Attachment of Enterohemorrhagic Escherichia coli to Host Cells Reduces O Antigen Chain Length at the Infection Site That Promotes Infection.

Many enteropathogenic bacteria express a needle-like type III secretion system (T3SS) that translocates effectors into host cells promoting infection. O antigen (OAg) constitutes the outer layer of Gram-negative bacteria protecting bacteria from host immune responses. Shigella constitutively shortens the OAg molecule in its three-dimensional conformation by glucosylation, leading to enhanced T3SS function. However, whether and how other enteropathogenic bacteria shorten the OAg molecule that probably facilitates infection remain unknown. For the first time, we report a smart mechanism by which enterohemorrhagic Escherichia coli specifically reduces the size of the OAg molecule at the infection site upon sensing mechanical signals of intestinal epithelial cell attachment via the membrane protein YgjI. YgjI represses expression of the OAg chain length regulator gene fepE via the global regulator H-NS, leading to shortened OAg chains and injection of more T3SS effectors into host cells. However, bacteria express long-chain OAg in the intestinal lumen benefiting their survival. Animal experiments show that blocking this regulatory pathway significantly attenuates bacterial virulence. This finding enhances our understanding of interactions between the surfaces of bacterial and host cells and the way this interaction enhances bacterial pathogenesis. IMPORTANCE Little is known about the regulation of cell wall structure of enteropathogenic bacteria within the host. Here, we report that enterohemorrhagic Escherichia coli regulates its cell wall structure during the infection process, which balances its survival in the intestinal lumen and infection of intestinal epithelial cells. In the intestinal lumen, bacteria express long-chain OAg, which is located in the outer part of the cell wall, leading to enhanced resistance to antimicrobial peptides. However, upon epithelial cell attachment, bacteria sense this mechanical signal via a membrane protein and reduce the OAg chain length, resulting in enhanced injection into epithelial cells of T3SS effectors that mediate host cell infection. Similar regulation mechanisms of cell wall structure in response to host cell attachment may be widespread in pathogenic bacteria and closely related with bacterial pathogenesis.

The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Shiga toxin remodels the intestinal epithelial transcriptional response to Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes diarrheal disease and the potentially lethal hemolytic uremic syndrome. We used an infant rabbit model of EHEC infection that recapitulates many aspects of human intestinal disease to comprehensively assess colonic transcriptional responses to this pathogen. Cellular compartment-specific RNA-sequencing of intestinal tissue from animals infected with EHEC strains containing or lacking Shiga toxins (Stx) revealed that EHEC infection elicits a robust response that is dramatically shaped by Stx, particularly in epithelial cells. Many of the differences in the transcriptional responses elicited by these strains were in genes involved in immune signaling pathways, such as IL23A, and coagulation, including F3, the gene encoding Tissue Factor. RNA FISH confirmed that these elevated transcripts were found almost exclusively in epithelial cells. Collectively, these findings suggest that Stx potently remodels the host innate immune response to EHEC.

The Canonical Long-Chain Fatty Acid Sensing Machinery Processes Arachidonic Acid To Inhibit Virulence in Enterohemorrhagic Escherichia coli.

