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

Anja Sander

Prof. Karen A. Krogfelt Statens Serum Institut

Prof. Beth A. McCormick UMASS Medical School, USA

PhD Erik Juncker Boll Statens Serum Institut

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EAEC and its pathogenesis

Results

Methods

Conclusions

Future perspectives

Outline

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Enteroaggregative Escherichia coli (EAEC)

From Huang et al. (2006)

HEp-2 cell

One of several diarrheagenic E. coli pathotypes

Does not secrete the heat labile (LT) or heat stable (ST) toxins of ETEC

Characteristic autoaggregative adherence pattern

Very heterogeneous

Chronic inflammation of the guts in the absence of diarrhea

German outbreak

EAEC and its pathogenesis

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EAEC pathogenesis

Modified after Harrington et al. (2006)

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EAEC and inflammation – what is known?

Epidemiological studies:

Increased levels of fecal inflammatory markers such as IL-8, lactoferrin and leukocytes in response to EAEC infection (Greenberg et al., 2002, Steiner et al., 1998)

In vitro studies:

EAEC aggregative adherence fimbriae (AAF) cause basolateral release of IL-8 from intestinal epithelial cells (Harrington et al. 2005)

EAEC disrupts the intestinal epithelial barrier by modifying and delocalizing tight junctions proteins (Strauman et al., 2010)

Liévin-Le Moal and Servin, 2006

IL-8

Tight junction

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Principal adhesins

Semiflexible bundle-forming structure

Four variants of the major AAF fimbrial subunits identified based on their sequence

Genetically very diverse

Encoded on virulence plasmids

No definitive receptors identified yet

Sheik et al. (2002)

AAF

Boisen et al. 2008

Aggregative adherence fimbriae (AAF)

AAF

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Inflammatory properties of AAFs

pEJB01 (AAF/I) / pEJB03 (AAF/III)

plac

rep (pMB1) Bla (AmpR)

lacZ

Gene A

Gene C

Gene D

Gene B Plasmid encoding the gene cluster for AAF/I and AAF/III of EAEC JM221 and 55898

Transferred into the afimbrial strain E. coli HB101, these plasmids encoding AAF/I and AFF/III induce PMN transmigration in vitro (Boll et al., 2011)

From Erik Juncker Boll

Major and minor fimbrial subunit genes

Accessory genes

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Benefits (fighting pathogens):

A protective attempt by the organism to remove invading pathogens

Phagocytosis and respiratory burst

Cost:

Unspecific response

Tissue damage and loss of epithelial barrier function

Neutrophil recruitment is a key event in the development of inflammatory diseases such as inflammatory bowel disease (IBD)

Polymorphonuclear neutrophil

The role of neutrophils in inflammation

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Pathogen-induced transepithelial migration of neutrophils

Neutrophil migration

LUMEN

BASOLATERAL

BloodTight junction

1 Host signaling response

2IL-8

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Bacteria

PMNs

Virulence factors trigger

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In vitro assay: Transepithelial migration of polymorphonuclear neutrophils (PMNs)

Colorimetric assay: Myeloperoxidase

Transwell inserts

Human colon cancer-derived epithelial cells

Methods

96-well microtiter plate

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Results

Part I and II: Pro-inflammatory properties of AFF

Inhibition studies of the signaling pathway underlying EAEC-induced PMN migration

Experimental controls:

Positive control: fLMP (induces PMN migration pathogen-independent)

Negative control: buffer HBSS+

Data presentation:

All 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

P-values <0.05, illustrated as * were considered statistically significant

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Example results for PMN migration assay

Measures the number of transmigrated PMNs, reflecting the inflammatory response induced by EAEC infection

Positive control

EAEC strains tested

Negative control

Mean ± St.Dev.

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Expression of AAFs is required for triggering PMN migration

042: EAEC prototype strain expressing AAF/II (Nataro et al. 1985)

JM221: EAEC prototype strain expressing AAF/I (Mathewson et al. 1986)

C555-91: UTI isolate expressing AAF/I (Olesen et al. 1994)

AIM: Do other AAF variants than 042 AAF/II have pro-inflammatory properties and trigger PMN migration?

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Expression of AAFs is required for triggering PMN migration

042: EAEC prototype strain expressing AAF/II

55989: EAEC prototype strain expressing AAF/III – not constructed

C1010-00: EAEC prototype strain expressing AAF/IV (Olesen et al. 1994)

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AAF/II biogenesis genes encoded on pEJB02

Region 1

aggR: transcription activator

aafA: major fimbrial subunit

aafD: accessory gene

Region 2

AafC: accessory gene

AafB: minor fimbrial subunit

From Erik Juncker Boll

Aim: To directly characterize the pro-inflammatory properties of plasmid-encoded AAFs

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Expression of plasmid-encoded AAF required for triggering PMN migration

In a model of human intestinal xenografts in immuno-deficient mice,HB101/pEJB02 caused PMN migration and tissue damage to the same extent as 042 (Boll et al. 2011)

HB101: afimbrial E. coli laboratory strain

Plasmid constructs from Boll et al. 2011 and Boisen et al. 2008

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Hypothesized model of EAEC-induced inflammation – Based on drug inhibitor studies

Results part 2: Signaling pathwayAIM: Do other EAEC's than 042 trigger the signaling pathway

underlying EAEC induced inflammation?

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The inhibitor baicalein blocks 12/15-LOX activity

Drug inhibitor study to characterize the signaling pathway underlying EAEC-induced PMN migration

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Using an RNA interference-based approach to test the hypothesized model of EAEC-induced inflammation

Key enzymes

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Transfection

Vector expressing

siRNA

The HCT-8 cell line is a transformed polarized human colon cancer-derived epithelial cell line

High transfection efficiency in contrast to T84 cell lines

Phospholipase A2 (PLA2)

mRNAsiRNA

RNA-inference is an alternative approach to classical drug inhibitor studies

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siRNA generated against mRNA of PLA2G6 reduces the

expression of this enzyme

iPLA2 is involved in the host signaling pathway underlying EAEC 042-induced PMN transepithelial migration

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12/15-LOX and MRP2 are involved in the host signaling pathway underlying EAEC-induced PMN migration

siRNA generated against mRNA of ALOX15 reduces the expression of these enzymessiRNA generated against mRNA of MRP2 reduces the expression of these enzymes

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Part I: AAF fimbriae of EAEC play a key role in

triggering EAEC-induced PMN migration

The inflammatory property of AAFs are conserved among different AAF-producing EAEC strains

Conclusions

Part II:

EAEC 042-induced epithelial inflammation is mediated through a conserved 12/15-lipoxygenase host cell signaling pathway

An arachidonic acid-derived neutrophil chemoattractant, presumably HXA3, is apically secreted into the intestinal lumen upon EAEC infection

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Further characterization of the role of AAFs in inflammation Identification of AAF receptors Identification of missing steps in the signaling pathway underlying EAEC-

induced inflammation

Investigate how EAEC potentially benefits from triggering this host innate immune response

S.Typhimurium modifies tight junction protein complexes to gain access to the basolateral compartment

Future perspectives in studying EAEC pathogenesis

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Statens Serum Institut (SSI) Professor Karen A. Krogfelt

PhD Erik Juncker Boll

McCormick Lab, UMASS Professor Beth A. McCormick

Everyone else in the lab

Acknowledgements