Biosynthesis of the O antigen of pathogenic Escherichia coli O157:H7. Characterization of α1,4-Fuc-transferase WbdO.

The O157:H7 strain of Escherichia coli is responsible for frequent outbreaks of hemorrhagic colitis worldwide. Its lipopolysaccharide is a virulence factor and contains an O antigen having repeating units with the tetrasaccharide structure [2-D-PerNAcα1-3-L-Fucα1-4-D-Glcß1-3-D-GalNAcα1-]n. Genes encoding glycosyltransferases WbdN, WbdO, and WbdP are responsible for the biosynthesis of this repeating unit. We have previously characterized the second enzyme in the pathway, WbdN, which transfers Glc in ß1-3 linkage to GalNAcα-O-PO3-PO3-(CH2)11-O-Ph (GalNAc-PP-PhU). In this work, Fuc-transferase WbdO from E. coli O157:H7 expressed in BL21 bacteria was characterized using the product of WbdN as the acceptor substrate. We showed that WbdO is specific for GDP-ß-L-Fuc as the donor substrate. Compounds that contained terminal Glc or Glcß1-3GalNAc structures but lacked the diphosphate group did not serve as acceptor substrates. The structure of the WbdO product was identified by mass spectrometry and Nuclear magnetic resonance (NMR) as L-Fucα1-4-D-Glcß1-3-D-GalNAc PP-PhU. WbdO is an unusual bivalent metal ion-dependent Fuc-transferase classified as an inverting GT2 family enzyme that has 2 conserved sequences near the N-terminus. The Asp37 residue within the 36VDGGSTD42 sequence was found to be essential for catalysis. Mutation of Asp68 to Ala within the conserved 67YDAMNK72 sequence resulted in a 3-fold increase in activity. These studies show that WbdOO157 is a highly specific Fuc-transferase with little homology to other characterized Fuc-transferases.

IKKß phosphorylation regulates RPS3 nuclear translocation and NF-κB function during infection with Escherichia coli strain O157:H7.

NF-κB is a major gene regulator in immune responses, and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription. However, it is unknown how nuclear translocation of RPS3 is regulated. Here we report that phosphorylation of RPS3 Ser209 by the kinase IKKß was crucial for nuclear localization of RPS3 in response to activating stimuli. Moreover, virulence protein NleH1 of the foodborne pathogen Escherichia coli strain O157:H7 specifically inhibited phosphorylation of RPS3 Ser209 and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but resulting in less mortality in a gnotobiotic piglet-infection model. Thus, the IKKß-dependent modification of a specific amino acid in RPS3 promoted specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.

Lcn2 deficiency accelerates the infection of Escherichia coli O157:H7 by disrupting the intestinal barrier function.

Bacterial infections result in intestinal inflammation and injury, which affects gut health and nutrient absorption. Lipocalin 2 (Lcn2) is a protein that reacts to microbial invasion, inflammatory responses, and tissue damage. However, it remains unclear whether Lcn2 has a protective effect against bacterial induced intestinal inflammation. Therefore, this study endeavors to investigate the involvement of Lcn2 in the intestinal inflammation of mice infected with Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7). Lcn2 knockout (Lcn2-/-) mice were used to evaluate the changes of inflammatory responses. Lcn2 deficiency significantly exacerbated clinical symptoms of E. coli O157:H7 infection by reducing body weight and encouraging bacterial colonization of. Compared to infected wild type mice, infected Lcn2-/- mice had significantly elevated levels of pro-inflammatory cytokines in serum and ileum, including interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α), as well as severe villi destruction in the jejunum. Furthermore, Lcn2 deficiency aggravated intestinal barrier degradation by significantly reducing the expression of tight junction proteins occludin and claudin 1, the content of myeloperoxidase (MPO) in the ileum, and the number of goblet cells in the colon. Our findings indicated that Lcn2 could alleviate inflammatory damage caused by E. coli O157:H7 infection in mice by enhancing intestinal barrier function.

Adhesion of Escherichia coli O157:H7 during sublethal injury and resuscitation: Importance of pili and surface properties.

