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

• 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)

Inner core(Conserved)

Phosphorylcholine or phosphocholine (ChoP)

ChoP is a part of mammalian structures

Cell membrane

(Phospholipids)

Phosphatidylcholine

Platelet Activating Factor

Phosphorylcholine (ChoP)

ChoP is a part of mammalian structures

Phosphatidylcholine

Platelet Activating Factor

Phosphorylcholine (ChoP)

OH

CH3

O

OOH

OP

O

CH3

CH3

CH3N+

CH2CH2

OOH

OP

O

CH3

CH3

CH3N+

CH2

OOH

OP

O

CH3

CH3

CH3N+

H

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: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 negativesGram negatives

• Both cause respiratory and systemic diseaseBoth cause respiratory and systemic disease

• Similar growth and biochemical characteristicsSimilar growth and biochemical characteristics

• H. somniH. somni was initially classified as a was initially classified as a HaemophilusHaemophilus-like organism and named -like organism and named Haemophilus somnusHaemophilus somnus

• H. influenzaeH. influenzae is a good model for studying is a good model for studying H. H. somni somni and vice versaand vice versa

Antigenic (phase) variation of ChoP

• Unpredictable

• Reversible

• High rate

Study objectives

1- How do they occur?

ChoP+ChoP+ ChoP-ChoP-

2- What happens in the host?

Expression of ChoP Antigenic variation of ChoP

Molecular level: DNA and LOS composition

Virulence and pathogenesis

Study objectives

ChoP+ChoP+ ChoP-ChoP-

2- What happens in the host?

Expression of ChoP Antigenic variation of ChoP

1- How do they occur?

Molecular level: DNA and LOS composition

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?

H. influenzae

Background(H. influenzae)

lic1A lic1B lic1C lic1Dlic1

Choline

Phosphate Choline

Choline kinase

ChoP

CMP

Pyrophosphorylase

CMP-ChoP

CMP

CMP-ChoP transporter

Choline transporter

H. influenzae lic1A

(CAAT)n

H. influenzae

H. somni genome

lic1A lic1B lic1C lic1D

lic1A lic1C lic1Dlic1B

choline kinase

H. somni lic1A

Objective 1: Genetic expression of ChoPin H. somni

(AACC)n

H. somni lic1A

Choline kinase

(AACC)n

1

386

771

1156

1541

1926

2311

2696

3081

3466

minus 5'-(AACC)-3'lic1A

EcoRI 237

HindIII 1205

f1 ori

T7 prom oter

T7 term inator

6X His tag

pUC ori

pSE3

(am picillin resistance)bla

nm

ol/

min

/mg

pro

tein

0

2

4

6

8

10

12

Control pSE3 (Lic1A) CKI1 (Yeast cholinekinase)

Objective 1: Genetic expression of ChoPin H. somni (functional assay)

In collaboration with Avula Sreenivas Dr. George Carman

• Restriction endonuclease digestion

• Polymerase chain reactions

• Ligation

• Screening by sequencing

Objective 1: Genetic expression of ChoPin H. somni

lic1A lic1B lic1C lic1D

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

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:

1 2 3 4

1 2 3 4 5

222ATG 333 AAC CAA CCA ACC444 555 666 888777 TGA

1 2 3Start codon

Termination codon

On

Off

Off

On

222ATG 333 444 AAC C55 566 677 8TG788 AXXAAC CAA CCA ACC

222ATG 333 444 CAA 556 667 88T778 GAXCC5AACAAC CAA CCA ACC

222ATG 333 444

1 2 3 4 5

CCA ACC

6

555 666 888777 TGACAAAACAAC CAA CCA ACC

(AACC)n

Objective 1: Antigenic variation of ChoP expression

ChoP expression variant

Proposed composition# of 5’-AACC-3’ repeatsFunctional gene product

PositiveChoPChoP, 2Hex, 2PE, 2Hep, 2Kdo, LipA-OHYes

NegativeSial, HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH

No HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH

3Hex, 2PE, 2Hep, 2Kdo, LipA-OH

ChoP expression variant

Proposed composition# of 5’-AACC-3’ repeatsFunctional gene

product

PositiveChoPChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

Yes HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH

NegativeChoPChoP, HexNAc, 4Hex, PE, 2Hep, 2Kdo, LipA-OH

Yes

ChoPChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 4Hex, PE, 2Hep, 2Kdo, LipA-OH

HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH

Strain 738

Strain 124P

Objective 1: Antigenic variation of ChoP expression

In c

oll

abo

rati

on

wit

h F

ran

k S

t. M

ich

ael

and

Dr.