The mammalian gastrointestinal tract is a complex biochemical organ that generates a diverse milieu of host- and microbe-derived metabolites. In this environment, bacterial pathogens sense and respond to specific stimuli, which are integrated into the regulation of their virulence programs. Previously, we identified the transcription factor FadR, a long-chain fatty acid (LCFA) acyl coenzyme A (acyl-CoA) sensor, as a novel virulence regulator in the human foodborne pathogen enterohemorrhagic Escherichia coli (EHEC). Here, we demonstrate that exogenous LCFAs directly inhibit the locus of enterocyte effacement (LEE) pathogenicity island in EHEC through sensing by FadR. Moreover, in addition to LCFAs that are 18 carbons in length or shorter, we introduce host-derived arachidonic acid (C20:4) as an additional LCFA that is recognized by the FadR system in EHEC. We show that arachidonic acid is processed by the acyl-CoA synthetase FadD, which permits binding to FadR and decreases FadR affinity for its target DNA sequences. This interaction enables the transcriptional regulation of FadR-responsive operons by arachidonic acid in EHEC, including the LEE. Finally, we show that arachidonic acid inhibits hallmarks of EHEC disease in a FadR-dependent manner, including EHEC attachment to epithelial cells and the formation of attaching and effacing lesions. Together, our findings delineate a molecular mechanism demonstrating how LCFAs can directly inhibit the virulence of an enteric bacterial pathogen. More broadly, our findings expand the repertoire of ligands sensed by the canonical LFCA sensing machinery in EHEC to include arachidonic acid, an important bioactive lipid that is ubiquitous within host environments.IMPORTANCE Polyunsaturated fatty acids (PUFAs) play important roles in host immunity. Manipulation of lipid content in host tissues through diet or pharmacological interventions is associated with altered severity of various inflammatory diseases. Our work introduces a defined host-pathogen interaction by which arachidonic acid, a host-derived and dietary PUFA, can impact the outcome of enteric infection with the human pathogen enterohemorrhagic Escherichia coli (EHEC). We show that long-chain fatty acids including arachidonic acid act as signaling molecules that directly suppress a key pathogenicity island in EHEC following recognition by the fatty acyl-CoA-responsive transcription factor FadR. Thus, in addition to its established effects on host immunity and its bactericidal activities against other pathogens, we demonstrate that arachidonic acid also acts as a signaling molecule that inhibits virulence in an enteric pathogen.

Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

Molecular basis of bile-salt- and iron-induced enterohaemorrhagic E. coli resistance to cationic antimicrobial peptides.

Colonization of the gastrointestinal tract by enterohaemorrhagic Escherichia coli (EHEC) is critically dependent on its ability to sense and respond to various microenvironments within the host. EHEC exposure to physiologically relevant levels of bile salts upregulates the two-component system, pmrAB, and the arnBCADTEF operon, resulting in lipopolysaccharide modification and increased resistance to the cationic antimicrobial peptide, polymyxin B (PMB). A similar pmrAB- and arn-dependent PMB resistance has been observed in Salmonella enterica in the presence of ferric iron. Limiting magnesium levels and mild acid can also induce Salmonella resistance to PMB through another two-component system, PhoPQ and the connector protein, PmrD. This study aims to evaluate the relative contributions of a bile-salt mix (BSM), iron, limiting magnesium as well as the roles of pmrAB, phoPQ and pmrD to EHEC's resistance to PMB. Killing assays show that EHEC treatment with the BSM or iron under excess magnesium and neutral pH conditions induces a pmrAB-dependent, phoP-independent PMB resistance. By contrast, exposure to limiting magnesium triggers a pmrB-, phoP- and pmrD-dependent PMB resistance. The iron-induced PMB resistance is independent of phoP and pmrD under limiting magnesium conditions while the bile-salt-induced PMB resistance is independent of pmrD only under non-PhoP-inducing conditions. GFP-pmrD transcriptional reporter studies reveal that the limiting magnesium enhances pmrD expression, which is repressed upon additional exposure to either BSM or iron. Our results also show that exposure to mild acid enhances PMB resistance in a pmrD-independent manner and GFP reporter results confirm minimal expression of pmrD at this pH regardless of the magnesium level. This study provides novel insights into how EHEC differentially employs PmrAB, PhoPQ and PmrD to monitor and respond to bile salts, iron, acidic pH and magnesium typically encountered within the gastrointestinal tract in order to modulate its survival against cationic antimicrobial peptides.

Enterohaemorrhagic Escherichia coli activates nitrate respiration to benefit from the inflammatory response for initiation of microcolony-formation.

