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By Shaadi Elswaifi

The Molecular Characterization of Phosphorylcholine (ChoP) on Histophilus somni Lipooligosaccharide: Contribution of ChoP to Bacterial Virulence and Pathogenesis. By Shaadi Elswaifi. Histophilus somni (Background). Old name was Haemophilus somnus

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By Shaadi Elswaifi

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  1. The Molecular Characterization of Phosphorylcholine (ChoP) on Histophilus somni Lipooligosaccharide: Contribution of ChoP to Bacterial Virulence and Pathogenesis ByShaadi Elswaifi

  2. Histophilus somni(Background) • Old name was Haemophilus somnus • H. somni is a gram negative pleomorphic coccobacillus • Originally isolated from casses of vervious disease in cattle: Thrombotic meningoencephalitis (TME) • Currently a respiratory disease that may also cause septicemia and other systemic diseases (Shipping fever) • Causes millions of dollars in loss in the cattle industry in North America

  3. Histophilus somni(virulence factors) • Immunoglobulin binding • Survival following phagocytosis • Induction of apoptosis in endothelial cells • Biofilm formation ? • Lipooligosaccharide (LOS) • Endotoxin • Variation in composition and structure. (Decoration with ChoP and sialylation)

  4. Histophilus somni LOS Hexose units. Outer core (Variable) Phosphorylcholine or phosphocholine (ChoP) Inner core (Conserved)

  5. ChoP is a part of mammalian structures Phosphorylcholine (ChoP) Cell membrane (Phospholipids) Phosphatidylcholine Platelet Activating Factor

  6. H C 3 C H 3 + H C 3 N H C C H + 3 3 C H H C N 3 + 3 N H C 3 H C 3 O O O O P O P O O H O H O P O O H O H O H O C H 3 H C 2 H C H C 2 2 ChoP is a part of mammalian structures Phosphorylcholine (ChoP) Phosphatidylcholine Platelet Activating Factor

  7. ChoP is expressed on surface of other bacterial pathogens • Haemophilus influenzae • Streptococcus pneumoniae • Neisseria species • Actinobacillus actinomycetemcomitans • Pseudomonas aeruginosa • Mycoplasma pneumoniae

  8. ChoP on H. influenzae Contribution to virulence: • ChoP correlates with persistence in the nasopharynx in an infant rat model. • ChoP binds to Platelet Activating Factor receptor (PAF-R) and plays a role in pathogenesis. • ChoP binds to C reactive protein (CRP) and activates complement, so H. influenzae loses it to cause systemic infections. • Similarity of ChoP to mammalian cell wall structure render H. influenzae less susceptible to host antimicrobial peptides in the nasopharynx.

  9. H. somni has similarities to H. influenzae • Gram negatives • Both cause respiratory and systemic disease • Similar growth and biochemical characteristics • H. somni was initially classified as a Haemophilus-like organism and named Haemophilus somnus • H. influenzae is a good model for studying H. somni and vice versa

  10. Antigenic (phase) variation of ChoP • Unpredictable • Reversible • High rate

  11. ChoP+ Study objectives ChoP- Expression of ChoP Antigenic variation of ChoP 1- How do they occur? • 2- What happens in the host? Molecular level: DNA and LOS composition Virulence and pathogenesis

  12. ChoP+ 1- How do they occur? Molecular level: DNA and LOS composition Study objectives ChoP- Expression of ChoP Antigenic variation of ChoP • 2- What happens in the host? Virulence and pathogenesis

  13. Objective 1: Molecular characterization of ChoP expression • What genes are responsible for expression of ChoP? • How does antigenic variation of ChoP expression occur?

  14. Choline transporter Choline kinase Pyrophosphorylase CMP-ChoP transporter Background(H. influenzae) lic1 lic1A lic1B lic1C lic1D Choline Choline Phosphate CMP CMP ChoP CMP-ChoP H. influenzae

  15. lic1A lic1A (CAAT)n H. influenzae lic1A (AACC)n H. somni lic1A Objective 1: Genetic expression of ChoPin H. somni lic1B lic1C lic1D H. influenzae H. somni genome lic1B lic1D lic1C choline kinase

  16. (AACC)n H. somni lic1A Objective 1: Genetic expression of ChoPin H. somni (functional assay) • Restriction endonuclease digestion • Polymerase chain reactions • Ligation • Screening by sequencing Choline kinase • In collaboration with Avula Sreenivas Dr. George Carman

  17. Objective 1: Genetic expression of ChoPin H. somni lic1 lic1A lic1B lic1C lic1D H. somni • H. somni genes homologous to ChoP genes in H. influenzae • Strain 129Pt has an interruption in lic1A and does not express ChoP • The gene lic1A encodes a choline kinase

  18. Objective 1: Molecular characterization of ChoP expression • What genes are responsible for expression of ChoP? • How does antigenic variation of ChoP expression occur?

  19. 1 2 3 555 556 566 555 666 667 677 666 ATG ATG 778 777 ATG 777 ATG 788 222 88T 222 8TG 222 888 222 888 333 333 TGA GAX 333 AXX TGA 333 (AACC)n Objective 1: Antigenic variation of ChoP expression How the 5’-AACC-3’ repeats work as a translational switch: Termination codon Start codon 444 AAC CAA CCA ACC On 1 2 3 4 444 AAC AAC C55 CAA CCA ACC Off 1 2 3 4 5 AAC 444 AAC CAA CCA ACC CAA CC5 Off 1 2 3 4 5 6 444 AAC AAC ACC CAA CCA ACC CCA CAA On

  20. Objective 1: Antigenic variation of ChoP expression Strain 124P 27 29 Strain 738 • In collaboration with Frank St. Michael and Dr. Andrew Cox 24 24

  21. Objective 1: Antigenic variation of ChoP expression(Steric interference) ChoP + ChoP - Strain 738 LOS profile – SDS PAGE • Howard et. al. Journal of Clinical Microbiology, 2000, p. 4412–4419

  22. Objective 1: Antigenic variation of ChoP expression(Steric interference) 5 4 4 ChoP ChoP 3 3 Inner core Inner core 1 1 2 2 Strain 738 ChoP+ Strain 738 ChoP- • Howard et. al. Journal of Clinical Microbiology, 2000, p. 4412–4419

  23. Objective 1: Antigenic variation of ChoP expression Strain 124P 27 29 Strain 738 • In collaboration with Frank St. Michael and Dr. Andrew Cox 24 24

  24. Objective 1: Molecular characterization of ChoP expression • What genes are responsible for expression of ChoP? • How does antigenic variation of ChoP expression occur?

  25. Study objectives Expression of ChoP Antigenic variation of ChoP 1- How do they occur? • 2- What happens in the host? Molecular level: DNA and LOS composition Virulence and pathogenesis

  26. ChoP+ Objective 2: Background(H. influenzae) Colonization of the respiratory tract Switching Rodent models ChoP- Systemic disease

  27. ChoP+ Objective 2: Background(H. influenzae) Platelet Activating Factor Receptor (PAF-R) Adhesion/Invasion (Respiratory colonization) Killing (Systemic spread) C-Reactive Protein (CRP) ChoP-

  28. Objective 2: Role of ChoP in H. somni disease H. somniChoP?

  29. ChoP+ ChoP+ ChoP+ ChoP+ Objective 2: Role of ChoP in H. somni disease

  30. Objective 2: Role of ChoP in H. somni disease ChoP- ChoP- ChoP- ChoP- ChoP- ChoP-

  31. ChoP+ ChoP+ ChoP+ ChoP+ Objective 2: Role of ChoP in H. somni disease ChoP- ChoP- ChoP- ChoP- ChoP- Antigenic variation ChoP-

  32. ChoP+ ChoP- ChoP+ Objective 2: Role of ChoP in H. somni disease ChoP- Always On Always Off (Gene knockout)

  33. Switching Recover ChoP+ Challenge ChoP+ Objective 2: Role of ChoP in H. somni disease Challenge ChoP- Switching Recover ChoP-

  34. Objective 2: Role of ChoP in H. somni disease

  35. ChoP+ Objective 2: Role of ChoP in H. somni disease

  36. Objective 2: Role of ChoP in H. somni disease

  37. ChoP- Objective 2: Role of ChoP in H. somni disease

  38. ChoP+ Objective 2: Role of ChoP in H. somni disease Switching ChoP-

  39. ChoP+ Objective 2: Role of ChoP in H. somni diseaseThe role of Platelet Activating Factor Receptor (PAF-R) Platelet Activating Factor ChoP Platelet Activating Factor Receptor (PAF-R)

  40. Objective 2: Role of ChoP in H. somni diseaseThe role of Platelet Activating Factor Receptor (PAF-R) ChoP+ ChoP – ChoP + ChoP- x250 • In collaboration with Drs. Chris Kuckleburg and Charles Czuprynski. Accepted in Infection and Immunity, November 2006

  41. Objective 2: Role of ChoP in H. somni diseaseAdhesion to bovine turbinate cells in vitro 10 9 8 7 6 ChoP+ 5 Mean score 4 3 2 1 0 738- 738+ ChoP- Does ChoP assist in adhesion to respiratory epithelial cells?

  42. Role of ChoP in H. somni diseaseIs this biofilm ? Does ChoP play a role in biofilm? Light microscopy Electron microscopy

  43. Study objectives Expression of ChoP Antigenic variation of ChoP 1- How do they occur? • 2- What happens in the host? Molecular level: DNA and LOS composition Virulence and pathogenesis

  44. lic1A lic1B lic1D lic1C ChoP+ Summary • The lic1ABCD and genes are responsible for expression and antigenic variation of ChoP on H. somni LOS. • ChoP expression plays a role in colonization of the bovine respiratory tract, probably through binding to PAF-R • Loss of ChoP expression correlates with systemic infection • There are two mechanisms of antigenic variation of ChoP expression on H. somni LOS ChoP-

  45. Acknowledgements

  46. Thank you Questions?

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