Use of a proteomic approach to identify
secreted proteins of Neisseria gonorrhoeae
J. Edwards1, Gibson, B. W. 2, Scheffler, N.K.2 and M. A.Apicella1
Dept. of Microbiology, The University of Iowa1
Department of Chemistry, Buck Institute, Novato, CA2
Supported by NIAID
Biology of the Gonococcus
* Gram-negative diplococcus
* Primary site of infection is mucosa of the urogenital tract
* Exclusive human pathogen
* Putative virulence factors include:
Pilus
Lipooligosaccharide (LOS, contains oligosaccharide
vs the polysaccharide determinant of LPS
of enteric organisms)
Opacity Associated (Opa) Outer Membrane Proteins
Porin (P.IA or P.IB isotypes, stable expression)
Natural History of Gonorrhea in Men
• James Boswell (1740 - 1795) An English bon vivant and essayist
kept a detailed diary which recounted his life.
• His journals record 19 episodes of gonococcal urethritis at least
12 being “fresh” infections. (Non-immunizing infection)
• His first attack lasted 10 weeks.
• His second attack lasted four months. (? persistence of infections)
• He recounts an infection acquired from a women who had no
symptoms but had gonorrhea three years earlier. (? asymptomatic
infection in women)
• He married and because of indiscretions acquired gonorrhea
several more times. His wife by whom he had four children (and
five miscarriages) never was reported to have had symptomatic
gonorrhea. (? asymptomatic infection in women)
Boswell’s Clap,
JAMA, 212:91-95,1970
Neisseria gonorrhoeae
Human Adaptation
• No animal model except human males.
• A sustained infection cannot be initiated in chimpanzees.
• Surface of the bacteria is covered with glycolipid surrogates
of human erythrocyte antigens.
• Virulence factors undergo phase and antigenic variation at a
high rate.
• Relative few regulatory genes.
• Human immune response is a black box after thirty years of
intensive studies.
• The organisms is highly transformable – each strain is a
clone
Pathogenesis of Gonococcal Infection in
Men
TEM Analysis of Urethral Exudates from Males with Gonorrhea
Pedestal formation and intimate membrane association between
invading gonococci and apical surfaces of epithelial cells
N. Gonorrhoeae infected urethral exudate
0.5 mm
0.5 mm
Confocal reconstruction of a human urethral epithelial
cell infected with N. gonorrhoeae
Pedestal formation and close membrane association between
invading gonococci and apical surfaces of epithelial cells
Urethral exudate
Primary Human
Urethral epithelial cells
0.5 mm
0.5 mm
Association/Invasion Assays
Infect primary cells
with gonococci
Rinse and kill extracellular
bacteria with gentamicin
(invasion) or omit gentamicin
(total association)
Plate bacteria to
quantitate
colony forming units
Lyse cervical cells
to release viable
intracellular bacteria
LOS is important for invasion of urethral epithelial cells
Gal 14GlcNac 13Gal14Glc14Hep15KDO2Lipid A
3.5
GlcNAc1

(Ac)0-1
3.0
3

1
2Hep

PEA
2.5
%
Invasion
2.0
1.5
P=.0002
1.0
P<.0001
P<.0001
P=.0002
0.5
0
1291
lgtB
lgtA
lgtE
pgm
Human Asialoglycoprotein
Receptor (ASGP-R)
Hetero-oligomer of 2 subunits, H1 and H2
Cytoplasmic N-terminus, hydrophobic membrane
spanning region, extracellular C-terminus
Originally found on hepatocytes
Constitutively recycling lectin which functions
in the removal of glycoproteins from serum via
clathrin-dependent receptor-mediated endocytosis
Binds ligands with terminal galactose or
N-acetylgalactosamine residues
Co-localization of ASGP-R (green) and
gonococci (red)
4 hour
challenge
Vertical z-series of area
indicated by arrow
Co-localization of clathrin (green)
and gonococci (red)
A
B
out
in
C
A
B
C
Clathrin coat
Clathrin
triskelions
Molecular basis of Gonococcal Infection in Men
Gonococcus
Urethral Epithelial Cell
Clathrin Complex
Lipooligosaccharide
Asialoglycoprotein
Galactose
Receptor
Asialoglycoprotein
Galactose
Receptor
Pathogenesis of Gonococcal Infection in Women
John Hunter’s Treatise on Venereal
Diseases
1786
“On Gonorrhea in Women”
“It may be asked, what there is of a woman
having a gonorrhea when she is not
sensible of having one symptom of the
disease, and none appears to the surgeon
on exam?”…..
Kindly provided to me by Dr. Kevin Ault
Ultrastructural Comparison of Natural Gonococcal Infection in
Women and Men
Cervical
Biopsy
(Provided by Brian
Evans, MD)
Male
Urethral
exudate
Ectocervical Cells
Attachment
Endocervical Cells
Invasion
Complement receptor 3
Fact: Gonococcal infection of the cervical epithelia is
frequently asymptomatic (50-70% of patients)
Question: Is a receptor that is involved in down
regulation of the inflammatory response a factor in
the infective process?
Basis of Hypothesis:
Complement Receptor type 3 (CR3) down-regulates the
inflammatory response.
CR3 has been shown to be present on rectal epithelial cells.
Cervical epithelia and rectal epithelia are derived from the
same embryonic precursor.
Leukocyte Integrin CD11b/CD18 (CR3)
CD11b
iC3b
ICAM-1, 2
Fibrinogen
NIF
Fibrinogen
Heparin
N
C
a
I Domain
Metal Binding
Domains
Transmembrane
Region
CD18
N
(Putative I-like Domain)
C

