Enterohemorrhagic Escherichia coli (EHEC) are important foodborne pathogens with a bovine reservoir. For many years, research and regulations have focused on the EHEC serogroup most commonly associated with severe human illness, EHEC O157. However, six additional EHEC serogroups have been identified as important human foodborne pathogens and have been declared adulterants in raw, non-intact ground beef by the United States Department of Agriculture’s Food Safety Inspection Service. Collectively these seven organisms are referred to as EHEC-7. With the addition of these six pathogens, epidemiological studies are needed to estimate the probability for cattle to carry them and to identify risk factors associated with their presence in samples of bovine origin. In addition, the potential for pre-harvest control of EHEC-7 in feedlot cattle, particularly by dietary intervention, is a knowledge gap that needs to be addressed. Finally, detection methods of EHEC-7 have not been validated, and there is no “gold-standard” test. The first study included in this dissertation was a cross-sectional study estimating the prevalence and risk factors associated with hide contamination by EHEC-7 on the hides of market beef cows at slaughter. The second study was a longitudinal analysis of EHEC-7 from fecal samples from cow-calf herds in Mississippi and Nebraska. The third study was a randomized controlled trial evaluating the effects of fiber from distillers grains on the probability to detect EHEC-7 in samples from the rectoanal mucosa of feedlot steers. The fourth study included in this dissertation was a Bayesian latent class analysis estimating the diagnostic performance of three EHEC-7 detection methods, specifically modeling the performance and prevalence of EHEC O157 in fecal samples from beef cow-calf herds.

Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics. The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections. VI In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract. In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken VII together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs. Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs. / Meeresschwamm-assoziierte Actinomyceten stellen ein Reservoir für verschiedene natürliche Produkte mit neuartigen biologischen Aktivitäten dar. Ihr antibiotisches Potenzial gegenüber einer Reihe von Gram-negativen und -positiven Bakterien ist bereits intensiv erforscht worden. Wenig ist allerdings über ihre antiinfektive und antivirulente Wirksamkeit gegenüber menschlichen Pathogenen bekannt. Ziel dieser Doktorarbeit war es, die antiinfektiven Fähigkeiten (anti-Shiga-Toxin und anti-Biofilm) der aus Schwämmen isolierten Actinobakterien zu untersuchen. Hierfür wurden bioaktive Metabolite der Actinobakterien identifiziert und isoliert und abschließend wurde ihr Wirkmechanismus mit Hilfe einer Transkriptomanalyse charakterisiert. Diese Arbeit ist in drei Studien gegliedert, welche alle zum Ziel hatten die antiinfektive Wirksamkeit von aus Actinomyceten gewonnenen Extrakten und Komponente(n), welche möglicherweise als zukünftige Therapeutika dienen könnten, zu untersuchen. Die erste Studie befasst sich mit den anti-Shiga-Toxin Effekten der Meeresschwamm- assoziierten Actinomyceten. Durchfallinfektionen stellen in vielen Entwicklungsländern aber auch in Industrieländern eine große Gefahr dar. Durchfallerkrankungen die durch enterohämorrhagische Escherichia coli (EHEC) hervorgerufen werden, können sich zu lebensbedrohlichen Komplikationen wie Gastroenteritis oder dem hämolytisch urenischen Syndrom (HUS) weiterentwickeln. Das von den EHEC Stämmen produzierte Shiga-Toxin (Stx) stellt hierbei den Haupt Virulenz Faktor dar, welcher die eukaryotische Proteinsynthese menschlicher Zellen negativ beeinflusst, was wiederum den Zelltod durch Apoptose