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
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
What is the molecular basis for these differences in virulence?
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)
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)
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
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
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)
Phosphorylation of STAT3/6 20 hours post-infection differs between the predominant Toxoplasma Types!
Saeij et al. Nature 2007
Saeij et al., Nature, 2007
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
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
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
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
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
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
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
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
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
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
Toxoplasma MitoTracker
Human foreskin fibroblasts
Association of Toxoplasma-RH and host mitochondria occurs in all cells tested.
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
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
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
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
What is the reason for these differences in virulence?
Reminder of the life cycle
carnivorism
grazing
Intermediate Hosts
Bradyzoites (tissue cysts)
Tachyzoites
ASEXUAL
Definitive Host
Gametes
Oocysts
SEXUAL
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)
So, repeat these studies in avian cells – different from results in mammals?
Ong et al., PLoS ONE, 2011
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
What is the clinical relevance of these strain-specific differences in
virulence?
Strain-type may explain some differences in disease-outcome in humans
J. Inf. Dis. 2001
Europe
Brazil
Gilbert et al. PLoS NTD 2008
years
Frac
tion
free
of e
ye le
sion
s
Gilbert et al., PLoS NTD, 2008
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:
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! !
THANKS!
Moritz Treeck Lena Pernas Michael Reese Anjali Shastri
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: