Master Thesis Anja Sander

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 Institut

Department 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 acknowledgments

This report is the result of the master thesis carried out from February to July 2011. This work is
presented 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 the
University of Massachusetts Medical School, USA.

Firstly, I would like to thank my supervisor Professor Karen A. Krogfelt for her support and guidance
with this project, especially for encouraging me to carry out my experimental work abroad in the
McCormick laboratory at UMASS Medical School, USA, a laboratory known for its excellent
research 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 great
opportunity to visit her laboratory and to tremendously extend my experimental skills and scientific
knowledge.

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 experimental
work during my stay, by answering and discussing questions and experimental details with great
commitment.

A very special thanks goes to my supervisor Erik Juncker Boll, whose pre-doctoral work in EAEC
pathogenesis I continued at the McCormick laboratory. Thank you for your invaluable support and
guidance, your dedication and good spirit during the entire course of my master thesis!

Roskilde, September 2011

Anja Sander

i

Summary

Enteroaggregative Escherichia coli (EAEC) is a worldwide emerging diarrheagenic pathogen that
causes enteric and food-borne infectious diseases. A key role for inflammation in EAEC pathogenesis
has been suggested by contemporary research. However, the factors and mechanisms by which EAEC
triggers the innate immune response of its host are not known with certainty. In this study, it was
found 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) of
EAEC 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 mucosal
adherence, 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-induced
PMN transepithelial migration is mediated through a conserved host signaling pathway involving the
apical release of an arachidonic acid-derived PMN chemoattractant. This lipid is generated from
arachidonic acid through a conserved 12/15-lipoxygenase pathway.

A better understanding of the inflammatory aspects of EAEC pathogenesis may potentially
contribute to the design of more targeted and effective anti-inflammatory therapies for the treatment
of diverse mucosal inflammatory conditions such as inflammatory bowel diseases (IBD).

ii

Resumé

Enteroaggregative Escherichia coli (EAEC) er en globalt fremspirende patogen, som forårsager
mave-tarm og fødevarebårne infektioner. En hovedrolle for inflammation i EAEC patogenesen er
blevet påpeget af nyere forskning. Alligevel er faktorerne og mekanismerne igennem hvilke EAEC
udløser inflammation hidtil ikke kendt med sikkerhed. I dette projekt blev der påvist, at EAEC
enteroaggregative 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 at
inducere polymorfonukleare neutrofil (PMN) transmigration og at disse pro-inflammatoriske
egenskaber er blevet konservet blandt forskellige AAF-producerende EAEC prototype stammer.
Disse resultater understreger, at AAFs ikke blot er de principielle EAEC adhesiner, som er ansvarlige
for tilhæftningen til tarmslimhinden, men at de spiller også en fremtrædende rolle i de
inflammatoriske 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, som
involverer apikal sekretion af et lipid med evnen til at tiltrække PMN. Dette lipid dannes fra frigjort
arakindonsyre via en 12/15-lipoxygenase signalvej.

En bedre forståelse af de inflammatoriske aspekter i EAEC patogenesen kunne potentielt bidrage til
udviklingen af mere målrettede og effektive anti-inflammatoriske terapier til behandling af diverse
slimhinde-associerede inflammatoriske sygdomme som kronisk inflammatorisk tarmsygdom (IBD).

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List of abbreviations
AA: Aggregative adherence
AAF: Aggregative adherence fimbriae
CD: Crohn´s disease
CF: Cystic fibrosis
COPD: Chronic obstructive pulmonary disease
DAEC: Diffusely adherent E. coli
DEC: Diarrheagenic E.coli
HG-DMEM: High Glucose - Dulbecco´s Modified Eagle Medium
EAEC: Enteroaggregative E. coli
ECM: Extracellular matrix proteins
EHEC: Enterohemorrhagic E. coli
EPEC: Enteropathogenic E. coli
ETEC: Enterotoxigenic E. coli
ExPEC: Extraintestinal pathogenic E. coli
fLMP: N-formylmethionyl-leucyl-phenylalanine
LT: Heat labile toxin
HUS: Hemolytic uremic syndrome
HXA3: Hepoxilin A3
IBD: Inflammatory bowel diseases
IL-8: Interleukin-8
LPS: Lipopolysaccharide
12/15-LOX: 12/15-lipoxygenase
MNEC: Meningitis associated E. coli
PKC: Protein kinase C
PLA2: Phospholipase A2
PMN: Polymorphonuclear neutrophil
SCID-HU-INT mice: Human intestinal xenografts in severe-combined immunodeficient mice
SEM: Scanning electron microscope
siRNA: Small interfering RNA
SPATEs: Serine protease autotransporters of Enterobacteriaceae
ST: Heat stable enterotoxin
TEER: Transepithelial electrical resistance
TJ: Tight junction
WT: Wild type

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Table of content

Preface and acknowledgments i
Summary ii
Resumé iii
List of abbreviations iv
Table of content v

INTRODUCTION

Introduction and aims 1
Escherichia coli - an overview 2
Enteroaggregative Escherichia coli (EAEC) 4
The history of EAEC discovery 4
Definition and identification 4
Epidemiology of EAEC infections 6
Clinical symptoms of EAEC infection 7

EAEC pathogenesis 8
Step 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 13
Step 2: Biofilm formation 13
Step 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 17
Animal models to study EAEC pathogenesis 18

MATERIALS AND METHODS

Bacterial strains, growth conditions and preparation 20
Growth conditions 21
Preparation 21
Cell line cultures and preparation of T84 cell monolayers 21
Media 21
Growth 21
Preparation 22
Isolation and purification of PMNs 22
In vitro model of transepithelial migration of PMNs 23
Procedure 23
Quantification 24
Inhibitor treatments of T84 cell monolayers 24
Presentation of data and statistical analysis 24

RESULTS

Part 1: Investigation of the molecular mechanisms by which EAEC triggers transepithelial
migration of PMNs in vitro

A: AAF/II play a key role in triggering EAEC 042-induced PMN transepithelial migration in
vitro, but expression of the fimbriae itself is not sufficient for triggering this inflammatory
event.
Aim and hypothesis 26
Results 27

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B: Expression of AAF/I, AAF/II and AAF/IV is indispensable for PMN transmigration induced
by other EAEC type strains
Results 29

Part 2: Host cell pathway underlying EAEC-induced transepithelial migration of PMNs

A: Arachidonic acid, a precursor for lipid-derived PMN chemoattractants, is released by PKC-
activated PLA2 during EAEC-induced inflammation
Results 32

B: An arachidonic acid-derived metabolite generated through the 12/15- LOX pathway plays a
role in regulating EAEC 042-induced PMN transepithelial migration
Results 33

C: EAEC 042-induced PMN transepithelial migration is facilitated by the MRP2 efflux
transporter
Results 36

Part 3: Additional PMN transmigration experiments

A: Hra1, an accessory EAEC 042 colonization factor, does not trigger EAEC 042-induced PMN
transmigration
Results 39

B: The probiotic E. coli strain Nissle DSM 6601 does not trigger inflammation in the PMN
transmigration model
Results 40

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DISCUSSION 42

CONCLUSIONS AND FUTURE PERSPECTIVES 49

REFERENCE LIST 51

Appendix

CD-Rom Data-Results 64
Solutions, buffers and detergents 64

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

Inflammation

INTRODUCTION

Introduction and aims

Enteroaggregative Escherichia coli (EAEC) is a large heterogeneous subgroup of
diarrheagenic E. coli (DEC). EAEC is among the most commonly isolated diarrheagenic
bacterial species with isolation rates similar to Campylobacter jejuni and higher than
Salmonella sp. (Chattaway et al., 2011; Nataro et al., 2006; Tompkins et al., 1999). EAEC
infection mainly causes persistent diarrhea in developing countries (Bhan et al., 1989c; Okeke
et 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 morbidity
and mortality (Clarke, 2001). The recent massive E. coli outbreak in Germany of hemolytic
uremic syndrome (HUS), caused by a Shiga-toxin producing E. coli strain with common
virulence properties of EAEC, highlights that highly pathogenic strains easily can emerge
through novel combinations of virulence factors of EAEC and other DEC strains (Chattaway
et al., 2011; Frank et al., 2011; Rasko et al., 2011; Scheutz et al., 2011) making it more
challenging to fight this emerging pathogen.

Besides the significant association with diarrhea, EAEC infections are generally regarded as
mildly inflammatory. EAEC infection may incite an asymptomatic colonization leading to
chronic 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 disease
mechanisms underlying intestinal inflammation caused by this organism. Several enteric
bacterial pathogens trigger inflammatory responses during infection of the intestinal mucosa
and some bacteria utilize these innate defense mechanisms for their own benefit to
successfully colonize the host. The hallmark of inflammation is neutrophil infiltration at the
site of infection. Neutrophils do, however, not only have beneficial actions, but are also
involved in the pathogenesis of many tissue-damaging inflammatory diseases. A detailed
understanding of the inflammatory mechanisms in EAEC pathogenesis may potentially

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contribute to the design of more targeted and effective anti-inflammatory therapies for the
treatment of diverse mucosal inflammatory conditions such as salmonellosis, shigellosis,
inflammatory bowel diseases (IBD), pneumonia, cystic fibrosis (CF), and chronic obstructive
pulmonary disease (COPD).

This work is focused on studying the inflammatory aspects of EAEC pathogenesis, more
specifically 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 overview

Escherichia coli belongs to the family of Enterobacteriaceae, taxonomically placed within the
gamma subdivision of the Proteobacteria phylum, and was discovered by German pediatrician
and bacteriologist Theodor Escherich in 1885. Other prominent members of this family are
Shigella and Salmonella (Madigan et al., 2006).

Phenotypically, E. coli is characterized as a Gram-negative rod-shaped facultative anaerobic
bacterium, which is non-sporulating, non-motile or motile by peritrichous flagella. This
organism 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 comprising
the E. coli core genome are shared by all strains (Lukjancenko et al., 2010). This great genetic
diversity makes E. coli challenging to study.

Human E. coli strains are broadly classified into three major groups based on clinical and
genetic criteria: 1.) commensal strains, 2.) intestinal pathogenic strains and 3.) extraintestinal
pathogenic strains (Russo & Johnson, 2000). Most E. coli strains are harmless commensals of

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the human gastrointestinal tract colonizing the infantile intestinal mucosa within a few hours
after 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 the
most abundant facultative anaerobic bacteria of the human colonic microflora. Non-pathogenic
E. coli strains rarely cause disease except in individuals with a weakened immune system or
when the gastrointestinal barrier gets disrupted (Kaper et al., 2004; Nataro & Kaper, 1998). In
these cases, a non-specific E. coli infection can lead to severe clinical complications such as
meningitis or sepsis. In contrast, pathogenic E. coli strains cause disease in otherwise healthy
individuals. This is possible because these pathogens have acquired special sets of virulence
factors, such as enterotoxins, adhesins or invasion factors, all of which can be encoded on
mobile 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 DEC
pathotypes: 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 the
third 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 classification
system is based on the distribution of the antigenic structures expressed on the surface of the
bacteria comprising lipopolysaccharide (LPS) or somatic (O) antigens, the capsular (K)
antigens, the flagellar (H) antigens and the fimbrial (F) antigens, as detected in agglutination
assays with specific rabbit antibodies (Kaper et al., 2004; Orskov et al., 1977)

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Enteroaggregative Escherichia coli (EAEC)

The history of EAEC discovery
In the early 1960´s, different serotypes of E. coli were associated with outbreaks of
diarrhea for the first time (Ewing et al., 1963). Strains belonging to these serotypes were
referred to as EPEC. In 1979, an in vitro assay based on the adhesion of the bacteria to HEp-2
cells was described by Cravioto et al. (1979). The assay identified EPEC strains binding to the
cells in a localized pattern. Later it was discovered that adherent non-EPEC strains were
associated 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) who
identified two different phenotypes among the enteroadherent strains by their adherence
pattern to HEp-2 cells; described as diffuse and aggregative adherence, respectively. This was
the first time EAEC was both described and associated with diarrheal disease as part of an
epidemiological study of pediatric diarrhea in Santiago, Chile (Nataro et al., 1987).

This finding was shortly after confirmed by three other studies linking EAEC to persistent
diarrhea among children (Bhan et al., 1989b, 1989c; Cravioto et al., 1991). EAEC
pathogenicity was initially seriously questioned, because many early studies failed to show
significant association of EAEC with disease (Echeverria et al., 1992; Gomes et al., 1989) The
role of EAEC as an etiological agent of diarrhea has later been proven more definitively
through two volunteer studies (Mathewson et al., 1986; Nataro et al., 1995) and a number of
outbreaks (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 of
diarrhea 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 pattern
in which the bacteria adhere to each other in a ‘stacked-brick’formation to HEp-2 cells and
glass cover slips (FIG. 1.). This definition, however, likely encompasses both pathogenic and
non-pathogenic strains (Nataro & Kaper, 1998; Nataro et al., 1987).

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To distinguish pathogenic from non-pathogenic strains, EAEC is further subdivided into
typical and atypical EAEC strains. “Typical” EAEC strains refer to those harboring the AggR
regulon on the pAA-virulence plasmid; these strains have been linked to diarrhea (Sarantuya
et al., 2004). In contrast, “atypical” EAEC lack the AggR regulon and can not be linked with
certainty to diarrheal disease (Nataro, 2005).

FIG. 1. EAEC adherence. This HEp-2 cell adherence assay shows the EAEC “stacked-brick” aggregative
adherence 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. EAEC
colonization is detected by isolating E. coli from stool samples and demonstrating the AA
pattern in the HEp-2 assay. However, this test is unsuitable as a diagnostic tool, as it is difficult
to 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 display
such great genetic diversity (Jenkins et al., 2006a, 2006b). Instead, DNA-based methods are
gaining ground. A DNA probe has been developed, CVD432, targeting the conserved aatA
gene on the otherwise heterogeneous pAA virulence plasmid (Baudry et al., 1990). EAEC is
currently identified at Statens Serum Institut by using PCR analysis targeting the pAA-

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encoded genes aatA, aggR and the chromosomal aaiC gene.