Conserved Region
Transmembrane
Region
Other Ligands for CR3
Filamentous Hemagglutinin
of B. Pertussis
Leishmania: LPG, gp63,
Slide courtesy of L. Schlesinger
E. coli LPS
Co-localization of CR3 and gonococci in patient 2 with
documented Gonococcal Cervicitis
CR3 (red) Co-localizes with Gonococci (green)
in Cervical Cells
30 min infection
90 min infection
3 hour infection
Endocervical Cells
30 min infection
90 min infection
Ectocervical Cells
3 hour infection
N. gonorrhoeae (green) with Trucated LOS
Co-localize with CR3 (red) and Elicit Membrane Ruffling
90 min infection
3 h infection
Gal14GlcNac13Gal14Glc14 Hep15KDO2Lipid A
GlcNAc1

(Ac)0-1
3

1
2Hep

PEA
CR3
D
C3
fI
fB
C3
fB
P
fH
P
fB
D
C3
fI
C3
fH
CR3
D
Pilus
LOS
Porin
I
H
Pilus
LOS
Porin
Gonococcus-Induced CR3-Mediated Ruffling
Gonococcal Outer
Cell Membrane
Porin
Pilus
LOS
I-domain
iC3b
CR3
Cervical Cell
Surface and
Intracellular space
Ruffling
Gonococci
Gonococci-induced Membrane Ruffling is Expedited upon
Cervical Cell Infection with Primed Infection Inocula
Endocervical Cells
0.5 hour infection with a
4.5 hour infection medium
1 micron
Ectocervical Cells
0.5 hour infection with a
4.5 hour infection medium
2 microns
Radiolabel
Gonococci
35S-Met-Cys
Infect CycloheximideTreated Cervical Cells
With Radiolabeled Gonococci
35 S -Met-Cys
Pulse label
Collect Supernatants,
Remove Gonococci by Filtration
Separate Supernatants
By SDS-PAGE
Visualize Bacterial
Products by
Autoradiography
Gonococcal Products are Released With Cervical Cell Infection
kDa 202
Ectocervical
Cells
Endocervical
Cells
90 min 3 h
90 min 3 h
202 kDa
133
133
71
71
41.8
41.8
30.6
30.6
17.8
17.8
6.9
6.9
Minimal Gonococcal Products are Released
upon Infection of Male Urethral Cells
kDa
209
134
84
40.6
31.9
18.5
7.2
No cells
Uninfected
control
90 min infection
3 h infection
Proteomic Analysis of Gonococcal
Products Released with Cervical Cell Infection
Ectocervical
Cells
Endocervical
Cells
kDa 216
Controls
P177 FHA
129
129
PilC
91
216 kDa
91
p88
p64
43.2
33
p55 PLD
43.2
p46
PorB
33
PilE
18.8
7.7
18.8
3h
infection
90 min
infection
3h
infection
90 min
infection
Uninfected
cervical
cells
Gonococci
without
cervical
cells
7.7
Protein Candidates - ProFound
Rank
p
Protein Description
MW
kDa
1
5.4e-.01
Phospholipase D family protein
Neisseria meningitidis
59
2
1.7e-.01
Cardiolipin synthetase family protein
Neisseria meningitidis
57
3
1.1e-.03
Transposase mycoplasma mycoides
57
4
7.0e-.02
Tail fiber protein E. coli 0157
64
5
4.6e-.02
Putative integrase
62
Phospholipase D
* Superfamily: PLD (I, II)
Cardiolipin Synthase (III)
Viral protein (V)
Nuclease (VI(?),VII)
PS Synthase (IV)
Helicase (VIII)
Bacterial PLDs: < 600 AAs
Virulence Factors for Yersinia pestis and Corynebacterium
Eukaryotic PLDs: > 1000 AAs
Accessory/Regulatory Domains
Diverse Function
Phosphatidyltransferase Motif: HxKx4Dx6GG/SxN (HKD)
Heterogeneous: Sequence outside the active site
Cellular localization
Function
N. gonorrhoeae Phospholipase D
Signal Sequence
524 AA
Hydrophobic Region
Secreted
pI 9.1
HKD Active Site Motif
pH Optimum 7.5 (?)
Sequence Homology:
NmA hypothetical PLD (525 AA)
0.0
NmB Cardiolipin Synthase Family (508 AA)
0.0
E. coli hypothetical protein
411 e- 113
E. coli putative Synthase
410 e- 113
S. flexneri putative Synthase
410 e- 113
S. typhi putative Phospholipase
406 e- 112
Hydrolytic Activity of Phospholipase D
PLD
R1-O
O
O-P-O-CH2- CH2-N+-(CH3)3
O-
-
=
R2-O
PLD
Phosphatidylcholine
(PtC)
Hydrolysis
H2O
Phosphatidic Acid (PA)
Choline
Phosphatidyltransferase Activity of Phospholipase D
PLD
Phosphatidylcholine
