waltz or rave? how polymorphic effectors influence ... · waltz or rave? how polymorphic effectors...
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Waltz or Rave? How polymorphic effectors influence Toxoplasma’s dance with its host
John Boothroyd Stanford University
at Zoobiquity April. 5, 2014
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Or Rave…
J.P.J. Saeij, J.P. Boyle et al. Infection & Immunity 2005
Waltz
500 Toxoplasma strain S22 inoculated
50 Toxoplasma strain S23 inoculated
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What is the molecular basis for these differences in virulence?
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Genetic crosses allow the mapping of Toxoplasma genes responsible for a phenotype
Gametes
Oocysts
carnivorism
Clone, map and phenotype recombinants.
>109 progeny
ME49 (Type II)
CEP (Type III)
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F1 progeny of a cross between Types II and III show virulence is a quantitative trait
F1 progeny from Pfefferkorn; mapped by Sibley et al., Genetics, 1992 and Khan et al., NAR, 2005
< 10 E7 S23~ 100 A5 STH1 STG2 STF3 STC8 CL11 CL16 CL19~1000 C12 H6 STH10 S30~10,000 S2910,000-100,000 STG4 STG10 CL17 CL12 STD10>100,000 STD3 S28 CL13 S21 S25 S27 STH5 S2T CL18 S1T
B4 STE1 STD2 STC7 STE7 STE10 STH11 S22 S26 CL15
Recombinant F1 progeny from II x III cross: ~LD50:
Grigg et al Science 2001 Saeij et al. Science 2006
(II)
(III)
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Or Rave
J.P.J. Saeij, J.P. Boyle et al. Infection & Immunity 2005 S22 S23
II III
F1-S23 50 parasites
Waltz
F1-S22 500 parasites 500 Toxoplasma strain S22 inoculated
50 Toxoplasma strain S23 inoculated
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Toxoplasma is an obligate intracellular parasite that injects rhoptry proteins (ROPs) early in invasion!
B. Nicholls (1983); S. Hakansson & D. Sibley (2001)
Kimata and Tanabe (1987)
Anti-ROP9
Discharging rhoptries
1 µm
Dense granules
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Dance Step 1: ROP16 is a polymorphic tyrosine kinase that directly phosphorylates host STAT3/6
Susan Coller, Ching Ong, Jeroen Saeij, Jon Boyle, and Anjali Shastri
(STAT = Signal Transducer and Activator of Transcription)
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Phosphorylation of STAT3/6 20 hours post-infection differs between the predominant Toxoplasma Types!
Saeij et al. Nature 2007
Saeij et al., Nature, 2007
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RH (Type I)
ROP16 is a JAK2 mimic responsible for the (VERY) rapid ���tyrosine phosphorylation of STATs
HFFs, 1 min p.i.
RHΔrop16
phase DAPI pSTAT6 (Y705) ROP 2/3/4 ROP 2/3/4
Yamamoto et al., JEM, 2009; Ong et al., JBC, 2010 Ong et al., J. Biol. Chem., 2010
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Jeroen Saeij, Jon Boyle and Michael Reese
+Sibley lab +Howard lab
Dance steps 2 and 3: ROP5 and ROP18 are polymorphic ROPs that collaborate to block a key
effect of Interferon-gamma
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ROP18 is a polymorphic ser/thr kinase that localizes to the parasitophorous vacuole membrane (PVM)
• Type I/II are ROP18+; Type III are ROP18-
Type I (ROP18+) Type III (ROP18-)
pvm pvm
n n
J-F Dubremetz
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ROP18 phosphorylates IRGs at the parasitophorous vacuole membrane (PVM)
• IRGs are small GTPases induced by IFNγ that attack the PVM
Steinfeldt et al., 2010; Fentress et al., 2010
I
IRGs attack PVM No IRGs at PVM No IRGs at PVM (ROP18+) (ROP18-)
PVM PVM
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Steinfeldt et al., 2010; Fentress et al., 2010
I
IRGs attack PVM No IRGs at PVM No IRGs at PVM (ROP18+) (ROP18-)
PVM PVM
ROP18 phosphorylates IRGs at the parasitophorous vacuole membrane (PVM)
• IRGs are small GTPases induced by IFNγ that attack the PVM
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Steinfeldt et al., 2010; Fentress et al., 2010
I
IRGs attack PVM No IRGs at PVM No IRGs at PVM (ROP18+) (ROP18-)
PVM PVM
ROP18 phosphorylates IRGs at the parasitophorous vacuole membrane (PVM)
• IRGs are small GTPases induced by IFNγ that attack the PVM
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ROP5
Unbound IRG
T102/T108 (phosphosites)
ROP5 is a pseudokinase that binds and alters Irga6 conformation, exposing target phospho-sites of ROP18
M. L. Reese, unpublished
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T102/T108 (phosphosites)
M. L. Reese, unpublished
Bound IRG
ROP5
ROP5 is a pseudokinase that binds and alters Irga6 conformation, exposing target phospho-sites of ROP18
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ROP5
IRG
Strain-specific polymorphic residues in green Isoform-specific polymorphic residues in yellow
ROP5 pseudoactive site
ROP5 polymorphisms cluster at IRG-binding site
M. L. Reese, unpublished
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Lena Pernas
Anjali Shastri
Moritz Treeck
Polymorphonuclear cell isolated from mouse peritoneum 6 dpi with Toxoplasma - RH Polymorphonuclear cell isolated from mouse peritoneum 6 dpi with Toxoplasma
� µ � �
Toxo
M N
N
N
M
M
M
M
Dance step 4: Polymorphic MAF1 mediates strain-specific association of Toxoplasma with host mitochondria
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Toxoplasma MitoTracker
Human foreskin fibroblasts
Association of Toxoplasma-RH and host mitochondria occurs in all cells tested.