Epidemiology of EAEC infections

Since the first original description by Nataro et al. (1987), EAEC has attracted attention as a
worldwide emerging pathogen in many clinical settings, encompassing endemic childhood
diarrhea 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-infected
patients (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 that
EAEC 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) caused
by 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). Diarrheal
outbreaks, 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 the
most likely one. The evidence for infection sources is usually of epidemiological nature rather
than 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 non
human infection source: from milk in infant feeding bottles (Morais et al., 1997) and from
cattle (Sandhu et al., 1999). EAEC outbreaks can be epidemiologically linked to contaminated
water and food (Itoh et al., 1997; Pai et al., 1997). Risk factors for EAEC infection include
travel to developing countries, ingestion of contaminated food and water, poor hygiene, host
susceptibility, and possibly immunosuppression (e.g. HIV infection) (Huang & Dupont, 2004;

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Huang et al., 2004). Especially children are considered as a risk group, although there is little
agreement in the age-specific prevalence for EAEC infection. Some studies report that
children 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 later
childhood (<5 years). The reason for these different observations could be strain or host
heterogeneity (Okeke et al., 2000).

Clinical symptoms of EAEC infection

The clinical manifestations of EAEC infections vary considerably among individuals due to
complex 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 those
individuals having a single nucleotide polymorphism (SNP) in the interleukin (IL)-8 promoter
region (Jiang et al., 2003).

Although not all EAEC infections result in symptomatic illness (Adachi et al., 2001), most
studies show that EAEC infection results in gastrointestinal disease. The incubation period of
EAEC diarrheal illness ranges from 8 to 18 hours (Huang et al., 2004) The common clinical
features of EAEC infection are well established in outbreaks, sporadic cases and volunteer
studies. 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 usually
self-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 of
patients (Cravioto et al., 1991; Steiner et al., 1998). In children, EAEC-induced enteric
inflammation is marked by raised levels of the pro-inflammatory cytokines IL-8 and IL-1β and
fecal lactoferrin (indicative of neutrophil infiltration) (Steiner et al., 1998). In adults, fecal
lactoferrin levels are increased as well as reported by Boukenooghe et al. (Bouckenooghe et

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al., 2000). Similar inflammation can arise in patients even without clinical diarrhea, and this
seemingly 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 to
malabsorption of nutrients caused by the inability to repair the mucosal damage induced by the
inflammatory host response (Huang et al., 2004; Petri et al., 2008). These long-term effects of
EAEC infection may be even more important than the short-term morbidity associated with
diarrheal illness.

In addition, severe complications such as HUS, causing high mortality and morbidity, can
develop 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 pathogenesis

The pathogenic mechanisms underlying EAEC infections are not completely understood yet.
The reasons behind this include the great EAEC strain heterogeneity meaning that no single
virulence factor is common for all strains (Nataro, 2005), the existence of both pathogenic and
non-pathogenic strains (Elias et al., 2002) and a lack of well established in vivo animal disease
models.

EAEC pathogenesis is thought to comprise three basic steps; 1.) adherence to the intestinal
mucosa by aggregative adherence fimbriae (AAF) and other adherence factors 2.) formation of
a mucus-containing biofilm on the intestinal surface; and 3.) release of toxins and elicitation of
inflammatory responses, mucosal toxicity and intestinal secretion (Harrington et al., 2006;
Huang & Dupont, 2004; Nataro, 2005). The virulence factors involved in each of these steps
will be described in this section.

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FIG. 2. Model of EAEC pathogenesis. Stages 1 to 3 shown in yellow, illustrate the three major steps in EAEC
pathogenesis. See text for details. Modified after Harrington et al. (2006)

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Step 1: Adherence to the intestinal mucosa by aggregative adherence fimbriae (AAF)

The first challenge to potential luminal pathogens is to successfully attach to the intestinal
surface to resist the fluid flow of the luminal contents and the peristaltic movements of
intestinal contraction.

Aggregative adherence fimbriae (AAF) – principal adhesins of EAEC
EAEC adherence displays a characteristic aggregative adherence (AA) pattern, when
attached to intestinal epithelial cells in culture or to intestinal mucosa. The EAEC defining AA
pattern is mediated primarily by fimbrial adhesins termed aggregative adherence fimbriae
(AAF). The AAFs of EAEC prototype strain 042 exhibit a semi-flexible bundle-forming
structure under the scanning electron microscope (SEM) (FIG. 3.) Four variants of the AAF
major 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 organization
similar to the genes of the Dr superfamily of fimbrial adhesins, which also comprise adhesins

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of 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, which
requires a periplasmic chaperone, an outer membrane usher protein, and two surface-expressed
subunits: a major and minor pilin subunit. AAF and Dr adhesins display a high level of
conservation of the usher and chaperone genes but greater divergence of the fimbrial subunit
genes (Servin, 2005). Since each of the four known AAF variants is only present in a subset of
strains 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 has
been shown to bind to extracellular matrix components (ECM) like fibronectin, laminin and
type IV collagen. Although ECM proteins are generally localized to the basement membrane,
interaction with bacterial enteric pathogens can occur during inflammation or opening of tight
junctions (TJ). It is suggested that binding to fibronectin by EAEC may activate host cell
signaling pathways (Farfan et al., 2008).

In addition to mediating adherence to intestinal mucosa and epithelium, AAFs also facilitate
hemagglutination of human erythrocytes and play an important role in biofilm formation on
abiotic surfaces (Boisen et al., 2008; Czeczulin et al., 1997), thus stressing their apparent
multi-functionality. Importantly, AAF adhesins also seem to be involved in triggering host
inflammatory responses (Harrington et al., 2005; Strauman et al., 2010). This topic will be
described 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 key
virulence regulator in EAEC (Nataro et al., 1994). AggR is encoded on the pAA virulence
plasmid 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 genes
including dispersin (aap), the dispersin transporter system (aatPABCD) and a chromosomal
type 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 for
inversion stimulation (FIS). FIS is also involved in EAEC biofilm formation (See Biofilm

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section). To down regulate AggR expression, the repressive actions of the nucleoid-associated
protein H-NS are needed. When EAEC is grown in media resembling the nutrient-rich
gastrointestinal environment (e.g. high glucose concentration and high osmolarity) the
expression of the positive regulators is favored, whereas expression of the negative regulator
HN-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 regulon
shortly after entering the gastrointestinal tract.

Dispersin and its transporter system
Dispersin is a secreted low-molecular weight protein encoded on the EAEC virulence
plasmid pAA (Sheikh et al., 2002). Secretion of this protein requires an ATP-binding cassette
(ABC) transporter complex called Aat (designated Aat-PABCD). This protein complex
consists 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 decreasing
bacterial autoaggregation, thus allowing for a more effective adherence and aggregation. This
effect is thought to be mediated by neutralization of the negatively charged LPS on the
bacterial surface, so that positively charged AAFs can stick out from the bacterium and bind to
distant 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 a
chromosomal pheU pathogeneicity island in EAEC prototype strain 042, which is also under
the 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 be
further studied, but a screening study showed a 74% prevalence of aaiC among worldwide
EAEC isolates (Dudley et al., 2006). The aaiC gene is part of the PCR analysis used to
identify EAEC at Statens Serum Institut (SSI).

