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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 - PowerPoint PPT Presentation
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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?