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Mitotracker
BUT, not all strains of Toxoplasma show mitochondrial association!
Type II
Type III
Mitotracker
Mitotracker
Type I
Pernas et al., PLoS Biology, in press
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MAF1 (Mitochondrial Association Factor 1) is necessary for host mitochondrial association (HMA):
Knock-out of MAF1 in a Type I strain converts to HMA-
5µm
Merge
µ
α� � � � � � � � � � � � � � � � � � �RFP
RH Δmaf1 RFP+ RH wt
Phase
Pernas et al., PLoS Biology, in press
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MAF1 acts alone: HA-tagged MAF1 expressed in mouse embryonic fibroblasts localizes to the mitochondria
*
*
*
* * *
*
* *
αTom20 αHA � � � � � � µ � �
Pernas et al., PLoS Biology, in press
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host plasma membrane!
Stat ROP16
pStat [active]
pStat
host nucleus!
rhoptries dense granules
Summary of the Dance Moves
And: ROP38 – down-regulates MAPK; Peixoto et al. 2010 GRA15 – activates NF-kB; Rosowski et al., 2011 GRA24 – activates p38 MAPK; Braun et al., 2013
ROP16 ROP5 ROP18
parasitophorous!vacuole!
!
IRG
pIRG [inactive] pIRG
ROP18
ROP5
MAF1
Modulation of NF-kB, IRF3, IRF7 signaling?
MAF1
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What is the reason for these differences in virulence?
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Reminder of the life cycle
carnivorism
grazing
Intermediate Hosts
Bradyzoites (tissue cysts)
Tachyzoites
ASEXUAL
Definitive Host
Gametes
Oocysts
SEXUAL
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Hypothesis: Do strain-specific differences exist to optimize infection of different strains in different host species??
STAT6 (murine/human)
ROP16 (Type I/III)
ROP16 (Type II)
STATx (species X)
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So, repeat these studies in avian cells – different from results in mammals?
Ong et al., PLoS ONE, 2011
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Maybe it’s not the host species but the host ecology that selects for different ROP16 alleles?
STAT6 (murine)
ROP16 (Type I/III)
Inflammatory response needs to be counter-balanced by upping Th2 (via STAT6 activation)?
ROP16 (Type II)
STAT6 (murine)
Th2 already dominates because of worms so not good to push further in that direction?
P
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What is the clinical relevance of these strain-specific differences in
virulence?
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Strain-type may explain some differences in disease-outcome in humans
J. Inf. Dis. 2001
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Europe
Brazil
Gilbert et al. PLoS NTD 2008
years
Frac
tion
free
of e
ye le
sion
s
Gilbert et al., PLoS NTD, 2008
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1. Strains differ dramatically in the character of their dance with the host.
S22 S23
3. Host range is likely the driver of these strain-specific differences but maybe it’s the overall “ecosystem” of the host, not host species, that is the variable
Summary
4. Strain-specific differences appear to matter in human infections, as well.
Brazil
Europe
years
Frac
tion
free
of e
ye le
sion
s
2. Strain-specific differences in virulence are a result of huge differences in the effectors introduced by different strains.
Type I Type II
HMA+ HMA-
In Summary:
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Colleagues and Collaborators:
Original crosses: Elmer Pfefferkorn – Dartmouth!Mapping: David Sibley – Washington Univ.!
Genome Sequencing: Sequencing Consortium and the ToxoDB team!ROP5/Irga6: Jonathan Howard, Tobias Steinfeldt and Martin Fleckenstein,
Cologne!MAF1: Jon Boyle and Yaw Adomako-Ankomah – Pittsburgh!
Michele Tonkin and Marty Boulanger – Victoria! !
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THANKS!
Moritz Treeck Lena Pernas Michael Reese Anjali Shastri
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1. Strains differ dramatically in the character of their dance with the host.
S22 S23
3. Host range is likely the driver of these strain-specific differences but maybe it’s the overall “ecosystem” of the host, not host species, that is the variable
Summary
4. Strain-specific differences appear to matter in human infections, as well.
Brazil
Europe
years
Frac
tion
free
of e
ye le
sion
s
2. Strain-specific differences in virulence are a result of huge differences in the effectors introduced by different strains.
Type I Type II
HMA+ HMA-
In Summary: