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Master Thesis Anja Sander
 

Master Thesis Anja Sander

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The Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative

The Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative
Escherichia coli (EAEC)-Induced Epithelial Inflammation

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    • Master Thesis Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC)-Induced Epithelial Inflammation Anja Sander B.Sc., s091919 Denmarks Technical University (DTU) in collaboration with Department of Microbiological Surveillance and Research, Statens Serum InstitutDepartment of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA Supervisors: Professor Karen A. Krogfelt, PhD (Eng) Professor Beth A. McCormick, PhD Erik J. Boll, PhD
    • Preface and acknowledgmentsThis report is the result of the master thesis carried out from February to July 2011. This work ispresented at Denmarks Technical University (DTU) and was carried out at Statens Serum Institut(SSI) in close collaboration with the Department of Microbiology and Physiological Systems at theUniversity of Massachusetts Medical School, USA.Firstly, I would like to thank my supervisor Professor Karen A. Krogfelt for her support and guidancewith this project, especially for encouraging me to carry out my experimental work abroad in theMcCormick laboratory at UMASS Medical School, USA, a laboratory known for its excellentresearch within the fields of mucosal inflammation, host:pathogen interactions, and cancer biology.Therefore, I also would like to thank professor Beth A. McCormick for offering me the greatopportunity to visit her laboratory and to tremendously extend my experimental skills and scientificknowledge.A special thanks goes to all other members of the McCormick laboratory: Ana Luisa, Kelly, Zach,Regino, Shrikanth and Terence. They all supported and contributed enormously to my experimentalwork during my stay, by answering and discussing questions and experimental details with greatcommitment.A very special thanks goes to my supervisor Erik Juncker Boll, whose pre-doctoral work in EAECpathogenesis I continued at the McCormick laboratory. Thank you for your invaluable support andguidance, your dedication and good spirit during the entire course of my master thesis!Roskilde, September 2011Anja Sander i
    • SummaryEnteroaggregative Escherichia coli (EAEC) is a worldwide emerging diarrheagenic pathogen thatcauses enteric and food-borne infectious diseases. A key role for inflammation in EAEC pathogenesishas been suggested by contemporary research. However, the factors and mechanisms by which EAECtriggers the innate immune response of its host are not known with certainty. In this study, it wasfound that the fimbriae of EAEC trigger epithelial transmigration of polymorphonuclear neutrophils(PMN), the hallmark of inflammation, through a conserved host signaling pathway.By using an in vitro model, it could be shown that the aggregative adherence fimbriae (AAF) ofEAEC are indispensable for triggering PMN transepithelial migration and that these pro-inflammatory properties are conserved among different AAF-producing EAEC prototype strains.These findings highlight that AAFs are not only the principal adhesins of EAEC mediating mucosaladherence, but also play a key role in the inflammatory aspects of EAEC pathogenesis.Furthermore, by using an RNA interference-based approach, it was demonstrated that EAEC-inducedPMN transepithelial migration is mediated through a conserved host signaling pathway involving theapical release of an arachidonic acid-derived PMN chemoattractant. This lipid is generated fromarachidonic acid through a conserved 12/15-lipoxygenase pathway.A better understanding of the inflammatory aspects of EAEC pathogenesis may potentiallycontribute to the design of more targeted and effective anti-inflammatory therapies for the treatmentof diverse mucosal inflammatory conditions such as inflammatory bowel diseases (IBD). ii
    • ResuméEnteroaggregative Escherichia coli (EAEC) er en globalt fremspirende patogen, som forårsagermave-tarm og fødevarebårne infektioner. En hovedrolle for inflammation i EAEC patogenesen erblevet påpeget af nyere forskning. Alligevel er faktorerne og mekanismerne igennem hvilke EAECudløser inflammation hidtil ikke kendt med sikkerhed. I dette projekt blev der påvist, at EAECenteroaggregative adherence fimbriae (AAF) inducerer epithelial transmigration af neutrofiler,inflammationens kendetegn, igennem en konserveret signal-kaskade i tarmepitelcellerne.Ved brug af en in vitro model blev det derudover klarlagt, at EAEC AAFs er uundværgelige for atinducere polymorfonukleare neutrofil (PMN) transmigration og at disse pro-inflammatoriskeegenskaber er blevet konservet blandt forskellige AAF-producerende EAEC prototype stammer.Disse resultater understreger, at AAFs ikke blot er de principielle EAEC adhesiner, som er ansvarligefor tilhæftningen til tarmslimhinden, men at de spiller også en fremtrædende rolle i deinflammatoriske aspekter af EAEC patogenesen.Derudover blev der ved brug af en RNA interference baseret tilgangsmåde demonstreret, at EAEC-induceret PMN transepitelial migration er formidlet via en konserveret signal-kaskade, sominvolverer apikal sekretion af et lipid med evnen til at tiltrække PMN. Dette lipid dannes fra frigjortarakindonsyre via en 12/15-lipoxygenase signalvej.En bedre forståelse af de inflammatoriske aspekter i EAEC patogenesen kunne potentielt bidrage tiludviklingen af mere målrettede og effektive anti-inflammatoriske terapier til behandling af diverseslimhinde-associerede inflammatoriske sygdomme som kronisk inflammatorisk tarmsygdom (IBD). iii
    • List of abbreviationsAA: Aggregative adherenceAAF: Aggregative adherence fimbriaeCD: Crohn´s diseaseCF: Cystic fibrosisCOPD: Chronic obstructive pulmonary diseaseDAEC: Diffusely adherent E. coliDEC: Diarrheagenic E.coliHG-DMEM: High Glucose - Dulbecco´s Modified Eagle MediumEAEC: Enteroaggregative E. coliECM: Extracellular matrix proteinsEHEC: Enterohemorrhagic E. coliEPEC: Enteropathogenic E. coliETEC: Enterotoxigenic E. coliExPEC: Extraintestinal pathogenic E. colifLMP: N-formylmethionyl-leucyl-phenylalanineLT: Heat labile toxinHUS: Hemolytic uremic syndromeHXA3: Hepoxilin A3IBD: Inflammatory bowel diseasesIL-8: Interleukin-8LPS: Lipopolysaccharide12/15-LOX: 12/15-lipoxygenaseMNEC: Meningitis associated E. coliPKC: Protein kinase CPLA2: Phospholipase A2PMN: Polymorphonuclear neutrophilSCID-HU-INT mice: Human intestinal xenografts in severe-combined immunodeficient miceSEM: Scanning electron microscopesiRNA: Small interfering RNASPATEs: Serine protease autotransporters of EnterobacteriaceaeST: Heat stable enterotoxinTEER: Transepithelial electrical resistanceTJ: Tight junctionWT: Wild type iv
    • Table of contentPreface and acknowledgments iSummary iiResumé iiiList of abbreviations ivTable of content vINTRODUCTIONIntroduction and aims 1Escherichia coli - an overview 2Enteroaggregative Escherichia coli (EAEC) 4 The history of EAEC discovery 4 Definition and identification 4Epidemiology of EAEC infections 6Clinical symptoms of EAEC infection 7EAEC pathogenesis 8Step 1: Adherence to the intestinal mucosa by aggregative adherence fimbriae (AAF) 10 Aggregative adherence fimbriae (AAF) – principal adhesins of EAEC 10 AggR – the global EAEC virulence regulator 11 Dispersin and its transporter system 12 AaiC 12 Other adhesins 13 Flagellin 13Step 2: Biofilm formation 13Step 3: Elaboration of toxins and elicitation of inflammatory responses 14 Toxins - plasmid and chromosomal encoded 14 Serine protease autotransporters of Enterobacteriaceae (SPATEs) 14 Pet 14 Pic 14 ShET1 15 EAST1 15 v
    • Inflammatory aspects in EAEC pathogenesis 15 Pro-inflammatory bacterial factors and host signaling molecules in EAEC inflammation 15 Modulating tight junctions – breaching the epithelial barrier 16 Host-pathogen communication – The signaling pathway underlying pathogen-induced PMN transmigration 17Animal models to study EAEC pathogenesis 18MATERIALS AND METHODSBacterial strains, growth conditions and preparation 20 Growth conditions 21 Preparation 21Cell line cultures and preparation of T84 cell monolayers 21 Media 21 Growth 21 Preparation 22Isolation and purification of PMNs 22In vitro model of transepithelial migration of PMNs 23 Procedure 23 Quantification 24Inhibitor treatments of T84 cell monolayers 24 Presentation of data and statistical analysis 24RESULTSPart 1: Investigation of the molecular mechanisms by which EAEC triggers transepithelialmigration of PMNs in vitroA: AAF/II play a key role in triggering EAEC 042-induced PMN transepithelial migration invitro, but expression of the fimbriae itself is not sufficient for triggering this inflammatoryevent. Aim and hypothesis 26 Results 27 vi
    • B: Expression of AAF/I, AAF/II and AAF/IV is indispensable for PMN transmigration inducedby other EAEC type strains Aim and hypothesis 28 Results 29Part 2: Host cell pathway underlying EAEC-induced transepithelial migration of PMNsA: Arachidonic acid, a precursor for lipid-derived PMN chemoattractants, is released by PKC-activated PLA2 during EAEC-induced inflammation Aim and hypothesis 31 Results 32B: An arachidonic acid-derived metabolite generated through the 12/15- LOX pathway plays arole in regulating EAEC 042-induced PMN transepithelial migration Aim and hypothesis 33 Results 33C: EAEC 042-induced PMN transepithelial migration is facilitated by the MRP2 effluxtransporter Aim and hypothesis 36 Results 36Part 3: Additional PMN transmigration experimentsA: Hra1, an accessory EAEC 042 colonization factor, does not trigger EAEC 042-induced PMNtransmigration Aim and hypothesis 39 Results 39B: The probiotic E. coli strain Nissle DSM 6601 does not trigger inflammation in the PMNtransmigration model Aim and hypothesis 40 Results 40 vii
    • DISCUSSION 42CONCLUSIONS AND FUTURE PERSPECTIVES 49REFERENCE LIST 51AppendixCD-Rom Data-Results 64Solutions, buffers and detergents 64 viii
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationINTRODUCTIONIntroduction and aimsEnteroaggregative Escherichia coli (EAEC) is a large heterogeneous subgroup ofdiarrheagenic E. coli (DEC). EAEC is among the most commonly isolated diarrheagenicbacterial species with isolation rates similar to Campylobacter jejuni and higher thanSalmonella sp. (Chattaway et al., 2011; Nataro et al., 2006; Tompkins et al., 1999). EAECinfection mainly causes persistent diarrhea in developing countries (Bhan et al., 1989c; Okekeet al., 2000) and acute diarrhea in industrialized countries (Bhan et al., 1989c; Bhatnagar et al.,1993). Infectious diarrhea is still a major health problem worldwide causing high morbidityand mortality (Clarke, 2001). The recent massive E. coli outbreak in Germany of hemolyticuremic syndrome (HUS), caused by a Shiga-toxin producing E. coli strain with commonvirulence properties of EAEC, highlights that highly pathogenic strains easily can emergethrough novel combinations of virulence factors of EAEC and other DEC strains (Chattawayet al., 2011; Frank et al., 2011; Rasko et al., 2011; Scheutz et al., 2011) making it morechallenging to fight this emerging pathogen.Besides the significant association with diarrhea, EAEC infections are generally regarded asmildly inflammatory. EAEC infection may incite an asymptomatic colonization leading tochronic intestinal inflammation in the absence of diarrhea ultimately leading to malnutrition,and impaired growth and development in children (Steiner et al., 1998).Despite an increasing focus on EAEC pathogenesis, little is yet known about the diseasemechanisms underlying intestinal inflammation caused by this organism. Several entericbacterial pathogens trigger inflammatory responses during infection of the intestinal mucosaand some bacteria utilize these innate defense mechanisms for their own benefit tosuccessfully colonize the host. The hallmark of inflammation is neutrophil infiltration at thesite of infection. Neutrophils do, however, not only have beneficial actions, but are alsoinvolved in the pathogenesis of many tissue-damaging inflammatory diseases. A detailedunderstanding of the inflammatory mechanisms in EAEC pathogenesis may potentially 1
