RpoS acts as a global repressor of virulence gene expression in E. coli O104:H4 and enteroaggregative E. coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

The global burden of antimicrobial resistance - urinary tract infections.

Antimicrobial resistance (AMR) has emerged as a significant global healthcare problem. Antibiotic use has accelerated the physiologic process of AMR, particularly in Gram-negative pathogens. Urinary tract infections (UTIs) are predominantly of a Gram-negative nature. Uropathogens are evolutionarily highly adapted and selected strains with specific virulence factors, suggesting common mechanisms in how bacterial cells acquire virulence and AMR factors. The simultaneous increase in resistance and virulence is a complex and context-dependent phenomenon. Among known AMR mechanisms, the plenitude of different ß-lactamases is especially prominent. The risk for AMR in UTIs varies in different patient populations. A history of antibiotic consumption and the physiology of urinary flow are major factors that shape AMR prevalence. The urinary tract is in close crosstalk with the microbiome of other compartments, including the gut and genital tracts. In addition, pharmacokinetic properties and the physiochemical composition of urinary compartments can contribute to the emergence of AMR. Alternatives to antibiotic treatment and a broader approach to address bacterial infections are needed. Among the various alternatives studied, antimicrobial peptides and bacteriophage treatment appear to be highly promising approaches. We herein summarize the present knowledge of clinical and microbiological AMR in UTIs and discuss innovative approaches, namely new risk prediction tools and the use of non-antibiotic approaches to defend against uropathogenic microbes.

Energetics of Spike Protein Opening of SARS-CoV-1 and SARS-CoV-2 and Its Variants of Concern: Implications in Host Receptor Scanning and Transmission.

The receptor binding domain(s) (RBD) of spike (S) proteins of SARS-CoV-1 and SARS-CoV-2 (severe acute respiratory syndrome coronavirus) undergoes closed to open transition to engage with host ACE2 receptors. In this study, using multi atomistic (equilibrium) and targeted (non-equilibrium) molecular dynamics simulations, we have compared energetics of RBD opening pathways in full-length (modeled from cryo-EM structures) S proteins of SARS-CoV-1 and SARS-CoV-2. Our data indicate that amino acid variations at the RBD interaction interface can culminate into distinct free energy landscapes of RBD opening in these S proteins. We further report that mutations in the S protein of SARS-CoV-2 variants of concern can reduce the protein-protein interaction affinity of RBD(s) with its neighboring domains and could favor its opening to access ACE2 receptors. The findings can also aid in predicting the impact of future mutations on the rate of S protein opening for rapid host receptor scanning.

Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain.

Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5-day-old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain-associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain.

A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K-12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K-12, the 4.88 Mbp Fec10 genome is characterized by distinct single-nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two-component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand-alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI-Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI-SG17M. Furthermore, we noted a unique contribution of ClpGGI-Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food-borne heat-resistant strains in the human gut.

Dietary conjugated linoleic acid links reduced intestinal inflammation to amelioration of CNS autoimmunity.

A close interaction between gut immune responses and distant organ-specific autoimmunity including the CNS in multiple sclerosis has been established in recent years. This so-called gut-CNS axis can be shaped by dietary factors, either directly or via indirect modulation of the gut microbiome and its metabolites. Here, we report that dietary supplementation with conjugated linoleic acid, a mixture of linoleic acid isomers, ameliorates CNS autoimmunity in a spontaneous mouse model of multiple sclerosis, accompanied by an attenuation of intestinal barrier dysfunction and inflammation as well as an increase in intestinal myeloid-derived suppressor-like cells. Protective effects of dietary supplementation with conjugated linoleic acid were not abrogated upon microbiota eradication, indicating that the microbiome is dispensable for these conjugated linoleic acid-mediated effects. Instead, we observed a range of direct anti-inflammatory effects of conjugated linoleic acid on murine myeloid cells including an enhanced IL10 production and the capacity to suppress T-cell proliferation. Finally, in a human pilot study in patients with multiple sclerosis (n = 15, under first-line disease-modifying treatment), dietary conjugated linoleic acid-supplementation for 6 months significantly enhanced the anti-inflammatory profiles as well as functional signatures of circulating myeloid cells. Together, our results identify conjugated linoleic acid as a potent modulator of the gut-CNS axis by targeting myeloid cells in the intestine, which in turn control encephalitogenic T-cell responses.

Gut microbiome and plasma metabolome changes in rats after oral gavage of nanoparticles: sensitive indicators of possible adverse health effects.

BACKGROUND: The oral uptake of nanoparticles is an important route of human exposure and requires solid models for hazard assessment. While the systemic availability is generally low, ingestion may not only affect gastrointestinal tissues but also intestinal microbes. The gut microbiota contributes essentially to human health, whereas gut microbial dysbiosis is known to promote several intestinal and extra-intestinal diseases. Gut microbiota-derived metabolites, which are found in the blood stream, serve as key molecular mediators of host metabolism and immunity. RESULTS: Gut microbiota and the plasma metabolome were analyzed in male Wistar rats receiving either SiO2 (1000 mg/kg body weight/day) or Ag nanoparticles (100 mg/kg body weight/day) during a 28-day oral gavage study. Comprehensive clinical, histopathological and hematological examinations showed no signs of nanoparticle-induced toxicity. In contrast, the gut microbiota was affected by both nanoparticles, with significant alterations at all analyzed taxonomical levels. Treatments with each of the nanoparticles led to an increased abundance of Prevotellaceae, a family with gut species known to be correlated with intestinal inflammation. Only in Ag nanoparticle-exposed animals, Akkermansia, a genus known for its protective impact on the intestinal barrier was depleted to hardly detectable levels. In SiO2 nanoparticles-treated animals, several genera were significantly reduced, including probiotics such as Enterococcus. From the analysis of 231 plasma metabolites, we found 18 metabolites to be significantly altered in Ag-or SiO2 nanoparticles-treated rats. For most of these metabolites, an association with gut microbiota has been reported previously. Strikingly, both nanoparticle-treatments led to a significant reduction of gut microbiota-derived indole-3-acetic acid in plasma. This ligand of the arylhydrocarbon receptor is critical for regulating immunity, stem cell maintenance, cellular differentiation and xenobiotic-metabolizing enzymes. CONCLUSIONS: The combined profiling of intestinal microbiome and plasma metabolome may serve as an early and sensitive indicator of gut microbiome changes induced by orally administered nanoparticles; this will help to recognize potential adverse effects of these changes to the host.

Fimbriae reprogram host gene expression - Divergent effects of P and type 1 fimbriae.

Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-ß and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors.

Comparative phenotypic characterization of hybrid Shiga toxin-producing / uropathogenic Escherichia coli, canonical uropathogenic and Shiga toxin-producing Escherichia coli.

Hybrid Shiga toxin (Stx)-producing Escherichia coli (STEC) and uropathogenic E. coli (UPEC) strains are phylogenetically positioned between STEC and UPEC and can cause both diarrhea and urinary tract infections (UTIs). However, their virulence properties and adaptation to different host milieu in comparison to canonical UPEC and STEC strains are unknown. We determined phenotypes of the STEC/UPEC hybrid with respect to virulence including acid resistance, motility, biofilm formation, siderophore production, and adherence to human colonic Caco-2 and bladder T24 cells and compared to phenotypes of commensal strain MG1655, UPEC strain 536, and STEC strains B2F1 and Sakai. Moreover, we assessed the adaptation of the hybrid to artificial urine medium (AUM) and simulated colonic environment medium (SCEM). Overall acid resistance at pH 2.5 was high except in strains B2F1 and hybrid 05-00787 which showed reduced and extremely low acid resistance, respectively. Motility was reduced in hybrid 05-00787 and 09-05501 but strong in the remaining hybrids. While some hybrids showed high biofilm formation in LB, overall biofilm formation in SCEM and AUM were low and non-existent, respectively. All strains tested showed siderophore activity at equilibrium. All strains except MG1655 adhered to Caco-2 cells with the hybrid having similar adherence when compared to 536 but exhibited 2 and 3 times lower adherence when compared to B2F1 and Sakai, respectively. All Stx-producing strains adhered stronger to T24 cells than strains 536 and MG1655. Overall growth in LB, SCEM and AUM was consistent within the hybrid strains, except hybrid 05-00787 which showed significantly different growth patterns. Our data suggest that the hybrid is adapted to both, the intestinal and extraintestinal milieu. Expression of phenotypes typical of intestinal and extraintestinal pathogens thereby supports its potential to cause diarrhea and UTI.

