Bacterial Neuraminidase
Evaluation of the Role of Neuraminidase in the
Pathogenesis of Adherent and Invasive Escherichia
C...
Oh god…. E. coli
Discover, Understand, Treat:
Why Translational Science is Awesome
Rosemary Cullander
University of Glasgow Class of 2014
C...
Overview of Inflammatory Bowel
Disease (IBD)
- Inflammatory bowel disease (IBD) is considered a consequence of
uncontrolle...
Overview of Inflammatory Bowel
Disease (IBD)
- Inflammatory bowel disease (IBD) is considered a consequence of
uncontrolle...
Role of gut microbiota in IBD
•

•
•

Studies have found selective enrichment of E. coli
relative to Firmicutes in patient...
What is Neuraminidase (neu)?

•
•
•

Bacterial vs Viral
Cleaves sialic acid (C9) residues
o

also known as NAN/ NANA/ Neu5...
Genetic Organization of E.E. coli nan-pathway of sialate catabolism
Genetic organization of coli nan pathway of sialate ca...
Aims of our Study

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

• Does neuraminidase pre-treatment o...
Adhesion Assay- Initial Results
• Neuraminidase pretreatment facilitated adhesion
of AIEC to Caco-2 intestinal epithelial ...
Chromogenic Assessment of
Neuraminidase Activity
•
•
•
•

Is the enzyme working?
Is activity affected by
pH?
What concentr...
Cell Viability- Live/Dead Assay
CCM (no serum) pH 6 control

CCM pH 6 1hr neuraminidase treatement

PBS pH 6 control

PBS ...
Fluorescent in situ
hybridization (FISH)

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

cells
Infected with 1x10...
How do AIEC adhere?
• FimB= switch

fimbriae off  on

• FimH= adhesin
• NanR- controls FimB
• NanR- nan repressor
o Derep...
FimH Mediated Adhesion

• Is AIEC adhesion
mediated by
FimH?

• Does neu influence

fim+ (strong agglutination) E.
coli +/...
Activity of neu inhibitors

•
•
•

Block function of neuraminidase protein

•

Sialic acid residues remain intact

difficu...
Cell Viability 2 - Inhibitor

• What effect does the inhibitor have on cells?
CCM ph6 1hr inhibitor

CCM ph6 1hr inhibitor...
Clinical Relevance of
Results
• Repress adhesion of these pathogenic

•

bacteria
Decrease microbe interaction in adhesion...
Clinical Relevance of
Results Why do we care?
• Repress adhesion of these pathogenic

•

PARVO

bacteria
Decrease microbe ...
Questions?
Acknowledgements
Simpson Lab
Kenneth W Simpson, Mark Rishniw
Deepali Herlekar, Belgin Dogan, Suzanne
Klaessig
Francis Davi...
Further (Fun!) Reading
•

Remission of histiocytic ulcerative colitis in Boxer dogs correlates with eradication of invasiv...
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....
Sialic acid utilization by bacterial pathogens- E. Severi et al

http://mic.sgmjournals.org/content/153/9/2817.full
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Governors Prize 2012- Neurominidase

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Governors Prize 2012- Neurominidase

  1. 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. 2. Oh god…. E. coli
  3. 3. Discover, Understand, Treat: Why Translational Science is Awesome Rosemary Cullander University of Glasgow Class of 2014 Cornell Leadership Program Scholar 2012
  4. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 17. Cell Viability 2 - Inhibitor • What effect does the inhibitor have on cells? CCM ph6 1hr inhibitor CCM ph6 1hr inhibitor + neu
  18. 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. 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. 20. Questions?
  21. 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 • • • • •
  22. 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. 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. 24. Sialic acid utilization by bacterial pathogens- E. Severi et al http://mic.sgmjournals.org/content/153/9/2817.full

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