zur Folge hat. Die Behandlung der EHEC-Patienten mit Antibiotika wird nicht empfohlen, da dies zu einem Anstieg von freigesetztem Stx der zersetzen Bakterien führen könnte, wodurch das Risiko für die Entwicklung des HUS ansteigt. Aus diesem Grund werden effektive Medikamente dringen benötigt, welche die Stx Produktion blockieren ohne das Wachstum der Bakterien zu beeinflussen. In dieser Studie wurden 194 Extrakte und einige isolierte Komponenten von aus Schwämmen gewonnenen Actinomyceten auf ihren negativen Einfluss auf die Stx Produktion des EHEC Stammes EDL933 mit der Hilfe des Ridascreen® Verotoxin ELISA Kits untersucht. Es konnte gezeigt werden, dass die Zugabe der Extrakte keinen signifikanten Einfluss auf die Stx Produktion hatte. Strepthonium A auf der anderen Seite, welches aus Streptomyces sp. SBT345 isoliert wurde (vom mediterranen Schwamm Agelas oroides) konnte die Stx Produktion von EDL933 bei einer Konzentration von 80 µM reduzieren ohne das Wachstum des EHEC Stammes zu beeinflussen. Die Struktur von Strepthonium A wurde mittels spektroskopischer Analyse (1D- und 2D-NMR), sowie mittels ESI-HRMS und ESI-HRMS2 Experimenten entschlüsselt. Basierend auf diesen Ergebnissen könnte Strepthonium A eine mögliche Alternative oder Zusatz in der Behandlung einer EHEC Infektion darstellen. In der zweiten Studie wurde der Einfluss der Meeresschwamm-assoziierten Actinomyceten auf die Biofilmbildung von Staphylokokken bewertet. Medizinische Produkte wie Kontakt Linsen, metallische Implantate, Katheter, Herzschrittmacher, usw. stellen optimale ökologische Nischen für die Ausbildung von bakteriellen Biofilmen dar, wodurch Infektionen im Menschen hervorgerufen werden können. Bakterien in einem Biofilm sind deutlich toleranter gegenüber der Immunantwort ihres Wirtes sowie gegenüber konventionellen Antibiotika und sind daher schwer zu bekämpfen. In dieser Studie wurde das anti-Biofilm Potential eines organischen Extrakts der flüssigen Fermentation von Streptomyces sp. SBT343 (vom mediterranen Schwamm Petrosia ficiformis) ermittelt. In vitro Ergebnisse zeigten, dass das organische Extrakt anti-Biofilm (gegenüber Staphylococci) Fähigkeiten besitzt und nicht toxisch für Maus Makrophagen (J774.1), Fibroblasten (NIH/3T3) und humane korneale Epithelzellen ist. Zudem konnte gezeigt werden, dass das SBT343 Extrakt die Ausbildung eines Biofilms von Staphylokokken auf den Oberflächen von Polystyrol, Glass und Kontaktlinsen unterbinden konnte ohne das bakterielle Wachstum zu beeinflussen. Die hochauflösende Fouriertransformation-Massenspektrometrie (HR-MS) Analyse konnte die Komplexität sowie die chemische Vielfalt an Komponenten im Extrakt aufzeigen. Eine vorläufige, physio-chemische Charakterisierung deutet darauf hin, dass die aktive Komponente im Extrakt hitzestabil und nicht proteinartiger Natur ist. Abschließend konnte durch Fraktionierungsexperimente gezeigt werden, dass die biologische Aktivität auf synergistischen Effekten mehrerer Komponenten im Extrakt beruht. In einer dritten Studie wurden 50 organische Extrakte, welche aus fester und flüssiger Fermentierung von 25 verschiedenen aus Meeresschwämmen isolierten Actinomyceten gewonnen wurden, auf anti-Biofilm-Aktivität untersucht. Hierbei wurde die anti-Biofilm Aktivität des organischen Extrakts der Festkultur von Streptomyces sp. SBT348 (vom mediterranen Schwamm Petrosia ficiformis) identifiziert. Eine Bioassay gestützte Fraktionierung führte zu der Identifikation der aktiven Fraktion Fr 7 im SBT348 Extrakt. Durch weitere Aufreinigung des Extrakts mit einer semipräparativen HPLC, konnten die bioaktiven Sub-Fraktionen SKC1, SKC2, SKC3, SKC4 und SKC5 isoliert werden. Die Sub- Fraktion SKC3 hatte den stärksten anti-Biofilm Effekt und bestand aus einer reinen Verbindung mit BIC90 und MIC Werten von 3,95 µg/ml und 31,25 µg/ml gegen S. epidermidis RP62A. SKC3 