Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

Transcriptomic and proteomic analysis of the virulence inducing effect of ciprofloxacin on enterohemorrhagic Escherichia coli.

Enterohemorrhagic E. coli (EHEC) is considered to be the most dangerous pathotype of E. coli, as it causes severe conditions such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Antibiotic treatment of EHEC infections is generally not recommended since it may promote the production of the Shiga toxin (Stx) and lead to worsened symptoms. This study explores how exposure to the fluoroquinolone ciprofloxacin reorganizes the transcriptome and proteome of EHEC O157:H7 strain EDL933, with special emphasis on virulence-associated factors. As expected, exposure to ciprofloxacin caused an extensive upregulation of SOS-response- and Stx-phage proteins, including Stx. A range of other virulence-associated factors were also upregulated, including many genes encoded by the LEE-pathogenicity island, the enterohemolysin gene (ehxA), as well as several genes and proteins involved in LPS production. However, a large proportion of the genes and proteins (17 and 8%, respectively) whose expression was upregulated upon ciprofloxacin exposure (17 and 8%, respectively) are not functionally assigned. This indicates a knowledge gap in our understanding of mechanisms involved in EHECs response to antibiotic-induced stress. Altogether, the results contribute to better understanding of how exposure to ciprofloxacin influences the virulome of EHEC and generates a knowledge base for further studies on how EHEC responds to antibiotic-induced stress. A deeper understanding on how EHEC responds to antibiotics will facilitate development of novel and safer treatments for EHEC infections.

The NMR structure of the Ea22 lysogenic developmental protein from lambda bacteriophage.

The ea22 gene resides in a relatively uncharacterized region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed upon infection. In lambda and Shiga toxin-producing phages found in enterohemorrhagic E. coli (EHEC) associated with food poisoning, Ea22 favors a lysogenic over lytic developmental state. The Ea22 protein may be considered in terms of three domains: a short amino-terminal domain, a coiled-coiled domain, and a carboxy-terminal domain (CTD). While the full-length protein is tetrameric, the CTD is dimeric when expressed individually. Here, we report the NMR solution structure of the Ea22 CTD that is described by a mixed alpha-beta fold with a dimer interface reinforced by salt bridges. A conserved mobile loop may serve as a ligand for an unknown host protein that works with Ea22 to promote bacterial survival and the formation of new lysogens. From sequence and structural comparisons, the CTD distinguishes lambda Ea22 from homologs encoded by Shiga toxin-producing bacteriophages.

Genomic island-encoded regulatory proteins in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is an important zoonotic pathogen that is a major cause of foodborne diseases in most developed and developing countries and can cause uncomplicated diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome. O islands (OIs), which are unique genomic islands in EHEC O157:H7, are composed of 177 isolated genomic features and harbour 26% of the total genes that are absent in the non-pathogenic E. coli K-12 genome. In the last twenty years, many OI-encoded proteins have been characterized, including proteins regulating virulence, motility, and acid resistance. Given the critical role of regulatory proteins in the systematic and hierarchical regulation of bacterial biological processes, this review summarizes the OI-encoded regulatory proteins in EHEC O157:H7 characterized to date, emphasizing OI-encoded regulatory proteins for bacterial virulence, motility, and acid resistance. This summary will be significant for further exploration and understanding of the virulence and pathogenesis of EHEC O157:H7.

Enterohaemorrhagic E. coli utilizes host- and microbiota-derived L-malate as a signaling molecule for intestinal colonization.

The mammalian gastrointestinal tract is a complex environment that hosts a diverse microbial community. To establish infection, bacterial pathogens must be able to compete with the indigenous microbiota for nutrients, as well as sense the host environment and modulate the expression of genes essential for colonization and virulence. Here, we found that enterohemorrhagic Escherichia coli (EHEC) O157:H7 imports host- and microbiota-derived L-malate using the DcuABC transporters and converts these substrates into fumarate to fuel anaerobic fumarate respiration during infection, thereby promoting its colonization of the host intestine. Moreover, L-malate is important not only for nutrient metabolism but also as a signaling molecule that activates virulence gene expression in EHEC O157:H7. The complete virulence-regulating pathway was elucidated; the DcuS/DcuR two-component system senses high L-malate levels and transduces the signal to the master virulence regulator Ler, which in turn activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence to epithelial cells of the large intestine. Disruption of this virulence-regulating pathway by deleting either dcuS or dcuR significantly reduced colonization by EHEC O157:H7 in the infant rabbit intestinal tract; therefore, targeting these genes and altering physiological aspects of the intestinal environment may offer alternatives for EHEC infection treatment.