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationcontribute to the design of more targeted and effective anti-inflammatory therapies for thetreatment of diverse mucosal inflammatory conditions such as salmonellosis, shigellosis,inflammatory bowel diseases (IBD), pneumonia, cystic fibrosis (CF), and chronic obstructivepulmonary disease (COPD).This work is focused on studying the inflammatory aspects of EAEC pathogenesis, morespecifically the complex interactions between the bacteria and its host, by using a well-established in vitro model of transepithelial migration of polymorphonuclear neutrophils(PMNs).Escherichia coli - an overviewEscherichia coli belongs to the family of Enterobacteriaceae, taxonomically placed within thegamma subdivision of the Proteobacteria phylum, and was discovered by German pediatricianand bacteriologist Theodor Escherich in 1885. Other prominent members of this family areShigella and Salmonella (Madigan et al., 2006).Phenotypically, E. coli is characterized as a Gram-negative rod-shaped facultative anaerobicbacterium, which is non-sporulating, non-motile or motile by peritrichous flagella. Thisorganism is one of the most common inhabitants of the intestinal tract of humans and warm-blooded animals. It has an optimal growth temperature of 37°C and grows well on non-selective media, usually by fermenting lactose and other sugars (Greenwood et al., 2007;Madigan et al., 2006).Genetically, the E. coli spp. are very heterogeneous, since only 20% of the genes comprisingthe E. coli core genome are shared by all strains (Lukjancenko et al., 2010). This great geneticdiversity makes E. coli challenging to study.Human E. coli strains are broadly classified into three major groups based on clinical andgenetic criteria: 1.) commensal strains, 2.) intestinal pathogenic strains and 3.) extraintestinalpathogenic strains (Russo & Johnson, 2000). Most E. coli strains are harmless commensals of 2
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationthe human gastrointestinal tract colonizing the infantile intestinal mucosa within a few hoursafter birth. Thereby, the organism can establish a lifelong symbiotic relationship with its host.As a highly successful competitor within that densely populated niche, E. coli becomes themost abundant facultative anaerobic bacteria of the human colonic microflora. Non-pathogenicE. coli strains rarely cause disease except in individuals with a weakened immune system orwhen the gastrointestinal barrier gets disrupted (Kaper et al., 2004; Nataro & Kaper, 1998). Inthese cases, a non-specific E. coli infection can lead to severe clinical complications such asmeningitis or sepsis. In contrast, pathogenic E. coli strains cause disease in otherwise healthyindividuals. This is possible because these pathogens have acquired special sets of virulencefactors, such as enterotoxins, adhesins or invasion factors, all of which can be encoded onmobile genetical elements, enabling them to colonize new niches (Russo & Johnson, 2000).Clinical symptoms may include diarrheal disease, urinary tract infection or sepsis/meningitis(Nataro & Kaper, 1998). Enteric disease itself may be caused by at least six distinct DECpathotypes: 1.) enteropathogenic E. coli (EPEC), 2.) enterohaemorrhagic E. coli (EHEC), 3.)enterotoxigenic E. coli (ETEC), 4.) enteroaggregative E. coli (EAEC), 5.) enteroinvasive E.coli (EIEC) and 6.) diffusely adherent E. coli (DAEC) (Kaper et al., 2004) Extraintestinal E.coli-associated infections like urinary tract infections or sepsis/meningitis are caused by thethird major group, recently described as extraintestinal pathogenic E. coli (ExPEC) (Russo &Johnson, 2000): uropathogenic E. coli (UPEC) and meningitis-associated E. coli (MNEC)(Kaper et al., 2004).Strains within each pathotype can be further subdivided by serotyping. This classificationsystem is based on the distribution of the antigenic structures expressed on the surface of thebacteria comprising lipopolysaccharide (LPS) or somatic (O) antigens, the capsular (K)antigens, the flagellar (H) antigens and the fimbrial (F) antigens, as detected in agglutinationassays with specific rabbit antibodies (Kaper et al., 2004; Orskov et al., 1977) 3
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationEnteroaggregative Escherichia coli (EAEC)The history of EAEC discovery In the early 1960´s, different serotypes of E. coli were associated with outbreaks ofdiarrhea for the first time (Ewing et al., 1963). Strains belonging to these serotypes werereferred to as EPEC. In 1979, an in vitro assay based on the adhesion of the bacteria to HEp-2cells was described by Cravioto et al. (1979). The assay identified EPEC strains binding to thecells in a localized pattern. Later it was discovered that adherent non-EPEC strains wereassociated with diarrhea as well and these strains were termed enteroadherent E. coli(Cravioto et al., 1991; Mathewson et al., 1985, 1986). It was Nataro et al. (1987) whoidentified two different phenotypes among the enteroadherent strains by their adherencepattern to HEp-2 cells; described as diffuse and aggregative adherence, respectively. This wasthe first time EAEC was both described and associated with diarrheal disease as part of anepidemiological study of pediatric diarrhea in Santiago, Chile (Nataro et al., 1987).This finding was shortly after confirmed by three other studies linking EAEC to persistentdiarrhea among children (Bhan et al., 1989b, 1989c; Cravioto et al., 1991). EAECpathogenicity was initially seriously questioned, because many early studies failed to showsignificant association of EAEC with disease (Echeverria et al., 1992; Gomes et al., 1989) Therole of EAEC as an etiological agent of diarrhea has later been proven more definitivelythrough two volunteer studies (Mathewson et al., 1986; Nataro et al., 1995) and a number ofoutbreaks (Boudailliez et al., 1997; Cobeljić et al., 1996; Czeczulin et al., 1999; Itoh et al.,1997; Morabito et al., 1998; Pai et al., 1997; Smith et al., 1997). It is likely that outbreaks ofdiarrhea and diarrheal illness due to EAEC infection are still underdiagnosed (Huang et al.,2004).Definition and identification EAEC is a DEC pathotype defined as E. coli that does not secrete the heat labile (LT)or heat stable (ST) toxins of ETEC and by its characteristic autoaggregative adherence patternin which the bacteria adhere to each other in a ‘stacked-brick’formation to HEp-2 cells andglass cover slips (FIG. 1.). This definition, however, likely encompasses both pathogenic andnon-pathogenic strains (Nataro & Kaper, 1998; Nataro et al., 1987). 4
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationTo distinguish pathogenic from non-pathogenic strains, EAEC is further subdivided intotypical and atypical EAEC strains. “Typical” EAEC strains refer to those harboring the AggRregulon on the pAA-virulence plasmid; these strains have been linked to diarrhea (Sarantuyaet al., 2004). In contrast, “atypical” EAEC lack the AggR regulon and can not be linked withcertainty to diarrheal disease (Nataro, 2005).FIG. 1. EAEC adherence. This HEp-2 cell adherence assay shows the EAEC “stacked-brick” aggregativeadherence to other bacteria, the coverslip, and the HEp-2 cells. From Okeke & Nataro (2001)The gold standard for EAEC identification remains the HEp-2 cell adhesion assay. EAECcolonization is detected by isolating E. coli from stool samples and demonstrating the AApattern in the HEp-2 assay. However, this test is unsuitable as a diagnostic tool, as it is difficultto perform and requires specialized facilities like a reference laboratory (Okeke & Nataro,2001). Serotyping is well suited to identify several pathogenic E. coli strains other than EAEC.However, many EAEC strains are difficult to serotype with this method, because they displaysuch great genetic diversity (Jenkins et al., 2006a, 2006b). Instead, DNA-based methods aregaining ground. A DNA probe has been developed, CVD432, targeting the conserved aatAgene on the otherwise heterogeneous pAA virulence plasmid (Baudry et al., 1990). EAEC iscurrently identified at Statens Serum Institut by using PCR analysis targeting the pAA- 5
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationencoded genes aatA, aggR and the chromosomal aaiC gene.Epidemiology of EAEC infectionsSince the first original description by Nataro et al. (1987), EAEC has attracted attention as aworldwide emerging pathogen in many clinical settings, encompassing endemic childhooddiarrhea in developing countries (Bardhan et al., 1998; Bhan et al., 1989a, 1989b; Fang et al.,1995) and industrialized countries (Bhatnagar et al., 1993; Chan et al., 1994; Huppertz et al.,1997; Tompkins et al., 1999), diarrhea in adults including traveler´s disease (Adachi et al.,2001; Gascón et al., 1998; Schultsz et al., 2000), as well as persistent diarrhea in HIV-infectedpatients (Durrer et al., 2000; Germani et al., 1998; Mathewson et al., 1998; Mayer & Wanke,1995; Mwachari et al., 1998; Wanke et al., 1998). A meta-analysis by Huang et al. shows thatEAEC is a cause of acute diarrheal disease globally among the above mentioned sub-populations (Huang et al., 2006)Of high interest are the extraordinary outbreaks of hemolytic uremic syndrome (HUS) causedby Shiga-toxin-producing EAEC strains in France (Boudailliez et al., 1997; Morabito et al.,1998) and recently in Germany (Bielaszewska et al., 2011; Frank et al., 2011). Diarrhealoutbreaks, mostly food borne, were also reported in UK, France, Japan, Switzerland and India(Cobeljić et al., 1996; Czeczulin et al., 1999; Itoh et al., 1997; Knutton et al., 2001; Pai et al.,1997; Smith et al., 1997)The mode of EAEC transmission is not yet fully understood, but the fecal-oral route is themost likely one. The evidence for infection sources is usually of epidemiological nature ratherthan microbiological and EAEC has only rarely been cultured from a non-human source(Huppertz et al., 1997; Okeke & Nataro, 2001). In two cases, EAEC was isolated from a nonhuman infection source: from milk in infant feeding bottles (Morais et al., 1997) and fromcattle (Sandhu et al., 1999). EAEC outbreaks can be epidemiologically linked to contaminatedwater and food (Itoh et al., 1997; Pai et al., 1997). Risk factors for EAEC infection includetravel to developing countries, ingestion of contaminated food and water, poor hygiene, hostsusceptibility, and possibly immunosuppression (e.g. HIV infection) (Huang & Dupont, 2004; 6
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationHuang et al., 2004). Especially children are considered as a risk group, although there is littleagreement in the age-specific prevalence for EAEC infection. Some studies report thatchildren are more likely to be affected in the first few months of life (González et al., 1997;Haider et al., 1991; Knutton et al., 2001), whereas others found most cases to emerge in laterchildhood (<5 years). The reason for these different observations could be strain or hostheterogeneity (Okeke et al., 2000).Clinical symptoms of EAEC infectionThe clinical manifestations of EAEC infections vary considerably among individuals due tocomplex host-pathogen interactions, including factors like EAEC strain heterogeneity,different amounts of ingested bacteria, host immune responses and host susceptibility (Okeke& Nataro, 2001) Genetic susceptibility to EAEC diarrhea is reported to be increased in thoseindividuals having a single nucleotide polymorphism (SNP) in the interleukin (IL)-8 promoterregion (Jiang et al., 2003).Although not all EAEC infections result in symptomatic illness (Adachi et al., 2001), moststudies show that EAEC infection results in gastrointestinal disease. The incubation period ofEAEC diarrheal illness ranges from 8 to 18 hours (Huang et al., 2004) The common clinicalfeatures of EAEC infection are well established in outbreaks, sporadic cases and volunteerstudies. Typical illness is characterized by watery, mucoid, secretory, often protracted diarrhea,which can be associated with abdominal pain, nausea, borborygymi and low-grade fever(Bhan et al., 1989c; Huppertz et al., 1997; Paul et al., 1994). EAEC infections are usuallyself-limiting and responsive to oral rehydration therapy in otherwise healthy individuals(Huang et al., 2004).In many cases EAEC diarrhea is inflammatory leading to bloody diarrhea in up to a third ofpatients (Cravioto et al., 1991; Steiner et al., 1998). In children, EAEC-induced entericinflammation is marked by raised levels of the pro-inflammatory cytokines IL-8 and IL-1β andfecal lactoferrin (indicative of neutrophil infiltration) (Steiner et al., 1998). In adults, fecallactoferrin levels are increased as well as reported by Boukenooghe et al. (Bouckenooghe et 7
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammational., 2000). Similar inflammation can arise in patients even without clinical diarrhea, and thisseemingly symptom-less carriage can lead to malnutrition and growth impairment over time(Steiner et al., 1998). Persistent diarrhea leads to malnutrition and growth impairment as well,especially in malnourished children living in developing countries. This is owed tomalabsorption of nutrients caused by the inability to repair the mucosal damage induced by theinflammatory host response (Huang et al., 2004; Petri et al., 2008). These long-term effects ofEAEC infection may be even more important than the short-term morbidity associated withdiarrheal illness.In addition, severe complications such as HUS, causing high mortality and morbidity, candevelop from infections with novel emerging hyper-virulent strains such as the shiga toxin-producing EAEC outbreak strain in Germany (Chattaway et al., 2011; Rasko et al., 2011;Scheutz et al., 2011).EAEC pathogenesisThe pathogenic mechanisms underlying EAEC infections are not completely understood yet.The reasons behind this include the great EAEC strain heterogeneity meaning that no singlevirulence factor is common for all strains (Nataro, 2005), the existence of both pathogenic andnon-pathogenic strains (Elias et al., 2002) and a lack of well established in vivo animal diseasemodels.EAEC pathogenesis is thought to comprise three basic steps; 1.) adherence to the intestinalmucosa by aggregative adherence fimbriae (AAF) and other adherence factors 2.) formation ofa mucus-containing biofilm on the intestinal surface; and 3.) release of toxins and elicitation ofinflammatory responses, mucosal toxicity and intestinal secretion (Harrington et al., 2006;Huang & Dupont, 2004; Nataro, 2005). The virulence factors involved in each of these stepswill be described in this section. 8
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 2. Model of EAEC pathogenesis. Stages 1 to 3 shown in yellow, illustrate the three major steps in EAECpathogenesis. See text for details. Modified after Harrington et al. (2006) 9
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationStep 1: Adherence to the intestinal mucosa by aggregative adherence fimbriae (AAF)The first challenge to potential luminal pathogens is to successfully attach to the intestinalsurface to resist the fluid flow of the luminal contents and the peristaltic movements ofintestinal contraction.Aggregative adherence fimbriae (AAF) – principal adhesins of EAEC EAEC adherence displays a characteristic aggregative adherence (AA) pattern, whenattached to intestinal epithelial cells in culture or to intestinal mucosa. The EAEC defining AApattern is mediated primarily by fimbrial adhesins termed aggregative adherence fimbriae(AAF). The AAFs of EAEC prototype strain 042 exhibit a semi-flexible bundle-formingstructure under the scanning electron microscope (SEM) (FIG. 3.) Four variants of the AAFmajor structural subunit are identified by now: AggA (AAF/I), AafA (AAF/II), Agg-3A(AAF/III) and Agg4A (AAF/IV) (Bernier et al., 2002; Boisen et al., 2008; Czeczulin et al.,1997; Nataro et al., 1992).FIG. 3. SEM photograph of EAEC prototype strain 042. White arrow indicates AAF/II. From Sheik et al.(2002)The AAF-encoding genes are encoded on high-molecular weight virulence plasmids,designated pAA (Nataro et al., 1992). The AAF biogenesis genes feature an organizationsimilar to the genes of the Dr superfamily of fimbrial adhesins, which also comprise adhesins 10
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationof uropathogenic E. coli (UPEC) and diffusely adherent E. coli (DAEC) (Boisen et al., 2008).Members of this adhesin family employ the chaperone-usher secretion pathway, whichrequires a periplasmic chaperone, an outer membrane usher protein, and two surface-expressedsubunits: a major and minor pilin subunit. AAF and Dr adhesins display a high level ofconservation of the usher and chaperone genes but greater divergence of the fimbrial subunitgenes (Servin, 2005). Since each of the four known AAF variants is only present in a subset ofstrains it is likely that more AAF variants remain to be identified (Nataro et al., 1995).While a definitive receptor for AAFs has yet to be identified, AAF/II of EAEC strain 042 hasbeen shown to bind to extracellular matrix components (ECM) like fibronectin, laminin andtype IV collagen. Although ECM proteins are generally localized to the basement membrane,interaction with bacterial enteric pathogens can occur during inflammation or opening of tightjunctions (TJ). It is suggested that binding to fibronectin by EAEC may activate host cellsignaling pathways (Farfan et al., 2008).In addition to mediating adherence to intestinal mucosa and epithelium, AAFs also facilitatehemagglutination of human erythrocytes and play an important role in biofilm formation onabiotic surfaces (Boisen et al., 2008; Czeczulin et al., 1997), thus stressing their apparentmulti-functionality. Importantly, AAF adhesins also seem to be involved in triggering hostinflammatory responses (Harrington et al., 2005; Strauman et al., 2010). This topic will bedescribed in more detail in the section about inflammatory aspects of EAEC pathogenesis.AggR – the global EAEC virulence regulator AggR is a member of the AraC/XylS family of transcriptional activators and a keyvirulence regulator in EAEC (Nataro et al., 1994). AggR is encoded on the pAA virulenceplasmid like the AAF genes and is associated with diarrheal illness (Jiang et al., 2002).AggR positively regulates the biogenesis of AAFs as well as other virulence-associated genesincluding dispersin (aap), the dispersin transporter system (aatPABCD) and a chromosomaltype IV secretion system (aaiA-Y). AggR expression is regulated by a positive feedback loop,where AggR itself enhances its own expression and by the E. coli global regulator factor forinversion stimulation (FIS). FIS is also involved in EAEC biofilm formation (See Biofilm 11
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationsection). To down regulate AggR expression, the repressive actions of the nucleoid-associatedprotein H-NS are needed. When EAEC is grown in media resembling the nutrient-richgastrointestinal environment (e.g. high glucose concentration and high osmolarity) theexpression of the positive regulators is favored, whereas expression of the negative regulatorHN-S is induced in nutrient-poor media like Luria Bertani (LB) broth (Morin et al., 2010).This regulatory scheme likely assures the rapid and high expression of the AggR regulonshortly after entering the gastrointestinal tract.Dispersin and its transporter system Dispersin is a secreted low-molecular weight protein encoded on the EAEC virulenceplasmid pAA (Sheikh et al., 2002). Secretion of this protein requires an ATP-binding cassette(ABC) transporter complex called Aat (designated Aat-PABCD). This protein complexconsists of five proteins, comprising an inner-membrane permease (AatP), an ABC protein(AatC) and a secreted outer membrane protein (AatA) (Nishi et al., 2003).The dispersin coat supports the dispersal of EAEC along the intestinal mucosa by decreasingbacterial autoaggregation, thus allowing for a more effective adherence and aggregation. Thiseffect is thought to be mediated by neutralization of the negatively charged LPS on thebacterial surface, so that positively charged AAFs can stick out from the bacterium and bind todistant sites e.g. mucosal surfaces or other bacteria (Harrington et al., 2006; Sheikh et al.,2002).AaiC This chromosomally encoded gene is part of a gene cluster (aaiA-Y) within achromosomal pheU pathogeneicity island in EAEC prototype strain 042, which is also underthe control of the AggR regulator. This gene cluster encodes a type VI secretion system (T6SS)through which AaiC is secreted. The potential role of AaiC in EAEC pathogenesis needs to befurther studied, but a screening study showed a 74% prevalence of aaiC among worldwideEAEC isolates (Dudley et al., 2006). The aaiC gene is part of the PCR analysis used toidentify EAEC at Statens Serum Institut (SSI). 12
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationOther adhesins Afimbrial adhesins have been identified in EAEC strains. Heat-resistant agglutinin 1(Hra1), described by Bhargava et al. (2009) is such an outer membrane protein (OMP) thatfunctions as an accessory colonization factor in autoaggregation, biofilm formation andaggregative adherence. Another example is a galactose-specific adhesin found in EAEC strainT7 (Grover et al., 2007).Flagellin Flagellin (fliC) is expressed by several enteric pathogens and commonly known for itspro-inflammatory effects. In EAEC, flagellin has been shown to cause release of the importantpro-inflammatory chemokine IL-8 from intestinal epithelial cells (IECs) in culture, therebycontributing to EAEC inflammation as well (Steiner et al., 1998, 2000).Step 2: Biofilm formationThe second stage of EAEC pathogenesis is characterized by formation of a mucus-containingbiofilm by the bacteria above the intact brush boarder on the enterocytes. A study by Sheikh etal. (2001) suggests that biofilm formation in EAEC is dependent on two proteins: Fis, achromosomal gene encoding a DNA-binding protein involved in growth-phase-dependentregulation, and YafK, a secreted protein with a yet unknown role. Fis contributes to biofilmformation via AAF/II biogenesis, activating AggR expression. Biofilm formation is inducedunder conditions with high glucose concentrations and high osmolarity resembling theintestinal environment (Sheikh et al., 2001). 13