An

dre

w C

ox

27

29

24

24

Howard et. al. Journal of Clinical Microbiology, 2000, p. 4412–4419

Strain 738 LOS profile – SDS PAGE

Ch

oP

Ch

oP

+ + C

ho

PC

ho

P- -

Objective 1: Antigenic variation of ChoP expression(Steric interference)

12

3

4

ChoP

Inner core

Strain 738 ChoP+

12

3

45

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)

ChoP expression variant

Proposed composition# of 5’-AACC-3’ repeatsFunctional gene product

PositiveChoPChoP, 2Hex, 2PE, 2Hep, 2Kdo, LipA-OHYes

NegativeSial, HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH

No HexNAc, 3Hex, 2PE, 2Hep, 2Kdo, LipA-OH

3Hex, 2PE, 2Hep, 2Kdo, LipA-OH

ChoP expression variant

Proposed composition# of 5’-AACC-3’ repeatsFunctional gene

product

PositiveChoPChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

Yes HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH

NegativeChoPChoP, HexNAc, 4Hex, PE, 2Hep, 2Kdo, LipA-OH

Yes

ChoPChoP, HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 4Hex, PE, 2Hep, 2Kdo, LipA-OH

HexNAc, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 3Hex, PE, 2Hep, 2Kdo, LipA-OH

ChoPChoP, 2Hex, PE, 2Hep, 2Kdo, LipA-OH

Strain 738

Strain 124P

Objective 1: Antigenic variation of ChoP expression

In c

oll

abo

rati

on

wit

h F

ran

k S

t. M

ich

ael

and

Dr.

An

dre

w C

ox

27

29

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

Molecular level: DNA and LOS composition

Virulence and pathogenesis

Objective 2: Background(H. influenzae)

ChoP-ChoP-

ChoP+ChoP+Colonization of the

respiratory tract

Systemic disease

Rodent models

Switching

Platelet Activating

Factor Receptor (PAF-R)

C-Reactive

Protein (CRP)

Adhesion/Invasion(Respiratory colonization)

Killing(Systemic spread)

ChoP+ChoP+

ChoP-ChoP-

Objective 2: Background(H. influenzae)

H. somni ChoP?

Objective 2: Role of ChoP in H. somni disease

Objective 2: Role of ChoP in H. somni disease

ChoP+ChoP+

ChoP+ChoP+

ChoP+ChoP+

ChoP+ChoP+

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-

Objective 2: Role of ChoP in H. somni disease

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-

ChoP-ChoP-Antigenic variation

ChoP+ChoP+

ChoP+ChoP+

ChoP+ChoP+

ChoP+ChoP+

Objective 2: Role of ChoP in H. somni disease

ChoP-ChoP-

ChoP-ChoP+

Always Off(Gene knockout)

Always On

ChoP+ChoP+

Objective 2: Role of ChoP in H. somni disease

Sw

itch

in

g

Challenge ChoP-

Recover ChoP+

Challenge ChoP+

Recover ChoP-

Sw

itch

in

g

Objective 2: Role of ChoP in H. somni disease

0

10

20

30

40

50

60

70

80

90

100

Pre-challenge 24 hours post-challenge

96 hour post-challenge

%

Objective 2: Role of ChoP in H. somni disease

0

10

20

30

40

50

60

70

80

90

100

Pre-challenge 24 hours post-challenge

96 hour post-challenge

%

ChoP+

Objective 2: Role of ChoP in H. somni disease

0

10

20

30

40

50

60

70

80

90

100

Pre-challenge Post-challenge

%

Objective 2: Role of ChoP in H. somni disease

0

10

20

30

40

50

60

70

80

90

100

Pre-challenge Post-challenge

%

ChoP-

Objective 2: Role of ChoP in H. somni disease

ChoP-ChoP-Switching

ChoP+ChoP+

Objective 2: Role of ChoP in H. somni disease

Platelet Activating

Factor Receptor (PAF-R)

Platelet Activating

Factor

ChoP

ChoP+ChoP+

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

x250

ChoP-ChoP-ChoP+ChoP+

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

ChoP – ChoP – 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. somni disease Adhesion to bovine turbinate cells in vitro

Mean score

0

1

2

3

4

5

6

7

8

9

10

738+ 738-

ChoP-ChoP-ChoP+ChoP+

Does ChoP assist in adhesion to respiratory epithelial cells?

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

Molecular level: DNA and LOS composition

Virulence and pathogenesis

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-

ChoP+ChoP+

lic1A lic1C lic1Dlic1B

AcknowledgementsAdvisory 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 SandalDr. Michael HowardDr. Jane DuncanGretchen BergDr. Farzana AhmedDr. Shivakumara Siddaramappa Dr. Rajiv BalyanKristin KnightAnna ChampionJulie TuckerCheryl RyderWang Xiaoshan

Kusum JasujaGerald SniderDr. Abey BandaraDr. Manas MandalDr. Jiaxin LiJennifer Murdoch

CMMID Dr. Stephen BoyleDr. Andrea ContrerasDr. Selen OlgunKerry WaiteDenis Guenette Dr. Mohamed Naguieb SeleemKey CarlsonMary Mainous

Dr. Geoffrey SaundersDr. François ElvingerDr. Thomas Caceci

Collaborators:Dr. Chris KuckleburgDr. Charles CzuprynskiFrank St. MichaelDr. Andrew CoxAvula SreenivasDr. George Carman

Dr. Gerhardt Schurig Dr. John LeeDr. Roger AveryDr. Ludeman Eng

Technical support:Kevin WeaverChris WakleyCharles (Chip) Aardema Kathy Lowe

Samer DaghashFriends and family

Thank you

Questions?