zeigte weder erkennbare Toxizität gegenüber Zelllinien in vitro noch gegenüber den Larven der großen Wachsmotte Galleria melonella in vivo. SKC3 war Hitze- und Enzym-resistent, was auf eine nicht proteinartige Natur hindeutet. Eine HR-MS Analyse ergab, dass die Masse von SKC3 1258,3 Da beträgt. Die Strukturanalyse von SKC3 durch 1D und 2D-NMR ist zurzeit in Bearbeitung. Um weiteres Verständnis über den anti-Biofilm Wirkmechanismus von SKC3 auf S. epidermidis RP62A zu erlangen, wurde eine RNA Sequenzierungsanalyse durchgeführt. Die Transkriptomanalyse zeigte, dass SKC3 von RP62A nach einer 20-minütigen Inkubationszeit erkannt wird und dass SKC3 den zentralen Metabolismus des Staphylokokken Stammes nach 3 h negativ beeinflusst. Zusammengenommen deuten die Ergebnisse darauf hin, dass SKC3 als Leitstruktur für die Entwicklung neuer anti- Staphylokokken Medikamente dienen könnte. Zusammenfassend heben die Ergebnisse dieser Arbeit die antiinfektiven Eigenschaften der Meeresschwamm-assoziierte Actinomyceten hervor und bieten eine Möglichkeit für die Nutzung dieser in Wirkstoffentwicklungsprogrammen.

As gastroenterites ainda são um importante problema de saúde pública nos países em desenvolvimento e as Escherichia colí são as principais causas de diarréias. Adultos brasileiros apresentam anticorpos reativos com E. colí enteropatogênica (EPEC), que apresenta muitas semelhanças com E. colí enterohemorágica (EHEC) e pode ser responsável por complicações como a síndrome hemolítica urêmica e a colite hemorrágica. Ambas as bactérias apresentam um mecanismo patogênico comum: a formação da lesão \"attaching and effacing\" nos microvilus dos enterócitos, mediadas por fatores de virulência, como Intimina, Tir e as Esps. Os lipopolissacarídeos (LPS) são componentes da membrana externa das bactérias gram-negativas, incluindo E. colí. A infecção por EHEC 0157 resulta na produção de anticorpos séricos antiLPS 0157, que geralmente são indicativos de infecção recente. Neste trabalho, nós investigamos a presença de anticorpos séricos IgG e IgM anti-EHEC 0157:H7, EHEC 0111:H- e EPEC 0111:H- em adultos brasileiros, da Grande São Paulo. Amostras de soro foram coletadas de 200 adultos saudáveis, doadores de sangue, e um pool foi formado com 100 amostras. Anticorpos foram determinados em 100 amostras individuais por ELlSA com bactérias íntegras e anti-LPS para a determinação de anticorpos específicos anti-LPS 0111 e 0157 com o \"pool\" de soro como controle, em relação às concentrações de IgG e IgM totais determinadas simultaneamente. Os resultados foram submetidos a análise estatística. O repertório de anticorpos IgG e IgM para as três bactérias foi investigado por \"immunoblotting\" (18). A presença de anticorpos anti-bacterianos e anti-LPS foi confirmada. Existe uma correlação positiva entre os anticorpos reativos com as três bactérias, e entre os anticorpos anti-bactéria e anti-LPS. As concentrações de IgM anti-LPSs foi mais alta que IgG, o que está de acordo com o mecanismo imune esperado para um antígeno timo-independente. As concentrações de anti-LPS 0157 foram altas, apesar da baixa freqüência da bactéria 0157 em nosso meio. Os ensaios de 18 mostraram anticorpos reativos com bandas antigênicas sugestivas de fatores de virulência. A origem destes em nossa população poderia ser o contato com bactérias da microbiota ou do ambiente, de animais ou alimentos. Alternativamente, nossa população pode estar exposta à EHEC mais freqüentemente do que imaginado até agora. / Gastroenteritis is still an important public health problem in developing countries and Escherichia coli are frequent agents of diarrhea. Brazilian adults present antibodies reactive with the principal virulence factors of enteropathogenic E. coli (EPEC), which have many genetic and antigenic similarities with enterohemorrhagic E. coli (EHEC), that may