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationStep 3: Elaboration of toxins and elicitation of inflammatory responsesThe third stage of EAEC pathogenesis involves release of EAEC toxins and triggering ofinflammatory responses, mucosal toxicity and intestinal secretion.Toxins - plasmid and chromosomally encoded Numerous pAA- or chromosomally encoded toxins and other secreted effector proteinshave been identified in EAEC strains. The pAA2 plasmid of EAEC 042 harbors the genesencoding the enteroaggregative E. coli heat stable enterotoxin (EAST1), and the plasmid-encoded toxin (Pet). The protein involved in colonization (Pic) and Shigella enterotoxin 1(ShET1) are encoded on the chromosome of EAEC 042. The following section describes thesefour virulence factors.Serine protease autotransporters of Enterobacteriaceae (SPATEs) Pic and Pet belong to the family of serine protease autotransporters (SPATES),characterized by a self-contained type V secretion system (T5SS). Members of this SPATEfamily are also found in Shigella spp., uropathogenic E .coli (UPEC) and other pathotypes ofdiarrheagenic E. coli (DEC) (Henderson et al., 2004). More than 20 SPATEs with diversefunctions have by now been described.Pet Pet, a 108 kDa protein, exerts its cytotoxic effects by altering the host cell cytoskeletonthrough proteolytic degradation of the membrane cytoskeletal protein spectrin, leading to cellrounding, detachment and cell death. Although Pet may play a role in EAEC pathogenesis, it isonly present in a minority of strains (Czeczulin et al., 1999)Pic Pic, a 116 kDa protein, cleaves mucin and induces hypersecretion of mucus, therebycontributing to biofilm formation and persistent colonization by EAEC (Navarro-Garcia &Elias, 2010). Moreover, Pic has been shown to confer a slight growth advantage in a mousemodel of gastrointestinal colonization (Harrington et al., 2009). 14
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationShET1 This protein is encoded within the pic gene, but on the complimentary strand. The roleof ShET1 in EAEC pathogenesis has not been studied yet, but in Shigella flexneri, ShET1induces time and dose dependent intestinal secretion in a rabbit model and may therebycontribute to the development of diarrheal illness (Fasano et al., 1997).EAST1 EAEST1, encoded by the astA gene, is widely found among both commensal anddiarrheagenic E. coli strains. EAST1 is often compared to E. coli STa (heat-Stable Toxin a),which is known to induce secretory diarrhea, and EAST1 is speculated to contribute todiarrheal disease as well (Ménard & Dubreuil, 2002).Inflammation in EAEC pathogenesisBacterial pathogens continually attack the epithelial barriers of the host. Although mucosalsurfaces are generally impermeable to most pathogens, many microorganisms have developedsophisticated strategies to breach or alter this barrier. In general, an array of bacterialpathogens including Shigella, Salmonella, and the E. coli pathotypes DAEC, EPEC and ETEC(Bétis et al., 2003a; Hurley et al., 2001; McCormick et al., 1993a, 1998; Savkovic et al.,1996) have evolved the capacity to engage their host cells in very complex interactionscommonly involving the exchange of biochemical signals, the net result of which is often thetriggering of host inflammatory responses.Pro-inflammatory bacterial factors and host signaling molecules in EAEC inflammation Epidemiological reports have shown that diarrhea, caused by a variety of inflammatorybacterial enteropathogens, is associated with the occurrence of cytokines in diarrheal stools(Greenberg et al., 2002).In the case of EAEC, several virulence factors (e.g. AggR and AafA)have been associated with increased levels of fecal inflammatory markers such as interleukinIL-8, IL-1β, lactoferrin, leukocytes and occult blood (Cennimo et al., 2009; Greenberg et al.,2002; Huang et al., 2004; Jiang et al., 2002). 15
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationIL-8 is an important pro-inflammatory chemokine, which is basolaterally secreted from theintestinal epithelium and directs neutrophils from the microvasculature to the immediatesubepithelial space. However, additional activation signals are necessary for full activation anddegranulation of neutrophils and complete transepithelial migration (Kucharzik et al., 2005;Madara et al., 1992; McCormick et al., 1995; Sansonetti et al., 1999). IL-8 levels weresignificantly higher in the feces of patients infected with EAEC strains harboring the pAA-plasmid borne virulence genes compared to those infected with virulence factor-negativeEAEC strains (Jiang et al., 2002).Lactoferrin is an iron-binding glycoprotein secreted from the intestinal mucosa and thesecondary granules of neutrophils to reduce microbial adhesion and proliferation (Legrand etal., 2005). IL-1β is secreted by mononuclear phagocytes and regulates multiple inflammatoryresponses, including neutrophil granula release and chemotaxis (Maloff et al., 1989; Smith etal., 1986).In vitro studies have shown that IL-8 is also released from non-polarized Caco-2 intestinalepithelial cells (IECs), when infected with EAEC 042. Later the same investigators identifiedan EAEC flagellin protein as the proposed major pro-inflammatory stimulus (Steiner et al.,1998, 2000). A study by Harrington et al. (2005) demonstrated that polarized T84 colonicepithelial cells release IL-8 even when infected with EAEC 042 mutated in the major flagellarsubunit FliC. The AafB minor subunit of AAF/II in EAEC 042 was since identified as a pro-inflammatory factor (Harrington et al., 2005). The host response to flagellin is mediated byToll-like receptor 5 (TLR5), which signals through a mitogen-activating protein kinase(MAPK) and nuclear factor-κB (NF-κB) to induce transcription of pro-inflammatory cytokinesfrom epithelial and monocytic cells (Khan et al., 2004). These findings suggest that multiplefactors contribute to EAEC inflammation in this in vitro model.Modulating tight junctions – breaching the epithelial barrier The establishment of tight junctions (TJ) between columnar epithelial cells contributesto the functional impermeability of the epithelial barrier, which enteroinvasive pathogens haveto overcome. EAEC prototype strains have the ability to induce AAF-dependent disruption ofthe epithelial barrier of T84 cell monolayers by causing delocalization of TJ proteins claudin-1 16
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationand occludin. As the epithelial barrier gets disrupted, bacterial proteins or the bacteriathemselves gain access to the intestinal submucosa or to basolateral receptors like ECMproteins (Strauman et al., 2010). At the same time a modification of TJs may also allow fortransepithelial migration of neutrophils as observed for other pathogens such as Salmonellaenterica serovar Typhimurium, Shigella flexneri, Pseudomonas aeruginosa and E. coli DAEC(Hurley et al., 2004; Köhler et al., 2007; McCormick et al., 1998; Peiffer et al., 2000).Neutrophils traverse epithelia by migrating through the paracellular space and crossingintercellular tight junctions (TJ).Host-pathogen communication – The signaling pathway underlying pathogen-induced PMNtransmigration Studies addressing the mechanisms underlying migration of polymorphonuclearneutrophils (PMNs) across model intestinal epithelia have crucially contributed to a betterunderstanding of the molecular and cellular events underlying PMN infiltration in reponse toenteric pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) andShigella flexneri (Köhler et al., 2002; McCormick et al., 1993a). Based on this knowledge, itis possible to make assumptions about the host signaling pathway underlying the inflammatoryresponses to EAEC infection.For S. Typhimurium-induced PMN transmigration it has been shown, that this process istriggered by a type III secretion system-translocated effector protein SipA. SipA initiates anADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directsactivation of protein kinase C α (PKC-α) (Criss et al., 2001; Silva et al., 2004). In a less wellunderstood process, activated PKC-α phosphorylates downstream targets finally leading toactivation of calcium-independent phospholipase A2 (iPLA2) (McCormick, 2007; Mumy et al.,2008b). S. flexneri-induced PMN transmigration requires activation of a mitogen-activatedprotein kinase (MAPK) signal transduction pathway, involving extracellular signal-regulatedkinase (ERK 1/2) and the upstream ERK kinase (MEK) (Köhler et al., 2002)The host signaling pathways underlying PMN transmigration triggered by these two entericpathogens converge when arachidonic acid is released from cell membranes by iPLA 2- andcPLA2 activity, respectively. Arachidonic acid is metabolized through a pathway involving 17
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammation12/15-lipoxygenase (12/15-LOX) leading to the synthesis and release of hepoxilin A3 (HXA3;8-hydroxy-11,12-epoxy-eicosatetraesanoic acid), a potent neutrophil chemoattractant (Mrsnyet al., 2004; Mumy et al., 2008a; Sutherland et al., 2000). Secretion of HXA3 on the apicalsurface is facilitated by the apically restricted efflux ATP-binding cassette (ABC) proteintransporter multidrug resistance associated protein 2 (MRP2). Thereby, HXA 3 establishes aparacellular chemotactic gradient through the tight junctional complex, guiding PMNmovement from the submucosa across the epithelium to the luminal site of infection, the finalstep in PMN recruitment (Chan et al., 2004; Mrsny et al., 2004; Pazos et al., 2008)Animal models to study EAEC pathogenesisIn order to study the pathogenic effects of EAEC toxins, animal species like rabbits and ratshave been used as in vivo and ex vivo animal model systems (Fasano et al., 1997; Navarro-García et al., 1998; Savarino et al., 1991). For EAEC colonization and disease studies, rabbitand gnotobiotic piglet models have been employed. While EAEC infection did not causedisease in rabbits, the piglets developed diarrhea in the absence of inflammation (Kang et al.,2001; Tzipori et al., 1992).Streptomycin-treated mice are a well established small animal model to study EAECcolonization factors, but since these mice do not develop any characteristic pathologicallesions or signs of inflammation in response to EAEC infection, this model is not well suitedfor studying EAEC disease (Harrington et al., 2009).A model with neonatal and weaned mice used to study the malnutritional effects of EAECinfection has been established as well and it could be demonstrated that EAEC infection inthese mice led to growth impairment and mild inflammation (Roche et al., 2010)As none of the existing animal model systems are able to reproduce all aspects of EAECpathogenesis, an in vivo model of human intestinal xenografts in severe-combinedimmunodeficient (SCID-HU-INT) mice has been established by Boll et al. (2011a) to studyEAEC pathogenesis and inflammation. This model is already well established for studyinginnate immune responses to enteric pathogens such as Salmonella enterica serovar 18
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationTyphimurium (Bertelsen et al., 2003). In this in vivo model, the xenografted tissue is of humanorigin, while the immune cells are of murine origin allowing to study EAEC inflammation inintact and morphologically fully developed human intestinal tissue (Savidge et al., 1995). 19