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Inflammation

Other 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) that
functions as an accessory colonization factor in autoaggregation, biofilm formation and
aggregative adherence. Another example is a galactose-specific adhesin found in EAEC strain
T7 (Grover et al., 2007).

Flagellin
Flagellin (fliC) is expressed by several enteric pathogens and commonly known for its
pro-inflammatory effects. In EAEC, flagellin has been shown to cause release of the important
pro-inflammatory chemokine IL-8 from intestinal epithelial cells (IECs) in culture, thereby
contributing to EAEC inflammation as well (Steiner et al., 1998, 2000).

Step 2: Biofilm formation

The second stage of EAEC pathogenesis is characterized by formation of a mucus-containing
biofilm by the bacteria above the intact brush boarder on the enterocytes. A study by Sheikh et
al. (2001) suggests that biofilm formation in EAEC is dependent on two proteins: Fis, a
chromosomal gene encoding a DNA-binding protein involved in growth-phase-dependent
regulation, and YafK, a secreted protein with a yet unknown role. Fis contributes to biofilm
formation via AAF/II biogenesis, activating AggR expression. Biofilm formation is induced
under conditions with high glucose concentrations and high osmolarity resembling the
intestinal environment (Sheikh et al., 2001).

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Inflammation

Step 3: Elaboration of toxins and elicitation of inflammatory responses

The third stage of EAEC pathogenesis involves release of EAEC toxins and triggering of
inflammatory responses, mucosal toxicity and intestinal secretion.

Toxins - plasmid and chromosomally encoded
Numerous pAA- or chromosomally encoded toxins and other secreted effector proteins
have been identified in EAEC strains. The pAA2 plasmid of EAEC 042 harbors the genes
encoding 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 these
four 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 SPATE
family are also found in Shigella spp., uropathogenic E .coli (UPEC) and other pathotypes of
diarrheagenic E. coli (DEC) (Henderson et al., 2004). More than 20 SPATEs with diverse
functions have by now been described.

Pet
Pet, a 108 kDa protein, exerts its cytotoxic effects by altering the host cell cytoskeleton
through proteolytic degradation of the membrane cytoskeletal protein spectrin, leading to cell
rounding, detachment and cell death. Although Pet may play a role in EAEC pathogenesis, it is
only present in a minority of strains (Czeczulin et al., 1999)

Pic
Pic, a 116 kDa protein, cleaves mucin and induces hypersecretion of mucus, thereby
contributing 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 mouse
model of gastrointestinal colonization (Harrington et al., 2009).

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Inflammation

ShET1
This protein is encoded within the pic gene, but on the complimentary strand. The role
of ShET1 in EAEC pathogenesis has not been studied yet, but in Shigella flexneri, ShET1
induces time and dose dependent intestinal secretion in a rabbit model and may thereby
contribute to the development of diarrheal illness (Fasano et al., 1997).

EAST1
EAEST1, encoded by the astA gene, is widely found among both commensal and
diarrheagenic 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 to
diarrheal disease as well (Ménard & Dubreuil, 2002).

Inflammation in EAEC pathogenesis

Bacterial pathogens continually attack the epithelial barriers of the host. Although mucosal
surfaces are generally impermeable to most pathogens, many microorganisms have developed
sophisticated strategies to breach or alter this barrier. In general, an array of bacterial
pathogens 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 interactions
commonly involving the exchange of biochemical signals, the net result of which is often the
triggering 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 inflammatory
bacterial 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 interleukin
IL-8, IL-1β, lactoferrin, leukocytes and occult blood (Cennimo et al., 2009; Greenberg et al.,
2002; Huang et al., 2004; Jiang et al., 2002).

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Inflammation

IL-8 is an important pro-inflammatory chemokine, which is basolaterally secreted from the
intestinal epithelium and directs neutrophils from the microvasculature to the immediate
subepithelial space. However, additional activation signals are necessary for full activation and
degranulation 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 were
significantly higher in the feces of patients infected with EAEC strains harboring the pAA-
plasmid borne virulence genes compared to those infected with virulence factor-negative
EAEC strains (Jiang et al., 2002).

Lactoferrin is an iron-binding glycoprotein secreted from the intestinal mucosa and the
secondary granules of neutrophils to reduce microbial adhesion and proliferation (Legrand et
al., 2005). IL-1β is secreted by mononuclear phagocytes and regulates multiple inflammatory
responses, including neutrophil granula release and chemotaxis (Maloff et al., 1989; Smith et
al., 1986).

In vitro studies have shown that IL-8 is also released from non-polarized Caco-2 intestinal
epithelial cells (IECs), when infected with EAEC 042. Later the same investigators identified
an 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 colonic
epithelial cells release IL-8 even when infected with EAEC 042 mutated in the major flagellar
subunit 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 by
Toll-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 cytokines
from epithelial and monocytic cells (Khan et al., 2004). These findings suggest that multiple
factors 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 contributes
to the functional impermeability of the epithelial barrier, which enteroinvasive pathogens have
to overcome. EAEC prototype strains have the ability to induce AAF-dependent disruption of
the epithelial barrier of T84 cell monolayers by causing delocalization of TJ proteins claudin-1

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Inflammation

and occludin. As the epithelial barrier gets disrupted, bacterial proteins or the bacteria
themselves gain access to the intestinal submucosa or to basolateral receptors like ECM
proteins (Strauman et al., 2010). At the same time a modification of TJs may also allow for
transepithelial migration of neutrophils as observed for other pathogens such as Salmonella
enterica 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 crossing
intercellular tight junctions (TJ).

Host-pathogen communication – The signaling pathway underlying pathogen-induced PMN
transmigration
Studies addressing the mechanisms underlying migration of polymorphonuclear
neutrophils (PMNs) across model intestinal epithelia have crucially contributed to a better
understanding of the molecular and cellular events underlying PMN infiltration in reponse to
enteric pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) and
Shigella flexneri (Köhler et al., 2002; McCormick et al., 1993a). Based on this knowledge, it
is possible to make assumptions about the host signaling pathway underlying the inflammatory
responses to EAEC infection.

For S. Typhimurium-induced PMN transmigration it has been shown, that this process is
triggered by a type III secretion system-translocated effector protein SipA. SipA initiates an
ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs
activation of protein kinase C α (PKC-α) (Criss et al., 2001; Silva et al., 2004). In a less well
understood process, activated PKC-α phosphorylates downstream targets finally leading to
activation of calcium-independent phospholipase A2 (iPLA2) (McCormick, 2007; Mumy et al.,
2008b). S. flexneri-induced PMN transmigration requires activation of a mitogen-activated
protein kinase (MAPK) signal transduction pathway, involving extracellular signal-regulated
kinase (ERK 1/2) and the upstream ERK kinase (MEK) (Köhler et al., 2002)

The host signaling pathways underlying PMN transmigration triggered by these two enteric
pathogens converge when arachidonic acid is released from cell membranes by iPLA 2- and
cPLA2 activity, respectively. Arachidonic acid is metabolized through a pathway involving

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Inflammation

12/15-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 (Mrsny
et al., 2004; Mumy et al., 2008a; Sutherland et al., 2000). Secretion of HXA3 on the apical
surface is facilitated by the apically restricted efflux ATP-binding cassette (ABC) protein
transporter multidrug resistance associated protein 2 (MRP2). Thereby, HXA 3 establishes a
paracellular chemotactic gradient through the tight junctional complex, guiding PMN
movement from the submucosa across the epithelium to the luminal site of infection, the final
step in PMN recruitment (Chan et al., 2004; Mrsny et al., 2004; Pazos et al., 2008)

Animal models to study EAEC pathogenesis

In order to study the pathogenic effects of EAEC toxins, animal species like rabbits and rats
have 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, rabbit
and gnotobiotic piglet models have been employed. While EAEC infection did not cause
disease 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 EAEC
colonization factors, but since these mice do not develop any characteristic pathological
lesions or signs of inflammation in response to EAEC infection, this model is not well suited
for studying EAEC disease (Harrington et al., 2009).
A model with neonatal and weaned mice used to study the malnutritional effects of EAEC
infection has been established as well and it could be demonstrated that EAEC infection in
these 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 EAEC
pathogenesis, an in vivo model of human intestinal xenografts in severe-combined
immunodeficient (SCID-HU-INT) mice has been established by Boll et al. (2011a) to study
EAEC pathogenesis and inflammation. This model is already well established for studying
innate immune responses to enteric pathogens such as Salmonella enterica serovar

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Inflammation

Typhimurium (Bertelsen et al., 2003). In this in vivo model, the xenografted tissue is of human
origin, while the immune cells are of murine origin allowing to study EAEC inflammation in
intact and morphologically fully developed human intestinal tissue (Savidge et al., 1995).

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Inflammation

MATERIALS AND METHODS

This section includes an overview of bacterial strains used in this work (TABLE 1), as well as
a detailed description of the main assay used during experimental work. In addition the
statistical 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 et
042 ΔaafA 3.4.14
locus encoding the AAF/II organelle. al., 1997)
(Mathewson
JM221 EAEC prototype strain JM221 (O92:H33) expressing AAF/I
et al., 1986)
EAEC type strain with kanamycin resistance cassette inserted into gene (Strauman et
JM221 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 et
042 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 et
042 SB/pBJ1
SspI and SphI sites of pBR322 al., 2009)
Mutaﬂor®/
Nissle DSM 6601 Probiotic non-pathogenic E. coli strain Ardeypharm,
Germany

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Inflammation

Growth conditions
Agitated overnight cultures of bacteria were grown in 3 ml Luria-Bertani (LB; BD
Difco, Becton Dickinson) broth at 37°C for at least 16 hours. Thereafter EAEC and
commensal strains were diluted 1:100 in respectively 10 ml preheated Dulbecco´s Modified
Eagle`s Medium (DMEM; D1145, Sigma) with 4500 mg glucose/L or in LB medium, and than
incubated 4 hours at 37°C under static conditions to reach exponential phase. Ampillicin (100
µg/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, washed
and 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 ml
overnight culture for PMN transepithelial migration assays.

Cell line cultures and preparation of T84 cell monolayers

Media
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 15
mM HEPES, 14 mM NaHCO3, 40 µg/ml penicillin, 80 µg/ml ampicillin, 90 µg/ml
streptomycin and 7.5% fetal calf serum.

Growth
Cell monolayers were grown on 0.33 cm2 suspended-collagen-coated permeable
polycarbonated transwell filters with pore sizes of 5 µm (Costar, Cambridge, MA). Inverted
monolayers used for PMN transmigrations were prepared as previously described (McCormick
et al., 1995; Nash et al., 1987; Parkos et al., 1992). Monolayers were utilized after 7 to 14
days once having reached a confluent, polarized, and differentiated state. This was determined
by measuring steady-state transepithelial cell resistance (TEER) using a Millicell ERS
voltohmmeter (World Precision Instruments, New Haven, CT) and all cell monolayers used

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Inflammation

reached a TEER value of at least 800 Ω per cm2.

Preparation
Prior to infection, insert-monolayers assemblies were lifted from the wells, drained of
media 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 were
then 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 PMNs

Human peripheral PMNs were purified from whole blood (anticoagulated with anticoagulation
detergent. See appendix section) collected by venipuncture from healthy human volunteers of
both 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 of
erythrocytes were removed by using a 2% gelatine sedimentation technique. Residual
erythrocytes were lysed in cold red cell lysis buffer containing NH 4Cl and removed after
centrifugation at 1200 rpm at 4°C. This technique allows for the rapid isolation of functionally
active PMN at greater than 90% purity (Cohen et al., 1983; McCormick et al., 1993b; Mrsny
et al., 2004). Finally PMNs were resuspended in modified HBSS- (without Ca2+) before being
added on cell monolayers.

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Inflammation

In vitro model of transepithelial migration of PMNs

The physiologically directed (basolateral-to-apical) PMN transepithelial migration assay using
cell 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 is
graphically 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 were
apically infected with 25 µl of bacterial suspensions for 90 minutes at 37°C and 5% CO 2 at a
multiplicity of infection (MOI) of approximately 100 bacteria per epithelial cell. After
infection, the cells were extensively washed and transferred with their apical side facing down
to 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 µl
of HBSS+ was placed on the basolateral surface of the monolayers followed by 20 µl of
prepared human PMNs (1 x 106). The monolayers were incubated at 37°C and 5% CO2 for 2½
hours after which the inserts were gently removed leaving only those PMNs in the bottom
wells 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.

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Inflammation

Quantification
PMN transmigration was quantified by assaying for the PMN azurophilic granule
marker myeloperoxidase (MPO) as previously described (Parkos et al., 1992). Briefly, PMN
myeloperoxidase (MPO) was released by adding 50 µl Triton X-100 -containing HBSS and
the pH was adjusted to 4.2 with 50 µl citrate buffer pH 4.2. Color development was assayed at
405 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 monolayers

Inhibitor 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 at
1 mM in dimethyl sulfoxide [DMSO]) in cell culture medium for 48 hours at 37°C. For
inhibitors not diluted in HBSS+, identical monolayers were incubated in the presence of
DMSO in the medium at the same concentration as during treatment to serve as vehicle
controls. Following treatment, inhibitors/media were thoroughly washed away and the cell
monolayers were equilibrated in HBSS+ for 30 minutes at 37°C prior to infection. All
inhibitors were purchased from Enzo Life Sciences.

TABLE 2. Inhibitor treatments used in this study.

Target Inhibitor Solvent Incubation period
Pan-PLA2 ONO-RS-082 DMSO 3 hours
12/15- LOX Baicalein DMSO 48 hours
+
P-glycoprotein Verapamil HBSS 2 hours

Presentation of data and statistical analysis
Since variation exists in both transepithelial resistance between groups of monolayers
(baseline resistance range 800-2.500 Ω x cm2) and between PMNs obtained from different
donors, individual experiments were performed using large numbers of cell monolayers

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Inflammation

performed in triplicate and PMNs from single blood donors on individual days. PMN isolation
was 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 experiment
performed in triplicate repeated at least three times. Data were compared by Student’s t-test
and p-values <0.05 were considered statistically significant.

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Inflammation

RESULTS

Part 1: Investigation of the molecular mechanisms by which EAEC triggers
transepithelial migration of PMNs in vitro

AAF adhesins play a diverse role in multiple aspects of EAEC pathogenesis. Not only do they
mediate adherence to mucosal surfaces, but they are also involved in biofilm formation on
abiotic surfaces (Boisen et al., 2008; Sheikh et al., 2001). Importantly AAF adhesins are
involved in mediating pro-inflammatory stimuli during EAEC infection (see Introduction
section). The first objective of this study was therefore to examine the role of AAFs in
triggering PMN transepithelial migration in vitro.

A: AAF/II play a key role in triggering EAEC 042-induced PMN transepithelial
migration in vitro but expression of the fimbriae itself is not sufficient for triggering this
inflammatory event

Aim and hypothesis
Boll et al. (2011a) demonstrated previously that EAEC prototype strain 042 promotes
transepithelial migration of PMNs. The ability to induce epithelial barrier disruption and
basolateral release of IL-8 from polarized T84 cell monolayers was shown to be confered to
the 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 PMN
migration and could show that HS carrying pAA2 induced significant PMN transmigration to
the same extent as EAEC 042, demonstrating that EAEC-specific factors encoded on pAA2
are sufficient to induce an inflammatory response. By screening of a large mutant bank of
EAEC 042 strains harboring mutations in potential pAA2-encoded virulence genes, Boll et al.
(2011a) identified the specific pAA-encoded genes involved in triggering these inflammatory
responses. Mutations in the genes encoding the AafA major pilin protein of AAF/II or
transcription factor AggR almost entirely abolished EAEC 042-induced PMN migration. In

26


Inflammation

contrast, mutations in the genes encoding EAEC virulence factors such as the AafB minor
pilin protein of AAF/II, dispersin, the toxins Pet and EAST1 or the chromosomally encoded
flagellin all induced PMN transmigration to the same extent as wild-type EAEC 042 did (Boll
et al., 2011a). These results suggest that the AAF/II organelle plays a key role in triggering
EAEC 042-induced PMN transmigration without requiring a functional minor pilin subunit of
042 AAF/II.

Here, to further address the role of AAF/II in EAEC-induced inflammation, the non-fimbriated
laboratory E. coli strain HB101 carrying a plasmid construct (pEJB02) harboring the genes
encoding 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), HB101
WT or HB101/pEJB02. The potent chemoattractant fLMP served as positive control, because
fLMP-induced PMN transepithelial migration takes place by a mechanism independent of
pathogen-induced signaling (McCormick et al., 1993b). The results in (FIG. 5.) show clearly
that HB101/pEJB02 failed to induce PMN transmigration in the in vitro model as shown for a
representative experiment.

The results indicate that HB101/pEJB02 might not express fully functional AAF/II structures
on its surface or that other accessory factors necessary for AAF/II-dependent PMN
transmigration are not present in HB101 (an afimbrial laboratory strain) in contrast to EAEC
042 (a clinical isolate).

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Inflammation

FIG. 5. HB101/pEJB02 failed to induce PMN transepithelial migration. T84 cell monolayers were apically
infected with EAEC prototype strain 042 WT, HB101 WT or HB101/pEJB02. (-); HBSS+ served as negative
control, whereas the PMN chemoattractant fLMP served as positive control for PMN transmigration. The data are
expressed as the mean ± SD of an individual experiment performed in triplicate repeated at least three times with
similar results.

B: Expression of AAF/I, AAF/II and AAF/IV is indispensable for PMN transmigration
induced by other EAEC type strains

Aim and hypothesis
After having investigated the role of AAF/II in triggering PMN infiltration, the next
objective 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 in
the 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 as
references. 55989 (AAF/III) was planned to be tested as well, but due to the multi-resistant
nature of this strain, it was not possible for E.J. Boll to construct an isogenic AAF/III mutant
of this strain.

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Inflammation

Results
As shown in (FIG. 6.), all four EAEC wild-type strains induced significant PMN
transmigration 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, 042
and C1010-00.

These findings suggest that the pro-inflammatory properties of the AAF organelles are
conserved among different AAF-producing EAEC prototype strains.

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Inflammation

FIG. 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 induced
by EAEC WT strain 042 and 042 ΔAAF/II. (C) PMN transmigration induced by EAEC WT strain C1010-00 and
its AAF/IV mutant. (D) PMN transmigration induced by EAEC WT strain C555-91 and its AAF/I mutant. The
data are expressed as the mean ± SD of an individual experiment performed in triplicate repeated at least three
times with similar results. (-), HBSS + only. fLMP served as positive control for PMN transmigration.***, p <
0.001.

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Inflammation

Part 2: Host cell pathway underlying EAEC-induced transepithelial migration of PMNs

Extensive studies in the McCormick laboratory have led to much insight into the host cell
signaling pathways underlying PMN transmigration induced by the enteric inflammatory
pathogens Salmonella Typhimurium and Shigella flexneri (see Introdution section). Using
drug inhibitors targeting specific host cell proteins, Boll et al. (2011b) has started to unravel
that EAEC-induced PMN transmigration is mediated through a similiar pathway as by these
other two enteric pathogens.

The objective of this part of the study is to characterize further the molecular mechanisms
underlying EAEC-induced PMN transepithelial migration using the in vitro model.

A: Arachidonic acid, a precursor for lipid-derived PMN chemoattractants, is released by
PKC-activated PLA2 during EAEC-induced inflammation

Aim and hypothesis
Animal and in vitro infection models have suggested an association between activated
PKC 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 the
membrane fraction of T84 cells in response to EAEC infection, implying a role for PKC-δ in
mediating the cellular response to EAEC-induced PMN transmigration.

PKC isoforms have been shown to activate PLA2 leading to the release of arachidonic acid
from 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 a
precursor in the production of eicosanoid lipids, which have either pro- or anti-inflammatory
effects (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 inhibitor
ONO-RS-082 for 3 hours prior to infection with EAEC WT strains 042 or JM221. Next, as an

31


Inflammation

alternative to the pharmacological approach, previously constructed HCT-8 cell lines were
applied harboring a plasmid construct expressing small interfering RNAs (siRNA) directed
against the mRNA molecules of PLA2G6, encoding human iPLA2, to decrease the expression of
this enzyme (Mumy et al., 2008a).

Results
When conducting the inhibitor drug study using ONO-RS-082 to inhibit PLA 2 in
EAEC WT strain 042 or JM221 infected T84 cell monolayers, experimental difficulties (data
not shown) were experienced.

In contrast, EAEC 042 infection of HCT-8 cells generating siRNA against PLA 2 mRNA lead
to a reduction in PMN transmigration by ~70% in comparison to infection of HCT-8
monolayers 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 being
less effective at forming a tight epithelial barrier than T84 cell monolayers. This inflammatory
response was, however, independent of a reduction in iPLA2 activity.