Umbelliferone Decorated Water-soluble Zinc(II) Phthalocyanines - In Vitro Phototoxic Antimicrobial Anti-cancer Agents.

In this contribution we report on the synthesis, characterization and application of water-soluble zinc(II) phthalocyanines, which are decorated with four or eight umbelliferone moieties for photodynamic therapy (PDT). These compounds are linked peripherally to zinc(II) phthalocyanine by a triethylene glycol linker attached to pyridines, leading to cationic pyridinium units, able to increase the water solubility of the system. Beside their photophysical properties they were analyzed concerning their cellular distribution in human hepatocyte carcinoma (HepG2) cells as well as their phototoxicity towards HepG2 cells, Gram-positive (S. aureus strain 3150/12 and B. subtilis strain DB104) and Gram-negative bacteria (E. coli strain UTI89 and E. coli strain Nissle 1917). At low light doses and concentrations, they exhibit superb antimicrobial activity against Gram-positive bacteria as well as anti-tumor activity against HepG2. They are even capable to inactivate Gram-negative bacteria, whereas the dark toxicity remains low. These unique water-soluble compounds can be regarded as all-in-one type photosensitizers with broad applications ranges in the future.

Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ Escherichia coli strains.

BACKGROUND: Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101. METHODS: Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 µM, 25 µM and 125 µM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis. RESULTS: Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 µM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells. CONCLUSION: Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.

Spontaneous onset of TNFα-triggered colonic inflammation depends on functional T lymphocytes, S100A8/A9 alarmins, and MHC H-2 haplotype.

Recently, we established a doxycycline-inducible human tumor necrosis factor alpha (TNFα)-transgenic mouse line, ihTNFtg. Non-induced young and elderly mice showed low but constitutive expression of hTNFα due to promoter leakiness. The persistently present hTNFα stimulated endogenous pro-inflammatory mouse mS100A8/A9 alarmins. Secreted mS100A8/A9 in turn induced the expression and release of mouse mTNFα. The continuous upregulation of pro-inflammatory mTNFα and mS100A8/A9 proteins, due to their mutual expression dependency, gradually led to increased levels in colon tissue and blood. This finally exceeded the threshold levels tolerated by the healthy organism, leading to the onset of intestinal inflammation. Here, recombinant hTNFα functioned as an initial trigger for the development of chronic inflammation. Crossing ihTNFtg mice with S100A9KO mice lacking active S100A8/A9 alarmins or with Rag1KO mice lacking T and B lymphocytes completely abrogated the development of colonic inflammation, despite the still leaky hTNFα promoter. Furthermore, both the intensity of the immune response and the strength of immunosuppressive Treg induction was found to depend on the major histocompatibility complex (MHC) genetic composition. In summary, the onset of intestinal inflammation in elderly mice depends on at least four factors that have to be present simultaneously: TNFα upregulation, S100A8/A9 protein expression, functional T lymphocytes and genetic composition, with the MHC haplotype being of central importance. Only joint action of these factors leads to chronic intestinal inflammation, while absence of any of these determinants abrogates the development of the autoimmune disorder. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Sub-Inhibitory concentrations of SOS-Response inducing antibiotics stimulate integrase expression and excision of pathogenicity islands in uropathogenic Escherichia coli strain 536.

Urinary tract infections are one of the most common bacterial infections and a major public health problem. The predominant causative agents are uropathogenic Escherichia coli. These strains differ from commensal E. coli by the presence of additional horizontally acquired chromosomal material, so-called pathogenicity islands, which encode traits that promote efficient bacterial colonization of the urinary tract. Uropathogenic model strain E. coli 536 possesses six archetypal pathogenicity islands. Bacteriophage-like integrases encoded by each pathogenicity island contribute to island instability. To learn more about the stability of these six islands and factors controlling their stability we constructed two chromosomal reporter systems for the measurement of island loss, as well as for the measurement of the promoter activity of the six island-associated integrase genes at the population level. We used these reporter gene modules to analyze the role of SOS response in island instability. Tests with subinhibitory concentrations of different antibiotics, including many drugs commonly used for the treatment of urinary tract infection, indicated that only SOS response-inducing antibiotics led to an increased loss of islands which was always associated with an increase in the bacterial subpopulations showing high integrase promoter activity. This suggests that island excision correlates with the expression of the cognate integrase. Our reporter modules are valuable tools to investigate the impact of various growth conditions on genome plasticity. Furthermore, a better understanding of the conditions, which affect bacterial integrase expression may open ways to specifically manipulate the genome content of bacterial pathogens by increasing pathogenicity island deletion rates in infecting or colonizing bacteria, thus leading to the attenuation of bacterial pathogens.

π-Extended Donor-Acceptor Porphyrins and Metalloporphyrins for Antimicrobial Photodynamic Inactivation.

Free base, zinc and palladium π-extended porphyrins containing fused naphthalenediamide units were employed as photosensitizers in antimicrobial photodynamic therapy (aPDT). Their efficacy, assessed by photophysical and in vitro photobiological studies on Gram-positive bacteria, was found to depend on metal coordination, showing a dramatic enhancement of photosensitizing activity for the palladium complex.

Gut microbiome as a response marker for pancreatic enzyme replacement therapy in a porcine model of exocrine pancreas insufficiency.

BACKGROUND: Exocrine pancreatic insufficiency (EPI) is characterized by the loss of active pancreatic enzymes and a resulting severely reduced food digestion. EPI therapy requires orally applied pancreatic enzyme replacement. The gut microbiome is a known mediator of intestinal diseases and may influence the outcome of EPI and the effects of a pancreatic enzyme replacement therapy (PERT). Here, we analyzed the effects of EPI and PERT on the gut microbiome in the model of pancreatic duct ligated minipigs. RESULTS: The microbial community composition in pig feces was analyzed by next generation sequencing of 16S rRNA amplicons. The data were evaluated for α- and ß-diversity changes and changes at the different Operational Taxonomic Unit (OTU) levels by Shannon-Wiener and inverse Simpson index calculation as well as by Principal Coordinates Analysis based on Bray-Curtis dissimilarity. Microbial α-diversity was reduced after EPI induction and reverted to nearly healthy state after PERT. Analysis of microbial composition and ß-diversity showed distinctive clusters of the three study groups and a change towards a composition comparable to healthy animals upon PERT. The relative abundance of possible pathobionts like Escherichia/Shigella, Acinetobacter or Stenotrophomonas was reduced by PERT. CONCLUSION: These data demonstrate that EPI-induced dysbiosis could be reverted by PERT to a nearly healthy state. Elevated α-diversity and the reduction of bacterial overgrowth after PERT promises benefits for EPI patients. Non-invasive microbiome studies may be useful for EPI therapy monitoring and as marker for response to PERT.

PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1-4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) and Forssman GSL (GalNAcα1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) revealed balanced adhesion toward the three GSLs for PapG I-mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III-harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface-exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

Carriage of Shiga toxin phage profoundly affects Escherichia coli gene expression and carbon source utilization.

BACKGROUND: Enterohemorrhagic Escherichia coli (E. coli) are intestinal pathogenic bacteria that cause life-threatening disease in humans. Their cardinal virulence factor is Shiga toxin (Stx), which is encoded on lambdoid phages integrated in the chromosome. Stx phages can infect and lysogenize susceptible bacteria, thus either increasing the virulence of already pathogenic bacterial hosts or transforming commensal strains into potential pathogens. There is increasing evidence that Stx phage-encoded factors adaptively regulate bacterial host gene expression. Here, we investigated the effects of Stx phage carriage in E. coli K-12 strain MG1655. We compared the transcriptome and phenotype of naive MG1655 and two lysogens carrying closely related Stx2a phages: ϕO104 from the exceptionally pathogenic 2011 E. coli O104:H4 outbreak strain and ϕPA8 from an E. coli O157:H7 isolate. RESULTS: Analysis of quantitative RNA sequencing results showed that, in comparison to naive MG1655, genes involved in mixed acid fermentation were upregulated, while genes encoding NADH dehydrogenase I, TCA cycle enzymes and proteins involved in the transport and assimilation of carbon sources were downregulated in MG1655::ϕO104 and MG1655::ϕPA8. The majority of the changes in gene expression were found associated with the corresponding phenotypes. Notably, the Stx2a phage lysogens displayed moderate to severe growth defects in minimal medium supplemented with single carbon sources, e.g. galactose, ribose, L-lactate. In addition, in phenotype microarray assays, the Stx2a phage lysogens were characterized by a significant decrease in the cell respiration with gluconeogenic substrates such as amino acids, nucleosides, carboxylic and dicarboxylic acids. In contrast, MG1655::ϕO104 and MG1655::ϕPA8 displayed enhanced respiration with several sugar components of the intestinal mucus, e.g. arabinose, fucose, N-acetyl-D-glucosamine. We also found that prophage-encoded factors distinct from CI and Cro were responsible for the carbon utilization phenotypes of the Stx2a phage lysogens. CONCLUSIONS: Our study reveals a profound impact of the Stx phage carriage on E. coli carbon source utilization. The Stx2a prophage appears to reprogram the carbon metabolism of its bacterial host by turning down aerobic metabolism in favour of mixed acid fermentation.

