Ana Lina Pe ei a Rod igues
Disse a ion p esen ed o ob ain he Ph.D deg ee in Molecula Biosciences
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie | Uni e sidade No a de Lisboa
Inse he e an image
wi h ounded co ne s
Ea ly e en s o cell au onomous esponse
agains Toxoplasma gondii in he mouse
Oei as,
July, 2021
Ana Lina Pe ei a
Rod igues
Ea ly e en s o cell au onomous esponse agains Toxoplasma gondii
Oei as, July, 2021
Ana Lina Pe ei a Rod igues
Disse a ion p esen ed o ob ain he Ph.D deg ee in in Molecula Biosciences
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie | Uni e sidade No a de Lisboa
Oei as, July, 2021
Ea ly e en s o cell au onomous
esponse agains Toxoplasma
gondii in he mouse
Resea ch wo k coo dina ed by:
I
Co e : IFN gamma-induced mu ine ib oblas 1,5 hou s pos -
Toxoplasma gondii in ec ion. Ubiqui in ( ed), IRGs (g een) and
cell nucleus (blue) a e ma ked.

II
Decla a ion
This hesis is he esul o he wo k ha I de eloped as a PhD s uden
a he Ins i u o Gulbenkian de Ciência, supe ised by P o esso
Jona han Howa d unde he scope o he Pos G adua e P og am
Science o De elopmen (PGCD) om Oc obe 2015 o June 2021.
Au ho con ibu ions a e b ie ly desc ibed in a pa ag aph a e he i s
page o each chap e . My PhD wo k is suppo ed by Fundação pa a a
Ciência e Tecnologia, SFR/BD/114402/2016.
Ou labo a o y was suppo ed by cen al unds o he Ins i u o
Gulbenkian de Ciência, he Sonde o schungsbe eiche 670 and 680
and Schwe punk 1399 o he Deu sche Fo schungsgemeinscha .
III
Summa y
Toxoplasma gondii is a widely sp ead pa asi e wo ldwide among wa m-
blooded o ganisms hanks o i s abili y o su i e he immune sys em
esponse and ch onically es ablish i sel in i s hos s. The success o T.
gondii depends en i ely on he es ablishmen o i s in acellula su i al
niche, called he pa asi opho ous acuole (PV). The PV memb ane
(PVM) is de i ed om he hos plasma memb ane a he poin o en y.
Bo h a ime o en y and om wi hin he acuole, he pa asi es elease
i ulence ac o s in o he cy oplasm ha es ic he immune esponse.
The immune esponse o mice and humans depends on he cy okine
IFNγ, which induces he syn hesis o hund eds o p o eins in ol ed in
se e al modali ies o immuni y. This hesis ocuses on a amily o
in e e on-inducible GTPases, he IRG p o eins, ha a e essen ial o
immuni y agains T. gondii in mice. IRG p o eins ha e h ee unc ional
subg oups, e ec o s, ha bind di ec ly o he PVM, egula o s, ha
p e en e ec o s om ac i a ing on endogenous cellula o ganelle
memb anes, and decoys, ha in e ac di ec ly a he PVM wi h
i ulence p o eins om ype I i ulen s ains and p e en he
inac i a ion o e ec o IRGs, he eby a enua ing he i ulence. In mice,
e ec o IRGs cause des uc ion o he pa asi opho ous acuole leading
o elimina ion o he pa asi e. In human cells, howe e , IRG p o eins
a e absen and pa asi e elimina ion occu s by o he mechanisms,
possibly in ol ing a second amily o in e e on-inducible GTPases, he
Guanyla e-Binding P o eins, GBPs. Despi e his immune esponse, a
small numbe o pa asi es escape he ac ion o GTPases, su i e,
encys in he b ain, and a e esponsible o subsequen ansmission i
he cu en hos is p eda ed by a ca . How he pa asi e can balance
how many pa asi es a e elimina ed by he hos o p e en acu e
mo ali y, and a he same ime ensu e, in he mouse, ha a leas
some acuoles a e no elimina ed by IRG p o eins, is hus a key ac o
IV
in lea ning how he pa asi e pe sis s in hos s ha exploi he IRG
p o ein esis ance mechanism. This majo ques ion a ises equally in he
human case, and in all o he wa m-blooded hos species ha become
in ec ed by T. gondii, how does he pa asi e ind he ideal si ua ion, no
killing bu also no being ully elimina ed by he hos ?
