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

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Presentation on theme: "The Molecular Characterization of Phosphorylcholine (ChoP) on Histophilus somni Lipooligosaccharide: Contribution of ChoP to Bacterial Virulence and Pathogenesis."— Presentation transcript:

1 The Molecular Characterization of Phosphorylcholine (ChoP) on Histophilus somni Lipooligosaccharide: Contribution of ChoP to Bacterial Virulence and Pathogenesis By Shaadi 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) Inner core (Conserved) Phosphorylcholine or phosphocholine (ChoP)

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

6 ChoP is a part of mammalian structures PhosphatidylcholinePlatelet Activating Factor Phosphorylcholine (ChoP) OH CH 3 O O OH O P O CH 3 CH 3 CH 3 N + CH 2 CH 2 O OH O P O CH 3 CH 3 CH 3 N + CH 2 O OH O P O CH 3 CH 3 CH 3 N + H

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 negativesGram negatives Both cause respiratory and systemic diseaseBoth cause respiratory and systemic disease Similar growth and biochemical characteristicsSimilar growth and biochemical characteristics H. somni was initially classified as a Haemophilus-like organism and named Haemophilus somnusH. 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 versaH. influenzae is a good model for studying H. somni and vice versa

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

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

12 Study objectives ChoP+ ChoP- 2- What happens in the host? Expression of ChoPAntigenic variation of ChoP 1- How do they occur? Molecular level: DNA and LOS composition 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 H. influenzae Background (H. influenzae) lic1Alic1Blic1Clic1D lic1 Choline Phosphate Choline Choline kinase ChoP CMP Pyrophosphorylase CMP-ChoP CMP CMP-ChoP transporter Choline transporter

15 H. influenzae lic1A (CAAT)n H. influenzae H. somni genome lic1Alic1Blic1Clic1D lic1A lic1C lic1Dlic1B choline kinase H. somni lic1A Objective 1: Genetic expression of ChoP in H. somni (AACC)n

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

17 Objective 1: Genetic expression of ChoP in H. somni lic1Alic1Blic1Clic1D lic1 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 How the 5-AACC-3 repeats work as a translational switch: ATG333 AAC CAACCAACC TGA 123 Start codon Termination codon On Off On 222ATG AACC TG788AXX AAC CAACCAACC 222ATG CAA T778GAX CC5 AAC CAACCAACC 222ATG CCA ACC TGA CAA AAC CAACCAACC (AACC)n Objective 1: Antigenic variation of ChoP expression

20 Functional gene product # of 5-AACC-3 repeatsProposed composition ChoP expression variant Yes ChoP ChoP, 2Hex, 2PE, 2Hep, 2Kdo, LipA-OH Positive No Sial, HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH Negative HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH Functional gene product # of 5-AACC-3 repeats Proposed composition ChoP expression variant Yes ChoP ChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH Positive HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH Yes ChoP ChoP, HexNAc, 4Hex, PE, 2Hep, 2Kdo, LipA-OH Negative ChoP ChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 4Hex, PE, 2Hep, 2Kdo, LipA-OH HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH Strain 738 Strain 124P Objective 1: Antigenic variation of ChoP expression In collaboration with Frank St. Michael and Dr. Andrew Cox

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

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

23 Functional gene product # of 5-AACC-3 repeatsProposed composition ChoP expression variant Yes ChoP ChoP, 2Hex, 2PE, 2Hep, 2Kdo, LipA-OH Positive No Sial, HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH Negative HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH Functional gene product # of 5-AACC-3 repeats Proposed composition ChoP expression variant Yes ChoP ChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH Positive HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH Yes ChoP ChoP, HexNAc, 4Hex, PE, 2Hep, 2Kdo, LipA-OH Negative ChoP ChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 4Hex, PE, 2Hep, 2Kdo, LipA-OH HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 3Hex, PE, 2Hep, 2Kdo, LipA-OH ChoP ChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH Strain 738 Strain 124P Objective 1: Antigenic variation of ChoP expression In collaboration with Frank St. Michael and Dr. Andrew Cox

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 1- How do they occur? 2- What happens in the host? Expression of ChoPAntigenic variation of ChoP Molecular level: DNA and LOS compositionVirulence and pathogenesis

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

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

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

29 ChoP +

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

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

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

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

34

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

36

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

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

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

40 x250 ChoP- ChoP+ Objective 2: Role of ChoP in H. somni disease The role of Platelet Activating Factor Receptor (PAF-R) ChoP – ChoP + 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 disease Adhesion to bovine turbinate cells in vitro Mean score ChoP- ChoP+ Does ChoP assist in adhesion to respiratory epithelial cells?

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

43 Study objectives 1- How do they occur? 2- What happens in the host? Expression of ChoPAntigenic variation of ChoP Molecular level: DNA and LOS compositionVirulence and pathogenesis

44 1.The lic1ABCD and genes are responsible for expression and antigenic variation of ChoP on H. somni LOS. 2.ChoP expression plays a role in colonization of the bovine respiratory tract, probably through binding to PAF-R 3.Loss of ChoP expression correlates with systemic infection 4.There are two mechanisms of antigenic variation of ChoP expression on H. somni LOS Summary ChoP- ChoP + lic1A lic1C lic1Dlic1B

45 Acknowledgements Kusum Jasuja Gerald Snider Dr. Abey Bandara Dr. Manas Mandal Dr. Jiaxin Li Jennifer Murdoch CMMID Dr. Stephen Boyle Dr. Andrea Contreras Dr. Selen Olgun Kerry Waite Denis Guenette Dr. Mohamed Naguieb Seleem Key Carlson Mary Mainous Advisory committee members and external examiner: Drs. Thomas Inzana, Nammalwar Sriranganathan, Ansar Ahmed, Kent Scarratt, and David Popham. Dr. Edward Swords Inzana lab members: Dr. Indra Sandal Dr. Michael Howard Dr. Jane Duncan Gretchen Berg Dr. Farzana Ahmed Dr. Shivakumara Siddaramappa Dr. Rajiv Balyan Kristin Knight Anna Champion Julie Tucker Cheryl Ryder Wang Xiaoshan Dr. Gerhardt Schurig Dr. John Lee Dr. Roger Avery Dr. Ludeman Eng Technical support: Kevin Weaver Chris Wakley Charles (Chip) Aardema Kathy Lowe Samer Daghash Friends and family Dr. Geoffrey Saunders Dr. François Elvinger Dr. Thomas Caceci Collaborators: Dr. Chris Kuckleburg Dr. Charles Czuprynski Frank St. Michael Dr. Andrew Cox Avula Sreenivas Dr. George Carman

46 Thank you Questions?


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