be responsible for complications following diarrhea, as haemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Both bacteria present a common pathogenic mechanism, with the formation of “attaching and effacing" lesion in microvilis’ enterocytes, mediated by virulence factors codified by the patogenicity island LEE, as Intimin, Tir e Esps. Lipopolysaccharides (LPS) are components of outer membrane and important virulence factors of Gram-negative bacteria including E. coli. The infection with EHEC O157 results in the production of serum antibodies to the O157 LPS antigens, and usually they are indicators of recent infection. In this work we investigated IgG and IgM serum antibodies reactive with EHEC O157:H7, EHEC O111:H- and EPEC O111:H- antigens in healthy Brazilian adults, living in São Paulo. Serum samples were collected from 200 healthy adults (blood donors) and a pool was formed with 100 samples. The antibody levels were determined by ELISA for 100 individual serum samples by means of a whole cell ELISA with the three bacteria and an anti-LPS ELISA for the determination of the concentrations of specific antibodies anti-LPS O111 and O157, using the serum pool as control, in relation to the total IgG and IgM concentrations determined simultaneously. The results were submitted to statistical analysis. The repertoire of IgG and IgM antibodies to the three bacteria was investigated by immunoblotting (IB). The presence of anti-bacterial and anti-LPS seric antibodies was confirmed. There is a positive correlation between the titers of antibodies reactive with the three bacteria and between anti-bacteria and anti-LPS antibodies. The concentrations of IgM anti-LPSs were significantly higher than IgG, which is in accordance with the immune mechanism expected to a thymus-independent antigen. Surprisingly the concentrations of anti-LPS O157 were high taking into account the low frequency of O157 bacteria isolation in our country. The IB assays showed the presence of antibodies, mainly IgG, reactive with many antigenic bands suggestive of virulence factors. The origin of anti-EHEC antibodies in our population could be the contact with microbiota or environment bacteria, and animal or human E. coli strains, pathogenic or not. Alternatively, our people may be exposed to EHEC more frequently than previously thought.

Enterohemorrhagic E. coli, EHEC, is a verotoxin producing, zoonotic pathogen, which causes diseases in humans such as bloody or watery diarrhea. Microorganisms compete for limited living space, nutrients and other resources and therefore other microorganisms are EHECs biggest competitors. To avoid outbreaks and infections with EHEC, one possible approach is to use harmless but competitive bacteria as probiotics. Therefore, the aim of this study was to evaluate three probiotic E. coli strains and their ability to outcompete EHEC in bovine feces.     Ten different cattle fecal samples from three different farms were used to mix with the three probiotic and EHEC strains. The mixture was diluted and cultivated at 0 h as a control and then incubated for 48 h at 20°C and 37°C before dilution and cultivation on CT-SMaC. Colonies was counted and ratios between EHEC and probiotic E. coli before and after incubation were calculated. Kruskal-Wallis test with Dunn’s test as post hoc test were used to see if observed reductions of EHEC were significant or not.     In 37°C, strain 10 was the only strain producing a significant reduction of EHEC. In contrast, no significant reduction was observed at 20°C in any of the strains.     Future research studying other factors and performed on live cattle models are necessary to confirm the usefulness of the studied probiotic candidates. However, these results indicate probiotics can be a useful tool to avoid infections and big outbreaks of EHEC in the future.