A e T. gondii in ades a cell, he g oup o e ec o IRGs a ack he
PVM, leading o i s des uc ion. I is no ye known how hese p o eins
ecognize and bind o he memb ane o hese acuoles. Fu he mo e,
p e ious wo k has shown he accumula ion o some o he p o eins in
he acuole egion, including ubiqui in, whose unc ion in his con ex is
unknown. In he p esen s udy, we ocus on he ac o s ha de e mine
he accumula ion o IRGs as pionee s in he des uc ion o speci ic
(suscep ible) PVs, a oiding o he s ( esis an ) and i s ela ionship wi h
he ec ui men o ubiqui in.
We s a ed he s udy by analyzing how he concen a ion o IRG
p o eins a ailable in he cy oplasm de e mines i s ec ui men o PVs
and how di e en IRGs accumula e he e. We ound ha ega dless o
he supply o cy osolic IRG, each one independen ly binds o di e en
p opo ions o PVs summing up o ~80%. The des uc ion o he PV
esul s om he join ac ion o he IRGs. I gb6 loads on o he highes
p opo ion o acuoles bu alone is no su icien o des oy he PVM.
Then we ask i he du a ion o pa asi e pe sis ence in he cells be o e
loading o IRGs, which we he e call PVM ma u a ion, p e en s i s
a ge ing by IRGs, he eby accoun ing o he ~20% o PVMs ha do
no load wi h IRG p o eins. Using pa asi es lacking TgIST, a p o ein
ha inhibi s he in e e on esponse a he le el o he ch oma in, and
hus inhibi s he induc ion o IRG p o eins, we demons a ed ha he
esidence ime in he cell be o e IRGs bind does no make he PVMs
p og essi ely less likely o bind IRG p o eins. The e o e, acuola
ma u a ion does no explain he elimina ion- esis an acuoles.
V
We hen ocus on de e mining he link be ween ubiqui in accumula ion
in PV and he p esence o IRGs. We ound ha a e IRG ec ui men ,
ubiqui in is p esen in ~70% o PVs al eady co e ed by IRGs,
equi alen o hose loaded by I gb6, I ga6 and I gd, bu no hose
loaded by I gb6 alone. A de ailed analysis allowed us o e i y ha
ubiqui in does no use IRG p o eins as a subs a e, bu needs hei
ac ion in des oying PVMs o allow ubiqui in accumula ion in o on
pa asi es. We show ha ubiqui in is p esen exclusi ely on he pa asi e
and no on he PVM.
In conclusion, ou esul s sugges ha T.gondii elimina ion is ini ia ed
by he ec ui men o IRGs o PVs. The accumula ion o he di e en
e ec o IRG p o eins may be h ough he ecogni ion o speci ic
molecula a ge s on he cy osolic ace o he PVM ha a e ca ied in o
he PVM om he plasma memb ane a he ime o pa asi e en y.
Dis up ion o he PVM needs coope a ion be ween di e en IRG
e ec o s. Dis up ion o he PVM allows ubiqui in access o he pa asi e
memb ane, and ini ia es a p ocess leading o dea h o he pa asi e and
nec osis o he hos cell.
XII
Lis o abb e ia ions
AF
Alexa luo
APS
Ammonium pe oxydisul a e
BCA
Bicinchoninic acid
BL/6
Mouse s ain C57BL/6
BMM
Bone ma ow-de i ed mac ophages
BSA
Bo ine se um albumin
CAI
Cell Au onomous Immuni y
CLRs
C- ype lec in ecep o s
CO2
Ca bon dioxide
DAPI
4′,6-diamidino-2-phenylindole
DC
Dend i ic cells
DMEM
Dulbecco’s modi ied Eagle’s medium
DNA
desoxy ibonucleic acid
DUBs
Deubiqui ina ing enzymes
EDTA
E hylenediamine e aace ic acid
ER
endoplasma ic e iculum
FACS
Fluo escence-ac i a ed cell so ing
FBS
Fe al bo ine se um
FCS
Fe al cal se um
GABARAPs
g-aminobu y ic acid ecep o -associa ed p o ein
GAP
GTPase ac i a ing p o eins
GAS
IFNγ-ac i a ed si e
GBP
Guanyla e-binding p o ein
GDP
Guanosine diphospha e
GED
GTPase e ec o domain
GTP
Guanosine iphospha e
HEK293T
Human Emb yonic Kidney 293 cells
HeLa
Hen ie a Lacks
HFFs
Human o eskin ib oblas s
HI-FBS
Hea -inac i a ed e al bo ine se um

XIII
HMGB1
High-mobili y g oup box 1
hMSC
Human mesenchymal s omal cells
HUVECs
Human Umbilical Vein Endo helial Cells
IFNGR
In e e on (IFN)-gamma ecep o
IFNγ
In e e on (IFN)-gamma
IRG
Immuni y- ela ed GTPases
ISGs
In e e on-s imula ed gene
ISRE
In e e on-s imula ed esponse elemen
IVN
In a acuola ne wo k
JAK
Janus kinase
KO
Knockou
LIR
LC3-in e ac ing egion
MEFs
Mouse Emb yonic Fib oblas s
MHC
Majo His ocompa ibili y Complex
MIC
Mic onemal p o eins
MJ
Mo ing junc ion
MOI
Mul iplici y o in ec ion
MX1
Myxo i us esis ance p o ein 1
NDP52
Nuclea do p o ein 52 kDa
NK
Na u al kille cells
NLPR
Py in domain PYD-con aining p o eins
NLR
NOD-like ecep o s
OPTN
Op ineu in
PAMPs
Pa hogen-associa ed molecula pa e ns
PBS
Phospha e-bu e ed saline
PBS
Phospha e-bu e ed saline
PBST
Phospha e-bu e ed saline Tween
PCR
Polyme ase chain eac ion
PFA
Pa a o maldehyde
PRR
Pa e n ecogni ion ecep o s
PV
Pa asi opho ous acuole
XIV
PVDF
Poly inylidine di luo ide
PVM
Pa asi opho ous acuole memb ane
RLR
RIG-I-like ecep o s
RNA
ibonucleic acid
RON
Rhop y neck p o ein
ROP
Rhop y bulb p o ein
RT
Room empe a u e
SCV
Salmonella-con aining acuole
SDS-PAGE
sodium dodecyl sul a e-polyac ylamide gel
elec opho esis
SQSTM
seques osome 1
STAT1
Signal T ansduce And Ac i a o O T ansc ip ion 1
T.gondi, Tg
Toxoplasma gondii
TLR
Toll-like ecep o s
TRAF6
TNF ecep o -associa ed ac o 6
TRIM21
T ipa i e mo i con aining-21
TVN
Tubulo esicula ne wo k
UB
Ubiqui in
UBD
Ubiqui in Binding domain
UV
Ul a iole
W/d
Wi hou
WT
Wild ype
XV
Table o con en s
1. In oduc ion ....................................................................................... 1
1.1. Cell au onomous immune esponse ........................................... 2
1.2. In e e on induc ion o cell au onomous Immuni y. ..................... 3
1.3. In e e on-inducible GTPases: ................................................... 6
1.3.1. Immuni y- ela ed GTPases (IRGs) ....................................... 7
1.3.2. Guanyla e-binding p o eins (GBPs) .................................... 14
1.3.3. Pa asi e con ol by he IRG and GBP esis ance sys ems . 16
1.4. Ubiqui ina ion as a mechanism o cellula immuni y agains
in acellula pa hogens. ...................................................................... 22
1.4.1. S uc u e o ubiqui ina ion p ocess ..................................... 22
1.4.2. Role o ubiqui in in immuni y agains pa hogens ................ 24
1.5. Toxoplasma e ec o s an agonis s o hos IFNγ- Inducible gene
exp ession. ......................................................................................... 27
1.6. Thesis aims. ............................................................................. 29
2. Mechanisms o IFNγ-inducible GTPases in he cell-au onomous
ea ly esponse agains Toxoplasma gondii. .......................................... 50
2.1. Abs ac .................................................................................... 52
2.2. In oduc ion .............................................................................. 53
2.3. Ma e ial and Me hods ............................................................... 55
2.3.1. Ma e ial ............................................................................... 55
2.3.2. Cell-biological me hods ...................................................... 57