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationMATERIALS AND METHODSThis section includes an overview of bacterial strains used in this work (TABLE 1), as well asa detailed description of the main assay used during experimental work. In addition thestatistical methods used for data analysis are described.Bacterial strains, growth conditions and preparation TABLE 1. Strains used in this work.Strain Description Reference (Nataro et al.,042 EAEC prototype strain (O44:H18) expressing AAF/II 1985) (Nataro et al.,042 ΔaggR EAEC strain 042 with kanamycin resistance cassette inserted into aggR. 1994) EAEC strain 042 with kanamycin resistance cassette inserted into gene (Czeczulin et042 ΔaafA 3.4.14 locus encoding the AAF/II organelle. al., 1997) (MathewsonJM221 EAEC prototype strain JM221 (O92:H33) expressing AAF/I et al., 1986) EAEC type strain with kanamycin resistance cassette inserted into gene (Strauman etJM221 ΔaggDCBA locus encoding the AAF/II organelle. al., 2010) (Olesen et al.,C1010-00 EAEC prototype strain C1010-00 (Orough:H1) expressing AAF/IV 1994) EAEC strain C1010-00 with kanamycin resistance cassette inserted into (Boisen et al.,C1010-00 Δagg4A gene locus encoding the AAF/IV organelle. 2008) UPEC/EAEC strain with kanamycin resistance cassette inserted into gene (Olesen et al.,C555-91 locus encoding the AAF/I organelle. 1994) (Boyer & Roulland-HB101 Non-fimbriated laboratory E. coli K-12/B hybrid strain Dussoix, 1969) E. coli strain harboring cloning vector pACYC184 with aafA, aafB, aafC Boll et al.,HB101/pEJB02 and aafD genes, an IS1 element and aggR of EAEC strain 042. (2011a) (Cohen et al.,F-18 Commensal E. coli strain 1983) (Bhargava et042 SB EAEC strain 042 with hra1 isogenic mutant; hra1::aphA-3 al., 2009) EAEC strain 042 containing cloning vector pBJ1 with hra1 cloned into (Bhargava et042 SB/pBJ1 SspI and SphI sites of pBR322 al., 2009) Mutaflor®/Nissle DSM 6601 Probiotic non-pathogenic E. coli strain Ardeypharm, Germany 20
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationGrowth conditions Agitated overnight cultures of bacteria were grown in 3 ml Luria-Bertani (LB; BDDifco, Becton Dickinson) broth at 37°C for at least 16 hours. Thereafter EAEC andcommensal strains were diluted 1:100 in respectively 10 ml preheated Dulbecco´s ModifiedEagle`s Medium (DMEM; D1145, Sigma) with 4500 mg glucose/L or in LB medium, and thanincubated 4 hours at 37°C under static conditions to reach exponential phase. Ampillicin (100µg/ml) or chloramphenicol (30 µg/ml) were added where needed.Preparation of bacteria for in vitro or in vivo assays Bacteria were pelleted by centrifugation at 8500 rpm for 10 minutes at 4°C, washedand suspended in Hanks balanced salt solution containing Mg 2¨+, Ca2¨+ and 10 mM HEPES(HBSS +; pH 7.4; Sigma, St. Louis, MO) to the appropriate concentration of 300 µl per 10 mlovernight culture for PMN transepithelial migration assays.Cell line cultures and preparation of T84 cell monolayersMedia The human colon cancer-derived epithelial cell lines T84 (passages 57 to 77) and HCT-8 (passages 30 to 40) were maintained in Dulbecco’s modified Eagle’s Medium (DMEM;D1145, Sigma) and Ham´s F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 15mM HEPES, 14 mM NaHCO3, 40 µg/ml penicillin, 80 µg/ml ampicillin, 90 µg/mlstreptomycin and 7.5% fetal calf serum.Growth Cell monolayers were grown on 0.33 cm2 suspended-collagen-coated permeablepolycarbonated transwell filters with pore sizes of 5 µm (Costar, Cambridge, MA). Invertedmonolayers used for PMN transmigrations were prepared as previously described (McCormicket al., 1995; Nash et al., 1987; Parkos et al., 1992). Monolayers were utilized after 7 to 14days once having reached a confluent, polarized, and differentiated state. This was determinedby measuring steady-state transepithelial cell resistance (TEER) using a Millicell ERSvoltohmmeter (World Precision Instruments, New Haven, CT) and all cell monolayers used 21
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationreached a TEER value of at least 800 Ω per cm2.Preparation Prior to infection, insert-monolayers assemblies were lifted from the wells, drained ofmedia by inverting, and gently washed by immersion in a beaker containing HBSS +(containing Ca2+ and Mg 2+, with 10 mM HEPES, pH 7.4, Sigma, St. Louis, MO). Inserts werethen placed into a new well with 600 µl HBSS+ in the lower (outer) well and 100 µl HBSS+added to the upper (inner) well and allowed to equilibrate for 25 minutes at 37°C and 5% CO 2(McCormick et al., 1993b).Isolation and purification of PMNsHuman peripheral PMNs were purified from whole blood (anticoagulated with anticoagulationdetergent. See appendix section) collected by venipuncture from healthy human volunteers ofboth sexes as previously described (Nash et al., 1987; Parkos et al., 1992, 1991).Briefly, the buffy coat was separated by centrifugation at 2200 rpm at room temperature (RT).The plasma and mononuclear cells were removed by aspiration, and the majority oferythrocytes were removed by using a 2% gelatine sedimentation technique. Residualerythrocytes were lysed in cold red cell lysis buffer containing NH 4Cl and removed aftercentrifugation at 1200 rpm at 4°C. This technique allows for the rapid isolation of functionallyactive PMN at greater than 90% purity (Cohen et al., 1983; McCormick et al., 1993b; Mrsnyet al., 2004). Finally PMNs were resuspended in modified HBSS- (without Ca2+) before beingadded on cell monolayers. 22
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationIn vitro model of transepithelial migration of PMNsThe physiologically directed (basolateral-to-apical) PMN transepithelial migration assay usingcell culture inserts of inverted T84 cell monolayers was performed as previously described(Parkos et al., 1992, 1991) and modified (McCormick et al., 1998). The experiment isgraphically outlined in (FIG. 4.).FIG. 4. Outline of PMN transepithelial migration assay showing the basic steps. See text for details.Procedure Briefly, inverted polarized T84 cell monolayers seeded on 0.33-cm 2 filters wereapically infected with 25 µl of bacterial suspensions for 90 minutes at 37°C and 5% CO 2 at amultiplicity of infection (MOI) of approximately 100 bacteria per epithelial cell. Afterinfection, the cells were extensively washed and transferred with their apical side facing downto their original well in the 24-well plate containing 600 µl of Hank’s balanced salt solution(HBSS+) or inhibitor solution (see Inhibitor treatment section) in the bottom chamber. 100 µlof HBSS+ was placed on the basolateral surface of the monolayers followed by 20 µl ofprepared human PMNs (1 x 106). The monolayers were incubated at 37°C and 5% CO2 for 2½hours after which the inserts were gently removed leaving only those PMNs in the bottomwells that had migrated through the monolayers. As a positive control for PMN transmigration,10 µl 0.2 µM of the potent PMN chemoattractant N-formyl-methionyl-leucyl-phenylalanine(fMLP) (Sigma, St. Louis, MO) was added to the bottom chamber. 23
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationQuantification PMN transmigration was quantified by assaying for the PMN azurophilic granulemarker myeloperoxidase (MPO) as previously described (Parkos et al., 1992). Briefly, PMNmyeloperoxidase (MPO) was released by adding 50 µl Triton X-100 -containing HBSS andthe pH was adjusted to 4.2 with 50 µl citrate buffer pH 4.2. Color development was assayed at405 nm with SoftMax® Pro software on a microtiter plate reader (Bio-Rad laboratories,Richmond, CA), after mixing equal parts of sample and solution containing 1 mM 2,2-azino-di-(3-ethyl) di-thiazoline sulfonic acid and 10 mM H2O2 in 100 mM citrate buffer pH 4.2(Parkos et al., 1991)Inhibitor treatments of T84 cell monolayersInhibitor treatments used in this study are described in TABLE 2. For 12/15-LOX inhibition,T84 cell monolayers were incubated in the presence of 2 µM baicalein (stock concentration at1 mM in dimethyl sulfoxide [DMSO]) in cell culture medium for 48 hours at 37°C. Forinhibitors not diluted in HBSS+, identical monolayers were incubated in the presence ofDMSO in the medium at the same concentration as during treatment to serve as vehiclecontrols. Following treatment, inhibitors/media were thoroughly washed away and the cellmonolayers were equilibrated in HBSS+ for 30 minutes at 37°C prior to infection. Allinhibitors were purchased from Enzo Life Sciences. TABLE 2. Inhibitor treatments used in this study.Target Inhibitor Solvent Incubation periodPan-PLA2 ONO-RS-082 DMSO 3 hours12/15- LOX Baicalein DMSO 48 hours +P-glycoprotein Verapamil HBSS 2 hoursPresentation of data and statistical analysisSince variation exists in both transepithelial resistance between groups of monolayers(baseline resistance range 800-2.500 Ω x cm2) and between PMNs obtained from differentdonors, individual experiments were performed using large numbers of cell monolayers 24
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationperformed in triplicate and PMNs from single blood donors on individual days. PMN isolationwas restricted to 10 different donors (repetitive donations) over the course of these studies.Values are expressed as the mean ± standard deviation (SD) of an individual experimentperformed in triplicate repeated at least three times. Data were compared by Student’s t-testand p-values <0.05 were considered statistically significant. 25
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationRESULTSPart 1: Investigation of the molecular mechanisms by which EAEC triggerstransepithelial migration of PMNs in vitroAAF adhesins play a diverse role in multiple aspects of EAEC pathogenesis. Not only do theymediate adherence to mucosal surfaces, but they are also involved in biofilm formation onabiotic surfaces (Boisen et al., 2008; Sheikh et al., 2001). Importantly AAF adhesins areinvolved in mediating pro-inflammatory stimuli during EAEC infection (see Introductionsection). The first objective of this study was therefore to examine the role of AAFs intriggering PMN transepithelial migration in vitro.A: AAF/II play a key role in triggering EAEC 042-induced PMN transepithelialmigration in vitro but expression of the fimbriae itself is not sufficient for triggering thisinflammatory eventAim and hypothesis Boll et al. (2011a) demonstrated previously that EAEC prototype strain 042 promotestransepithelial migration of PMNs. The ability to induce epithelial barrier disruption andbasolateral release of IL-8 from polarized T84 cell monolayers was shown to be confered tothe commensal E. coli strain HS by acquisition of the pAA2 virulence plasmid of EAEC 042(Harrington et al., 2005; Strauman et al., 2010).Therefore, Boll et al. (2011a) tested the role of the pAA2 virulence plasmid in triggering PMNmigration and could show that HS carrying pAA2 induced significant PMN transmigration tothe same extent as EAEC 042, demonstrating that EAEC-specific factors encoded on pAA2are sufficient to induce an inflammatory response. By screening of a large mutant bank ofEAEC 042 strains harboring mutations in potential pAA2-encoded virulence genes, Boll et al.(2011a) identified the specific pAA-encoded genes involved in triggering these inflammatoryresponses. Mutations in the genes encoding the AafA major pilin protein of AAF/II ortranscription factor AggR almost entirely abolished EAEC 042-induced PMN migration. In 26
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationcontrast, mutations in the genes encoding EAEC virulence factors such as the AafB minorpilin protein of AAF/II, dispersin, the toxins Pet and EAST1 or the chromosomally encodedflagellin all induced PMN transmigration to the same extent as wild-type EAEC 042 did (Bollet al., 2011a). These results suggest that the AAF/II organelle plays a key role in triggeringEAEC 042-induced PMN transmigration without requiring a functional minor pilin subunit of042 AAF/II.Here, to further address the role of AAF/II in EAEC-induced inflammation, the non-fimbriatedlaboratory E. coli strain HB101 carrying a plasmid construct (pEJB02) harboring the genesencoding AAF/II and AggR was tested in the in vitro model of PMN transepithelial migration(Boll et al., 2011a).Results Polarized T84 cells were apically infected with EAEC 042 wild-type (WT), HB101WT or HB101/pEJB02. The potent chemoattractant fLMP served as positive control, becausefLMP-induced PMN transepithelial migration takes place by a mechanism independent ofpathogen-induced signaling (McCormick et al., 1993b). The results in (FIG. 5.) show clearlythat HB101/pEJB02 failed to induce PMN transmigration in the in vitro model as shown for arepresentative experiment.The results indicate that HB101/pEJB02 might not express fully functional AAF/II structureson its surface or that other accessory factors necessary for AAF/II-dependent PMNtransmigration are not present in HB101 (an afimbrial laboratory strain) in contrast to EAEC042 (a clinical isolate). 27
