan, using glycan arrays. E. coli expressing F9 fimbriae had a positive advantage for adherence to spinach hemicellulose extract and tissues, which have galactosylated oligosaccharides as recognized by LM24 and LM25 antibodies. As fimbriae are multimeric structures with a molecular pattern, we investigated whether F9 fimbriae could induce a transcriptional response in model plant Arabidopsis thaliana, compared with flagella and another fimbrial type, E. coli common pilus (ECP), using DNA microarrays. F9 induced the differential expression of 435 genes, including genes involved in the plant defence response. The expression of F9 at environmentally relevant temperatures and its recognition of plant xyloglucan adds to the suite of adhesins EHEC has available to exploit the plant niche.

Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies., 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. IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

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 ofEscherichia coliO157:H7 andSalmonella entericaserovar 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 ofS. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants ofE. coliO157:H7 andS. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella fromE. coliO157 (H7), EPEC O127 (H6) andS. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increasein vitroactin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion forE. coliO157:H7 andS. Typhimurium, respectively.

EnterohemorrhagicEscherichia coli(EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC on host tissues is the first step enabling bacterial colonization. Adhesins like fimbriae and flagella mediate this mechanism. 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 non-flagellated 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. Flagella-mediated adhesion to plasma membranes has broad implications to host-pathogen interactions.ImportanceBacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues and form biofilm. EnterohemorrhagicE. coliO157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E.coliflagella-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E.coliflagella adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts are important in bacterial adhesion. These findings significantly strengthen plasma membrane adhesion via bacterial flagella in an important human pathogen. This mechanism represents a promising target for the development of novel anti-adhesion therapies.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of ligands and mechanisms of binding are still debated. In this study, the molecular basis ofE. coliO157:H7 andS. 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. SEM revealed flagella disappearing into cellular surfaces and TEM ofS. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants ofE. coliO157:H7 andS. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella fromE. coliO157 (H7), EPEC O127 (H6), andS. Typhimurium (P1 & P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerisation rates.

Flax (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3). This polyunsaturated fatty acid is well known for its nutritional role in human and animal diets. Understanding storage lipid biosynthesis in developing flax embryos can lead to an increase in seed yield via marker-assisted selection. While a tremendous amount of work has been done on different plant species to highlight their metabolism during embryo development, a comprehensive analysis of metabolic flux in flax is still lacking. In this context, we have utilized in vitro cultured developing embryos of flax and determined net fluxes by performing three complementary parallel labeling experiments with 13C-labeled glucose and glutamine. Metabolic fluxes were estimated by computer-aided modeling of the central metabolic network including 11 cofactors of 118 reactions of the central metabolism and 12 pseudo-fluxes. A focus on lipid storage biosynthesis and the associated pathways was done in comparison with rapeseed, arabidopsis, maize and sunflower embryos. In our hands, glucose was determined to be the main source of carbon in flax embryos, leading to the conversion of phosphoenolpyruvate to pyruvate. The oxidative pentose phosphate pathway (OPPP) was identified as the producer of NADPH for fatty acid biosynthesis. Overall, the use of 13C-metabolic flux analysis provided new insights into the flax embryo metabolic processes involved in storage lipid biosynthesis. The elucidation of the metabolic network of this important crop plant reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems.

Flaxseed (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3), this polyunsaturated fatty acid is well known for its nutritional role in human and animal diet. Understanding storage lipid biosynthesis in developing flaxseed embryos can lead to an increase in seed yield. While a tremendous amount of work has been done on different plant species to highlight their metabolism during embryos development, flaxseed metabolic flux analysis is still lacking. In this context, we have developed an in vitro cultured developing embryos of flaxseed and determined net fluxes by performing three complementary parallel labeling experiments with 13C-labeled glucose and glutamine. Metabolic fluxes were estimated by computer- aided modeling of the central metabolic network including 11 cofactors of 118 reactions of the central metabolism, 12 pseudo fluxes. A focus on lipid storage biosynthesis and the associated pathways was done in comparison with rapeseed, arabidopsis, maize and sunflower embryos. In our conditions, glucose was the main source of carbone of flaxseed embryos, leading to the conversion of phosphoenolpyruvate to pyruvate. The oxidative pentose phosphate pathway (OPPP) was identified as the producer of NADPH for fatty acid biosynthesis. Overall, the use of 13C-metabolic flux analysis provided new insight into flaxseed embryos metabolic processes involved in storage lipids biosynthesis. The elucidation of the metabolic network of this important crop plant reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems.