2.3.3. Immuno luo escence .......................................................... 59
2.3.4. Analyses o cellula p o eins ............................................... 60
2.3.5. Mic oscopy and image analyses ........................................ 61
2.3.6. Quan i ica ion o IRG p o eins and GBP1 on he PVM ....... 62
2.3.7. S a is ical analysis .............................................................. 64
2.4. Resul s ..................................................................................... 65
2.4.1. IRG p o eins a ge ype II T. gondii PVs in an In e e on-γ
dependen manne . ........................................................................ 65
XVI
2.4.2. The accumula ion o IRG p o eins occu s in cha ac e is ic
p opo ions bu does no co e 100% o PVs. ................................ 68
2.4.3. The ec ui men o I ga6 o PVs is independen o he
ec ui men o I gb6 ........................................................................ 71
2.4.4. The binding o I gb6 is also no c ucial o ec ui men o
o he GTPases o he PVs. ............................................................. 73
2.4.5. IRG p o ein accumula ion on PVs s a s ea ly and
inc eases o e ime. ....................................................................... 76
2.4.6. The cellula concen a ion o IRGs is no a limi ing ac o in
i s ec ui men o acuoles. ............................................................. 79
2.4.7. Toxoplasma gondii in ec ion in e e es wi h IFNγ–induced
IRG exp ession. .............................................................................. 83
2.4.8. Toxoplasma gondii educes IRG p o ein exp ession and
loading in he absence o TgIST ..................................................... 88
2.4.9. GBP1 is ec ui ed o T. gondii PVs and colocalizes o some
ex en wi h IRG p o eins. ................................................................ 93
2.5. Discussion ................................................................................ 96
2.6. Acknowledgmen s .................................................................. 100
2.7. Supplemen a y ma e ial ......................................................... 101
3. Ubiqui ina ion as a mechanism o con ol o Toxoplasma gondii
in ec ion. .............................................................................................. 111
3.1. Abs ac .................................................................................. 113
3.2. In oduc ion ............................................................................ 115
3.3. Ma e ial and Me hods ............................................................ 117
3.3.1. Ma e ial ............................................................................. 117
3.3.2. Cell-biological me hods .................................................... 121
3.3.3. Immuno luo escence ........................................................ 123
3.3.4. Nec osis assay using PI ................................................... 124
3.3.5. Nec osis and T. gondii assays by Flow Cy ome y ........... 124
3.3.6. Mic oscopy and image analyses ...................................... 125
3.3.7. Blinding and unbiased analysis ........................................ 127
XVII
3.3.8. Quan i ica ion o IRG, GBP1 and Ubiqui in on he PVM .. 127
3.3.9. Quan i ica ion o nec o ic and li e cells by Image J, Fiji ... 129
3.3.10. S a is ical analysis ............................................................ 130
3.4. Resul s ................................................................................... 130
3.4.1. Ubiqui in is ec ui ed o Type II Toxoplasma gondii PVs in
an In e e on-γ dependen manne . .............................................. 130
3.4.2. Ubiqui in is ec ui ed o PVs deco a ed by IRG p o eins. . 133
3.4.3. The p esence o he amily o e ec o IRGs, no single
IRGs on he PVM, is c ucial o i s ubiqui ina ion. .......................... 136
3.4.4. Ubiqui in colocalizes wi h mislocalized IRG e ec o p o eins
in I gm1 + I gm3 de icien cells. .................................................... 139
3.4.5. Ubiqui in accumula es on PVs deco a ed wi h GBP1. ..... 143
3.4.6. Ubiqui in ec ui men o he PV is dependen on IRG
