Bacterial Neuraminidase

Evaluation of the Role of Neuraminidase in the Pathogenesis of Adherent and Invasive Escherichia

Coli (AIEC)

Rosemary Cullander University of Glasgow Class of 2014

Cornell Leadership Program Scholar 2012

Oh god…. E. coli

Discover, Understand, Treat: Why Translational Science is Awesome

Rosemary Cullander

University of Glasgow Class of 2014 Cornell Leadership Program Scholar 2012

Overview of Inflammatory Bowel Disease (IBD)

- Inflammatory bowel disease (IBD) is considered a consequence of

uncontrolled intestinal inflammation in response to a combination of

environmental, microbial, and immunological factors in genetically

susceptible individuals, though its exact etiology is not entirely clear.

Crohn's Disease (CD) Ulcerative Colitis (UC)

Overview of Inflammatory Bowel Disease (IBD)

- Inflammatory bowel disease (IBD) is considered a consequence of

uncontrolled intestinal inflammation in response to a combination of

environmental, microbial, and immunological factors in genetically

susceptible individuals, though its exact etiology is not entirely clear.

Crohn's Disease (CD) Ulcerative Colitis (UC)

Role of gut microbiota in IBD

• Studies have found selective enrichment of E. coli relative to Firmicutes in patients with ileal CD, and a correlation between the severity of ileal inflammation and the density of E. coli colonization.

• Enteric flora is increasingly implicated in the pathogenesis of IBD, offering a potential therapeutic target for disease management.

• Neuraminidase is an enzyme implicated in cell-cell interaction as well as between host and pathogen

What is Neuraminidase (neu)?

• Bacterial vs Viral

• Cleaves sialic acid (C9) residues o also known as NAN/ NANA/ Neu5Ac

• Can be extracellular/ intracellular

Bacterial

Aldolase

(nanA)

Pyruvate

ManNAc N-Acetylneuraminic acid

Neu5Ac

(Sialic acid)

Genetic organization of E. coli nan pathway of sialate catabolism.

Kalivoda K A et al. J. Bacteriol. 2003;185:4806-4815

Aldolasae

Nan (N-acyl neuraminate) operon

SIALIC ACID

N-acetylneuraminic acid

derepressed if

grown on sialic

acid-only medium 2ndary transporter

Genetic Organization of E. coli nan-pathway of sialate catabolism

Aims of our Study

• Do AIEC strains express neuraminidase? o Exogenous? Endogenous?

• Does neuraminidase pre-treatment of intestinal epithelium facilitate E. coli strain adhesion? o What factor facilitates binding? FimH?

• Does this translate to clinical treatments?

http://www.ecoliblog.com/2006/05/

Adhesion Assay- Initial Results

• Neuraminidase pretreatment facilitated adhesion of AIEC to Caco-2 intestinal epithelial cells

3

3.5

4

4.5

5

5.5

6

6.5

Adhesion of E.coli Strains to Caco-2 cells With and Without Neuraminidase-PBS Pre-treatment

Neu control

Neu +

Chromogenic Assessment of Neuraminidase Activity

• Is the enzyme working?

• Is activity affected by pH?

• What concentration of inhibitor is needed?

• Is there exogenous/ endogenous activity of neuramnidase by the bug?

5-Bromo-4-chloro-3-indolyl α-D-N-

acetylneuraminic acid sodium salt

Fig 1

Fig 2

Fig 3

Cell Viability- Live/Dead Assay CCM (no serum) pH 6 control

CCM pH 6 1hr neuraminidase treatement

PBS pH 6 control

PBS pH 6 1hr neuraminidase treatement

Fluorescent in situ hybridization (FISH)

• 7-day maturation of Caco-2 intestinal epithelial cells

• Infected with 1x10^8 Bacteria/ well

• MOI~ 200 (#of bacteria/ #of Caco-2 Cells)

Infection after 1 hr Control Treatment (CCM) Infection after 1hr neuraminidase treatment (CCM)

How do AIEC adhere?

• FimB= switch fimbriae off on

• FimH= adhesin

• NanR- controls FimB

• NanR- nan repressor

oDerepressed if grown on sialic acid medium

Readout of FimH Expression:

Yeast Agglutination

FimH Mediated Adhesion

• Is AIEC adhesion mediated by FimH?

• Does neu influence FimH mediated adhesion?

3

3.5

4

4.5

5

5.5

6

601-1 545-4 NC101 fim 41CB-2

fim- (no agglutination) E. coli strains +/- neuraminidase pre-

treatment Caco-2 cells

control

N+

33.5

44.5

55.5

66.5

fim+ (strong agglutination) E. coli +/- neuraminidase pre-treatment of Caco-2 cells

control

n+

Activity of neu inhibitors

• Block function of neuraminidase protein • Sialic acid residues remain intact

• difficult to get stocks of viral neuraminidase inhibitors for experimental use

• Competative Inhibitor

N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid

Cell Viability 2 - Inhibitor

• What effect does the inhibitor have on cells?

CCM ph6 1hr inhibitor CCM ph6 1hr inhibitor + neu

Clinical Relevance of Results • Repress adhesion of these pathogenic

bacteria

• Decrease microbe interaction in adhesion o Neu produced by E. coli

o Neu produced by other intestinal flora

Do these bacteria “potentiate” E. coli?

o Reduce E. coli energy source

• Could be a novel target for further microbial disease conditions?

Clinical Relevance of Results • Repress adhesion of these pathogenic

bacteria

• Decrease microbe interaction in adhesion o Neu produced by E. coli

o Neu produced by other intestinal flora

Do these bacteria “potentiate” E. coli?

o Reduce E. coli energy source

• Could be a novel target for further microbial disease conditions?

PARVO

FOOD ALLERGIES

Why do we care?

Ulcerative Colitis

Questions?

Acknowledgements Simpson Lab

• Kenneth W Simpson, Mark Rishniw

• Deepali Herlekar, Belgin Dogan, Suzanne Klaessig

• Francis Davis, Alison Manchester, Brendan Chandler, VisheshKothari, Alyssa Chandler

Leadership Program

• Dr Fraser, Dr McGregor, Dr Parker

Mentors

• Dr Marshall, Dr Jonsson, Dr Evans

Further (Fun!) Reading

• Remission of histiocytic ulcerative colitis in Boxer dogs correlates with eradication of invasive

intramucosal Escherichia coli J Vet Intern Med. 2009 Sep-Oct;23(5):964-9. Epub 2009 Aug 11.

• Granulomatous colitis of boxer dogs Vet Clin North Am Small Anim Pract. 2011 Mar;41(2):433-45.

• Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxerdogs.

Infect Immun. 2006 Aug;74(8):4778-92.

• Adherent-invasive Escherichia coli and Crohn's disease. Curr Opin Gastroenterol. 2007

Jan;23(1):16-20.

• Adherent-invasive Escherichia coli in inflammatory bowel disease. Inflamm Bowel Dis. 2007

Oct;13(10):1277-83.

• Antimicrobial resistance impacts clinical outcome of granulomatouscolitis in boxer dogs. J Vet

Intern Med. 2010 Jul-Aug;24(4):819-24. Epub 2010 May 11.

• Healthy: 470-1, 601-1

• Colonic crohns disease: 524-2, 584-1, 355-5, 355-1

• Illeal crohns disease: all the rest…

• Dog: I-9, T-75

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3.5

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4.5

5

5.5

6

6.5

Adhesion of E.coli Strains to Caco-2 cells With and Without Neuraminidase-PBS Pre-treatment

Neu control

Neu +

Sialic acid utilization by bacterial pathogens- E. Severi et al http://mic.sgmjournals.org/content/153/9/2817.full