BACKGROUND: For successful colonization, enterohaemorrhagic Escherichia coli (EHEC) injects virulence factors, called effectors, into target cells through the type three secretion system (T3SS), which is composed of a needle and basal body. Under anaerobic conditions, the T3SS machinery remains immature and does not have a needle structure. However, activation of nitrate respiration enhances the completion of the T3SS machinery. Because nitric oxide released by the host inflammatory response increases nitrate concentration, we sought to determine the effect of the inflammatory response on initiation of EHEC microcolony-formation. RESULTS: The colony-forming capacity was increased in accordance with the increase of nitrate in the medium. The addition of the nitric oxide-producing agent NOR-4 also enhanced the adherence capacity, which was dependent on nitrate reductase encoded by the narGHJI genes. Culture supernatant of epithelial cells, which was stimulated by a cytokine mixture, enhanced the colony-forming capacity of wild-type EHEC but not of the narGHJI mutant. Finally, colony formation by wild-type EHEC on epithelial cells, which were preincubated with heat-killed bacteria, was higher than the narGHJI mutant, and this effect was abolished by aminoguanidine hydrochloride, which is an iNOS (inducible nitric oxide synthase) inhibitor. CONCLUSIONS: These results indicate that the inflammatory response enhances EHEC adherence by increasing nitrate concentration.

PAMP protects intestine from Enterohemorrhagic Escherichia coli infection through destroying cell membrane and inhibiting inflammatory response.

Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.

Enterococcus faecalis Enhances Expression and Activity of the Enterohemorrhagic Escherichia coli Type III Secretion System.

The gut microbiota can significantly impact invading pathogens and the disease they cause; however, many of the mechanisms that dictate commensal-pathogen interactions remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a potentially lethal human intestinal pathogen that uses microbiota-derived molecules as cues to efficiently regulate virulence factor expression. Here, we investigate the interaction between EHEC and Enterococcus faecalis, a common human gut commensal, and show that E. faecalis affects both expression and activity of the EHEC type III secretion system (T3SS) via two distinct mechanisms. First, in the presence of E. faecalis there is increased transcription of genes encoding the EHEC T3SS. This leads to increased effector translocation and ultimately greater numbers of pedestals formed on host cells. The same effect was observed with several strains of enterococci, suggesting that it is a general characteristic of this group. In a mechanism separate from E. faecalis-induced transcription of the T3SS, we report that an E. faecalis-secreted protease, GelE, cleaves a critical structural component of the EHEC T3SS, EspB. Our data suggest that this cleavage actually increases effector translocation by the T3SS, supporting a model where EspB proteolysis promotes maximum T3SS activity. Finally, we report that treatment of EHEC with E. faecalis-conditioned cell-free medium is insufficient to induce increased T3SS expression, suggesting that this effect relies on cell contact between E. faecalis and EHEC. This work demonstrates a complex interaction between a human commensal and pathogen that impacts both expression and function of a critical virulence factor.IMPORTANCE This work reveals a complex and multifaceted interaction between a human gut commensal, Enterococcus faecalis, and a pathogen, enterohemorrhagic E. coli We demonstrate that E. faecalis enhances expression of the enterohemorrhagic E. coli type III secretion system and that this effect likely depends on cell contact between the commensal and the pathogen. Additionally, the GelE protease secreted by E. faecalis cleaves a critical structural component of the EHEC type III secretion system. In agreement with previous studies, we find that this cleavage actually increases effector protein delivery into host cells by the secretion system. This work demonstrates that commensal bacteria can significantly shape expression and activity of pathogen virulence factors, which may ultimately shape the progression of disease.

Pre-Harvest Survival and Post-Harvest Chlorine Tolerance of Enterohemorrhagic Escherichia coli on Lettuce.