Escherichia coli O157:H7 can recover from sublethally injured (SI) state, which causes threat of foodborne illness. Adhesion plays a key role in the carriage of pathogens in food. In this study, we investigated the adhesion ability of SI and recovered E. coli O157:H7 wildtype and its three pili-deficient mutants (curli, type 1 fimbriae, and type IV pili) on six food-related surfaces. Plate counting was used to determine adhesion population after washing and oscillating the surfaces. Spinach exhibited the stronger adhesion population of E. coli O157:H7 than the other fresh produces (p < 0.05). In addition, at least one key pili dominated adhesion on these surfaces, and curli was always included. The adhesion population and contribution of different types of pili were jointly affected by surface and physiological state. This can be attributed to high hydrophobicity and positive charge density on surface and different expression levels of csgB, fimA, fimC and ppdD in SI and recovered cells. Among glucose, mannose, maltose, fructose, lactose, and sucrose, addition of 0.5% mannose could reduce adhesion of cells at all physiological states on stainless steel. Overall, this research will provide support for controlling adhesion of SI and recovered E. coli O157:H7.

The E. coli transcription factor GrlA is regulated by subcellular compartmentalization and activated in response to mechanical stimuli.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that colonizes the gastrointestinal tract and has evolved intricate mechanisms to sense and respond to the host environment. Upon the sensation of chemical and physical cues specific to the host's intestinal environment, locus of enterocyte effacement (LEE)-encoded virulence genes are activated and promote intestinal colonization. The LEE transcriptional activator GrlA mediates EHEC's response to mechanical cues characteristic of the intestinal niche, including adhesive force that results from bacterial adherence to epithelial cells and fluid shear that results from intestinal motility and transit. GrlA expression and release from its inhibitor GrlR was not sufficient to induce virulence gene transcription; mechanical stimuli were required for GrlA activation. The exact mechanism of GrlA activation, however, remained unknown. We isolated GrlA mutants that activate LEE transcription, independent of applied mechanical stimuli. In nonstimulated EHEC, wild-type GrlA associates with cardiolipin membrane domains via a patch of basic C-terminal residues, and this membrane sequestration is disrupted in EHEC that expresses constitutively active GrlA mutants. GrlA transitions from an inactive, membrane-associated state and relocalizes to the cytoplasm in response to mechanical stimuli, allowing GrlA to bind and activate the LEE1 promoter. GrlA expression and its relocalization in response to mechanical stimuli are required for optimal virulence regulation and colonization of the host intestinal tract during infection. These data suggest a posttranslational regulatory mechanism of the mechanosensor GrlA, whereby virulence gene expression can be rapidly fine-tuned in response to the highly dynamic spatiotemporal mechanical profile of the gastrointestinal tract.

Isolation, Characterization, and Comparative Genomic Analysis of Bacteriophage Ec_MI-02 from Pigeon Feces Infecting Escherichia coli O157:H7.

The most significant serotype of Shiga-toxigenic Escherichia coli that causes foodborne illnesses is Escherichia coli O157:H7. Elimination of E. coli O157:H7 during food processing and storage is a possible solution. Bacteriophages have a significant impact on bacterial populations in nature due to their ability to lyse their bacterial host. In the current study, a virulent bacteriophage, Ec_MI-02, was isolated from the feces of a wild pigeon in the United Arab Emirates (UAE) for potential future use as a bio-preservative or in phage therapy. Using a spot test and an efficiency of plating analysis, Ec_MI-02 was found to infect in addition to the propagation host, E. coli O157:H7 NCTC 12900, five different serotypes of E. coli O157:H7 (three clinical samples from infected patients, one from contaminated green salad, and one from contaminated ground beef). Based on morphology and genome analysis, Ec_MI-02 belongs to the genus Tequatrovirus under the order Caudovirales. The adsorption rate constant (K) of Ec_MI-02 was found to be 1.55 × 10-8 mL/min. The latent period was 50 min with a burst size of almost 10 plaque forming units (pfu)/host cell in the one-step growth curve when the phage Ec_MI-02 was cultivated using the propagation host E. coli O157:H7 NCTC 12900. Ec_MI-02 was found to be stable at a wide range of pH, temperature, and commonly used laboratory disinfectants. Its genome is 165,454 bp long with a GC content of 35.5% and encodes 266 protein coding genes. Ec_MI-02 has genes encoding for rI, rII, and rIII lysis inhibition proteins, which supports the observation of delayed lysis in the one-step growth curve. The current study provides additional evidence that wild birds could also be a good natural reservoir for bacteriophages that do not carry antibiotic resistance genes and could be good candidates for phage therapy. In addition, studying the genetic makeup of bacteriophages that infect human pathogens is crucial for ensuring their safe usage in the food industry.