FIG. 7. iPLA2 is involved in the host signaling pathway underlying EAEC-induced PMN transepithelial
migration. HCT-8 monolayers, transfected with a vector control or a vector modified to generate siRNAs aimed
at decreasing the expression of PLA2G6 mRNA, the gene encoding human iPLA2, were used to test the
involvement of iPLA2 in the host signaling pathway underlying EAEC 042 or commensal E. coli F-18-induced
PMN transepithelial migration. The data are expressed as the mean ± SD of an individual experiment performed
in triplicate repeated at least three times with similar results. (-), HBSS+ only. * P < 0.01

32


Inflammation

This finding demonstrates a role for iPLA2-released arachidonic acid in the host signaling
pathway underlying EAEC-induced inflammation.

B: An arachidonic acid-derived metabolite generated through the 12/15- LOX pathway
plays a role in regulating EAEC 042-induced PMN transepithelial migration

Aim and Hypothesis
Among the eicosanoids derived from arachidonic acid are the potent PMN-
chemoattractants leukotriene B4 (LTB4) and hepoxilin A3 (HXA3), respectively generated
through the 5-LOX and 12/15-LOX pathway (Funk, 2001; Mrsny et al., 2004). The
involvement of either the 5-LOX or 12/15-LOX pathway in EAEC strain 042-induced
inflammation was investigated using the inhibitor-based approach by Boll et al. (2011b) T84
cells 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-LOX
inhibitor caffeic acid was found not to affect 042-induced PMN transmigration, whereas the
12/15-LOX inhibitor baicalein reduced 042-induced PMN transmigration by ~50% (Boll et
al., 2011b).

To determine whether the 12/15-LOX pathway also is involved in the inflammatory responses
induced by other EAEC strains, the baicalein drug study was carried out here, this time using
EAEC prototype strain JM221. Moreover, to support the baicalein drug study data for EAEC
042, previously constructed HCT-8 cells were applied harboring a plasmid construct, which
expresses siRNAs directed against mRNA of ALOX15, encoding human 12/15-LOX, to reduce
the activity of this enzyme (Mumy et al., 2008a).

Results
Despite repeating the experiment many times, pretreatment of T84 cell monolayers
with baicalein for 48 hours prior to infection, did not lead to any definitive conclusions
regarding an effect on JM221-induced PMN transmigration. In the representative experiment
shown below (FIG. 8.), an attenuating effect of the inhibitor is observed at the lowest
concentration (0.5 µM baicalein). However, the same effect was not observed at the higher

33


Inflammation

concentrations. Instead, an increase in the PMN transmigration rate could be observed at
higher concentrations, presumably due to the inhibitor solvents possible cytotoxic effects on
T84 cell monolayers. It was not possible to repeat the previous EAEC 042 drug study data
from Boll et al. (2011b), indicating that other technical difficulties might have been involved
in this case.

FIG. 8. PMN transepithelial migration using the 12/15-LOX inhibitor baicalein and EAEC WT strain
JM221. (A) and (B) T84 cell monolayers were pretreated with 0.5 µM, 1.0 µM or 2.0 µM of the 12/15-LOX
inhibitor 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. fLMP
served as positive control for PMN migration. Control, HBSS+ only.

34


Inflammation

Next, the RNA inference-based approach was carried out employing the before mentioned
HCT-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 infected
apically with EAEC WT strain 042 or the commensal E. coli strain F-18. The decreased
expression of 12/15-LOX strongly attenuated EAEC-induced PMN transepithelial migration
by ~70% compared to control monolayers transfected with a vector expressing unspecific
siRNA (FIG. 9.). Reduction in 12/15-LOX activity did not have any effect on the extent of the
PMN transmigration rate induced by F-18 infection.

FIG. 9. 12/15-LOX is involved in the host signaling pathway underlying EAEC-induced PMN
transepithelial migration. Monolayers of HCT-8 cells, transfected with a vector control or a vector modified to
generate siRNAs targeting mRNA of ALOX15, the gene encoding human 12/15-LOX, were used to study the
involvement of 12/15-LOX in the host signaling pathway underlying EAEC WT strain 042 or commensal E. coli
F-18 induced transepithelial PMN migration. The data are expressed as the mean ± SD of an individual
experiment performed in triplicate repeated at least three times with similar results. (-), HBSS + only. * p < 0.01

In summary, an involvement of an arachidonic acid-derived lipid in the host signaling pathway
underlying EAEC-induced inflammation was demonstrated.

35


Inflammation

C: EAEC 042-induced PMN transepithelial migration is facilitated by the MRP2 efflux
transporter

Aim and hypothesis
The apical expression of the ABC efflux transporter MRP2 is up-regulated in response
to epithelial inflammation, and in Salmonella Typhimurium-induced PMN transmigration it
serves 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 the
synthesis 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 PMN
transmigration, it was next sought to determine which role MRP2 plays in this context. Boll et
al. (2011b) showed that the MRP2 inhibitor probenecid attenuated 042-induced PMN
transmigration. To further strengthen these findings, a study was carried out here using
previously constructed HCT-8 cell monolayers generating siRNA against mRNA of MRP2, the
gene 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 a
drastic reduction by ~60% of EAEC 042-induced PMN transmigration compared to siRNA
control monolayers. F-18-induced PMN transmigration was unaffected by MRP2 down-
regulation (FIG. 10a.).

36


Inflammation

FIG. 10a. MRP2 is also involved in the host signaling pathway underlying EAEC-induced PMN
transmigration. HCT-8 cell monolayers, transfected with a vector control or a vector modified to generate
siRNAs targeting mRNA of MRP2, the gene encoding human MRP2, were used to test the involvement of MRP2
in the host signaling pathway underlying EAEC WT strain 042 or commensal E. coli F-18-induced PMN
transepithelial migration. The data are expressed as the mean ± SD of an individual experiment performed in
triplicate 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 expressed
on the membrane of T84 cells, including P-glycoprotein (Chan et al., 2004). As a control
experiment to assess for the specificity of MRP2-directed HXA3 release, a drug inhibitor

experiment targeting P-glycoprotein was therefore carried out. The extent of EAEC 042-
induced PMN transmigration was totally unaffected by addition of the P-glycoprotein inhibitor
verapamil (FIG. 10b.). Therefore, HXA3 is likely excluded as a substrate for this transporter.

37


Inflammation

FIG. 10b. PMN transepithelial migration using the P-glycoprotein inhibitor verapamil and EAEC
prototype 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 as
the mean ± SD of an individual experiment performed in triplicate repeated at least three times with similar
results. fLMP served as positive control for PMN migration. Control, HBSS+ only.

38


Inflammation

Along with the previous results by Boll et al. (2011b), these findings suggest a role for MRP2
in the host signaling pathway underlying EAEC-induced inflammation.