In the field, foodborne pathogens such as enterohemorrhagic Escherichia coli (EHEC) are capable of surviving on produce over time, yet little is known about how these pathogens adapt to this environment. To assess the impact of pre-harvest environmental conditions on EHEC survival, we quantified survival on romaine lettuce under two relative humidity (75% and 45%) and seasonal conditions (March and June). Greenhouse-grown lettuce was spray-inoculated with EHEC and placed in a growth chamber, mimicking conditions typical for June and March in Salinas Valley, California. Bacteria were enumerated on days 0, 1, 3, and 5 post-inoculation. Overall, we found that the effect of relative humidity on EHEC survival depended on the seasonal conditions. Under June seasonal conditions, higher relative humidity led to lower survival, and lower relative humidity led to greater survival, five days post-inoculation. Under March seasonal conditions, the impact of relative humidity on EHEC survival was minimal over the five days. The bacteria were also tested for their ability to survive a chlorine decontamination wash. Inoculated lettuce was incubated under the June 75% relative humidity conditions and then washed with a 50 ppm sodium hypochlorite solution (40 ppm free chlorine). When incubated under June seasonal conditions for three to five days, EHEC strains showed increased tolerance to chlorine (adj. p < 0.05) compared to chlorine tolerance upon inoculation onto lettuce. This indicated that longer incubation on lettuce led to greater EHEC survival upon exposure to chlorine. Subsequent transcriptome analysis identified the upregulation of osmotic and oxidative stress response genes by EHEC after three and five days of incubation on pre-harvest lettuce. Assessing the physiological changes in EHEC that occur during association with pre-harvest lettuce is important for understanding how changing tolerance to post-harvest control measures may occur.

Genetics, Toxicity, and Distribution of Enterohemorrhagic Escherichia coli Hemolysin.

The ability to produce enterohemolysin is regarded as a potential virulence factor for enterohemorrhagic Escherichia coli (EHEC) and is frequently associated with severe human diseases such as hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). The responsible toxin, which has also been termed EHEC-hemolysin (EHEC-Hly, syn. Ehx), belongs to the Repeats in Toxin (RTX)-family of pore-forming cytolysins and is characterized by the formation of incomplete turbid lysis zones on blood agar plates containing defibrinated sheep erythrocytes. Besides the expression of Shiga toxins (Stx) and the locus of enterocyte effacement (LEE), EHEC-Hly is a commonly used marker for the detection of potential pathogenic E. coli strains, although its exact role in pathogenesis is not completely understood. Based on the current knowledge of EHEC-Hly, this review describes the influence of various regulator proteins, explains the different mechanisms leading to damage of target cells, discusses the diagnostic role, and gives an insight of the prevalence and genetic evolution of the toxin.

Differential transcriptome analysis of enterohemorrhagic Escherichia coli strains reveals differences in response to plant-derived compounds.

BACKGROUND: Several serious vegetable-associated outbreaks of enterohemorrhagic Escherichia coli (EHEC) infections have occurred during the last decades. In this context, vegetables have been suggested to function as secondary reservoirs for EHEC strains. Increased knowledge about the interaction of EHEC with plants including gene expression patterns in response to plant-derived compounds is required. In the current study, EHEC O157:H7 strain Sakai, EHEC O157:H- strain 3072/96, and the EHEC/enteroaggregative E. coli (EAEC) hybrid O104:H4 strain C227-11φcu were grown in lamb's lettuce medium and in M9 minimal medium to study the differential transcriptional response of these strains to plant-derived compounds with RNA-Seq technology. RESULTS: Many genes involved in carbohydrate degradation and peptide utilization were similarly upregulated in all three strains, suggesting that the lamb's lettuce medium provides sufficient nutrients for proliferation. In particular, the genes galET and rbsAC involved in galactose metabolism and D-ribose catabolism, respectively, were uniformly upregulated in the investigated strains. The most prominent differences in shared genome transcript levels were observed for genes involved in the expression of flagella. Transcripts of all three classes of the flagellar hierarchy were highly abundant in strain C227-11φcu. Strain Sakai expressed only genes encoding the basal flagellar structure. In addition, both strains showed increased motility in presence of lamb's lettuce extract. Moreover, strain 3072/96 showed increased transcription activity for genes encoding the type III secretion system (T3SS) including effectors, and was identified as a powerful biofilm-producer in M9 minimal medium. CONCLUSION: The current study provides clear evidence that EHEC and EHEC/EAEC strains are able to adjust their gene expression patterns towards metabolization of plant-derived compounds, demonstrating that they may proliferate well in a plant-associated environment. Moreover, we propose that flagella and other surface structures play a fundamental role in the interaction of EHEC and EHEC/EAEC with plants.