Intestinal Enteroid Monolayers Model the Human Intestinal Environment for Escherichia coli Infection.

Enterohemorrhagic Escherichia coli O157:H7 is an enteric pathogen responsible for bloody diarrhea, hemolytic uremic syndrome, and in severe cases, even death. The study of O157:H7 is difficult due to the high specificity of the bacteria for the human intestine, along with our lack of sufficiently complex human cell culture models. The recent development of human intestinal enteroids derived from intestinal crypt multipotent stem cells has allowed us to construct two-dimensional differentiated epithelial monolayers grown in transwells that mimic the human intestine. Unlike previous studies, saline was added to the apical surface, while maintaining culture media in the basolateral well. The monolayers continued to grow and differentiate with apical saline. Apical infection with O157:H7 or commensal E. coli resulted in robust bacterial growth from 105 to over 108 over 24 h. Despite this robust bacterial growth, commensal E. coli neither adhered to nor damaged the epithelial barrier over 30 h. However, O157:H7 was almost fully adhered (>90%) by 18 h with epithelial damage observed by 30 h. O157:H7 contains the locus of enterocyte effacement (LEE) pathogenicity island responsible for attachment and damage to the intestinal epithelium. Previous studies report the ability of nutrients such as biotin, d-serine, and L-fucose to downregulate LEE gene expression. O157:H7 treated with biotin or L-fucose, but not d-serine displayed both decreased attachment and reduced epithelial damage over 36 h. These data illustrate enteroid monolayers can serve as a suitable model for the study of O157:H7 pathogenesis, and identification of potential therapeutics. IMPORTANCE O157:H7 is difficult to study due to its high specificity for the human intestine and the lack of sufficiently complex human cell culture models. The recent development of human intestinal enteroids derived from intestinal crypt multipotent stem cells has allowed us to construct two-dimensional differentiated epithelial monolayers grown in transwells that mimic the human intestine. Our data illustrates enteroid monolayers can serve as a suitable model for the study of O157:H7 pathogenesis, and allow for identification of potential therapeutics.

Exopolysaccharide from Lactobacillus casei NA-2 attenuates Escherichia coli O157:H7 surface adhesion via modulation of membrane surface properties and adhesion-related gene expression.

The natural compound, exopolysaccharide from Lactobacillus casei NA-2 (EPS-cn2), has been shown to inhibit biofilm formation by Escherichia coli O157:H7. Although bacterial adhesion to substrate surfaces is a primary, indispensable step in this process, the mechanisms by which EPS-cn2 can block E. coli O157:H7 adhesion to biotic or abiotic surfaces remain unclear. In this study, investigation of E. coli O157:H7 response to EPS-cn2 revealed that 1 mg/mL EPS-cn2 can decrease adherence to polystyrene and confluent Caco-2 cell surfaces to 49.0% (P＜0.0001) and 57.0% (P＜0.01) of that in untreated E. coli O157:H7, respectively. Moreover, EPS-cn2 significantly reduced outer membrane hydrophobicity by 49.0% and decreased the electronegativity of the membrane surface charge by as much as 1.57 mV (P＜0.05) compared to untreated cells. High throughput RNA sequencing indicated that genes responsible for adhesion through extracellular matrix secretion, such as poly-N-acetyl-glucosamine (PNAG) biosynthesis, locus of enterocyte effacement (LEE) proteins and outer membrane protein (OmpT) were all down-regulated in response to EPS-cn2, while chemotaxis and motility-related flagellar assembly genes were differentially up-regulated, suggesting that the EPS-cn2 may serve as an extracellular signal to attenuate adhesion-related gene expression and alter bacterial surface properties in E. coli O157:H7. These findings support the further development of EPS-cn2 for pathogenic biofilm management in clinical and industrial settings, and suggests the further targeting of adhesion-related genes to limit the persistence of this highly pathogenic strain in sensitive environments.