loading 145
3.4.7. Ubiqui ina ion o PVs is associa ed wi h he e iciency wi h
which IRGs des oy he PVM. ...................................................... 147
3.4.8. Ubiqui ina ion o PVs in mouse CIM s ain cells equi es he
p esence o I gb2-b1 .................................................................... 153
3.4.9. Pa asi e memb anes a e he a ge o ubiqui ina ion. ...... 154
3.4.10. Nei he TRAF6 no TRIM21 E3 ligases a e indi idually
equi ed o ubiqui ina ion o PVs ................................................. 157
3.4.11. K48- and K63- ype ubiqui in linkages a e bo h in ol ed in
pa asi e ubiqui ina ion. ................................................................. 159
3.4.12. IRG and ubiqui in ec ui men o PVs is ollowed by hos
cell p og ammed nec osis. ........................................................... 161
3.5. Discussion .............................................................................. 163
3.6. Acknowledgmen s .................................................................. 167
3.7. Supplemen a y ma e ial ......................................................... 169
4. Gene al discussion ....................................................................... 184
4.1. Summa y o he indings. ....................................................... 186

XVIII
4.2. Coope a i e loading o e ec o IRG p o eins on he PVM a e
he key o he cell's au onomous esis ance o T. gondii .................. 189
4.3. Pa ial egula ion o IRG exp ession and ec ui men by TgIST
is impo an o pa asi e. .................................................................. 191
4.4. Ubiqui in a he pa asi e su ace ma ks he dis up ion o he
PVM . .............................................................................................. 194
4.5. Concluding ema ks and u u e pe spec i es ......................... 198
4.6. Supplemen a y ma e ial ......................................................... 200
XIX
Lis o igu es
Figu e 2.1. Quan i ica ion o I g p o eins p esen on he PVM o
a i ulen T. gondii in IFNg s imula ed MEFs ........................................ 64
Figu e 2.2. IRG p o eins p e e en ially a ge ype II T. gondii PVs in a
In e e on-γ dependen manne ........................................................... 67
Figu e 2.3. The accumula ion o IRG p o eins occu s in a cha ac e is ic
p opo ion ha does no co e 100% o PVs. ...................................... 69
Figu e 2.4. Di e en e ec o IRG co-localize o he same PVM. ......... 71
Figu e 2.5. I gb6 and I ga6 on PVM a e no co ela ed. ...................... 73
Figu e 2.6. The ec ui men o o he s GTPases is no g ea ly a ec ed
by he absence o I gb6. ...................................................................... 75
Figu e 2.7. Accumula ion o IRG p o eins on PVs s a s ea ly and
inc eases o e ime. ............................................................................ 78
Figu e 2.8The cy osolic concen a ion o IRG p o eins is no no mally a
limi ing ac o in hei ec ui men o acuoles. .................................... 80
Figu e 2.9. Loading o indi idual PVs in a cell by IRG p o eins a e no
co ela ed. ............................................................................................ 82
Figu e 2.10. The du a ion o hos cell p es imula ion wi h IFNγ in e e es
wi h IRG p o eins media ed esponse o T.gondii. ............................... 85
Figu e 2.11. Sho ime p es imula ion wi h IFN-γ allow T. gondii o
educe and supp ess IRG exp ession. ................................................ 87
Figu e 2.12. T. gondii impac he cy osolic I gb6 ia TgIST and TgIST
independen ac o , independen ly o ime. .......................................... 90
Figu e 2.13. T. gondii a ec he I gb6 ec ui men o he PVM pa ially
ia TgIST. ............................................................................................ 92
Figu e 2.14. GBP1 is ec ui ed o PVs independen o I ga6 and I gb6.
............................................................................................................. 95
Figu e 3.1. Li e-cell mic oscopy-based dual pa ame e acking o
IFNγ-dependen ib oblas nec osis in esponse o T. gondii in ec ion.