As gastroenterites ainda são um importante problema de saúde pública nos países em desenvolvimento e as Escherichia colí são as principais causas de diarréias. Adultos brasileiros apresentam anticorpos reativos com E. colí enteropatogênica (EPEC), que apresenta muitas semelhanças com E. colí enterohemorágica (EHEC) e pode ser responsável por complicações como a síndrome hemolítica urêmica e a colite hemorrágica. Ambas as bactérias apresentam um mecanismo patogênico comum: a formação da lesão \"attaching and effacing\" nos microvilus dos enterócitos, mediadas por fatores de virulência, como Intimina, Tir e as Esps. Os lipopolissacarídeos (LPS) são componentes da membrana externa das bactérias gram-negativas, incluindo E. colí. A infecção por EHEC 0157 resulta na produção de anticorpos séricos antiLPS 0157, que geralmente são indicativos de infecção recente. Neste trabalho, nós investigamos a presença de anticorpos séricos IgG e IgM anti-EHEC 0157:H7, EHEC 0111:H- e EPEC 0111:H- em adultos brasileiros, da Grande São Paulo. Amostras de soro foram coletadas de 200 adultos saudáveis, doadores de sangue, e um pool foi formado com 100 amostras. Anticorpos foram determinados em 100 amostras individuais por ELlSA com bactérias íntegras e anti-LPS para a determinação de anticorpos específicos anti-LPS 0111 e 0157 com o \"pool\" de soro como controle, em relação às concentrações de IgG e IgM totais determinadas simultaneamente. Os resultados foram submetidos a análise estatística. O repertório de anticorpos IgG e IgM para as três bactérias foi investigado por \"immunoblotting\" (18). A presença de anticorpos anti-bacterianos e anti-LPS foi confirmada. Existe uma correlação positiva entre os anticorpos reativos com as três bactérias, e entre os anticorpos anti-bactéria e anti-LPS. As concentrações de IgM anti-LPSs foi mais alta que IgG, o que está de acordo com o mecanismo imune esperado para um antígeno timo-independente. As concentrações de anti-LPS 0157 foram altas, apesar da baixa freqüência da bactéria 0157 em nosso meio. Os ensaios de 18 mostraram anticorpos reativos com bandas antigênicas sugestivas de fatores de virulência. A origem destes em nossa população poderia ser o contato com bactérias da microbiota ou do ambiente, de animais ou alimentos. Alternativamente, nossa população pode estar exposta à EHEC mais freqüentemente do que imaginado até agora. / Gastroenteritis is still an important public health problem in developing countries and Escherichia coli are frequent agents of diarrhea. Brazilian adults present antibodies reactive with the principal virulence factors of enteropathogenic E. coli (EPEC), which have many genetic and antigenic similarities with enterohemorrhagic E. coli (EHEC), that may be responsible for complications following diarrhea, as haemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Both bacteria present a common pathogenic mechanism, with the formation of “attaching and effacing” lesion in microvilis’ enterocytes, mediated by virulence factors codified by the patogenicity island LEE, as Intimin, Tir e Esps. Lipopolysaccharides (LPS) are components of outer membrane and important virulence factors of Gram-negative bacteria including E. coli. The infection with EHEC O157 results in the production of serum antibodies to the O157 LPS antigens, and usually they are indicators of recent infection. In this work we investigated IgG and IgM serum antibodies reactive with EHEC O157:H7, EHEC O111:H- and EPEC O111:H- antigens in healthy Brazilian adults, living in São Paulo. Serum samples were collected from 200 healthy adults (blood donors) and a pool was formed with 100 samples. The antibody levels were determined by ELISA for 100 individual serum samples by means of a whole cell ELISA with the three bacteria and an anti-LPS ELISA for the determination of the concentrations of specific antibodies anti-LPS O111 and O157, using the serum pool as control, in relation to the total IgG and IgM concentrations determined simultaneously. The results were submitted to statistical analysis. The repertoire of IgG and IgM antibodies to the three bacteria was investigated by immunoblotting (IB). The presence of anti-bacterial and anti-LPS seric antibodies was confirmed. There is a positive correlation between the titers of antibodies reactive with the three bacteria and between anti-bacteria and anti-LPS antibodies. The concentrations of IgM anti-LPSs were significantly higher than IgG, which is in accordance with the immune mechanism expected to a thymus-independent antigen. Surprisingly the concentrations of anti-LPS O157 were high taking into account the low frequency of O157 bacteria isolation in our country. The IB assays showed the presence of antibodies, mainly IgG, reactive with many antigenic bands suggestive of virulence factors. The origin of anti-EHEC antibodies in our population could be the contact with microbiota or environment bacteria, and animal or human E. coli strains, pathogenic or not. Alternatively, our people may be exposed to EHEC more frequently than previously thought.

Enterohemorrhagic E. coli (EHEC) infections are a leading cause of foodborne illness in the United States. Shiga-like toxins are produced that can cause hemorrhagic colitis and can lead to dangerous complications, such as acute kidney injury and hemolytic uremic syndrome (HUS). There are currently no specific treatments for HUS, and therefore more research into EHEC and HUS needs to be done. Our study focuses on Shiga-like toxin induction of endoplasmic reticulum (ER) stress in in vitro and in vivo systems, using human monocyte-like THP-1 cells and a non-human primate model of HUS. We used qPCR to determine the levels of ER stress marker expression induced by both Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) challenges. We also looked at ER stress marker expression in non-human primates that survived a lethal Stx2 challenge after being given a Stx2 binding tetravalent peptide. We expected to see increased ER stress marker expression in THP-1 cells challenged with both Shiga-like toxins and in animals that received lethal doses of the toxins. Although results were inconclusive for THP-1 cell experiments, our preliminary non-human primate data suggest that the timing of ER stress marker production is important, and Shiga-like toxins may suppress the unfolded protein response (UPR) in some baboon tissues. We also show that the therapeutic peptide TVP may reverse this UPR suppression and relieve ER stress leading to animal survival. Our study, along with the current literature, shows that Shiga-like toxin induced ER stress is a promising area for future study.

BACKGROUND: Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) are emerging food- and water- borne pathogens and a leading cause of acute renal failure in otherwise healthy children. Ribotoxic Shiga toxins are the primary virulence factors and are responsible for the potentially lethal EHEC complication of hemolytic uremic syndrome (HUS). HUS, defined clinically by microangiopathic hemolytic anemia, thrombocytopenia and thrombotic microangiopathy which contribute to acute kidney injury or renal failure, is associated with significant patient morbidity. No pathogen- or toxin- specific therapeutic exists, and antibiotic use is contraindicated. Understanding the molecular mechanisms of Stx toxicity could lead to the development of Stx specific therapies. HYPOTHESIS: Experimental evidence suggests a role for leukocytes in systemic Stx2 trafficking and in Stx2 mediated kidney pathology. Cell stress responses, such as the ER stress response and ribosomal stress response, are hypothesized to induce apoptosis, and ultimately cell death, contributing to kidney injury; however these processes have only been described in vitro. If leukocyte and kidney cell stress responses are playing significant roles in in vivo Stx2 kidney injury, then down-regulation of these processes may provide therapeutic benefit. RESULTS: Mice injected with Stx2 or infected with Stx2-producing bacteria developed lethal kidney injury as judged by biomarkers and histopathology. Experimentally induced leukopenia did not alter kidney injury in either model, but did cause striking increases in the intestinal bacterial colonization which was dependent on the presence of Stx2. No Stx binding capacity was observed for either murine or human leukocytes ex vivo. Transcriptional evidence of kidney ER stress and apoptotic biomarkers were observed in both models of Stx2-mediated kidney injury, but down-regulation of these processes did not yield therapeutic benefit. CONCLUSIONS: Contrary to the current disease paradigm, no major role for leukocytes in systemic Stx2 trafficking or kidney injury was observed in vivo, but a novel role for host immune responses to Stx2 in the control of intestinal colonization by Stx2-producing bacteria was identified. Cell stress and apoptosis is induced by Stx2 in vivo but prevention of these is not sufficient to appreciably alter organ injury or survival in the murine models.