Crystal structures of QseE and QseG: elements of a three-component system from Escherichia coli.

Bacteria regulate virulence by using two-component systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). TCSs respond to environmental signals and change gene-expression levels. The HK QseE and the RR QseF regulate the virulence of Enterobacteriaceae bacteria such as enterohemorrhagic Escherichia coli. The operon encoding QseE/QseF also contains a gene encoding an outer membrane lipoprotein, qseG. The protein product QseG interacts with QseE in the periplasmic space to control the activity of QseE and constitutes a unique QseE/F/G three-component system. However, the structural bases of their functions are unknown. Here, crystal structures of the periplasmic regions of QseE and QseG were determined with the help of AlphaFold models. The periplasmic region of QseE has a helix-bundle structure as found in some HKs. The QseG structure is composed of an N-terminal globular domain and a long C-terminal helix forming a coiled-coil-like structure that contributes to dimerization. Comparison of QseG structures obtained from several crystallization conditions shows that QseG has structural polymorphisms at the C-terminus of the coiled-coil structure, indicating that the C-terminus is flexible. The C-terminal flexibility is derived from conserved hydrophilic residues that reduce the hydrophobic interaction at the coiled-coil interface. Electrostatic surface analysis suggests that the C-terminal coiled-coil region can interact with QseE. The observed structural fluctuation of the C-terminus of QseG is probably important for interaction with QseE.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD.

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.

Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.

Acid tolerance of enterohemorrhagic Escherichia coli O157:H7 strain ATCC 43894 and its relationship with a large virulence plasmid pO157.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes a severe intestinal infection including hemolytic uremic syndrome in humans. Various factors contribute to its pathogenesis, including a large virulence plasmid pO157. This F-like 92-kb plasmid is isolated in virtually all clinical EHEC isolates, and is considered a hallmark of EHEC virulence. A previous report stated that removal of pO157 from EHEC ATCC 43894 induced overexpression of GadAB that are essential in glutamate-dependent acid resistance (GDAR) system, yet the mechanism remains elusive. Based on this observation, we surmised that pO157 is involved in the regulation of GDAR system. We comparatively analyzed 43894 and its pO157-cured (ΔpO157) mutant 277 for i) their acid resistance, ii) changes in the transcriptional profiles and iii) expression of GDAR associated genes/proteins. Survivability of 43894 upon exposure to acidic conditions was significantly lower than the ΔpO157 mutant. In addition, RNA-sequencing revealed that genes involved in GDAR were significantly down-regulated in 43894 when compared to the ΔpO157 mutant. Exogenous expression of GadE in 43894 led to expression of GadAB, suggesting possible intervention of pO157 in GDAR regulation. Despite these findings, reintroduction of pO157 into 277 did not reverted Gad overexpression. Likewise, removing pO157 from 43894 using the plasmid incompatibility method did not induce Gad overexpression as shown in 277. Taken together, the results suggest that variation in acid resistance among EHEC isolates exists, and the large virulence plasmid pO157 has no effect on weak acid resistance phenotype displayed in 43894.

The Epidemiological and Clinical Characteristics of the Largest Outbreak of Enterohemorrhagic Escherichia coli in Korea.

BACKGROUND: The largest outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 occurred at a preschool in South Korea from June 12 to 29, 2020. This study aimed to analyze the epidemiological and clinical characteristics of EHEC infection in this outbreak. METHODS: Epidemiological investigation was performed on all 184 children and 19 workers at the preschool using a standard questionnaire to assess symptoms, food intake, attendance, and special activity history. Pulsed-field gel electrophoresis analysis of confirmed cases was performed to determine genetic relevance. RESULTS: During this outbreak, 103 children were affected, whereas only one infection was identified in adults. Of the 103 pediatric patients, 85 had symptoms (82.5%), including diarrhea, abdominal pain, bloody stool, fever, and vomiting. Thirty-two patients (31.1%) were hospitalized, 15 (14.6%) were diagnosed with hemolytic uremic syndrome, and 4 (3.9%) received dialysis treatment. Pulsed-field gel electrophoresis analysis identified 4 genotypes with high genetic relevance (92.3%). Epidemiological investigation revealed that this outbreak might have occurred from ingesting foods stored in a refrigerator with a constant temperature above 10°C, which was conducive to bacterial growth. Despite several measures after outbreak recognition, new infections continued to appear. Therefore, the preschool was forced to close on June 19 to prevent further person-to-person transmission. CONCLUSION: Our findings from the response to the largest outbreak will help prepare countermeasures against future EHEC outbreak.

Recent Advancements in Technologies to Detect Enterohaemorrhagic Escherichia coli Shiga Toxins.

Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.

Enterohemorrhagic Escherichia coli responds to gut microbiota metabolites by altering metabolism and activating stress responses.

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

Another advantage of multi-locus variable-number tandem repeat analysis that can putatively subdivide enterohemorrhagic Escherichia coli O157 strains into clades by maximum a posteriori estimation.

Enterohemorrhagic Escherichia coli O157 (O157) strains can be subdivided into clades based on their single-nucleotide polymorphisms, but such analysis using conventional methods requires intense effort by laboratories. Although multi-locus variable-number tandem repeat analysis (MLVA), which can be performed with low laboratory burden, has been used as a molecular epidemiological tool, it has not been evaluated whether MLVA can be used the clade subdivision of O157 strains like it can for that of other pathogenic bacteria. This study aimed to establish a method for subdividing O157 strains into clades using MLVA data. The standardized index of association, ISA, for O157 strains isolated in Chiba prefecture, Japan (Chiba isolates) revealed the presence of unique tandem repeat patterns in each major clade (clades 2, 3, 7, 8, and 12). A likelihood database of tandem repeats for these clades was then constructed using the Chiba isolates, and a formula for maximum a posteriori (MAP) estimation was constructed. The ratio of the number of O157 strains putatively subdivided into a clade by MAP estimation from MLVA data relative to the number of O157 strains subdivided using single-nucleotide polymorphism analysis (designated as the concordance ratio [CR]) was calculated using the Chiba isolates and O157 strains isolated in Yamagata prefecture (Yamagata isolates). The CRs for the major Chiba and Yamagata isolate clades, other than clade 2, were 89%-100%. Although the CR for clade 2 Chiba isolates was >95%, that of the Yamagata isolates was only 78.9%. However, these clade 2 CRs were not significantly different from one another, indicating that clade 2 strains can be subdivided correctly by MAP estimation. In conclusion, this study expands the utility of MLVA, previously applied predominantly for molecular epidemiological analysis, into a low-laboratory-burden tool for subdividing O157 strains into phylogenetic groups.

A novel toxin-antitoxin system swpAB alters gene expression patterns and reduces virulence expression in enterohemorrhagic Escherichia coli.

Toxin-antitoxin (TA) systems are found widely among many bacteria, including enterohemorrhagic Escherichia coli (EHEC), but their functions are still poorly understood. In this study, we identified and characterized a novel TA system belonging to the relBE family, classified as a type II TA system, found in EHEC. The protein encoded by the toxin gene is homologous to RelE ribonuclease. Using various conditions for increasing the toxin activity, high-level induction of a toxin gene, and repression of an antitoxin gene in wild-type EHEC, we showed that the TA system, named swpAB (switching of gene expression profile), is involved in selective repression of a set of genes, including some virulence genes, and in the reduction of adherence capacity, rather than in suppression of bacterial growth. A detailed analysis of the profiles of RNA levels along sequences at 15 min after high expression of swpA revealed that two virulence genes, espA and tir, were direct targets of the SwpA toxin. These results suggested that the swpAB system can alter gene expression patterns and change bacterial physiological activity without affecting bacterial growth.

The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity.

