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 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

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)

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

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

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

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

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.

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

Antigenic (phase) variation of ChoP Unpredictable Reversible High rate

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Objective 2: Role of ChoP in H. somni disease

Objective 2: Role of ChoP in H. somni disease

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

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

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

Objective 2: Role of ChoP in H. somni disease

Objective 2: Role of ChoP in H. somni disease

Objective 2: Role of ChoP in H. somni disease

Objective 2: Role of ChoP in H. somni disease

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

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+

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

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?

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

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

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-

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

Thank you Questions?