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 5. HB101/pEJB02 failed to induce PMN transepithelial migration. T84 cell monolayers were apicallyinfected with EAEC prototype strain 042 WT, HB101 WT or HB101/pEJB02. (-); HBSS+ served as negativecontrol, whereas the PMN chemoattractant fLMP served as positive control for PMN transmigration. The data areexpressed as the mean ± SD of an individual experiment performed in triplicate repeated at least three times withsimilar results.B: Expression of AAF/I, AAF/II and AAF/IV is indispensable for PMN transmigrationinduced by other EAEC type strainsAim and hypothesis After having investigated the role of AAF/II in triggering PMN infiltration, the nextobjective was to assess the possible pro-inflammatory role of other AAF variants.Wild-type and AAF-mutant strains of the following EAEC strains were included for testing inthe PMN transepithelial migration model: JM221 (AAF/I), C555-91 (AAF/I) and C1010-00(AAF/IV). In addition, EAEC 042 (AAF/II) and its AAF mutant strain were included asreferences. 55989 (AAF/III) was planned to be tested as well, but due to the multi-resistantnature of this strain, it was not possible for E.J. Boll to construct an isogenic AAF/III mutantof this strain. 28
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationResults As shown in (FIG. 6.), all four EAEC wild-type strains induced significant PMNtransmigration in the in vitro model. In contrast, mutations in the genes encoding AAF/I,AAF/II or AAF/IV all attenuated PMN transmigration in EAEC strains JM221, C555-91, 042and C1010-00.These findings suggest that the pro-inflammatory properties of the AAF organelles areconserved among different AAF-producing EAEC prototype strains. 29
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 6. AAF variants I, II and IV are indispensable to induce PMN transepithelial migration in vitro. (A)PMN transmigration induced by EAEC WT strain JM221 and JM221 ΔAAF/I. (B) PMN transmigration inducedby EAEC WT strain 042 and 042 ΔAAF/II. (C) PMN transmigration induced by EAEC WT strain C1010-00 andits AAF/IV mutant. (D) PMN transmigration induced by EAEC WT strain C555-91 and its AAF/I mutant. Thedata are expressed as the mean ± SD of an individual experiment performed in triplicate repeated at least threetimes with similar results. (-), HBSS + only. fLMP served as positive control for PMN transmigration.***, p <0.001. 30
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationPart 2: Host cell pathway underlying EAEC-induced transepithelial migration of PMNsExtensive studies in the McCormick laboratory have led to much insight into the host cellsignaling pathways underlying PMN transmigration induced by the enteric inflammatorypathogens Salmonella Typhimurium and Shigella flexneri (see Introdution section). Usingdrug inhibitors targeting specific host cell proteins, Boll et al. (2011b) has started to unravelthat EAEC-induced PMN transmigration is mediated through a similiar pathway as by theseother two enteric pathogens.The objective of this part of the study is to characterize further the molecular mechanismsunderlying EAEC-induced PMN transepithelial migration using the in vitro model.A: Arachidonic acid, a precursor for lipid-derived PMN chemoattractants, is released byPKC-activated PLA2 during EAEC-induced inflammationAim and hypothesis Animal and in vitro infection models have suggested an association between activatedPKC and inflammatory disease (Chang et al., 2000; Jacobson et al., 1995; Savkovic et al.,2003). Boll et al. (2011b) showed a significant increase of phosphorylated PKC-δ in themembrane fraction of T84 cells in response to EAEC infection, implying a role for PKC-δ inmediating the cellular response to EAEC-induced PMN transmigration.PKC isoforms have been shown to activate PLA2 leading to the release of arachidonic acidfrom cell membrane phospholipids (Mumy et al., 2008a; van Rossum & Patterson, 2009;Steer et al., 2002). Activated arachidonic acid is a key inflammatory mediator and serves as aprecursor in the production of eicosanoid lipids, which have either pro- or anti-inflammatoryeffects (Serhan & Savill, 2005).To determine whether arachidonic acid is part of the signaling pathway underlying EAEC-induced PMN transmigration, T84 cell monolayers were treated with the pan-PLA2 inhibitorONO-RS-082 for 3 hours prior to infection with EAEC WT strains 042 or JM221. Next, as an 31
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationalternative to the pharmacological approach, previously constructed HCT-8 cell lines wereapplied harboring a plasmid construct expressing small interfering RNAs (siRNA) directedagainst the mRNA molecules of PLA2G6, encoding human iPLA2, to decrease the expression ofthis enzyme (Mumy et al., 2008a).Results When conducting the inhibitor drug study using ONO-RS-082 to inhibit PLA 2 inEAEC WT strain 042 or JM221 infected T84 cell monolayers, experimental difficulties (datanot shown) were experienced.In contrast, EAEC 042 infection of HCT-8 cells generating siRNA against PLA 2 mRNA leadto a reduction in PMN transmigration by ~70% in comparison to infection of HCT-8monolayers expressing non-specific mRNA (FIG. 7.) Notably, the commensal E. coli strain F-18 triggered some inflammatory response as well, presumably due to HCT-8 monolayers beingless effective at forming a tight epithelial barrier than T84 cell monolayers. This inflammatoryresponse was, however, independent of a reduction in iPLA2 activity.FIG. 7. iPLA2 is involved in the host signaling pathway underlying EAEC-induced PMN transepithelialmigration. HCT-8 monolayers, transfected with a vector control or a vector modified to generate siRNAs aimedat decreasing the expression of PLA2G6 mRNA, the gene encoding human iPLA2, were used to test theinvolvement of iPLA2 in the host signaling pathway underlying EAEC 042 or commensal E. coli F-18-inducedPMN transepithelial migration. The data are expressed as the mean ± SD of an individual experiment performedin triplicate repeated at least three times with similar results. (-), HBSS+ only. * P < 0.01 32
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationThis finding demonstrates a role for iPLA2-released arachidonic acid in the host signalingpathway underlying EAEC-induced inflammation.B: An arachidonic acid-derived metabolite generated through the 12/15- LOX pathwayplays a role in regulating EAEC 042-induced PMN transepithelial migrationAim and Hypothesis Among the eicosanoids derived from arachidonic acid are the potent PMN-chemoattractants leukotriene B4 (LTB4) and hepoxilin A3 (HXA3), respectively generatedthrough the 5-LOX and 12/15-LOX pathway (Funk, 2001; Mrsny et al., 2004). Theinvolvement of either the 5-LOX or 12/15-LOX pathway in EAEC strain 042-inducedinflammation was investigated using the inhibitor-based approach by Boll et al. (2011b) T84cells were pretreated with the 5-LOX inhibitor caffeic acid for 24 hours or with the 12/15-LOX inhibitor baicalein for 48 hours prior to infection with EAEC WT strain 042. The 5-LOXinhibitor caffeic acid was found not to affect 042-induced PMN transmigration, whereas the12/15-LOX inhibitor baicalein reduced 042-induced PMN transmigration by ~50% (Boll etal., 2011b).To determine whether the 12/15-LOX pathway also is involved in the inflammatory responsesinduced by other EAEC strains, the baicalein drug study was carried out here, this time usingEAEC prototype strain JM221. Moreover, to support the baicalein drug study data for EAEC042, previously constructed HCT-8 cells were applied harboring a plasmid construct, whichexpresses siRNAs directed against mRNA of ALOX15, encoding human 12/15-LOX, to reducethe activity of this enzyme (Mumy et al., 2008a).Results Despite repeating the experiment many times, pretreatment of T84 cell monolayerswith baicalein for 48 hours prior to infection, did not lead to any definitive conclusionsregarding an effect on JM221-induced PMN transmigration. In the representative experimentshown below (FIG. 8.), an attenuating effect of the inhibitor is observed at the lowestconcentration (0.5 µM baicalein). However, the same effect was not observed at the higher 33
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationconcentrations. Instead, an increase in the PMN transmigration rate could be observed athigher concentrations, presumably due to the inhibitor solvents possible cytotoxic effects onT84 cell monolayers. It was not possible to repeat the previous EAEC 042 drug study datafrom Boll et al. (2011b), indicating that other technical difficulties might have been involvedin this case.FIG. 8. PMN transepithelial migration using the 12/15-LOX inhibitor baicalein and EAEC WT strainJM221. (A) and (B) T84 cell monolayers were pretreated with 0.5 µM, 1.0 µM or 2.0 µM of the 12/15-LOXinhibitor baicalein for 48 hours prior to infection with EAEC JM221 WT. The data are expressed as the mean ±SD of an individual experiment performed in triplicate repeated at least three times with similar results. fLMPserved as positive control for PMN migration. Control, HBSS+ only. 34
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationNext, the RNA inference-based approach was carried out employing the before mentionedHCT-8 cell lines transfected with a vector that produces siRNAs targeting mRNA of ALOX15,encoding human 12/15-LOX (Mumy et al., 2008a) The HCT-8-transfected cells were infectedapically with EAEC WT strain 042 or the commensal E. coli strain F-18. The decreasedexpression of 12/15-LOX strongly attenuated EAEC-induced PMN transepithelial migrationby ~70% compared to control monolayers transfected with a vector expressing unspecificsiRNA (FIG. 9.). Reduction in 12/15-LOX activity did not have any effect on the extent of thePMN transmigration rate induced by F-18 infection.FIG. 9. 12/15-LOX is involved in the host signaling pathway underlying EAEC-induced PMNtransepithelial migration. Monolayers of HCT-8 cells, transfected with a vector control or a vector modified togenerate siRNAs targeting mRNA of ALOX15, the gene encoding human 12/15-LOX, were used to study theinvolvement of 12/15-LOX in the host signaling pathway underlying EAEC WT strain 042 or commensal E. coliF-18 induced transepithelial PMN migration. The data are expressed as the mean ± SD of an individualexperiment performed in triplicate repeated at least three times with similar results. (-), HBSS + only. * p < 0.01In summary, an involvement of an arachidonic acid-derived lipid in the host signaling pathwayunderlying EAEC-induced inflammation was demonstrated. 35