Immunogenic Evaluation of Bivalent Vaccine Candidate against Enterohemorrhagic and Enterotoxigenic Escherichia coli.

BACKGROUND: Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major causative agent of bloody diarrhea and hemorrhagic uremic syndrome, while ETEC is the most important cause of diarrhea in neonates and travelers. OBJECTIVES: To evaluate the immunologic properties of a subunit vaccine candidate comprising the main immunogenic epitopes from these two bacterial strains. METHODS: The construct comprised of LTB and CfaB antigens from ETEC, and Intimin and Stx2B antigens from EHEC, was designed, analyzed and synthesized using bioinformatics methods. The chimeric gene was sub-cloned in the expression vector and expressed in E. coli host. The purified chimera protein was injected subcutaneously into the experimental animals. The production of specific antibodies was confirmed by immunological methods, and the protection capacity was evaluated by the challenge of immunized mice with the pathogenic bacteria. RESULTS: Chimeric recombinant protein was able to increase IgG titer. Neutralization assay indicated that the antibodies generated against LtB moiety were able to neutralize ETEC toxin. In animal challenge study, all non-immune mice died within 3 days after the injection of toxin, but all immunized mice survived from Stx toxin. CONCLUSIONS: The immunity to both ETEC and EHEC bacteria is significant, and this structure can be considered as a candidate for vaccine production against these bacterial strains.

A nanobody targeting the translocated intimin receptor inhibits the attachment of enterohemorrhagic E. coli to human colonic mucosa.

Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection.

Effect of sodium chloride and chitosan on the inactivation of heat resistant or Shiga-toxin producing Escherichia coli during grilling of burger patties.

Cattle are a reservoir for enterohemorrhagic Escherichia coli (EHEC), and ground beef is a major vehicle for human infection with EHEC. Heat resistance of E. coli, including EHEC, is impacted by NaCl and other additives. This study aimed to evaluate the effect of NaCl and other additives on the heat resistance of E. coli in beef patties. E. coli AW1.7ΔpHR1(pLHR) with the locus of heat resistance (LHR), E. coli AW1.7ΔpHR1(pRK767) without LHR, or a 5-strain cocktail of EHEC were inoculated (107-108 CFU/g) into ground beef (15% fat) with NaCl (0-3%), marinade, carvacrol (0.1%), potassium lactate (3%) or chitosan (0.1%) following different protocols. Patties were grilled immediately, or stored in sterile bags for two days at 4 °C prior to grilling to a core temperature of 71 °C. Cell counts of LHR-positive E. coli AW1.7ΔpHR1(pLHR) were higher than that of the isogenic LHR-negative E. coli AW1.7ΔpHR1(pRK767) by >3 log10 (CFU/g) after cooking. Addition of 3% NaCl increased survival of E. coli AW1.7ΔpHR1(pRK767) and the EHEC cocktail while cell counts of the heat resistant strains were not changed. A protective effect of NaCl was not observed with E. coli AW1.7ΔpHR1(pRK767) or EHEC if cells of E. coli were cooled to 4 °C prior to mixing with cold meat and NaCl, indicating that the response of E. coli to osmotic shock contributes to this effect. Chitosan enhanced the thermal destruction of LHR-positive E. coli AW1.7ΔpHR1(pLHR) in ground beef stored at 4 °C for 2 days, while marinade, carvacrol, or potassium lactate had no such effect, indicating that chitosan can be characterized as an effective hurdle concept to reduce the potential risk of LHR-positive pathogen to meat safety.