In the context of the growing demand for α-linolenic acid due to its high nutritional value as a polyunsaturated fatty acid, we have investigated the contribution of 2-lysophosphatidic acid acyltransferase (LPAAT) enzymes from flax (Linum usitatissimum) in the accumulation of α-linolenic acid into the oil fraction of flax seed. We have isolated the cDNAs encoding three class A microsomal LPAAT2 isoforms from developing flax seeds. The three isoforms, denominated LPAAT2A, LPAAT2A2 and LPAAT2B, are able to complement the LPAAT deficient JC201 E. coli mutant, confirming their functionality. We have performed enzymatic assays showing that the specific activity of the LPAAT2A isoform is significantly higher than that of the LPAAT2A2 and LPAAT2B toward the unsaturated oleic, linoleic and linolenic acids. Moreover, LPAAT2A presents in vitro a high specificity and selectivity for linoleic and linolenic acids as compared to saturated fatty acids. The three isoforms are expressed during all the stages of seed development and in stem and leaf tissues, as shown by an analysis of the transcription level of the corresponding genes. The heterologous expression of LPAAT2A in Arabidopsis seeds leads to an increase in the accumulation of linoleic and linolenic acids in the oil fraction of the seeds from two transgenic lines.

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Plant acylcarnitines are present during anabolic processes of lipid metabolism. Their low contents relatively to the corresponding acyl-CoAs suggest that they are associated to specific pools of activated fatty acids. The non-proteinaceous amino acid carnitine exists in plants either as a free form or esterified to fatty acids. To clarify the biological significance of acylcarnitines in plant lipid metabolism, we have analyzed their content in plant extracts using an optimized tandem mass spectrometry coupled to liquid chromatography method. We have studied different developmental processes (post-germination, organogenesis, embryogenesis) targeted for their high requirement for lipid metabolism. The modulation of the acylcarnitine content was compared to that of the lipid composition and lipid biosynthetic gene expression level in the analyzed materials. Arabidopsis mutants were also studied based on their alteration in de novo fatty acid partitioning between the prokaryotic and eukaryotic pathways of lipid biosynthesis. We show that acylcarnitines cannot specifically be associated to triacylglycerol catabolism but that they are also associated to anabolic pathways of lipid metabolism. They are present during membrane and storage lipid biosynthesis processes. A great divergence in the relative contents of acylcarnitines as compared to the corresponding acyl-CoAs suggests that acylcarnitines are associated to very specific process(es) of lipid metabolism. The nature of their involvement as the transport form of activated fatty acids or in connection with the management of acyl-CoA pools is discussed. Also, the occurrence of medium-chain entities suggests that acylcarnitines are associated with additional lipid processes such as protein acylation for instance. This work strengthens the understanding of the role of acylcarnitines in plant lipid metabolism, probably in the management of specific acyl-CoA pools.

The way plants are grown and samples are harvested, prepared, and extracted has a profound impact on the output of a metabolomics experiment. In this chapter, we detail the experimental procedures from plant cultivation to extract preparation, in order to avoid difficulties that could result in contamination or undesired changes of the analytes. Two plant organs are mentioned as examples: tomato fruits (Solanum lycopersicum) and flax seeds (Linum usitatissimum). Extractions designed for the untargeted analysis of semipolar compounds by liquid chromatography-mass spectrometry (LC-MS) and targeted analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS) or gas chromatography with flame ionization detector (GC-FID) are described.

UMR BFP - Equipe Métabolisme; International audience; The way plants are grown and samples are harvested, prepared, and extracted has a profound impact on the output of a metabolomics experiment. In this chapter, we detail the experimental procedures from plant cultivation to extract preparation, in order to avoid difficulties that could result in contamination or undesired changes of the analytes. Two plant organs are mentioned as examples: tomato fruits (Solanum lycopersicum) and flax seeds (Linum usitatissimum). Extractions designed for the untargeted analysis of semipolar compounds by liquid chromatography-mass spectrometry (LC-MS) and targeted analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS) or gas chromatography with flame ionization detector (GC-FID) are described.

Helicobacter pylori is a Gram-negative bacterium that colonizes the mucus niche of the gastric mucosa and infects more than half of the world's human population. Chronic infection may cause gastritis, duodenal ulcer, intestinal metaplasia or gastric cancer. In the stomach, H. pylori interacts with O-glycans of gastric mucins but the mechanism by which the bacteria succeed in altering the mucosa remains mainly unknown. To better understand the physiopathology of the infection, inhibitory adhesion assays were performed with various O-glycans expressed by human gastric mucins, and topographic expression of gastric mucins MUC5AC and MUC6 was analyzed for healthy uninfected individuals, for infected asymptomatic individuals and for patients infected by H. pylori and having the incomplete type of intestinal metaplasia. The glycosylation of the gastric mucosa of asymptomatic individuals infected by H. pylori was determined and compared with the glycosylation pattern found for patients with the incomplete type of intestinal metaplasia. Results show that H. pylori manages to modulate host's glycosylation during the course of infection in order to create a favorable niche, whereas asymptomatic infected individuals seem to counteract further steps of infection development by adapting their mucus glycosylation.

Summary Bacterial attachment to plant and animal surfaces is generally thought to constitute the initial step in colonization, requiring adherence factors such as flagella and fimbriae. We describe the molecular mechanism underpinning flagella-mediated adherence to plant tissue for the foodborne pathogen, enterohaemorrhagic Escherichia coli. Escherichia coli H7 flagella interacted with a sulphated carbohydrate (carrageenan) on a glycan array, which occurred in a dose-dependent manner. Adherence of E. coli O157 : H-expressing flagella of serotype H7, H6 or H48 to plants associated with outbreaks from fresh produce and to Arabidopsis thaliana, was dependent on flagella interactions with phospholipids and sulpholipids in plasma membranes. Adherence of purified H7 and H48 flagella to carrageenan was reduced at higher concentrations of KH2PO4 or KCl, showing an ionic basis to the interactions. Purified H7 flagella were observed to physically interact with plasma membranes in spinach plants and in A. thaliana. The results show a specific interaction between E. coli H7, H6 and H48 flagella and ionic lipids in plant plasma membranes. The work extends our understanding of the molecular mechanisms underpinning E. coli flagella targeting of plant hosts and suggests a generic mechanism of recognition common in eukaryotic hosts belonging to different biological kingdoms.

Human gastric mucin MUC5AC is secreted in the colonic mucus of cancer patients and is a specific marker of precancerous lesions called aberrant crypt foci. Using MUC5AC as a specific marker can improve sensitivity in the detection of early colorectal cancer. Here we demonstrated that the accumulation of MUC5AC in xenograft and mouse stomach can be detected by magnetic resonance imaging (MRI). We used ultrasmall particles of iron oxide (USPIOs) conjugated with disulfide constrained heptapeptide that were identified using a screening phage display. To accomplish this, we employed positive selection of the phage display library on MUC5AC purified from fresh human colonic adenomas in combination with negative selection of the phage library on purified human MUC2, which is predominantly found in normal colorectal tissues. This conjugate was tested on human colorectal cancer cell lines that were either able or unable to secrete MUC5AC, both in vitro and in vivo. MUC5AC-USPIO contrast agent and USPIOs alone were not detected in cell lines unable to secrete MUC5AC. A combination of MRI and microscopy studies was performed to detect a specific accumulation of the contrast agent in vivo. Thus, the MUC5AC contrast agent enabled non-invasive detection of precancerous lesions and colorectal cancer, highlighting its potential use in diagnostics, in the early detection of colorectal cancer recurrences after treatment and in mechanistic studies implicating MUC5AC. Copyright © 2016 John Wiley & Sons, Ltd.