Origin and Evolution of Hybrid Shiga Toxin-Producing and Uropathogenic Escherichia coli Strains of Sequence Type 141.

Hybrid Shiga toxin-producing Escherichia coli (STEC) and uropathogenic E. coli (UPEC) strains of multilocus sequence type 141 (ST141) cause both urinary tract infections and diarrhea in humans and are phylogenetically positioned between STEC and UPEC strains. We used comparative genomic analysis of 85 temporally and spatially diverse ST141 E. coli strains, including 14 STEC/UPEC hybrids, collected in Germany (n = 13) and the United States (n = 1) to reconstruct their molecular evolution. Whole-genome sequencing data showed that 89% of the ST141 E. coli strains either were STEC/UPEC hybrids or contained a mixture of virulence genes from other pathotypes. Core genome analysis and ancestral reconstruction revealed that the ST141 E. coli strains clustered into two lineages that evolved from a common ancestor in the mid-19th century. The STEC/UPEC hybrid emerged ∼100 years ago by acquiring an stx prophage, which integrated into previously unknown insertion site between rcsB and rcsD, followed by the insertion of a pathogenicity island (PAI) similar to PAI II of UPEC strain 536 (PAI II536-like). The two variants of PAI II536-like were associated with tRNA genes leuX and pheU, respectively. Finally, microevolution within PAI II536-like and acquisition of the enterohemorrhagic E. coli plasmid were observed. Our data suggest that intestinal pathogenic E. coli (IPEC)/extraintestinal pathogenic E. coli (ExPEC) hybrids are widespread and that selection pressure within the ST141 E. coli population led to the emergence of the STEC/UPEC hybrid as a clinically important subgroup. We hypothesize that ST141 E. coli strains serve as a melting pot for pathogroup conversion between IPEC and ExPEC, contrasting the classical theory of pathogen emergence from nonpathogens and corroborating our recent phenomenon of heteropathogenicity among pathogenic E. coli strains.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.

Orthosipon stamineus extract exerts inhibition of bacterial adhesion and chaperon-usher system of uropathogenic Escherichia coli-a transcriptomic study.

Specific recognition and bacterial adhesion to host cells by uropathogenic Escherichia coli (UPEC) are the first steps towards infection of epithelial tissue of the human urogenital system. Therefore, targeting of UPEC virulence factors, relevant for adhesion, is a promising approach for prevention of recurrent urinary tract infections (UTI). A fully characterized plant-derived aqueous extract from the leaves of Orthosiphon stamineus (OWE), a plant traditionally used in clinical practice in Europe and Asia for UTI, has been shown to exert strong antiadhesive effects under in vitro and in vivo conditions. For improved understanding of the underlying mechanisms, transcriptome analysis of OWE-treated UPEC strain UTI89 by Illumina sequencing and cross-validation of these data by qPCR indicated significant downregulation of bacterial adhesins (curli, type 1-, F1C-, and P fimbriae) and of the chaperone-mediated protein folding/unfolding and pilus assembly process; in contrast, flagellar and motility-related genes were upregulated. We conclude that OWE transforms the sessile lifestyle of bacteria into a motile one and therefore disables bacterial attachment to the host cell. Additionally, the extract inhibited gene expression of multiple iron-acquisition systems (ent, fep, feo, fhu, chu, sit, ybt). The present study explains the antiadhesive and anti-infective effect of the plant extract by pinpointing specific biochemical and molecular targets.