Bacterial genomes contain an abundance of transposable insertion sequence (IS) elements that are essential for genome evolution and fitness. Among them, IS629 is present in most strains of enterohemorrhagic Escherichia coli O157 and accounts for many polymorphisms associated with gene inactivation and/or genomic deletions. The excision of IS629 from the genome is promoted by IS-excision enhancer (IEE) protein. Despite IEE has been identified in the most pathogenic serotypes of E. coli, its biochemical features that could explain its role in IS excision are not yet understood. We show that IEE is present in >30% of all available E. coli genome assemblies, and is highly conserved and very abundant within enterohemorrhagic, enteropathogenic and enterotoxigenic genomes. In vitro analysis of the recombinant protein from E. coli O157:H7 revealed the presence of a Mn2+-dependent error-prone DNA polymerase activity in its N-terminal archaeo-eukaryotic primase (AEP) domain able to promote dislocations of the primer and template strands. Importantly, IEE could efficiently perform in vitro an end-joining reaction of 3'-single-strand DNA overhangs with ≥4 bp of homology requiring both the N-terminal AEP and C-terminal helicase domains. The proposed role for IEE in the novel IS excision mechanism is discussed.

Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

QseC sensor kinase modulates the human microbiota during enterohemorrhagic Escherichia coli O157:H7 infection in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®).

Enterohemorrhagic Escherichia coli (EHEC) is an important gastrointestinal pathogen known for its ability to cause hemorrhagic colitis and induce hemolytic-uremic syndrome. The inner membrane QseC histidine kinase sensor has shown to be an important regulator of the locus of enterocyte effacement (LEE) island, where important EHEC key virulence genes are located. However, the QseC role during EHEC infection in human microbiota remains unknown. Herein, using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), we investigated whether the QseC sensor has a role in human microbiota modulation by EHEC in a dynamic model. Our data demonstrated that the QseC sensor modulates human microbiota during EHEC infection, and its absence leads to an increase in Lactobacillaceae and Bifidobacterium genus predominance, although non-effect on Bacteroides genus by EHEC strains was observed. In co-culture, the Lactobacillus acidophilus has affected EHEC growth and impaired the EHEC growth under space-niche competition, although no growth difference was observed in the QseC sensor presence. Also, differences in EHEC growth were not detected in competition with Bacteroides thetaiotaomicron and EHEC strains did not affect B. thetaiotaomicron growth either. When investigating the mechanisms behind the SHIME results, we found that hcp-2 expression for the type 6 secretion system, known to be involved in bacterial competition, is under QseC sensor regulation beneath different environmental signals, such as glucose and butyrate. Our findings broaden the knowledge about the QseC sensor in modulating the human microbiota and its importance for EHEC pathogenesis.

Escherichia coli O157:H7 senses microbiota-produced riboflavin to increase its virulence in the gut.

Riboflavin is produced by most commensal bacteria in the human colon, where enterohemorrhagic Escherichia coli (EHEC) colonizes and causes diseases. Sensing environmental signals to site-specifically express the type-III secretion system (T3SS), which injects effectors into host cells leading to intestinal colonization and disease, is key to the pathogenesis of EHEC. Here, we reveal that EHEC O157:H7, a dominant EHEC serotype frequently associated with severe diseases, acquired a previously uncharacterized two-component regulatory system rbfSR, which senses microbiota-produced riboflavin to directly activate the expression of LEE genes encoding the T3SS in the colon. rbfSR is present in O157:H7 and O145:H28 but absent from other EHEC serotypes. The binding site of RbfR through which it regulates LEE gene expression was identified and is conserved in all EHEC serotypes and Citrobacter rodentium, a surrogate for EHEC in mice. Introducing rbfSR into C. rodentium enabled bacteria to sense microbiota-produced riboflavin in the mouse colon to increase the expression of LEE genes, causing increased disease severity in mice. Phylogenic analysis showed that the O55:H7 ancestor of O157:H7 obtained rbfSR which has been kept in O157:H7 since then. Thus, acquiring rbfSR represents an essential step in the evolution of the highly pathogenic O157:H7. The expression of LEE genes and cell attachment ability of other EHEC serotypes in the presence of riboflavin significantly increased when rbfSR was introduced into them, indicating that those serotypes are ready to use RbfSR to increase their pathogenicity. This may present a potential public health issue as horizontal gene transfer is frequent in enteric bacteria.

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.

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.