Part 3: Additional PMN transmigration experiments

A: Hra1, an accessory EAEC 042 colonization factor, does not trigger EAEC 042-induced
PMN transmigration

Aim and hypothesis
EAEC strain 042 harbors a gene, hra1, encoding heat-resistant agglutinin 1 (Hra1), a
hemagglutinin originally reported from a porcine enterotoxigenic E. coli strain. Hra1 has also
been found in uropathogenic E. coli strains and in the neonatal meningitis E. coli strain
RS218, in these strains called Hek (Fagan & Smith, 2007). A role for the outer membrane
protein 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 with
professor Iruka N. Okeke from the Department of Biology, Haverford College, USA. The
objective of this part of the study was to test whether deletion of the hra1 gene altered the
extent 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 as
wild-type EAEC 042. Thus, while Hra1 might have more functions besides being an accessory
colonization factor, triggering inflammation is not one of them.

39


Inflammation

FIG. 11. Hra1, an EAEC accessory colonization factor, is not involved in EAEC042-induced PMN
epithelial transmigration. T84 cell monolayers were apically infected with EAEC WT strain 042 or the 042
strains SB1 (Δhra1) or SB1/pBJ1 (hra1-complemented strain). The data are expressed as the mean ± SD of an
individual experiment performed in triplicate repeated at least three times with similar results. fLMP served as
positive control for PMN migration. (-), HBSS+ only.

B: The probiotic E. coli strain Nissle DSM 6601 does not trigger inflammation in the
PMN transmigration model

Aim and hypothesis
E. coli Nissle DSM 6601 was isolated in 1917 from a German soldier based on its
potential to protect from presumably infectious gastroenteritis (Schultz, 2008). Later this strain
was mostly used for the treatment of chronic inflammatory disorders, mainly because of its
antagonistic effects towards competing intestinal microbiota and its lack of virulence factors
(Schultz, 2008). Due to its clinical application, the pro-inflammatory potential of Nissle DSM
6601 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 any
inflammatory response and therefore it is considered as a safe therapeutic agent, which can be
used as an aid in treating infectious gastroenteritis or chronic IBDs in clinical settings.

40


Inflammation

FIG. 12. The probiotic E. coli strain Nissle DSM 6601 does not induce PMN epithelial transmigration. T84
cell monolayers were apically infected with Nissle DSM 6601. The data are expressed as the mean ± SD of an
individual experiment performed in triplicate repeated at least three times with similar results. fLMP served as
positive control for PMN migration. (-), HBSS+ only

41


Inflammation

DISCUSSION

EAEC is one of several enteric pathogens causing inflammatory diarrhea leading to high
mortality and morbidity worldwide. Many lines of evidence suggest that inflammation is an
important aspect in EAEC pathogenesis, yet the factors and exact mechanisms by which this
pathogen 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 PMN
infiltration in vitro. By focusing on AAF-producing EAEC prototype strains, it was found that
expression 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 042
AAF/II and AggR mutant strains lost the ability to trigger PMN transmigration. Moreover, the
results of this study strongly indicate that AAF-dependent pro-inflammatory properties are
conserved among EAEC strains expressing different AAF variants. This is impressive,
considering the great genetic divergence of the fimbrial subunit genes (See Introduction
section) (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 was
tested further in the PMN transmigration model. For that purpose, the pEJB02 plasmid
encoding 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 was
surprising, given that Boll et al. (2011a) has showed that constructed plasmids encoding the
other three AAF variants are sufficient to trigger PMN transmigration in the HB101
background.

Nevertheless, Boll et al. (2011a) could verify that HB101/pEJB02 exhibits adherence to T84
cell 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, but
protruding fimbriae structures could not be detected under the electron microscope (Boll et al.,
2011a).

42


Inflammation

Taken together, these facts imply that the 042 AAF/II adhesins on the surface of HB101 seem
not to form a fully functional AAF/II organelle. Alternatively, other accessory factors
necessary for AAF/II-dependent PMN transmigration are not present in the afimbrial strain
HB101 in contrast to in EAEC 042. Such a missing accessory factor could be the bacterial
surface coating protein dispersin, which mediates proper AAF protrusion from the bacterial
surface of EAEC 042 (Sheikh et al., 2002). Interestingly, an EAEC 042 dispersin mutant was
found to fully induce PMN transmigration to the same extent as the 042 WT strain, suggesting
that 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 fimbriae
protrusion is important for triggering PMN transmigration in the model.

Surprisingly, when Boll et al. (2011a) tested HB101 WT, HB101/pEJB02 and EAEC WT
strain 042 in chimeric SCID-HU-INT mice, HB101/pEJB02 carrying the AAF/II-encoding
plasmid caused PMN infiltration and tissue damage to the same extent as wild-type EAEC
042. This is in marked contrast to the results of this study using the in vitro PMN
transmigration model, presumably reflecting the different physiological parameters of each
model system.

Expression of the AggR-regulon might be favored and induced by physiological factors in the
xenografted tissue, which very much resembles the human colonic environment, leading to
expression 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 bacterial
culture prior to infection and this fundamental difference may partly explain the lack of
AAF/II expression from this particular plasmid. Other major differences between these two
models are the time course of AAF expression (4 hours vs. possibly 24 hours) and EAEC
infection (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 to
induce AAF expression in HB101/pEJB02, since the mucus serves as a nutritional source for
intestinal bacteria by providing carbohydrates (Chang et al., 2004).

43


Inflammation

The fact, that AAF/II expressed by EAEC 042 and HB101/pEJB02 clearly triggered
inflammation and caused tissue damage in SCID-HU-INT mice, is in line with findings of this
study, in that AAFs play a key role in triggering the host signaling pathway leading to PMN
transmigration across intestinal epithelia. Previous in vitro studies support the inflammatory
role of AAF by demonstrating that IL-8 is basolaterally secreted from polarized T84 cell
monolayers in response to AAF-mediated adhesion (Harrington et al., 2005).

A second objective of this study was to investigate the host signaling pathway underlying
EAEC-induced inflammation, focusing on the role that PMN transmigration plays in this
context. 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 with
a conserved signaling pathway triggered by enteric pathogens such as Salmonella
Typhimurium, Shigella flexneri, Campylobacter species but also by pulmonal pathogens like
Pseudomonas aeruginosa (Hurley et al., 2006; Mrsny et al., 2004; Mumy et al., 2008a; Pazos
et 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 PMN
transmigration. This study was able to further substantiate and validate the previous 042 drug
inhibitor studies carried out by Boll et al. (2011b), showing as well that arachidonic acid, a
precursor for the lipid-derived PMN chemoattractant HXA 3 is released by a calcium-
independent phospholipase A2 (iPLA2) from the epithelial cell membrane. Interfering with
either synthesis or secretion of this PMN chemoattractant (presumably HXA 3) by blocking
12/15-LOX or MRP2 expression resulted in a marked reduction in PMN transmigration. The
data generated from these two different approaches support this lipid´s function as a potent
PMN chemoattractant, being apically secreted by MRP2 in this conserved signaling cascade.

The advantage of the RNA interference based-approach compared to the drug inhibitor-based
approach used by Boll et al (2011b)., is that siRNAs are able to bind more specifically to their
cellular targets, whereas it is generally well known, that results from experiments using
pharmacological 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

Master Thesis Anja Sander

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