A bacterial toxin co-opts caspase-3 to disable active gasdermin D and limit macrophage pyroptosis.

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1ß maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

Potassium diformate alleviated inflammation of IPEC-J2 cells infected with EHEC.

Potassium diformate (KDF) is a kind of formate, which possesses the advantages of antimicrobial activity, growth promotion and preventing diarrhea in weaned piglets. However, the researches of KDF in animal production mostly focused on apparent indexes such as growth performance and the mechanisms of KDF on intestinal health have not been reported. Thus, porcine small intestinal epithelial cells (IPEC-J2) infected with Enterohemorrhagic Escherichia coli (EHEC) was used to investigate the role of KDF on alleviating intestinal inflammation in this study. The 0.125 mg/mL KDF treated IPEC-J2 cells for 6 h and IPEC-J2 cells challenged with 5 × 107 CFU/mL EHEC for 4 h were confirmed as the optimum concentration and time for the following experiment. The subsequent experiment was divided into four groups: control group (CON), EHEC group, KDF group, KDF+EHEC group. The results showed that KDF increased the cell viability and the gene expression levels of SGLT3 and TGF-ß, while decreased the content of IL-1ß compared with the CON group. The cell viability and the gene expressions of SGLT1, SGLT3, GLUT2, Claudin-1, Occludin and TGF-ß, and the protein expression of ZO-1 in EHEC group were lower than those in CON group, whereas the gene expressions of IL-1ß, TNF, IL-8 and TLR4, and the level of phosphorylation NF-кB protein were increased. Pretreatment with KDF reduced the content of IgM and IL-1ß, the gene expressions of IL-1ß, TNF, IL-8 and TLR4 and the level of phosphorylation NF-кB protein, and increased the gene expression of TGF-ß and the protein expression of Occludin in IPEC-J2 cells infected EHEC. In conclusion, 0.125 mg/mL KDF on IPEC-J2 cells for 6 h had the beneficial effects on ameliorating the intestinal inflammation because of reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines through regulating NF-кB signaling pathway under the EHEC challenge.

The NMR structure of the Orf63 lytic developmental protein from lambda bacteriophage.

The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In lambda phage and Shiga toxin (Stx) producing phages found in enterohemorrhagic Escherichia coli (EHEC) associated with food poisoning, Orf63 expression reduces the host survival and hastens the period between infection and lysis thereby giving it pro-lytic qualities. The NMR structure of dimeric Orf63 reveals a fold consisting of two helices and one strand that all make extensive intermolecular contacts. Structure-based data mining failed to identify any Orf63 homolog beyond the family of temperate bacteriophages. A machine learning approach was used to design an amphipathic helical ligand that bound a hydrophobic cleft on Orf63 with micromolar affinity. This approach may open a new path towards designing therapeutics that antagonize the contributions of Stx phages in EHEC outbreaks.

[Redox Biology Involved in the Toxicity of Enterohemorrhagic Escherichia coli Toxin SubAB].

AB5 toxins of pathogenic bacteria enter host cells and utilize the retrograde trafficking pathway to translocate to the cytoplasm and exert its pathogenesis. Cholera toxin and Shiga toxin reach the endoplasmic reticulum (ER), and the A subunit undergoes redox regulation by ER proteins to become active fragments, which pass through the ER membrane and translocate to the cytoplasm. By acting on molecular targets in the cytoplasm, the normal function of host cells are disrupted, causing diseases. ER chaperone proteins such as protein disulfide isomerase (PDI) and binding immunoglobulin protein (BiP) induce conformational changes triggered by the reduction of disulfide bonds in the A subunit. This is thought to be dependent on cysteine thiol-mediated redox regulation, but the detailed mechanism remains unclear. On the other hand, subtilase cytotoxin (SubAB), produced by enterohemorrhagic Escherichia coli (EHEC), localizes to the ER without translocating to the cytoplasm and cleaves BiP as a substrate. Therefore, it is thought that ER stress-based cytotoxicity and intestinal bleeding occur without translocating to the cytoplasm. We reported that PDI is involved in BiP cleavage through SubAB localization to the ER. Like other AB5 toxins, this indicates the involvement of redox regulation via chaperone proteins in the ER, but also suggests that SubAB does not translocate to the cytoplasm because it cleaves BiP. Although there are few reports on the redox state of ER protein thiols, it is suggested that polysulfidation, which is discussed in this symposium, may be involved.

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.

Bacterial outer membrane vesicles provide an alternative pathway for trafficking of Escherichia coli O157 type III secreted effectors to epithelial cells.

IMPORTANCE: Bacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.

L-glutamine protects against enterohemorrhagic Escherichia coli infection by inhibiting bacterial virulence and enhancing host defense concurrently.

IMPORTANCE: The type 3 secretion system (T3SS) was obtained in many Gram-negative bacterial pathogens, and it is crucial for their pathogenesis. Environmental signals were found to be involved in the expression regulation of T3SS, which was vital for successful bacterial infection in the host. Here, we discovered that L-glutamine (Gln), the most abundant amino acid in the human body, could repress enterohemorrhagic Escherichia coli (EHEC) T3SS expression via nitrogen metabolism and therefore had potential as an antivirulence agent. Our in vitro and in vivo evidence demonstrated that Gln could decline EHEC infection by attenuating bacterial virulence and enhancing host defense simultaneously. We repurpose Gln as a potential treatment for EHEC infection accordingly.

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.

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.

Enterohemorrhagic Escherichia coli senses microbiota-derived nicotinamide to increase its virulence and colonization in the large intestine.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen that specifically colonizes and infects the human large intestine. EHEC O157:H7 engages intricate regulatory pathways to detect host intestinal signals and regulate virulence-related gene expression during colonization and infection. However, the overall EHEC O157:H7 virulence regulatory network in the human large intestine remains incompletely understood. Here, we report a complete signal regulatory pathway where the EvgSA two-component system responds to high-nicotinamide levels produced by microbiota in the large intestine and directly activates loci of enterocyte effacement genes to promote EHEC O157:H7 adherence and colonization. This EvgSA-mediated nicotinamide signaling regulatory pathway is conserved and widespread among several other EHEC serotypes. Moreover, disruption of this virulence-regulating pathway by the deletion of evgS or evgA significantly decreased EHEC O157:H7 adherence and colonization in the mouse intestinal tract, indicating that these genes could be potential targets for the development of new therapeutics for EHEC O157:H7 infection.

Genomic Island-Encoded Histidine Kinase and Response Regulator Coordinate Mannose Utilization with Virulence in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

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.

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.

Intestinal mucus-derived metabolites modulate virulence of a clade 8 enterohemorrhagic Escherichia coli O157:H7.

The human colonic mucus is mainly composed of mucins, which are highly glycosylated proteins. The normal commensal colonic microbiota has mucolytic activity and is capable of releasing the monosaccharides contained in mucins, which can then be used as carbon sources by pathogens such as Enterohemorrhagic Escherichia coli (EHEC). EHEC can regulate the expression of some of its virulence factors through environmental sensing of mucus-derived sugars, but its implications regarding its main virulence factor, Shiga toxin type 2 (Stx2), among others, remain unknown. In the present work, we have studied the effects of five of the most abundant mucolytic activity-derived sugars, Fucose (L-Fucose), Galactose (D-Galactose), N-Gal (N-acetyl-galactosamine), NANA (N-Acetyl-Neuraminic Acid) and NAG (N-Acetyl-D-Glucosamine) on EHEC growth, adhesion to epithelial colonic cells (HCT-8), and Stx2 production and translocation across a polarized HCT-8 monolayer. We found that bacterial growth was maximum when using NAG and NANA compared to Galactose, Fucose or N-Gal, and that EHEC adhesion was inhibited regardless of the metabolite used. On the other hand, Stx2 production was enhanced when using NAG and inhibited with the rest of the metabolites, whilst Stx2 translocation was only enhanced when using NANA, and this increase occurred only through the transcellular route. Overall, this study provides insights on the influence of the commensal microbiota on the pathogenicity of E. coli O157:H7, helping to identify favorable intestinal environments for the development of severe disease.

Crystal structures of WrbA, a spurious target of the salicylidene acylhydrazide inhibitors of type III secretion in Gram-negative pathogens, and verification of improved specificity of next-generation compounds.

The enterohemorrhagic Escherichia coli pathotype is responsible for severe and dangerous infections in humans. Establishment of the infection requires colonization of the gastro-intestinal tract, which is dependent on the Type III Secretion System. The Type III Secretion System (T3SS) allows attachment of the pathogen to the mammalian host cell and cytoskeletal rearrangements within the host cell. Blocking the functionality of the T3SS is likely to reduce colonization and therefore limit the disease. This route offers an alternative to antibiotics, and problems with the development of antibiotics resistance. Salicylidene acylhydrazides have been shown to have an inhibitory effect on the T3SS in several pathogens. However, the main target of these compounds is still unclear. Past work has identified a number of putative protein targets of these compounds, one of which being WrbA. Whilst WrbA is considered an off-target interaction, this study presents the effect of the salicylidne acylhydrazide compounds on the activity of WrbA, along with crystal structures of WrbA from Yersinia pseudotuberculosis and Salmonella serovar Typhimurium; the latter also containing parts of the compound in the structure. We also present data showing that the original compounds were unstable in acidic conditions, and that later compounds showed improved stability.