Shiga toxin 2 from enterohemorrhagic Escherichia coli induces reactive glial cells and neurovascular disarrangements including edema and lipid peroxidation in the murine brain hippocampus.

BACKGROUND: Shiga toxin 2 from enterohemorrhagic Escherichia coli is the etiologic agent of bloody diarrhea, hemolytic uremic syndrome and derived encephalopathies that may result to death in patients. Being a Gram negative bacterium, lipopolysaccharide is also released. Particularly, the hippocampus has been found affected in patients intoxicated with Shiga toxin 2. In the current work, the deleterious effects of Shiga toxin 2 and lipopolysaccharide are investigated in detail in hippocampal cells for the first time in a translational murine model, providing conclusive evidences on how these toxins may damage in the observed clinic cases. METHODS: Male NIH mice (25 g) were injected intravenously with saline solution, lipopolysaccharide, Shiga toxin 2 or a combination of Shiga toxin 2 with lipopolysaccharide. Brain water content assay was made to determine brain edema. Another set of animals were intracardially perfused with a fixative solution and their brains were subjected to immunofluorescence with lectins to determine the microvasculature profile, and anti-GFAP, anti-NeuN, anti-MBP and anti-Iba1 to study reactive astrocytes, neuronal damage, myelin dysarrangements and microglial state respectively. Finally, the Thiobarbituric Acid Reactive Substances Assay was made to determine lipid peroxidation. In all assays, statistical significance was performed using the One-way analysis of variance followed by Bonferroni post hoc test. RESULTS: Systemic sublethal administration of Shiga toxin 2 increased the expressions of astrocytic GFAP and microglial Iba1, and decreased the expressions of endothelial glycocalyx, NeuN neurons from CA1 pyramidal layer and oligodendrocytic MBP myelin sheath from the fimbria of the hippocampus. In addition, increased interstitial fluids and Thiobarbituric Acid Reactive Substances-derived lipid peroxidation were also found. The observed outcomes were enhanced when sublethal administration of Shiga toxin 2 was co-administered together with lipopolysaccharide. CONCLUSION: Systemic sublethal administration of Shiga toxin 2 produced a deterioration of the cells that integrate the vascular unit displaying astrocytic and microglial reactive profiles, while edema and lipid peroxidation were also observed. The contribution of lipopolysaccharide to pathogenicity caused by Shiga toxin 2 resulted to enhance the observed hippocampal damage.

Persistencia, internalización y translocación de Escherichia coli O157:H7, O157:H16 y O105ab en plantas y frutos de tomate (Solanum lycopersicum L.) / Persistence, internalization and translocation of Escherichia coli O157:H7, O157:H16 and O105ab in plants and tomato fruits (Solanum lycopersicum L.)

La presencia de bacterias patógenas, como Escherichia coli, afecta la calidad e inocuidad de las hortalizas que se consumen en fresco y se relaciona con graves problemas de salud. El objetivo de este trabajo fue determinar si 3 cepas diferentes de E. coli tienen la capacidad de penetrar y permanecer en plantas y frutos de tomate. Se siguió un diseño experimental completamente al azar, para lo cual se estableció un cultivo de tomate (variedad «Cid¼) en condiciones de invernadero y se evaluaron 3 tratamientos, T1 (E. coli O157: H7), T2 (E. coli de cultivo de tomate -#91;EcT-#93; O157: H16), T3 (E. coli de cultivo de espinaca -#91;EcH-#93; EcH O105ab) y un testigo T4, con 100 plantas cada uno y 4 formas de inoculación: en el sustrato, en el tallo, en el pecíolo y en el pedúnculo. Se realizaron muestreos en etapa vegetativa, floración, fructificación y madurez fisiológica para cuantificar en placa las UFC/g y saber si las bacterias lograban moverse y recuperarse en la raíz, el tallo, la flor y el fruto. Los grupos filogenéticos a los que correspondieron las bacterias recuperadas fueron confirmados mediante pruebas bioquímicas, serotipificación y PCR. A los 120 días la recuperación de bacterias en la planta fue del 23% (E. coli O157: H7), 28% (EcT O157: H16) y 55% (EcH O105ab) con la inoculación al sustrato, mientras que con la inoculación por punción la recuperación fue (en igual orden) del 5%, 3% y 4% a los 30 días; del 37%, 35% y 30% a los 90 días; y del 42%, 39% y 13% a los 65 días. Las cepas utilizadas mostraron la capacidad de entrar en la planta de tomate y de permanecer en ella y transportarse hasta llegar al fruto, sin producir síntomas que indiquen su presencia.