........................................................................................................... 130
XX
Figu e 3.2. Ubiqui in is ec ui ed o Type II Toxoplasma PVs in an
in e e on-γ dependen manne . ........................................................ 132
Figu e 3.3. Ubiqui in is ec ui ed o PVs deco a ed by IRG p o eins. 134
Figu e 3.4. Ubiqui ina ion may be sligh ly educed in he absence o
single I g p o eins. ............................................................................. 138
Figu e 3.5. Ubiqui in colocalizes wi h misloca ed IRG e ec o s in I gm1
and I gm3 de icien cells. ................................................................... 142
Figu e 3.6. Ubiqui in accumula ed on PVs deco a ed by GBP1. ....... 144
Figu e 3.7. Ubiqui in ec ui men o he PV is dependen on IRG
loading. .............................................................................................. 146
Figu e 3.8. The e is mo e accumula ion o ubiqui in in dis up ed PVs.
........................................................................................................... 148
Figu e 3.9. IRG p o eins in cells om he CIM mouse s ain a e able o
dis up ype I PVM. ............................................................................ 149
Figu e 3.10. Cells om he CIM mouse s ain a e able o ubiqui ina e
ype I PVM. ........................................................................................ 152
Figu e 3.11. The p esence o he I gb2-b1 allele is equi ed o
ubiqui ina ion o ype I T. gondii PV. .................................................. 154
Figu e 3.12. Ubiqui in is ec ui ed o he pa asi e memb ane dependen
on IRG loading and PVM dis up ion. ................................................. 157
Figu e 3.13. The ubiqui ina ion o PVs is no a ec ed in TRAF6 and
TRIM21 knockou s cells. ................................................................... 158
Figu e 3.14. Lysine K48 and K63- ype linkages bo h con ibu e o he
polyubiqui ina ion o PVs. .................................................................. 160
Figu e 3.15. IRG and ubiqui in ec ui men o PVs is ollowed by
nec o ic dea h o he hos cell. ........................................................... 162
Supplemen al Figu e 2. 1. I gb10 loca ion is a ec ed in I gb6KO cells
........................................................................................................... 101
Supplemen al Figu e 2. 2. Toxoplasma gondii in ec ion impac he
IFNγ–induced I gb6 exp ession. ........................................................ 102
XXI
Supplemen al Figu e 3. 1. Nec o ic cells dea h is ea u ed by
condensed nucleus ............................................................................ 169
Supplemen al Figu e 3. 2. Nec osis is IFNγ-induced cells is igge ed by
a i ulen T. gondii in ec ion ................................................................ 170
Supplemen al Figu e 3. 3. The IFNγ Induced cells unde go nec osis
a e a leas one included T. gondii dies. .......................................... 171
Supplemen al Figu e 3. 4. In ec ion dependen nec osis is pa ially
a ec ed in I gb6 KO cells. ................................................................. 171
Supplemen al Figu e 3. 6. o easome inhibi ion does no inc ease T.
gondii ubiqui ina ion. .......................................................................... 173
Supplemen al Figu e 3. 7. Ve i ica ion o TRAF6KO- cells by PCR .. 174
Supplemen al Figu e 3. 8. W and TRIM21KO geno yping PCR ....... 174
Supplemen al Figu e 3. 9. K63 Ubiqui ina ion is educed in TRIM21KO
cells. .................................................................................................. 175
Supplemen al Figu e 4. 1. Cells ha do no exp ess IRG ha bou mo e
PVs, han cells ha exp ess IRG ....................................................... 200
Supplemen al Figu e 4. 2 MYR-independen ac o impac I gb6
exp ession. T. gondii ......................................................................... 201
!
6
wa es o ansc ip ion e en s modula ed indi ec ly by IFNs, he
seconda y esponse genes 24. ISGs unc ion in sys emic esis ance
and/o au onomous esis ance o cells, and hose in ol ed in he s udy
pe o med in his hesis a e desc ibed below.
1.3. In e e on-inducible GTPases:
Among many o he a ge s, IFNγ-dependen signaling leads o
ansc ip ion o se e al amilies o la ge guanosine iphospha ases
(GTPases), which a e among he mos p ominen ly IFNγ-induced
p o eins and play impo an oles in he immune esponse o di e se
cell ypes agains pa hogens anging om bac e ia, o i uses, ungi
and pa asi es 29. Indeed i was s udy o in e e on-media ed induc ion o
human Guanyla e Binding P o ein 1 (GBP1) ha led o he iden i ica ion
and elucida ion o he JAK-STAT pa hway 30.
The in e e on-inducible GTPases a e enzymes wi h an N- e minal
globula GTPase domain (GD) ollowed by a C- e minal helical domain
(CTHD). The ancien conse ed domain, he G-domain o GD, is able
o bind and hyd olyze guanosine 5'- iphospha e (GTP) o guanosine 5'-
diphospha e (GDP) o guanosine 5'-monophospha e (GMP). These
p o eins wo k by swi ching be ween he wo in e con e ible bound
s a es, he inac i e GDP-bound o m and he ac i e GTP-bound o m,
media ing e ec o unc ions. They a e able o swi ch ON and OFF
be ween hese wo o ms.
In con as wi h small GTPases, in which he ansi ions be ween GDP
and GTP a e acili a ed by guanine nucleo ide exchange ac o s (GEFs)
and GTPase-ac i a ing p o eins (GAPs), la ge GTPases gene ally ha e
low a ini ies o nucleo ides and exchange GDP and GTP eely in he
cy oplasm. Ac i a ion is ini ia ed by dime iza ion be ween he
nucleo ide-binding domains, o en wi h subsequen oligome iza ion

7
apping he GTP-bound s a e and unc ioning as “GAP” ac i i y and
consequen ly acili a ed hyd olysis 29,31. Like dynamins, la ge GTPases
gene ally associa e wi h in acellula lipid memb anes and possess he
abili y o sel -assemble and allow p o ein–p o ein o p o ein–lipid
in e ac ions h ough he C- e minal helical domain 31–33.
The e a e 3 amilies o GTPases playing a ole in IFN-media ed
immune esis ance: he myxo i us esis ance p o eins (Mx), he
immuni y- ela ed GTPases (IRGs), and guanyla e-binding p o eins
(GBPs) 31. Bo h IRG and GBP p o eins can be ansc ibed in esponse
o ype I, II and III In e e on 29 while he Mx amily p o eins, a e no
induced by ype II In e e on31.
1.3.1. Immuni y- ela ed GTPases (IRGs)
The IRG p o eins a e s ongly ansc ibed only in esponse o IFN, wi h
IFNγ being hei majo induce ; IFNα / β also igge s IRG exp ession,
al hough o a lesse ex en 31. These 47kDa uni p o eins ( o me “p47
GTPases”) ha e been ex ensi ely s udied since he iden i ica ion o he
i s amily membe , I gd (as IRG47 in Gilly and Wall 1992), in he ea ly
1990s. In his hesis I will use he widely accep ed o mal nomencla u e
o IRG genes and p o eins in oduced by Bekpen e al (2005).
In he C57BL/6 mouse genome, he e a e 16 unc ional and possibly
unc ional genes: 9 unc ional IRG genes,namely he h ee IRGM
p o eins (I gm1, I gm2, I gm3) I ga6, I gb6, I gb6*, I gb10, I gd, and
I gb2b1, 7 possibly unc ional (I ga3, I ga4, I ga7, I ga8, I gb5*b3,
I gb5b4, I gb9b8), and 3 non- unc ional pseudogenes (I ga1, I ga2,
I ga5 and I gb7); all 16 a e dis ibu ed in 3 clus e s, wo clus e s
sepa a ed by 10Mb on Ch omosome 11 and one clus e on
ch omosome 18. An isola ed di e gen and highly conse ed IRG gene
on ch omosome 7 (I gc) has no immune ole, is exp essed exclusi ely
8
in he es is in haploid spe ma ids and is equi ed o no mal
spe ma ogenesis 34 (Howa d Lab unpublished esul s).