The gastrointestinal tract is lined by a thick and complex layer of mucus that protects the mucosal epithelium from biochemical and mechanical aggressions. This mucus barrier confers protection against pathogens but also serves as a binding site that supports a sheltered niche of microbial adherence. The carcinogenic bacteria Helicobacter pylori colonize the stomach through binding to host glycans present in the glycocalyx of epithelial cells and extracellular mucus. The secreted MUC5AC mucin is the main component of the gastric mucus layer, and BabA-mediated binding of H. pylori to MUC5AC confers increased risk for overt disease. In this study we unraveled the O-glycosylation profile of Muc5ac from glycoengineered mice models lacking the FUT2 enzyme and therefore mimicking a non-secretor human phenotype. Our results demonstrated that the FUT2 determines the O-glycosylation pattern of Muc5ac, with Fut2 knock-out leading to a marked decrease in a1,2-fucosylated structures and increased expression of the terminal type 1 glycan structure Lewis-a. Importantly, for the first time, we structurally validated the expression of Lewis-a in murine gastric mucosa. Finally, we demonstrated that loss of mucin FUT2-mediated fucosylation impairs gastric mucosal binding of H. pylori BabA adhesin, which is a recognized feature of pathogenicity. The authors thank Nuno Mendes for excellent technical support at the animal facility of IPATIMUP, University of Porto; Dr. Jacques Bara from the U-673 INSERM, CNRS, Paris, France for providing the PM7, 45M1 and 7LE antibodies; Dr Ingemar Carlstedt, University of Lund, Sweden for providing LUM6-3 and LUM5-1 antibodies and Dr. Ola Söderberg from the Uppsala Universitet, Sweden for support with the PLA experiments. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and the projects with the references FCOMP-01-0124-FEDER 028188 and FCOMP-01-0124-FEDER041276 (EXPL/CTM-BIO/0762/2013, PTDC/BBB-EBI/0786/2012 and PTDC/BBB-EBI/0567/2014). The authors acknowledge the support by Gastric Glyco Explorer Initial Training Network (Seventh Framework Programme GastricGlycoExplorer project, grant number 316929). AM received an individual grant (SFRH/BPD/75871/2011) from FCT, POPH (Programa Operacional Potencial Humano) and FSE (Fundo Social Europeu) and acknowledges EMBO for a Short-Term Fellowship (EMBO ASTF 330-212). The resources provided by the Consortium for Functional Glycomics were funded by NIGMS-GM62116. TB is supported by grants from Vetenskapsrådet/VR, Cancerfonden, and the J.C. Kempe and Seth M. Kempe Memorial Foundation and this work was in part performed within the Umeå Centre for Microbial Research (UCMR), and the Biochemical Imaging Center Umeå (BICU).

Helicobacter pylori infects more than half of the world's population. Although most patients are asymptomatic, persistent infection may cause chronic gastritis and gastric cancer. Adhesion of the bacteria to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori-related diseases and is mediated by mucin O-glycans. In order to define which glycans may be implicated in the binding of the bacteria to the gastric mucosa in humans, we have characterized the exact pattern of glycosylation of gastric mucins. We have identified that the major component was always a core 2-based glycan carrying two blood group H antigens, whatever was the blood group of individuals. We have also demonstrated that around 80% of O-glycans carried blood group A, B or H antigens, suggesting that the variation of gastric mucin glycosylation between individuals is partly due to the blood group status. This study will help better understanding the role of O-glycans in the physiology and homeostasis of gastric mucosa. Overall, the results reported here give us the necessary background information to begin studies to determine whether individuals who express certain carbohydrate epitopes on specific mucins are predisposed to certain gastric diseases.

Human gastric mucin MUC5AC is secreted in the colonic mucus of cancer patients and is a specific marker of precancerous lesions called aberrant crypt foci. Using MUC5AC as a specific marker can improve sensitivity in the detection of early colorectal cancer. Here we demonstrated that the accumulation of MUC5AC in xenograft and mouse stomach can be detected by magnetic resonance imaging (MRI). We used ultrasmall particles of iron oxide (USPIOs) conjugated with disulfide constrained heptapeptide that were identified using a screening phage display. To accomplish this, we employed positive selection of the phage display library on MUC5AC purified from fresh human colonic adenomas in combination with negative selection of the phage library on purified human MUC2, which is predominantly found in normal colorectal tissues. This conjugate was tested on human colorectal cancer cell lines that were either able or unable to secrete MUC5AC, both in vitro and in vivo. MUC5AC-USPIO contrast agent and USPIOs alone were not detected in cell lines unable to secrete MUC5AC. A combination of MRI and microscopy studies was performed to detect a specific accumulation of the contrast agent in vivo. Thus, the MUC5AC contrast agent enabled non-invasive detection of precancerous lesions and colorectal cancer, highlighting its potential use in diagnostics, in the early detection of colorectal cancer recurrences after treatment and in mechanistic studies implicating MUC5AC. Copyright © 2016 John WileySons, Ltd.

Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR-GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells.

Type 1 fimbriae (T1F) are well characterised cell surface organelles expressed by Escherichia coli and required for adherence to mannosylated host tissue. They satisfy molecular Koch's postulates as a virulence determinant and a host-adapted role has been reinforced by reports that T1F expression is repressed at sub-mammalian temperatures. Analysis of a group of 136 environmental and animal E. coli isolates that express T1F at 37 °C showed that 28% are also capable of expression at 20 °C, in a phase variable manner. The heterogeneous proportions varied widely, and although growth temperature impacted the total proportion expressing T1F, there was no direct correlation between growth at 37 and 20 °C, indicative of differences in thermoregulation of the genetic switch (fimS) that controls phase variation. Specificities of the adhesin (FimH) also varied between the isolates: most bound to α-(1-3) mannan and yeast extracts as expected, but some recognised β-(1-4)-mannans and N-linked glycoproteins from plants, and T1F from two of the isolates mediated binding to plant roots. The results expand our view of a well described adherence factor to show alternative expression profiles and adhesin specificities, which in turn may confer an advantage for certain isolates in alternative hosts and habitats.

