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?
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3.5
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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