SlyA regulates virulence gene expressions through activation of pchA regulatory gene in enterohemorrhagic Escherichia coli.

SlyA is a DNA-binding protein that alters the nucleoid complex composed of histone-like nucleoid-structuring protein (H-NS) and activates gene expression. In enterohemorrhagic Escherichia coli (EHEC), the expression of virulence genes is repressed by H-NS but is up-regulated in response to environmental factors by releasing a nucleoid complex. This study examined the effect of slyA deletion mutation in EHEC and discovered that the production of the locus of enterocyte effacement (LEE)-encoded EspB and Tir, as well as the cell adherence ability, was reduced in the mutant compared with the wild type. The promoter activity of the LEE1 operon, including the regulatory gene, ler, was reduced by slyA mutation, but tac promoter-controlled expression of pchA, which is a regulatory gene of LEE1, abolished the effect. The promoter activity of pchA was down-regulated by the slyA mutation. Furthermore, the coding region was required for its regulation and was bound to SlyA, which indicates the direct regulation of pchA by SlyA. However, the slyA mutation did not affect the butyrate-induced increase in pchA promoter activity. Additionally, the pchA promoter activity was increased via induction of lrp, a regulatory gene for butyrate response, in the slyA mutant and, conversely, by introducing high copies of slyA into the lrp mutant. These results indicate that SlyA is a positive regulator of pchA and is independent of the Lrp regulatory system. SlyA may be involved in the virulence expression in EHEC, maintaining a certain level of expression in the absence of a butyrate response.

Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli.

In bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs). To determine in vivo RNA sequence requirements for Hfq interactions, and to study riboregulation in a bacterial pathogen, Hfq was UV crosslinked to RNAs in enterohemorrhagic Escherichia coli (EHEC). Hfq bound repeated trinucleotide motifs of A-R-N (A-A/G-any nucleotide) often associated with the Shine-Dalgarno translation initiation sequence in mRNAs. These motifs overlapped or were adjacent to the mRNA sequences bound by sRNAs. In consequence, sRNA-mRNA duplex formation will displace Hfq, promoting recycling. Fifty-five sRNAs were identified within bacteriophage-derived regions of the EHEC genome, including some of the most abundant Hfq-interacting sRNAs. One of these (AgvB) antagonized the function of the core genome regulatory sRNA, GcvB, by mimicking its mRNA substrate sequence. This bacteriophage-encoded "anti-sRNA" provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.

Glycan-Adhering Lectins and Experimental Evaluation of a Lectin FimH Inhibitor in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 Strain EDL933.

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.

The legacy of bacterial invasions on soil native communities.

Soil microbial communities are often not resistant to the impact caused by microbial invasions, both in terms of structure and functionality, but it remains unclear whether these changes persist over time. Here, we used three strains of Escherichia coli O157:H7 (E. coli O157:H7), a species used for modelling bacterial invasions, to evaluate the resilience of the bacterial communities from four Chinese soils to invasion. The impact of E. coli O157:H7 strains on soil native communities was tracked for 120 days by analysing bacterial community composition as well as their metabolic potential. We showed that soil native communities were not resistant to invasion, as demonstrated by a decline in bacterial diversity and shifts in bacterial composition in all treatments. The resilience of native bacterial communities (diversity and composition) was inversely correlated with invader's persistence in soils (R2 = 0.487, p < 0.001). Microbial invasions also impacted the functionality of the soil communities (niche breadth and community niche), the degree of resilience being dependent on soil or native community diversity. Collectively, our results indicate that bacteria invasions can potentially leave a footprint in the structure and functionality of soil communities, indicating the need of assessing the legacy of introducing exotic species in soil environments.

The role of l-arabinose metabolism for Escherichia coli O157:H7 in edible plants.

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.

Antibacterial Activity of Caffeic Acid Combined with UV-A Light against Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes.

The aim of this study was to evaluate the antibacterial activity of caffeic acid (CA), which is a natural polyphenol, combined with UV-A light against the representative foodborne bacteria Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Data regarding the inactivation of these bacteria and its dependence on CA concentration, light wavelength, and light dose were obtained. E. coli O157:H7 and Salmonella Typhimurium were reduced to the detection limit when treated with 3 mM CA and UV-A for 3 J/cm2 and 4 J/cm2, respectively, and 5 J/cm2 treatment induced 3.10 log reduction in L. monocytogenes. To investigate the mechanism for inactivation of Salmonella Typhimurium and L. monocytogenes, measurement of polyphenol uptake, membrane damage assessment, enzymatic activity assay, and transmission electron microscopy (TEM) were conducted. It was revealed that CA was significantly (P < 0.05) absorbed by bacterial cells, and UV-A light allowed a higher uptake of CA for both pathogens. Additionally, CA plus UV-A treatment induced significant (P < 0.05) cell membrane damage. In the enzymatic activity assay, the activities of both pathogens were reduced by CA, and a greater reduction occurred by use of CA plus UV-A. Moreover, transmission electron microscopy (TEM) images indicated that CA plus UV-A treatment notably destroyed the intercellular structure. In addition, antibacterial activity was also observed in commercial apple juice, which showed results similar to those obtained from phosphate-buffered saline (PBS), resulting in a significant (P < 0.05) reduction for all three pathogens without any changes in color parameters (L*, a*, and b*), total phenolic compounds, and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity. IMPORTANCE Photodynamic inactivation (PDI), which involves photoactivation of a photosensitizer (PS), is an emerging field of study, as it effectively reduces various kinds of microorganisms. Although there are several PSs that have been used for PDI, there is a need to find naturally occurring PSs for safer application in the food industry. Caffeic acid, a natural polyphenol found in most fruits and vegetables, has recently been studied for its potential to act as a novel photosensitizer. However, no studies have been conducted regarding its antibacterial activity depending on treatment conditions and its antibacterial mechanism. In this study, we closely examined the effectiveness of caffeic acid in combination with UV-A light for inactivating representative foodborne bacteria in liquid medium. Therefore, the results of this research are expected to be utilized as basic data for future application of caffeic acid in PDI, especially when controlling pathogens in liquid food processing.

Models for factors influencing pathogen survival in low water activity foods from literature data are highly significant but show large unexplained variance.

Factors that control pathogen survival in low water activity foods are not well understood and vary greatly from food to food. A literature search was performed to locate data on the survival of foodborne pathogens in low-water activity (<0.70) foods held at temperatures <37 °C. Data were extracted from 67 publications and simple linear regression models were fit to each data set to estimate log linear rates of change. Multiple linear stepwise regression models for factors influencing survival rate were developed. Subset regression modeling gave relatively low adjusted R2 values of 0.33, 0.37, and 0.48 for Salmonella, E. coli and L. monocytogenes respectively, but all subset models were highly significant (p < 1.0e-9). Subset regression models showed that Salmonella survival was significantly (p < 0.05) influenced by temperature, serovar and strain type, water activity, inoculum preparation method, and inoculation method. E. coli survival was significantly influenced by temperature, water activity, and inoculum preparation. L. monocytogenes survival was significantly influenced by temperature, serovar and strain type, and inoculum preparation method. While many factors were highly significant (p < 0.001), the high degrees of variability show that there is still much to learn about the factors which govern pathogen survival in low water activity foods.

Salt, glucose, glycine, and sucrose protect Escherichia coli O157:H7 against acid treatment in laboratory media.

This study investigated the effects of combinations of acetic or malic acid and various solutes (salt, glucose, glycine, or sucrose) on the survival of Escherichia coli O157:H7 in laboratory broth. Additionally, the effectiveness of combining organic acids and various concentrations of salt (0-18%) or sucrose (0-100%) with different water activity values against E. coli O157:H7 were evaluated. For treatment of 1% malic acid, the addition of 3% salt showed synergistic effect. Whereas, when 3% salt, glucose, glycine, or sucrose was added to 1% acetic acid, the solutes antagonized the action of the acid against E. coli O157:H7. Acetic, lactic, or propionic acid combined with salt at either 7 or 9% or sucrose at 60, 80, or 100% resulted in the highest resistance of E. coli O157:H7. From a result of evaluating the membrane fatty acid (MFA) composition of cells, salt or sucrose significantly increased levels of saturated fatty acids (SFAs) or SFAs and cyclopropane fatty acids, respectively. From the results of this study, the addition of solutes and organic compounds may increase the tolerance of E. coli O157:H7 to acetic, lactic, and propionic acid treatments and that the salt or sucrose significantly affects cell MFA composition.

Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli.

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/ß domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most expanded effector family in the pathogenic arsenal of E. coli.

A high-throughput genomic screen identifies a role for the plasmid-borne type II secretion system of Escherichia coli O157:H7 (Sakai) in plant-microbe interactions.

Shiga-toxigenic Escherichia coli (STEC) is often transmitted into food via fresh produce plants, where it can cause disease. To identify early interaction factors for STEC on spinach, a high-throughput positive-selection system was used. A bacterial artificial chromosome (BAC) clone library for isolate Sakai was screened in four successive rounds of short-term (2 h) interaction with spinach roots, and enriched loci identified by microarray. A Bayesian hierarchical model produced 115 CDS credible candidates, comprising seven contiguous genomic regions. Of the two candidate regions selected for functional assessment, the pO157 plasmid-encoded type two secretion system (T2SS) promoted interactions, while a chaperone-usher fimbrial gene cluster (loc6) did not. The T2SS promoted bacterial binding to spinach and appeared to involve the EtpD secretin protein. Furthermore, the T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 °C, and etpD was expressed in planta by E. coli Sakai on spinach plants.

Dps Protects Enterohemorrhagic Escherichia coli against Acid-Induced Antimicrobial Peptide Killing.

Dps, a DNA-binding protein from starved cells in Escherichia coli, is part of the bacterial defense system that protects DNA against various cellular stresses. Our lab previously demonstrated that a novel antimicrobial peptide, WRWYCR, enhances acid-induced killing of enterohemorrhagic Escherichia coli (EHEC) and ameliorates infection in a Citrobacter rodentium mouse model of EHEC infection. WRWYCR has previously been shown to compromise DNA damage repair and to increase chelatable iron within the cell. These findings, combined with the effects of peptide and acid stress on DNA damage, suggest a key defense role for Dps in peptide-induced killing of EHEC. The goal of this study is to evaluate the role of Dps in peptide-induced killing of EHEC through survival assays and flow cytometric analyses of DNA damage and hydroxyl radical formation. Our results demonstrate that disruption of the dps gene in stationary-phase EHEC O157:H7 cells, but not in exponential-phase cells, enhances acid-, peptide-, and peptide-acid-induced killing relative to that of wild-type (WT) EHEC. Using flow cytometric analysis, we have also demonstrated increased levels of hydroxyl radicals in peptide-treated wild-type EHEC relative to those in the untreated control. Disruption of the dps gene further increases this. These findings indicate that peptide treatment of EHEC enhances the formation of hydroxyl radicals, likely through the Fenton reaction, thereby contributing to the killing action of the peptide, and that dps protects against peptide killing of EHEC. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials.IMPORTANCE The research presented in this paper explores the role of the DNA-binding protein Dps as a key defense mechanism of enterohemorrhagic Escherichia coli (EHEC) strains in protecting against killing by the novel antimicrobial peptide WRWYCR. Our results demonstrate that Dps protects against peptide-induced killing of EHEC through direct protection against acid stress and hydroxyl radical formation, both of which are mechanisms targeted by the antimicrobial peptide. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials through specific targeting of Dps in order to allow a more potent response to the antimicrobial WRWYCR.

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.