This document summarizes research on the role of the enzyme neuraminidase in the pathogenesis of adherent and invasive Escherichia coli (AIEC) in inflammatory bowel disease. The research aims to determine if AIEC strains express neuraminidase and if neuraminidase facilitates AIEC adhesion to intestinal epithelial cells. Initial results found that neuraminidase pretreatment increased AIEC adhesion. Further experiments assessed neuraminidase activity, the effect of inhibitors, and the role of the FimH adhesin in mediating adhesion. The results suggest neuraminidase and FimH play a role in AIEC pathogenesis and that neuraminidase inhibitors may help repress pathogenic bacterial adhesion and be a novel treatment for intestinal diseases involving dysbios

1. 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

2. Oh god…. E. coli

3. Discover, Understand, Treat:
Why Translational Science is Awesome
Rosemary Cullander
University of Glasgow Class of 2014
Cornell Leadership Program Scholar 2012

4. 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)

5. 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)

6. 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

7. 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)
N-Acetylneuraminic acid
Neu5Ac
(Sialic acid)
ManNAc
Pyruvate

8. Genetic Organization of E.E. coli nan-pathway of sialate catabolism
Genetic organization of coli nan pathway of sialate catabolism.
Nan (N-acyl neuraminate) operon
derepressed if
grown on sialic
acid-only medium
2ndary transporter
SIALIC ACID
N-acetylneuraminic acid
Kalivoda K A et al. J. Bacteriol. 2003;185:4806-4815
Aldolasae

9. 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/

10. Adhesion Assay- Initial Results
• Neuraminidase pretreatment facilitated adhesion
of AIEC to Caco-2 intestinal epithelial cells
Adhesion of E.coli Strains to Caco-2 cells With and Without
Neuraminidase-PBS Pre-treatment
6.5
6
5.5
5
4.5
Neu control
Neu +
4
3.5
3

11. 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?
Fig 1
Fig 2
Fig 3
5-Bromo-4-chloro-3-indolyl α-D-Nacetylneuraminic acid sodium salt

12. 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

13. 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)

14. How do AIEC adhere?
• FimB= switch
fimbriae off  on
• FimH= adhesin
• NanR- controls FimB
• NanR- nan repressor
o Derepressed if
grown on sialic
acid medium
Readout of FimH Expression:
Yeast Agglutination

15. FimH Mediated Adhesion
• Is AIEC adhesion
mediated by
FimH?
• Does neu influence
fim+ (strong agglutination) E.
coli +/- neuraminidase pretreatment of Caco-2 cells
6.5
6
5.5
5
4.5
4
3.5
3
FimH mediated
adhesion?
control
n+
fim- (no agglutination) E. coli
strains +/- neuraminidase pretreatment Caco-2 cells
6
5.5
5
control
4.5
N+
4
3.5
3
601-1
545-4
NC101 fim
41CB-2

16. 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

17. Cell Viability 2 - Inhibitor
• What effect does the inhibitor have on cells?
CCM ph6 1hr inhibitor
CCM ph6 1hr inhibitor + neu

18. 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?

19. Clinical Relevance of
Results Why do we care?
• Repress adhesion of these pathogenic
•
PARVO
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 Ulcerative
Colitis
• Could be a novel target for further microbial
disease conditions?
FOOD ALLERGIES

20. Questions?

21. 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
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22. 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.

23. Adhesion of E.coli Strains to Caco-2 cells With and Without
Neuraminidase-PBS Pre-treatment
6.5
6
5.5
5
Neu control
4.5
Neu +
4
3.5
3
•
•
•
•
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

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