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationC: EAEC 042-induced PMN transepithelial migration is facilitated by the MRP2 effluxtransporterAim and hypothesis The apical expression of the ABC efflux transporter MRP2 is up-regulated in responseto epithelial inflammation, and in Salmonella Typhimurium-induced PMN transmigration itserves as an efflux pump for apical secretion of the potent PMN chemoattractant hepoxilin A 3.Interestingly, the same study found that inhibition of 12/15-LOX, being critical for thesynthesis of HXA3, leads to a down-regulation of MRP2 expression (Pazos et al., 2008).Having demonstrated the involvement of 12/15-LOX pathway in EAEC-induced PMNtransmigration, it was next sought to determine which role MRP2 plays in this context. Boll etal. (2011b) showed that the MRP2 inhibitor probenecid attenuated 042-induced PMNtransmigration. To further strengthen these findings, a study was carried out here usingpreviously constructed HCT-8 cell monolayers generating siRNA against mRNA of MRP2, thegene encoding human MRP2, to reduce the expression of this protein (Pazos et al., 2008).Results siRNA-mediated decreased expression of MRP2 by HCT-8 monolayers resulted in adrastic reduction by ~60% of EAEC 042-induced PMN transmigration compared to siRNAcontrol monolayers. F-18-induced PMN transmigration was unaffected by MRP2 down-regulation (FIG. 10a.). 36
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 10a. MRP2 is also involved in the host signaling pathway underlying EAEC-induced PMNtransmigration. HCT-8 cell monolayers, transfected with a vector control or a vector modified to generatesiRNAs targeting mRNA of MRP2, the gene encoding human MRP2, were used to test the involvement of MRP2in the host signaling pathway underlying EAEC WT strain 042 or commensal E. coli F-18-induced PMNtransepithelial migration. The data are expressed as the mean ± SD of an individual experiment performed intriplicate repeated at least three times with similar results. (-), HBSS+ only. *, p < 0.01.In addition to MRP2, other intestinal apical efflux transporters are also known to be expressedon the membrane of T84 cells, including P-glycoprotein (Chan et al., 2004). As a controlexperiment to assess for the specificity of MRP2-directed HXA3 release, a drug inhibitorexperiment targeting P-glycoprotein was therefore carried out. The extent of EAEC 042-induced PMN transmigration was totally unaffected by addition of the P-glycoprotein inhibitorverapamil (FIG. 10b.). Therefore, HXA3 is likely excluded as a substrate for this transporter. 37
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 10b. PMN transepithelial migration using the P-glycoprotein inhibitor verapamil and EAECprototype strain 042 (A) and (B) T84 cell monolayers were pretreated with 20 µM, 40 µM or 100 µM of the P-glycoprotein inhibitor verapamil for 2 hours prior to infection with EAEC strain 042. The data are expressed asthe mean ± SD of an individual experiment performed in triplicate repeated at least three times with similarresults. fLMP served as positive control for PMN migration. Control, HBSS+ only. 38
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationAlong with the previous results by Boll et al. (2011b), these findings suggest a role for MRP2in the host signaling pathway underlying EAEC-induced inflammation.Part 3: Additional PMN transmigration experimentsA: Hra1, an accessory EAEC 042 colonization factor, does not trigger EAEC 042-inducedPMN transmigrationAim and hypothesis EAEC strain 042 harbors a gene, hra1, encoding heat-resistant agglutinin 1 (Hra1), ahemagglutinin originally reported from a porcine enterotoxigenic E. coli strain. Hra1 has alsobeen found in uropathogenic E. coli strains and in the neonatal meningitis E. coli strainRS218, in these strains called Hek (Fagan & Smith, 2007). A role for the outer membraneprotein Hra1 in adherence by neonatal meningitis E. coli has recently been defined (Fagan &Smith, 2007). It has been demonstrated that Hra1 is an accessory EAEC colonization factor(Bhargava et al., 2009). To further study the role of Hra1, a collaboration was carried out withprofessor Iruka N. Okeke from the Department of Biology, Haverford College, USA. Theobjective of this part of the study was to test whether deletion of the hra1 gene altered theextent of EAEC 042-elicited PMN transmigration.Results As (FIG. 11.) clearly demonstrates, EAEC 042 strains SB1 (Δhra1) and SB1/pBJ1(hra1-complemented strain) induced PMN transepithelial migration to the same extent aswild-type EAEC 042. Thus, while Hra1 might have more functions besides being an accessorycolonization factor, triggering inflammation is not one of them. 39
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 11. Hra1, an EAEC accessory colonization factor, is not involved in EAEC042-induced PMNepithelial transmigration. T84 cell monolayers were apically infected with EAEC WT strain 042 or the 042strains SB1 (Δhra1) or SB1/pBJ1 (hra1-complemented strain). The data are expressed as the mean ± SD of anindividual experiment performed in triplicate repeated at least three times with similar results. fLMP served aspositive control for PMN migration. (-), HBSS+ only.B: The probiotic E. coli strain Nissle DSM 6601 does not trigger inflammation in thePMN transmigration modelAim and hypothesis E. coli Nissle DSM 6601 was isolated in 1917 from a German soldier based on itspotential to protect from presumably infectious gastroenteritis (Schultz, 2008). Later this strainwas mostly used for the treatment of chronic inflammatory disorders, mainly because of itsantagonistic effects towards competing intestinal microbiota and its lack of virulence factors(Schultz, 2008). Due to its clinical application, the pro-inflammatory potential of Nissle DSM6601 was tested in the in vitro PMN transmigration model.Results As shown in (FIG. 12.) the probiotic E. coli Nissle strain DSM 6601 did not elicit anyinflammatory response and therefore it is considered as a safe therapeutic agent, which can beused as an aid in treating infectious gastroenteritis or chronic IBDs in clinical settings. 40
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 12. The probiotic E. coli strain Nissle DSM 6601 does not induce PMN epithelial transmigration. T84cell monolayers were apically infected with Nissle DSM 6601. The data are expressed as the mean ± SD of anindividual experiment performed in triplicate repeated at least three times with similar results. fLMP served aspositive control for PMN migration. (-), HBSS+ only 41
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationDISCUSSIONEAEC is one of several enteric pathogens causing inflammatory diarrhea leading to highmortality and morbidity worldwide. Many lines of evidence suggest that inflammation is animportant aspect in EAEC pathogenesis, yet the factors and exact mechanisms by which thispathogen triggers the innate immune response of its host are not known with certainty.The findings of this study demonstrate a key role for AAF in triggering EAEC-induced PMNinfiltration in vitro. By focusing on AAF-producing EAEC prototype strains, it was found thatexpression of AAF is required for triggering PMN transmigration in all four strains tested.Deletion of the AAF genes in these strains almost completely abolished PMN transmigration.This finding is consistent with the results from Boll et al. (2011a), showing that EAEC 042AAF/II and AggR mutant strains lost the ability to trigger PMN transmigration. Moreover, theresults of this study strongly indicate that AAF-dependent pro-inflammatory properties areconserved among EAEC strains expressing different AAF variants. This is impressive,considering the great genetic divergence of the fimbrial subunit genes (See Introductionsection) (Boisen et al., 2008).As part of a study, conducted by Boll et al. (2011a), to directly characterize the pro-inflammatory properties of different AAF variants, the pro-inflammatory role of AAF/II wastested further in the PMN transmigration model. For that purpose, the pEJB02 plasmidencoding AAF/II of EAEC 042 was transferred to the afimbrial E. coli strain HB101.Unfortunately, this strain failed to induce PMN migration in the in vitro model. This wassurprising, given that Boll et al. (2011a) has showed that constructed plasmids encoding theother three AAF variants are sufficient to trigger PMN transmigration in the HB101background.Nevertheless, Boll et al. (2011a) could verify that HB101/pEJB02 exhibits adherence to T84cell monolayers and forms biofilm in microtiter plates to the same extent as EAEC 042.Expression of AafA, the major pilin subunit, could be confirmed by immunostaining, butprotruding fimbriae structures could not be detected under the electron microscope (Boll et al.,2011a). 42
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationTaken together, these facts imply that the 042 AAF/II adhesins on the surface of HB101 seemnot to form a fully functional AAF/II organelle. Alternatively, other accessory factorsnecessary for AAF/II-dependent PMN transmigration are not present in the afimbrial strainHB101 in contrast to in EAEC 042. Such a missing accessory factor could be the bacterialsurface coating protein dispersin, which mediates proper AAF protrusion from the bacterialsurface of EAEC 042 (Sheikh et al., 2002). Interestingly, an EAEC 042 dispersin mutant wasfound to fully induce PMN transmigration to the same extent as the 042 WT strain, suggestingthat factors other than dispersin may contribute to proper AAF organelle function (Boll et al.,2011a).These findings highlight that both proper fimbriae/accessory factor expression and fimbriaeprotrusion is important for triggering PMN transmigration in the model.Surprisingly, when Boll et al. (2011a) tested HB101 WT, HB101/pEJB02 and EAEC WTstrain 042 in chimeric SCID-HU-INT mice, HB101/pEJB02 carrying the AAF/II-encodingplasmid caused PMN infiltration and tissue damage to the same extent as wild-type EAEC042. This is in marked contrast to the results of this study using the in vitro PMNtransmigration model, presumably reflecting the different physiological parameters of eachmodel system.Expression of the AggR-regulon might be favored and induced by physiological factors in thexenografted tissue, which very much resembles the human colonic environment, leading toexpression of AAF/II during the 24 hour infection period (Sheikh et al., 2002). In comparison,AAF/II expression in the in vitro model needs to be triggered whilst growing in bacterialculture prior to infection and this fundamental difference may partly explain the lack ofAAF/II expression from this particular plasmid. Other major differences between these twomodels are the time course of AAF expression (4 hours vs. possibly 24 hours) and EAECinfection (1.5 hours vs. 24 hours), for the in vitro and animal model, respectively. Moreover,the xenografted tissue becomes intensively vascularized and produces mucus (Savidge et al.,1995). Components of the mucus layer could be such physiological factors contributing toinduce AAF expression in HB101/pEJB02, since the mucus serves as a nutritional source forintestinal bacteria by providing carbohydrates (Chang et al., 2004). 43