(PtC)
Hydrolysis
H2O
Phosphatidic Acid (PA)
Choline
Transphosphatidylation
1o Alcohol (e. g. Ethanol)
Phosphatidylalcohol
PLD
(e. g. Phosphatidylethanol)
Gonococcal PLD Activity
1
0.9
Fluorescence Units
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
No EGTA
1291 WT
1291DPLD Streptomyces
20 mM EGTA
Negative Control
Exogenous Phosphatidylcholine and Ethanol Inhibit
Gonococcal Invasion of Primary Cervical Cells
Inhibition (%)
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
10
0.1
1
PC (mg/ml)
n=2
0.01
0
10
1
0.1
Ethanol (%)
n=4
0.01
Association/Invasion Assays
Infect primary cells
with gonococci
Rinse and kill extracellular
bacteria with gentamicin
(invasion) or omit gentamicin
(total association)
Plate bacteria to
quantitate
colony forming units
Lyse cervical cells
to release viable
intracellular bacteria
PLD-deficient Gonococci are Impaired in their
Ability to Adhere to and to Invade Primary Cervical Cells
Association (%)
Invasion (%)
30
3
25
2.5
20
2
15
1.5
10
1
5
0.5
0
0
1291-WT
1291DPLD
Does N. gonorrhoeae PLD
play a role in
CR3 recruitment to the
cervical cell surface?
Confocal Microscopy Suggests PLD-deficient Gonococci are
Impaired in their Ability to Elicit Increased Levels
of CR3 Surface Expression on Primary Cervical Cells
Uninfected Cervical Cells
3h infection: 1291DPLD
CR3: RED
Gonococci: Green
3h infection: 1291-WT
Wildtype, but not PLD-deficient,
Gonococci Elicit Increased Levels of
CR3 Surface Expression on Primary Cervical Cells
3
2.5
Mean
Absorbance
(490 nm)
2
1.5
Antibody: H5A4 (a-I-domain)
Dilution: 1/400
1
0.5
0
1291-WT
1291DPLD Uninfected
Control
No 1o
Antibody
Anti-Gonococcal PLD Immune Sera Inhibits PLD Activity and the
Association and Invasion of Cervical Epithelia by Gonococci
PLD Activity
Association (%)
1.6
Invasion (%)
30
3
1.2
1
0.8
0.6
0.4
0.2
0
Con 1291
WT
1291
DPLD
Ss
PLD
No Antibody
Pos. Con
W/ Pre-bleed
Neg. Con
w/ anti-PLD Immune Sera
Percent of Original Inoculum
Percent of Original Inoculum
1.4
Fluorescence Units
10
25
20
15
10
5
0
1291
WT
1291
DPLD
No Ab
2.5
2
1.5
1
0.5
0
1291
WT
1291
DPLD
With Ab
Does N. gonorrhoeae PLD
play a role in
cervical cell signal transduction?
SEM Analysis Suggests Gonococcal PLD may be Required
to Potentiate Membrane Ruffling of Primary Cervical Cells
3 hour Infection of Ectocervical Cells
1291DPLD
1291-WT
Tyrosine Kinase Activation Partially Rescues PLD Deficiency
CR3 Surface Expression
3
Invasion (%)
Percent of Original Inoculum
3
Percent of Original Inoculum
25
2
2.5
20
1.5
15
1
Con
1291
WT
No Treatment
1291
DPLD
1
0.5
5
0
2
1.5
10
0.5
0
Association (%)
30
2.5
Absorbance (490 nm)
12
1291
WT
1291
DPLD
Addition of pervanadate
(Tyr-Kinase Activation)
0
1291
WT
1291
DPLD
Addition of H2O2
(negative control)
13
Protein Kinase C Activation Rescues PLD Deficiency
CR3 Surface Expression
Association (%)
Percent of Original Inoculum
Absorbance (490 nm)
2.5
2
1.5
1
0.5
0
30
30
25
25
Percent of Original Inoculum
3
20
15
10
5
Con
1291
WT
No Treatment
1291
DPLD
Invasion (%)
0
20
15
10
5
1291
WT
Addition of PMA
(PKC Activation)
1291
DPLD
0
1291
WT
Addition of 4-a-phorbol
(negative control)
1291
DPLD
Conclusions:
* Using a proteomic approach, we have identified a N.
gonorrhoeae PLD
* Deletion of PLD impairs the association and invasion of
gonococci with PCCs
* Gonococcal PLD plays a role in recruitment of CR3 to the
cervical cell plasma membrane surface
•Antibodies to and inhibitors of PLD significantly reduce
gonococcal invasion of primary cervical epithelia
• Activation of tyrosine kinase and protein kinase C can rescue
PLD deficiency