Use of a proteomic approach to identify secreted proteins of Neisseria gonorrhoeae

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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
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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)
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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)
  Boswells Clap, JAMA, 21291-95,1970

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• 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

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Pathogenesis of Gonococcal Infection in Men
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TEM Analysis of Urethral Exudates from Males with
Gonorrhea
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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
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Confocal reconstruction of a human urethral
epithelial cell infected with N. gonorrhoeae
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Pedestal formation and close membrane association
between invading gonococci and apical surfaces of
epithelial cells
Primary Human Urethral epithelial cells
Urethral exudate
0.5 mm
0.5 mm
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Association/Invasion Assays
Infect primary cells with gonococci
Rinse and kill extracellular bacteria with
gentamicin (invasion) or omit gentamicin (total
association)
Lyse cervical cells to release viable intracellula
r bacteria
Plate bacteria to quantitate colony forming units
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LOS is important for invasion of urethral
epithelial cells
3.5
3.0 2.5
Invasion
2.0 1.5
P.0002
1.0 0.5
P.0002
Plt.0001
Plt.0001
0
1291
pgm
lgtE
lgtB
lgtA
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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
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Molecular basis of Gonococcal Infection in Men
Gonococcus
Urethral Epithelial Cell
Clathrin Complex
Lipooligosaccharide
Asialoglycoprotein
Galactose
Receptor
Asialoglycoprotein
Galactose
Receptor
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Pathogenesis of Gonococcal Infection in Women
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John Hunters 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
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Ultrastructural Comparison of Natural Gonococcal
Infection in Women and Men
Cervical Biopsy
(Provided by Brian Evans, MD)
Male Urethral exudate
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Endocervical Cells
Ectocervical Cells
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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 Complemen
t 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.
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Leukocyte Integrin CD11b/CD18 (CR3)
CD11b
iC3b
ICAM-1, 2
Fibrinogen
NIF
Fibrinogen
Heparin
N
C
a
Metal Binding
I Domain
Transmembrane
Region
Domains
CD18
(Putative I-like Domain)
N
C
b
Other Ligands for CR3
Transmembrane
Region
Conserved Region
Filamentous Hemagglutinin
of B. Pertussis
Leishmania

LPG, gp63,
Slide courtesy of L. Schlesinger
E. coli LPS
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Co-localization of CR3 and gonococci in patient 2
with documented Gonococcal Cervicitis
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CR3 (red) Co-localizes with Gonococci (green) in
Cervical Cells
Endocervical Cells
Ectocervical Cells
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CR3
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CR3
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Pilus
LOS
Porin
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Pilus
LOS
Porin
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Gonococcus-Induced CR3-Mediated Ruffling
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3
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Hydrolytic Activity of Phospholipase D
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Association/Invasion Assays
Infect primary cells with gonococci
Rinse and kill extracellular bacteria with
gentamicin (invasion) or omit gentamicin (total
association)
Lyse cervical cells to release viable intracellula
r bacteria
Plate bacteria to quantitate colony forming units
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PLD-deficient Gonococci are Impaired in their
Ability to Adhere to and to Invade Primary
Cervical Cells
Association ()
Invasion ()
1291DPLD
1291-WT
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Does N. gonorrhoeae PLD play a role in CR3
recruitment to the cervical cell surface?
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Does N. gonorrhoeae PLD play a role in cervical
cell signal transduction?
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Tyrosine Kinase Activation Partially Rescues PLD
Deficiency
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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