Cette thèse de doctorat s'est déroulée dans le cadre d'un projet européen FP7 (7th Framework Program) MSCA (Marie Sklodowska-Curie action) ITN (Initiale Training Network) EID (European Industrial Doctorates) appelé DISCo (a multidisciplinary Doctoral Industrial School on novel preventive strategies against Escherichia coli infections) coordonné par Mariagrazia Pizza et co-coordonné par Mickaël Desvaux. Ainsi, ce doctorat s'est déroulé pour moitié en Italie au centre de recherche GSK (GlaxoSmithKline) sur le site de Sienne sous la supervision de Roberto Rosini et la direction de Fabio Polticelli de Universita degli Studi Roma Tre. L'autre moitié de la thèse s'est déroulée en France à l'INRA, centre Auvergne-Rhône-Alpes sur le site de Theix sous la direction de Mickaël Desvaux et Grégory Jubelin comme co-encadrant. Cette thèse de doctorat participe au développement de nouvelles stratégies préventives aux infections aux E. coli pathogènes intestinaux (InPEC), en particulier E. coli entérohémorragiques (EHEC), par une stratégie vaccinale. Dans ce contexte, une approche de vaccinologie inverse a été mise en œuvre pour identifier de nouveaux antigènes candidats qui ont ensuite été délivrés par la technologie GMMA (Generalized Modules for Membrane Antigens). Par ailleurs, un domaine épitope potentiel chez les autotransporteurs, i.e. l'autochaperon, a été caractérisé par analyse des séquences protéiques et modélisation structurale. / Enterohemorrhagic E. coli (EHEC) are a major cause of large outbreaks mainly affecting developed countries. From 1982 to 2002, a total of 350 E. coli O157 outbreaks were reported in the United States. EHEC infection causes diarrheal disease often associated with clinical complications like hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although efforts focused on hygiene have been implemented in the food supply chain to reduce the risk of the foodborne E. coli O157 infection, outbreaks caused by this pathogen are still common. In addition, antibiotic-based therapy is discouraged for their potential undesirable effect in releasing shiga-toxin from the bacteria. Among non-antibiotic preventing strategies, vaccine development is warranted, still nowadays a licensed vaccine specific for human use against EHEC is not available. In this study, we used the Reverse Vaccinology approach applied on the EHEC O157:H7 genome to select new potential vaccine candidates. We identified a panel of 24 of potential protein antigens and we successfully expressed three of them in Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing these vaccine candidates resulted to be immunogenic, raising a specific antibody response for two of the selected antigens. In particular, immunization with MC001 candidate was able to reduce intestinal EHEC O157:H7 colonization lowering the bacterial count in feces, colon and ceacum tissues in mice. This candidate was found to be homologue to the Salmonella Typhimurium Lipid A deacylase enzyme (LpxR) and to our knowledge this study was the first report describing it as vaccine candidate. Also, gene distribution and sequence variability analysis showed that MC001 was mainly present and conserved in EHEC O157:H7 and in some EPEC. Given the high genetic variability among and within these pathotypes, the identification and inclusion of this conserved candidate in a vaccine might cover against major intestinal pathogenic strains. Furthermore, because it has been showed that during the infection process some autotransporters, as MC021 can be reactive, we also analysed molecular determinant with an important role for their proper secretion and folding, namely the autochaperon (AC) domain. It appeared the AC is a common feature of autotransporters but strictly associated with passenger domains exhibiting a –helix fold. Their exposition at the bacterial cell the surface further positions the AC as a potential antigenic target and/or development of new treatments. These findings further provide new research directions for the development of non-antibiotic preventive strategy against InPEC in human but also animal.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an attaching and effacing pathogen that upon attachment to host cells, induce characteristic attaching and effacing lesions and formation of F-actin rich pedestals beneath sites of bacterial attachment. EHEC harbors a Type III secretion system through which it delivers dozens of effectors into the host cell. The two secreted effectors critical for EHEC-mediated actin pedestal formation are the translocated intimin receptor (Tir) and EspFU. EspFU consists of an N-terminal secretion signal and a C-terminus containing six tandem 47-residue proline-rich repeats, each of which can bind and activate the actin nucleation promoting factor N-WASP. Structural and functional analyses described here have identified the mechanism of N-WASP activation by EspFU and the minimal domains and specific residues required for this activity. While EspFU and Tir are the only bacterial effectors required for F-actin pedestal formation, recruitment of EspFU to Tir is mediated by an unidentified putative host factor. To identify the host factor responsible for linking these two effectors, a combination of in vitro and functional assays were used to identify the host factor, IRTKS and the residues required for these interactions were defined. Further, the presence of at least two 47-residue repeats in all characterized clinical isolates of canonical EHEC strains led us to address the minimal requirements for EspFU functional domains to promote recruitment to Tir and N-WASP activation. Here we show that two proline-rich elements of EspFU are required for recruitment of EspFU by IRTKS to sites of bacterial attachment. Furthermore, once artificially clustered at the membrane, a single N-WASP binding element of EspFU can induce actin pedestal formation.

Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.