Mucin glycoproteins are secreted in large amounts by the intestinal epithelium and constitute an efficient component of innate immune defenses to promote homeostasis and protect against enteric pathogens. In this study, our objective was to investigate how the bacterial enteropathogen Shigella flexneri , which causes bacillary dysentery, copes with the mucin defense barrier. We report that upon in vitro infection of mucin-producing polarized human intestinal epithelial cells, virulent S. flexneri manipulates the secretion of gel-forming mucins. This phenomenon, which is triggered only by virulent strains, results in accumulation of mucins at the cell apical surface, leading to the appearance of a gel-like structure that favors access of bacteria to the cell surface and the subsequent invasion process. We identify MUC5AC, a gel-forming mucin, as a component of this structure. Formation of this gel does not depend on modifications of electrolyte concentrations, induction of trefoil factor expression, endoplasmic reticulum stress, or response to unfolded proteins. In addition, transcriptional and biochemical analyses of infected cells reveal modulations of mucin gene expression and modifications of mucin glycosylation patterns, both of which are induced by virulent bacteria in a type III secretion system-dependent manner. Thus, S. flexneri has developed a dedicated strategy to alter the mucus barrier by targeting key elements of the gel-forming capacity of mucins: gene transcription, protein glycosylation, and secretion.

Bacterial attachment to plant and animal surfaces is generally thought to constitute the initial step in colonization, requiring adherence factors such as flagella and fimbriae. We describe the molecular mechanism underpinning flagella-mediated adherence to plant tissue for the foodborne pathogen, enterohaemorrhagic Escherichia coli. Escherichia coli H7 flagella interacted with a sulphated carbohydrate (carrageenan) on a glycan array, which occurred in a dose-dependent manner. Adherence of E. coli O157 : H-expressing flagella of serotype H7, H6 or H48 to plants associated with outbreaks from fresh produce and to Arabidopsis thaliana, was dependent on flagella interactions with phospholipids and sulpholipids in plasma membranes. Adherence of purified H7 and H48 flagella to carrageenan was reduced at higher concentrations of KH2 PO4 or KCl, showing an ionic basis to the interactions. Purified H7 flagella were observed to physically interact with plasma membranes in spinach plants and in A.thaliana. The results show a specific interaction between E. coli H7, H6 and H48 flagella and ionic lipids in plant plasma membranes. The work extends our understanding of the molecular mechanisms underpinning E.coli flagella targeting of plant hosts and suggests a generic mechanism of recognition common in eukaryotic hosts belonging to different biological kingdoms.

International audience; DMBT1 (deleted in malignant brain tumor 1), a human mucin-like glycoprotein, belonging to the scavenger receptor cystein-rich (SRCR) superfamily, is mainly secreted from mucosal epithelia. It has been shown previously that interaction of hensin, the rabbit ortholog of DMBT1, with galectin 3, a β-galactoside-binding lectin, induces a terminal differentiation of epithelial cells. In this paper, we have used surface plasmon resonance (SPR), to analyse the binding of galectin 3 to two purified samples of human DMBT1:recombinant DMBT1 produced in CHO cells and DMBT1 isolated from intestinal tissues. Characterization of their glycosylation profile by nano-ESI-Q-TOF tandem mass spectrometry showed significant differences in O-glycans between the two DMBT1 samples. Results obtained by SPR demonstrated that the oligosaccharide side chains of DMBT1 are recognized by the carbohydrate-recognition domain (CRD) of galectin 3 and modification in the pattern of oligosaccharides modulates the binding parameters of DMBT1 with galectin 3. Moreover, using immunohistochemistry on paraffin-embedded colonic tissue sections, we could show a co-localisation of DMBT1 and galectin 3 in human intestine, suggesting a potential physiological interaction.

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.

The flagellum is the motility device of many bacteria and the long external filament is made of several thousand copies of a single protein, flagellin. While posttranslational modifications of flagellin are common among bacterial pathogens, the role of lysine methylation remained unknown. Here, we show that both flagellins ofSalmonella enterica, FliC and FljB, are methylated at surface-exposed lysine residues. ASalmonellamutant deficient in flagellin methylation was outcompeted for gut colonization in a gastroenteritis mouse model. In support, methylation of flagellin promoted invasion of epithelial cellsin vitro. Lysine methylation increased the surface hydrophobicity of flagellin and enhanced flagella-dependent adhesion ofSalmonellato phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization and successful infection of the host.

Animal pathogenic enterobacteria are major source of foodborne outbreaks. Among them, Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is known to lead to severe diseases like Haemolytic Uremic Syndrome (HUS). The adhesion on tissues, which is the first step initiating the colonisation, is mediated by cell surface proteins and organelles, including flagellum. This present work aims to study the interaction between the bacterial flagellum with lipid membrane into a relevant biological model, the Giant Unilamellar Vesicles (GUVs). GUVs are widely used as a membrane model, because of their similar size and curvature to the cell plasma membrane. A bacterial adhesion enhanced in the presence of flagella was confirmed on GUVs by microscopy, and precisely, by a newly developed assay for this work, allowing bacterial adhesion quantification on liposomes. By testing different head groups, the nature of the fatty acid chains or the liposome curvature, the lipid packing was found to be a key parameter for the adhesion. To test this, the incorporation of polyunsaturated lipids on HT29 cell line was used. The WT strain of EHEC O157:H7 was significantly more adherent than its non-flagellated mutant. The hydrophobicity was identified to influence the flagellum interaction with the lipid bilayer, by using a post-traductionnal modification (methylation) of the flagellin, the flagellum main component. With lipid packing, membrane fluidity leads to a heterogeneous bacterial adhesion on our models, mimicking the phase segregation, potentially existing on plasma membrane. It results that the flagellum is advantageous to reach potential lipid rafts. At the end of this work, a first mechanism of bacterial flagellum adhesion on lipid bilayer has been proposed and highlights the key parameters, but as well new questionings, especially the existence of an interaction with the cytoskeleton. A future precise knowledge of this mechanism represents a step toward understanding pathogen virulence, which is mandatory to develop alternative solutions to antibiotics.; Les entérobactéries pathogènes sont responsables d’épidémies majeures souvent d’origine alimentaire. Parmi elles, Escherichia coli entérohémorragique (EHEC) O157:H7 est connue pour conduire à de graves symptômes, tels que le syndrome hémolytique et urémique (HUS). L’adhésion sur un tissu, qui est la première étape initiant la colonisation, est orchestrée par des organites et protéines situés sur la paroi cellulaire bactérienne, en particulier le flagelle. Ce travail a pour objectif d’étudier l’interaction du flagelle bactérien avec les lipides de la membrane dans un système biologiquement pertinent, à savoir les liposomes unilamellaires géants (GUV, pour Giant Unilamellar Vesicles). Les GUVs sont largement utilisées en tant que modèle membranaire, car elles possèdent une taille et une courbure similaire à la membrane plasmique des cellules eucaryotes. Une meilleure adhésion bactérienne en présence du flagelle a été confirmée sur GUVs par microscopie, puis de manière précise par un essai développé dans le cadre de ce travail, permettant de quantifier l’adhésion bactérienne sur liposomes. En faisant varier la nature des têtes polaires, des chaînes carbonées et la courbure des liposomes, l’ordre lipidique s’est avéré être un paramètre clé de l’adhésion. Ceci a été vérifié à l’échelle cellulaire par l’incorporation de lipides polyinsaturés dans la membrane de cellules HT29, sur lesquelles la souche sauvage O157:H7 adhère significativement plus que son mutant non-flagellé. L’hydrophobicité du flagelle a été identifiée comme un facteur important pour l’interaction avec la membrane, grâce à une modification post-traductionnelle (méthylation) de la flagelline, composante majeure du flagelle. De concert avec l’ordre lipidique, la fluidité membranaire entraîne une adhésion bactérienne hétérogène sur notre modèle mimant la ségrégation de phases, qui existe potentiellement sur la membrane plasmique. Il en résulte que le flagelle est un avantage pour conquérir les potentiels radeaux lipidiques. A l’issue de ce travail, un premier mécanisme d’adhésion du flagelle bactérien sur la bicouche lipidique a été proposé et met en évidence les paramètres clés, mais aussi de nouvelles interrogations, notamment l’existence d’une interaction avec le cytosquelette. Une connaissance précise de ce mécanisme représente une étape vers la compréhension de la virulence des pathogènes, qui est indispensable pour développer des solutions alternatives aux antibiotiques.