Assay for Type III Secretion in Escherichia coli.

The type III secretion system (T3SS) is crucial for the virulence of several pathogenic Escherichia coli species as well as for other gram-negative bacterial strains. Therefore, the ability to monitor this system constitutes a valuable tool for assessing the involvement of different proteins in bacterial virulence, for identifying critical domains and specific mutations, and for evaluating the antivirulence activities of various drugs. The major advantage of the T3SS secretion assay for E. coli over assays for other gram-negative pathogens is that it does not necessarily require specific antibodies. Here, we describe how to grow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) strains under T3SS-inducing conditions, separate the supernatant fraction from the bacterial pellet, analyze this fraction on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and evaluate the level of T3SS activity. We describe a qualitative analysis using Coomassie staining and a quantitative assay using western blotting.

Improved Molecular Diagnosis and Culture of the Emerging Heteropathotype Enterohemorrhagic Escherichia coli O80:H2 Using Its Non-Melibiose-Fermenting and Antibiotic-Resistance Properties.

Enterohemorrhagic Escherichia coli (EHEC) O80:H2, belonging to sequence type ST301, is among the main causes of hemolytic and uremic syndrome in Europe, a major concern in young children. Aside from the usual intimin and Shiga toxin virulence factors (VFs), this emerging serotype possesses a mosaic plasmid combining extra-intestinal VF- and antibiotic resistance-encoding genes. This hybrid pathotype can be involved in invasive infections, a rare occurrence in EHEC infections. Here, we aimed to optimize its detection, improve its clinical diagnosis, and identify its currently unknown reservoir. O80:H2 EHEC strains isolated in France between 2010 and 2018 were phenotypically and genetically analyzed and compared with non-O80 strains. The specificity and sensitivity of a PCR test and a culture medium designed, based on the molecular and phenotypic signatures of O80:H2 EHEC, were assessed on a collection of strains and stool samples. O80:H2 biotype analysis showed that none of the strains (n = 137) fermented melibiose versus 5% of non-O80 EHEC (n = 19/352). This loss of metabolic function is due to deletion of the entire melibiose operon associated with the insertion of a 70-pb sequence (70mel), a genetic scar shared by all ST301 strains. This metabolic hallmark was used to develop a real-time PCR test (100% sensitivity, 98.3% specificity) and a melibiose-based culture medium including antibiotics, characterized by 85% specificity and sensitivity for clinical specimens. These new tools may facilitate the diagnosis of this atypical clone, help the food industry to identify the reservoir and improve our epidemiological knowledge of this threatening and emerging clone.

Citrobacter rodentium(ϕStx2dact), a murine infection model for enterohemorrhagic Escherichia coli.

The formation of attaching and effacing (A/E) lesions on intestinal epithelium, combined with Shiga toxin production, are hallmarks of enterohemorrhagic Escherichia coli (EHEC) infection that can lead to lethal hemolytic uremic syndrome. Although an animal infection model that fully recapitulates human disease remains elusive, mice orally infected with Citrobacter rodentium(ϕStx2dact), a natural murine pathogen lysogenized with an EHEC-derived Shiga toxin 2-producing bacteriophage, develop intestinal A/E lesions and toxin-dependent systemic disease. This model has facilitated investigation of how: (A) phage gene expression and prophage induction contribute to disease and are potentially triggered by antibiotic treatment; (B) virulence gene expression is altered by microbiota and the colonic metabolomic milieu; and (C) innate immune signaling is affected by Stx. Thus, the model provides a unique tool for accessing diverse aspects of EHEC pathogenesis.

Enhanced production of Shiga toxin 1 in enterohaemorrhagic Escherichia coli by oxygen.

Enterohaemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although stx1 and stx2 were found within the late operons of the Stx-encoding phages (Stx-phages), stx1 could mainly be transcribed from the stx1 promoter (PStx1), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of stx1. In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe2+-responsive transcription factor. The intracellular Fe2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe2+ availability drove the formation of less Fe2+-Fur, less DNA binding to the PStx1 region, and an increase in Stx1 production.

Computational and biochemical analysis of type IV pilus dynamics and stability.

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend and retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating nuclear magnetic resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability, and function, we performed a total of 108 ms all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH, and ionic strength is complemented with targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments reveal a conserved set of residues defining a calcium-binding site at the interface between three pilin subunits. Calcium binding enhances T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.