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)
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. ChoP is a part of mammalian structures2 O P 3 N + O H C 3 P N + 2 O H P C 3 N +
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. Study objectives 1- How do they occur? 2- What happens in the host?
ChoP+
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. Antigenic variation of ChoP
Study objectives
ChoP+
ChoP-
Expression of ChoP
Antigenic variation of ChoP
1- How do they occur?
Molecular level: DNA and LOS composition
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. Background (H. influenzae)
lic1
lic1A
lic1B
lic1C
lic1D
Choline transporter
Choline kinase
CMP-ChoP transporter
Pyrophosphorylase
Choline
Phosphate
Choline
CMP
CMP
ChoP
CMP-ChoP
H. influenzae

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

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

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

20. Objective 1: Antigenic variation of ChoP expression
Strain 124P
Functional gene product
# of 5’-AACC-3’ repeats
Proposed composition
ChoP expression variant
Yes
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 27 29
Strain 738
Functional gene product
# of 5’-AACC-3’ repeats
Proposed composition
ChoP expression variant
Yes
ChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH
Positive
HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH
ChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH
ChoP, HexNAc, 4Hex, PE, 2Hep, 2Kdo, LipA-OH
Negative
ChoP, 4Hex, PE, 2Hep, 2Kdo, LipA-OH
ChoP, 3Hex, PE, 2Hep, 2Kdo, LipA-OH
In collaboration with Frank St. Michael and Dr. Andrew Cox 24 24

21. Objective 1: Antigenic variation of ChoP expression (Steric interference)
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 2 1 2 1
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
Functional gene product
# of 5’-AACC-3’ repeats
Proposed composition
ChoP expression variant
Yes
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 27 29
Strain 738
Functional gene product
# of 5’-AACC-3’ repeats
Proposed composition
ChoP expression variant
Yes
ChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH
Positive
HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH
ChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH
ChoP, HexNAc, 4Hex, PE, 2Hep, 2Kdo, LipA-OH
Negative
ChoP, 4Hex, PE, 2Hep, 2Kdo, LipA-OH
ChoP, 3Hex, PE, 2Hep, 2Kdo, LipA-OH
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 1- How do they occur? 2- What happens in the host?
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. Objective 2: Background (H. influenzae)
Colonization of the respiratory tract
ChoP+
Switching
Rodent models
ChoP-
Systemic disease

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

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

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

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

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

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

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

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

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

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

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

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

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

40. Objective 2: Role of ChoP in H
Objective 2: Role of ChoP in H. somni disease The role of Platelet Activating Factor Receptor (PAF-R)
ChoP+
ChoP-
x250
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
Objective 2: Role of ChoP in H. somni disease Adhesion to bovine turbinate cells in vitro
Mean score 1 2 3 4 5 6 7 8 9 10
738+
738-
ChoP-
ChoP+
Does ChoP assist in adhesion to respiratory epithelial cells?

42. Role of ChoP in H. somni disease Is this biofilm
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 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. 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
lic1A
lic1C
lic1D
lic1B
ChoP+
ChoP-

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?