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationThe fact, that AAF/II expressed by EAEC 042 and HB101/pEJB02 clearly triggeredinflammation and caused tissue damage in SCID-HU-INT mice, is in line with findings of thisstudy, in that AAFs play a key role in triggering the host signaling pathway leading to PMNtransmigration across intestinal epithelia. Previous in vitro studies support the inflammatoryrole of AAF by demonstrating that IL-8 is basolaterally secreted from polarized T84 cellmonolayers in response to AAF-mediated adhesion (Harrington et al., 2005).A second objective of this study was to investigate the host signaling pathway underlyingEAEC-induced inflammation, focusing on the role that PMN transmigration plays in thiscontext. Extending on the findings of Boll et al. (20011b), it was found that the EAEC 042-induced host signaling pathway leading to PMN transmigration has key steps in common witha conserved signaling pathway triggered by enteric pathogens such as SalmonellaTyphimurium, Shigella flexneri, Campylobacter species but also by pulmonal pathogens likePseudomonas aeruginosa (Hurley et al., 2006; Mrsny et al., 2004; Mumy et al., 2008a; Pazoset al., 2008).A RNA interference-based approach was employed to probe into the involvement of iPLA 2,12/15-LOX and MRP2 in the host signaling pathway underlying EAEC 042-induced PMNtransmigration. This study was able to further substantiate and validate the previous 042 druginhibitor studies carried out by Boll et al. (2011b), showing as well that arachidonic acid, aprecursor for the lipid-derived PMN chemoattractant HXA 3 is released by a calcium-independent phospholipase A2 (iPLA2) from the epithelial cell membrane. Interfering witheither synthesis or secretion of this PMN chemoattractant (presumably HXA 3) by blocking12/15-LOX or MRP2 expression resulted in a marked reduction in PMN transmigration. Thedata generated from these two different approaches support this lipid´s function as a potentPMN chemoattractant, being apically secreted by MRP2 in this conserved signaling cascade.The advantage of the RNA interference based-approach compared to the drug inhibitor-basedapproach used by Boll et al (2011b)., is that siRNAs are able to bind more specifically to theircellular targets, whereas it is generally well known, that results from experiments usingpharmacological inhibitors have to be interpreted with caution because of their potential non-specific effects. Non-specific binding to other cellular proteins than the target enzyme itself 44
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationcould potentially lead to distortions of cellular functions.To investigate whether the 12/15-LOX host signaling pathway is also induced by AAF-expressing EAEC strains other than 042, drug inhibitor studies were carried out using EAECstrain JM221. Interestingly, when treating T84 cell with the 12/15.LOX inhibitor baicalein orthe PLA2-inhibitor ONO-RS-082, there were experimental difficulties with getting JM221 towork in this model. In contrast, when Boll et al. (2011b) carried out the same experimentsusing EAEC strain 042, significant attenuative effects of both inhibitors were observed.A possible explanation for these conflicting observations could be the possible cytotoxity ofDMSO towards T84 cells, when pretreated with baicalein dissolved in DMSO for 48 hours.DMSO has been shown to exert toxic effects on Caco2/TC7 cells, leading to alterations inapical membrane permeability, or on TJ complexes (Da Violante et al., 2002). But JM221 mayitself be part of the problem, since a study conducted by Strauman et al. (2010) has shown thatthe cell viability of T84 cell monolayers is significantly decreased after JM221 infection butunaffected in case of an infection with EAEC 042. The accumulating possible cytotoxic effectsexerted by DMSO and JM221 on T84 cell monolayers could explain the experimentaldifficulties with this particular inhibitor drug study.The data in (FIG. 8.) suggests that the cytotoxic effects might be reflected by the high PMNtransmigration rates despite increasing inhibitor concentrations, as well as by the loweredPMN transmigration rate for DMSO compared to the blank control. Ideally, a dose-dependentinhibition of PMN transmigration by the 12/15-LOX inhibitor baicalein would have beenobserved. There is also the possibility that EAEC JM221-induced PMN transmigration ismediated through a host signaling pathway other than the previously described 12/15-LOXpathway. 45
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationFIG. 13. Host signaling pathway underlying EAEC-induced inflammation – transcellular signaling withneutrophils.As depicted in (FIG. 13.), the findings of this study combined with those of Boll et al. (2011b)demonstrate that PMN movement is directed by two distinct PMN chemoattractants acting inconcert: IL-8 recruits PMNs from the bloodstream to the epithelial submucosa, while HXA 3 isa key factor for directing PMNs across the epithelium itself, the final step in recruiting PMNsto the mucosal lumen. This paracellular PMN movement is directed by establishing achemotactic HXA3 gradient across the epithelial tight junction (TJ) protein complexes. A 46
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationcritical role for HXA3 as potent PMN chemoattractant in both intestinal and pulmonaryinflammation, has been previously demonstrated (Hurley et al., 2004, 2001; McCormick,2007).Many enteric pathogens have evolved sophisticated strategies for modulating or exploiting thehosts immune response to their own benefit. For example, Salmonella survives withinphagocytic cells like macrophages by producing enzymes that directly inactivate reactiveoxygen and nitrogen species produced by the macrophage (De Groote et al., 1996). In the caseof Afa/Dr-expressing DAEC strains, increased PMN infiltration at the site of infectionpromotes up-regulation of the brush-border associated decay accelerating factor (DAF/CD55),the receptor for Afa/Dr adhesins, leading to increased mucosal adherence of the bacteria (Bétiset al., 2003b).To which extent EAEC exploits the host innate immune responses is an area of ongoingresearch. During the process of PMN migration TJ proteins between intestinal epithelial cellsare modified, allowing for an increased passage of neutrophils. This opening of the TJ proteinscould contribute to EAEC pathogenesis as observed in Salmonella Typhimurium by easing theaccess for the bacteria or its toxins to the basolateral compartment (Köhler et al., 2007;Strauman et al., 2010). After being transmigrated into the lumen during acute inflammation,PMNs secrete adenosine 5`-monophosphate (5`-AMP), which in turn is converted toadenosine by the apical epithelial cells. Adenosine subsequently activates intestinal fluidsecretion through adenosine receptors on the apical membrane of the intestinal epithelial cells.Enhanced fluid secretion is potentially leading to development of secretory diarrhea (Madaraet al., 1993; Strohmeier et al., 1997). Thereby, the inflammatory process resulting in secretorydiarrhea might aid in the spreading of EAEC by facilitating its rapid dissemination into theenvironment and, consequently, the infection of a new host.A deeper understanding of bacterial-induced inflammation potentially contributes to theunderstanding of the disease mechanisms behind major inflammatory bowel diseases (IBDs)such as Crohn´s disease (CD) and ulcerative colitis (UC), because several studies suggest thatthese inflammatory diseases are triggered by host responses to enteric pathogens, especiallymucosa-associated E. coli (Rhodes, 2007; Rolhion & Darfeuille-Michaud, 2007). Notable is 47
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationthe finding, that 12-LOX activity is increased under conditions such as IBD, suggesting adysregulation of HXA3, so a pharmacologic regulation of epithelial HXA 3 synthesis mightprovide an effective therapeutic strategy for treating epithelial inflammatory disorders(Shannon et al., 1993). Another promising option to reduce tissue damage caused byneutrophil release of reactive oxygen species (ROS) would be to block the ability of theneutrophils to respond/recognize HXA3, thereby limiting the severity of the disease (Mumy &McCormick, 2009). 48
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationCONCLUSIONS AND FUTURE PERSPECTIVESThe findings of this study contribute to a better understanding of the inflammatory aspects ofEAEC pathogenesis. The results demonstrate that the aggregative adherence fimbriae (AAF)of EAEC play a key role in triggering PMN transmigration, the hallmark of inflammation.The results from drug inhibitor studies by Boll et al. (2011b), probing into the host signalingpathway underlying EAEC-induced inflammation, where extended and verified here by usingan RNA interference based approach. These results underline that an arachidonic acid-derivedneutrophil chemoattractant is apically secreted into the intestinal lumen upon EAEC infection,thereby guiding the neutrophils across the intestinal epithelium to the site of infection.The ability of AAF-producing EAEC prototype strains to trigger PMN transepithelialmigration in vitro was shown to require the expression of these adhesins, further substantiatingtheir pro-inflammatory properties. These pro-inflammatory properties were found to beconserved among all four tested EAEC prototype strains expressing different AAF variants,despite the remarkable genetic diversity of the fimbrial subunit genes.For EAEC 042, it has been shown that its AAF/II binds to extracellular matrix (ECM) proteins(Farfan et al., 2008). More studies are needed to identify the epithelial surface receptorsinvolved in EAEC adherence, so the mechanisms by which EAEC initiates the host signalingpathway underlying PMN transepithelial migration can be better understood.New answers result in even more questions to be investigated: Do virulence factors other thanAAF contribute to this host inflammatory responses and if yes, which? How does EAECeventually benefit from triggering this host innate immune response? Several entericpathogens have developed advanced strategies for modulating the host immune responses.Infiltrating neutrophils at the site of infection likely kill invading pathogens by producingintracellular reactive oxygen species (ROS). Neutrophils, however, do not only have beneficialactions; tissue damage caused by neutrophil release of reactive oxygen species (ROS) couldfacilitate access of the bacteria or their products to the basolateral compartment but also leadto increased fluid secretion or perhaps to an alteration in gene expression e.g. of surfacereceptors, all of which potentially could contribute to successful EAEC colonization of the 49
    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial Inflammationhost.Moreover, further inhibitor studies are needed to determine whether the 12/15-LOX hostsignaling pathway is conserved among other EAEC prototype strains than 042, since druginhibitor studies with EAEC strain JM221 did not lead to conclusive results yet.To unravel the complex bacterial-epithelial “crosstalk” underlying EAEC-inducedinflammation, further studies are important steps towards a better overall understanding ofEAEC pathogenesis. This need is underscored by the recent massive outbreak of hemolyticuremic syndrome (HUS) in Germany caused by a Shiga-toxin and AAF-producing EAECstrain leading to high morbidity and mortality as well.Moreover, it may potentially lead to an increased understanding of the disease mechanismsunderlying inflammatory conditions such as salmonellosis, shigellosis, inflammatory boweldiseases (IBD), pneumonia, cystic fibrosis (CF), and chronic obstructive pulmonary disease(COPD). 50
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    • Molecular Mechanisms Underlying Diarrheagenic Enteroaggregative Escherichia coli (EAEC) - Induced Epithelial InflammationAPPENDIX • CD-Rom Data - Results includedSolutions, buffers and detergents (see Material and Methods section)Hanks Balanced Salt pH 7.4 for 10 LSolution (HBBS+)9.995 L dH2O23.8 g Hepes97.5 g HBSS powder (Sigma, St. Louis, MO)4.0 – 0.5 ml 10 M NaOH1.4 g CaClHanks Balanced Salt pH 7.2 - for 2 LSolution (HBBS ) filter sterilized1.993 L dH204.76 g Hepes19 g HBSS without CaCl powder (Sigma, St. Louis, MO)0.7 ml 10 M NaOHCitrate buffer for 500 ml pH 4.2400 ml dH2O73.5 g Sodium citrate52.5 g Citric acidAnticoagulation for 500 ml filter sterilizeddetergent (ACD)500 ml dH2O6.9 g Citric acid12.5 g Sodium citrate10 g DextroseRed Blood Cell Lysis for 1 L filter sterilizedBuffer1L dH2O8.29 g Ammonium chloride (NH4Cl)1g Sodium bibcarbonate (NaHCO3)0,038 g EDTA 64