The presence of pathogenic bacteria, such as Escherichia coli affects the quality and safety of vegetables that are consumed fresh and is associated with serious health problems. The objective of this study was to determine if three different strains of E. coli can penetrate and remain in plants and tomato fruits. A completely randomized experimental design was followed for which a tomato crop ("Cid" variety) was established under greenhouse conditions and three treatments were evaluated, T1 (E. coli O157: H7), T2 (E. coli from tomato cultivation -#91;EcT-#93; O157: H16), T3 (E. coli from spinach cultivation -#91;EcH-#93; O105ab) and a T4 control, with 100 plants each and four forms of inoculation: in the substrate, steam, petiole and the peduncle. Samples were carried out in vegetative stage, flowering, fruiting and physiological maturity to quantify in petri dish CFU/g and know if the bacteria managed to move around and recover in root, stem, flower and fruit. The phylogenetic groups that corresponded to the bacteria recovered were confirmed by biochemical tests, serotyping and PCR. At 120 days the recovery of bacteria in the plant was 23% (E. coli O157: H7), 28% (EcT O157: H16) and 55% (EcH O105ab) whit inoculation to the substrate while the inoculation by puncture the recovery was (in the same order) of 5%, 3%, and 4% at 30 days; 37%, 35% and 30% at 90 days; and 42%, 39% and 13% at 65 days. The strains submit the ability to enter the tomato plant and to stay in it and transported to the fruit, without producing that indicate their presence.

Pathogenic and non-pathogenic Escherichia coli colonization and host inflammatory response in a defined microbiota mouse model.

Most Escherichia coli strains in the human intestine are harmless. However, enterohemorrhagic Ecoli (EHEC) is a foodborne pathogen that causes intestinal disease in humans. Conventionally reared (CONV) mice are inconsistent models for human infections with EHEC because they are often resistant to Ecoli colonization, in part due to their gastrointestinal (GI) microbiota. Although antibiotic manipulation of the mouse microbiota has been a common means to overcome colonization resistance, these models have limitations. Currently, there are no licensed treatments for clinical EHEC infections and, thus, new tools to study EHEC colonization need to be developed. Here, we used a defined microbiota mouse model, consisting of the altered Schaedler flora (ASF), to characterize intestinal colonization and compare host responses following colonization with EHEC strain 278F2 or non-pathogenic Ecoli strain MG1655. Significantly higher (P<0.05) levels of both strains were found in feces and cecal and colonic contents of C3H/HeN ASF compared to C3H/HeN CONV mice. GI inflammation was significantly elevated (P<0.05) in the cecum of EHEC 278F2-colonized compared to E. coli MG1655-colonized C3H/HeN ASF mice. In addition, EHEC 278F2 differentially modulated inflammatory-associated genes in colonic tissue of C3H/HeN ASF mice compared to E. coli MG1655-colonized mice. This approach allowed for prolonged colonization of the murine GI tract by pathogenic and non-pathogenic Ecoli strains, and for evaluation of host inflammatory processes. Overall, this system can be used as a powerful tool for future studies to assess therapeutics, microbe-microbe interactions, and strategies for preventing EHEC infections.

Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells.

Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB5 toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galß4Glcß1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases.

CRISPR Screen Reveals that EHEC's T3SS and Shiga Toxin Rely on Shared Host Factors for Infection.

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen's key virulence factors.