Transcript the presentation - IUSTI Marrakesh 2016 :: Home

SYPHILIS PATHOGENESIS
Understanding the hostpathogen interaction for
the stealth pathogen
Treponema pallidum
Presented by
Professor David A. Lewis
Western Sydney Sexual Health Centre
Sydney Medical School-Westmead
The University of Sydney
Page 1
Introduction
Treponema pallidum subsp. pallidum (Tp)
– Member of the Spirochaetaceae family
– Gram negative – poor dyeability
– Can persist in vivo for decades
– Strains may be propagated in rabbits’ testicles
– May be cultured transiently in vitro in rabbit epithelial cells
– Genetic manipulation is not yet possible due to lack of robust
in vitro culture systems
– Paucity of immunological reagents for work in rabbits
Studying pathogenesis and the immune response is difficult
The University of Sydney
Ho & Lukehart, J. Clin. Invest. 2011;121(12):4584-4592
Page 2
T. pallidum Antigenic Structure
-
The University of Sydney
6-15 μm long
0.2 μm in diameter
Spiral shaped
Corkscrew motility
Page 3
T. pallidum Genome
– Tp genome is small (1.14 Mb) encoding 1,041 putative proteins
The University of Sydney
Fraser et al., Science 1998,281(5375):375-388
Page 4
Limited Metabolic Capability
– Lack of metabolic capability – Tp relies on glycolysis for energy
– Tp lacks TCA cycle enzymes and electron transport chain
– Amino acid and fatty acid synthesis pathways lacking
– Essential macromolecules obtained from host with subsequent use
of interconversion pathways
– Homologues for amino acid transporters, e.g. TpN32 lipoprotein
associated methionine transport system
– Several cation-specific transporters identified, e.g. tro operon
encoded ABC transporter system (zinc & manganese)
– Homologues for several sugar transporters also identified
The University of Sydney
LaFond & Lukehart, Clin. Micro. Rev. 2006;19(1):29-49
Page 5
Growth Characteristics
– Infectious capability lost within a few hours outside human host
– Remain viable in whole blood or plasma stored at 4oC for up to
24 hours
– Tp may remain motile for 3-6 days if suspended in appropriate
fluids containing reducing agents
– In vitro propagation only lasts for about seven generations
– Microaerophilic organism, surviving best at 1%-4% O2
– Doubling time is slow (30-33 hours) - reflects low energy status
– Sensitive to reactive oxygen species (catalase/oxidase absent)
– Poor stress response to heat – heat lability of some Tp enzymes
The University of Sydney
LaFond & Lukehart, Clin. Micro. Rev. 2006;19(1):29-49
Page 6
Vent Diseases (Rabbit Syphilis)
– Treponema paraluis cuniculi
– 3-6 weeks incubation
– Very contagious
– Genital area inflamed with ulcers
and blisters
– Facial sores in 15% rabbits
– Diagnosis made with darkground
microscopy and/or serology
– Treatment with procaine penicillin
or benzathine penicillin
The University of Sydney
http://www.raising-rabbits.com/rabbit-syphilis.html
Page 7
Experimental T. pallidum Infections in Rabbits
The University of Sydney
Baeslack, J. Infect. Dis. 1913;12(1):55-67
Page 8
T. pallidum Lacks Several Classical Virulence Factors
– Tp lacks endotoxin found in other Gram
negative bacteria
– There are no Tp homologues for type III
secretion systems
– Tp only produces cytotoxic effects
except in extremely high numbers
– Putative haemolysins identified in the Tp
genome but their function remains
unknown
Despite this, Tp causes a variety of disease
manifestations in many tissue types
Primary chancre
The University of Sydney
LaFond & Lukehart, Clin. Micro. Rev. 2006;19(1):29-49
Page 9
Invasive Nature of T. pallidum
– Tp rapidly invades a variety of tissues and organs
– Tp enters bloodstream of rabbits within minutes of intratesticular
or intradermal experimental inoculation
– Tp may be visualized in rabbit lymph nodes, brain, aqueous
humour and CSF from 18 hours post-intratesticular inoculation
– Organisms can persist in tissues during latent infections – lymph
node extracts from infected rabbits can infect naïve ones
– Observations in rabbits are mirrored in humans
– Tp frequently found in CSF of patients with early syphilis
– Tp found in gummatous lesions after decades by a variety of
methods (silver stain, immunofluorescence, PCR)
The University of Sydney
Cumberland & Turner, Am. J. Syphilis 1949;33:201-212; Kampmeier, Med. Clin. N. Am. 1964;48:667-697;
Zoechling et al., Br. J. Dermatol. 1997;136:683-686
Page 10
T. pallidum Attachment I
– Tp can attach to many cell types
(e.g. epithelial, fibroblasts,
endothelial cells)
– Tp can adhere to isolated
capillaries, kidney and abdominal
wall tissues in vitro
10 μm
5 μm
– Non-pathogenic T. phagedenis, T.
refringens and T. vincentii do not
adhere to cultured cells
Scanning EM of Tp incubated with capillaries.
A: 30 mins, B: 24 hours
Quist et al., Br. J. Vener. Dis. 1983;59:11-20
The University of Sydney
Quist et al., Br. J. Vener. Dis. 1983;59:11-20; FitzGerald et al., Infect. Immun.1977;18:467-478
Page 11
T. pallidum Attachment II
– Bacterial adhesins likely migrate
through the semi-fluid outer membrane
towards the tip to result in a ‘capping’
phenomenon
Scanning electron micrograph of the
spirochaete Treponema pallidum
attached to testicular cell membranes
– Integrin may act as host receptors and
binding may be mediated through
ECM components
– Attachment does not occur with heat-killed Tp or Tp rendered
non-motile by 23hr incubation at 37oC
– Tp binding is blocked by immune rabbit or human sera
LaFond & Lukehart, Clin. Micro. Rev. 2006;19(1):29-49;
http://www.britannica.com/science/Treponema-pallidum/images-videos/Scanning-electron-micrograph-of-the-spirochete-Treponema-pallidum-attached-to/7511
The University of Sydney
Page 12
T. pallidum Binding to Extracellular Matrix Components
– Tp attaches to fibronectin, laminin and collagen I – binding
blocked by antibodies against each component
– ECM-binding adhesins identified from Tp genome analysis
– Both Tp0155 and Tp0483 bind to matrix fibronectin (in tissues)
– Tp0483 also binds soluble fibronectin (in blood)
– Tp0751 binds to laminin – inhibited by anti-Tp0751 Abs
Host cell membrane
Fibronectin structure
The University of Sydney
FitzGerald et al., Br. J. Vener. Dis. 1984;60:357-363; Cameron et al., J. Bacteriol. 2004;186:7019-7022;
Cameron et al., Infect. Immun. 2003;71:2525-2533
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Motility
– Flagellar shaft made up of
several filament proteins
– FlaB1-B3 form the core
– FlaA subunits compose the
sheath
– Although immune responses to flagella are induced during
natural infection, immunization with flagellar subunits from Tp
and T. phagedenis does not provide protection
– Tp responds to nutrient gradients via transmembrane and
cytoplasmic chemotaxis proteins, e.g. crossing endothelial barrier
The University of Sydney
LaFond & Lukehart, Clin. Micro. Rev. 2006;19(1):29-49
Page 14
Inflammation I: MMP-1 & Neutrophils
– Tp induces matrix metalloproteinase-1(MMP-1) in dermal cells
– MMP-1 breaks down collagen and aids tissue penetration
– PMNs are the first cells to
infiltrate the infection site
although unable to control
disease process
– TpN17 and TpN47 dermal
injections can also produce
transient PMN infiltration
The University of Sydney
Chung et al., Acta Derm. Venereol. 2002;82:174-178; Riley et al., J. Infect. Dis. 1992;165:484-493
Page 15
Inflammation II: Dendritic Cells
– Dendritic cells (DCs) are stimulated by Tp lipopeptides through the
TLR2 pathway
– Immature DCs phagocytose Tp – express CD83 when mature
– Tp-driven mature DCs express cytokines (TNF-α, IL1-β, IL-6, IL-12)
– Same cytokine profile produced when
immature DCs are stimulated by the lipid
portion of TpN47
– Administration of synthetic TpN17 and TpN47
to human volunteers resulted in CD83+ DCs in
blister fluid induced by suction
– CD83+ DCs also found in secondary syphilis
skin lesions
The University of Sydney
Bouis et al., Infect. Immun. 2001;69:518-528; Sellati et al., J. Immunol. 2001;166:4131-4140
Page 16
Inflammation III: Macrophages
– Macrophages also produce TNF-α
in response to intact/sonicated Tp
or purified Tp lipopeptides
– As with maturing DCs, macrophages
express various cytokines (e.g. TNFα, IL1-β) after stimulation with
TpN15, TpN17 and TpN38
– TpN47 stimulates macrophages to
produce the T-cell chemo-attractant
cytokines MIP-1α and MIP-1β
The University of Sydney
Sellati et al., J. Immunol. 1999;163:2049-2056; Sellati et al., J. Infect. Dis. 2000;181:283-293
Page 17
Inflammation IV: Delayed-Type Hypersensitivity
– DCs present antigens to T cells in the lymph nodes
– Stimulated T cells migrate to the site of infection
– In a rabbit model, T cells were present at the site of infection by
3 days post-inoculation, macrophages only arrive at days 6-10
and the number of viable Tp disappears sharply at 13-17 days.
Delayed-type hypersensitivity is the likely mode of clearance
– T cells and macrophages are found in rabbit dermal lesions as
well as 1o/2o syphilis lesions
– Macrophages are activated and T cell proliferation is stimulated
by local production of IFN-γ and IL-2 – mRNA for these cytokines
is detected in 1o/2o syphilis lesions
The University of Sydney
Lukehart et al., J. Immunol. 1980;124:461-467; Sell et al., J. Investig. Dermatol. 1980;75:470-475;
Engelkens et al., Genitourin. Med. 1993;69:102-107; Van Voorhis et al., J. Infect. Dis. 1996;173:491-495
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Inflammation V: Phagocytosis
– Macrophages play a role in immune
clearance
– Intact and degraded TP found inside
their phagocytic vacuoles
– Rabbit peritoneal macrophages can
phagocytose opsonized Tp
– Abs and C3b act as opsonizing agents
– Tp92 and TprK proteins can induce
opsonic Ab production
– Abs against the VDRL reagent can
increase phagocytosis of Tp
Tp incubated with rabbit peritoneal
macrophages in presence of 10% heatinactivated normal or immune rabbit sera.
Salazar et al. Microbes Infect. 2002;4:1133-1140
Sell et al., Lab. Investig. 1982;46:355-364; Lukehart et al., J. Immunol. 1978;121:2014-2024; Salazar et al., Microbes Infect. 2002;4:1133-1140;
Cameron et al., J. Infect. Dis. 2000;181:1401-1413; Centurion-Lara et al., J. Exp. Med. 1999;189:647-656; Baker-Zander et al., J. Infect. Dis. 1993;167:1100-1105
The University of Sydney
Page 19
Inflammation VI: Antibody Production
– In rabbits, anti-Tp IgM and IgG
appear by day 6 post-infection
– Broad Ab response to surface
lipids, lipoproteins, flagellar
proteins, outer membrane Tpr
proteins, other proteins
– Abs work by opsonisation,
blocking Tp adhesin-mediated
binding, immobilization and
complement-based neutralization
Hanff et al., J. Immunol. 1983;131:1973-1977; Lukehart et al., Sex. Transm. Dis. 1986;13:9-15; Weiser et al., Infect. Immun. 1976;13:1402-1407
The University of Sydney
Page 20
Ho & Lukehart, Syphilis, in: The Prokaryotes – Human Microbiology 2013, Springer-Verlag DOI 10.1007/978-3-642-30144—5_109
The University of Sydney
Page 21
•
•
•
•
Eroded epithelium
Dense, plasma-cell-rich infiltrate
Neovascularization
Necrotizing vasculitic changes
with mural fibrin deposition
• Endarteritis obliterans
Primary syphilis
The University of Sydney
https://www.studyblue.com/notes/note/n/male-gu-lecture-4-penis/deck/5315986
Page 22
Evasion of the Host Immune Response
– Immune privileged sites (e.g. central nervous
system and eye) may harbour Tp and enable reseeding to other tissues
– Slow metabolism and low critical antigenic mass
may favour latency and immune evasion
– Tp avoids the host’s iron sequestration defence
mechanism by interacting with transferrin and
lactoferrin
– Tp has strategies to reduce the need for iron – it
lacks an electron transfer chain and possesses
enzymes that utilize other cations (e.g. Zn2+, Mn2+)
The University of Sydney
LaFond & Lukehart, Clin. Micro. Rev. 2006;19(1):29-49
Page 23
T. pallidum has Characteristics of a Stealth Pathogen
– Surface of Tp has low
antigenicity
– In Tp-infected animals,
Abs bind to physically
disrupted Tp but not
intact organisms
– Freeze-fracture EM of
bacteria/outer
membrane (OM) vesicles
confirms antigenic
inertness of Tp OM
The University of Sydney
Radolf et al., Proc. Natl. Acad. Sci. USA 1989;86:2051-2055; Blanco et al., J. Bacteriol. 1994;176:6088-6099;
Radolf et al., Infect. Immun. 1995;63:4244-4252; Salazar et al., Microbes Infect. 2002;4:1133-1140
Page 24
Putative Outer Membrane Proteins of T. pallidum
– Several putative OMPs identified in Tp genome on basis of
having cleavable signal sequences and transmembrane domains
– ECM-binding proteins Tp0155 (fibrinogen), Tp0483 (fibrinogen)
and Tp0751 (laminin) are thought to be OMPs
– T. pallidum rare outer membrane protein 2 (TROMP-2) is an
integral transmembrane protein with unknown function
– The Tp Repeat (Tpr) protein family is encoded by 12 tpr genes
separated into three subfamilies
– TrpF, TprI and TprK are thought be surface-exposed
– Surface exposed Tpr C and TprD have heterogeneity in their
sequences that remain unchanged during the course of infection
The University of Sydney
LaFond & Lukehart, Clin. Micro. Rev. 2006;19(1):29-49; Cameron & Lukehart, Vaccine 2014;32:1602-1609
Page 25
Characteristics of T. pallidum tprK
– tprK has seven discrete variable (V) regions, interspersed with
conserved regions
– Individual human/rabbit hosts are infected with several Tp
subpopulations with diverse tprK sequences
– DNA sequence cassettes that donate DNA sequences are located in
parts of the Tp genome removed from the tprK gene
– Antigenic diversity in trpK V
regions is produced by gene
conversion
(tprK)
(tprK)
The University of Sydney
– In experimental infection, Abs
target the V regions and T cells
recognize conserved TprK
epitopes
Centurion-Lara et al., Mol. Microbiol. 2004;52:1579-1596; Morgan et al., Infect. Immun. 2003;71:5605-5612;
Morgan et al., J. Immunol. 2002;169:952-957
Page 26
Latest Model of the T. pallidum Outer Membrane
– A novel approach encapsulating Tp in gel microdroplets and
then exposing them to syphilitic sera has recently reported that
TprK may be a periplasmic protein
– Cox et al. suggest that Tp OMPs are likely to have β-barrel
topology and have identified Tp0326 as a β-barrel assembly
machinery protein A (BamA) orthologue
– Highly conserved BamA
family members are
essential for OM biogenesis
Tp0453 is a pore-forming lipoprotein
anchored to the inner leaflet of the Tp OM
The University of Sydney
– These proteins enable
nascent OMPs (β-barrel
precursors) to be correctly
folded in the OM
Cox et al., Infect. Immuno. 2010;78(12):5178-5194; Desrosiers et al., Mol. Microbiol.
Page 27
Vaccine Development
– Miller generated complete protection against homologous
challenge in a rabbit model following 60 IV injections of
irradiated Tp over 37 weeks
– Slow development of immunity correlates with protein-poor
surface of Tp
– Treponemes are cleared naturally from lesions through a Th1
response (T cells, IFN-γ, DTH)
– Likely that long immunization schedules and careful selection of
Tp components and adjuvants likely to generate a Th1 response
are most likely to succeed
– Little known about immune correlates in humans
The University of Sydney
Miller, J. Immunol. 1973;110:1206-1215; Cameron & Lukehart, Vaccine 2014;32:1602-1609
Page 28
Summary
– Tp is a stealth pathogen characterized by a proteinpoor surface
– Characteristic corkscrew motility, attachment
mechanisms and binding to ECM components support
extensive dissemination within the host
– Clearance of bacteria at sites of infection relies on
DCs, macrophages, T cells and opsonizing antibodies
(Th1 response)
– Tp evades the immune system due to low bacterial
turnover, low bacterial load, limited surface Ag
exposure, antigenic diversity and invasion of immune
privileged sites
– Syphilis is a prime candidate for vaccine development
The University of Sydney
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