Cha ac e is ically, he 47 kDa open eading ames o IRG p o eins a e
encoded on single exons. Fou pai s o IRGB genes (I gb2-b1, I gb5*-
b3; I gb5-b4; and I gb9-b8) on ch omosome 11 encode “ andem” IRG
p o eins buil o wo exons each encoding he ypical 47 kDa IRG
p o ein s uc u e, gi ing a molecula weigh a ound 94 kDa 34,35
Figu e 1. 2. Linea o de o IRG gene clus e s on Ch omossome 11 and
Ch omossome 18 o C57BL/6 mouse s ain
Dis ibu ion o IRG genes in ch omosomes 11 and 18 including he posi ion o he IRG
coding uni s (black blocks) and he di ec ion o ansc ip ion (a owhead), he posi ion
on he ch omosome (numbe s), and he andem genes connec ed wi h a line. ψ
indica es pseudogenes, * ma ks he second copy o he I gb6 gene iden ical a he
p o ein le el (modi ied om Lilue e al. 2013).
In humans and highe p ima es he p47 GTPase amily is g ea ly
educed and p obably non- unc ional. The human genome has only wo
IRG genes, IRGC and he g ea ly unca ed IRGM, homologous o
mouse IRGM p o eins 34,36,37. The human IRGM agmen is no
inducible by in e e ons 38.
The mouse immune IRG p o eins, despi e g ea simila i y in sequence
and s uc u e can be subdi ided in o 3 unc ionally dis inc sub amilies:
e ec o s, egula o s and decoys (see sec ion 1.3.1.1.). The e ec o s
and decoys we e ini ially called GKS p o eins,31 and he egula o s
GMS p o eins because o a s iking sequence ea u e o he nucleo ide
9
binding si e. GKS IRG p o eins (e ec o s and decoys) possess he
uni e sally conse ed sequence (GX4GKS) o he G1 mo i o he GTP
binding si e and he GMS egula o p o eins con ain he non-canonical
GX4GMS sequence in he G1 mo i 31,39. I is s ill no ye known whe he
pu i ied GMS p o eins can hyd olyze GTP, al hough i has been
sugges ed om pull-down expe imen s ha I gm3 has GTPase ac i i y
40. Howe e , he G1 me hionine would seem o be incompa ible wi h
co ec posi ioning o he nucleo ide phospha es.
1.3.1.1. Molecula p ope ies o IRG p o eins: Biochemis y
and s uc u e
S udies o he GTPase ac i i y o he IRGs e idenced hyd olysis o
GTP o GDP we e ex ended wi h he s udy o he pu i ied I ga6 41,42.
The I ga6 p o ein (o iginally called IIGP1) is he only IRG p o ein ha
has i s s uc u e and chemical p ope ies cha ac e ized in de ail so a
43. Since IRG p o eins ha e high sequence homology, he I ga6
s uc u e de e mined in 2004 has been used as a model o he o he
IRG p o eins. This p o ein s uc u e b eaks down in o h ee egions: N-
and C- e minal helical domains, wi h a Ras-like G domain be ween
hem (Figu e 1.3).
Figu e 1. 3. C ys al 3D s u u e o I ga6 p o ein
10
Ribbon p esen a ion o he I ga6 c ys al a ached o GDP / Mg2 + is showing he helical
N- e minal egion (αA-C helix) in Cyan, he G domain (helix S1-H5) in ligh blue, he
linke helix (αE) in g ay and he helical C- e minal egion (αF-αL helix) in da k blue.
GDP and Mg2+ a e shown as a omic s ick igu e and yellow sphe e. The opology is
shown schema ically using he same colo code 43 .
I ga6 binds GDP wi h g ea e a ini y han GTP. I s basal GTPase
ac i i y inc eases wi h inc easing p o ein concen a ion sugges ing
coope a i e in e ac ions. I ga6 can be ound in i o in monome s (in
he absence o nucleo ides) and in GTP-dependen homo-oligome s,
accompanied by a con o ma ional change, ha a e esol ed a e GTP
hyd olysis 44. The oligome iza ion o I ga6 and accele a ed hyd olysis o
he GTP occu s h ough an in e ace o I ga6 wi hin he G domain and
includes he nucleo ide binding si e and he exchange egions o he
bound GTP subs a e. This in e ace is also esponsible o he
in e ac ion o I ga6 wi h o he s IRGs 42. The possibili y ha coope a i e
hyd olysis be ween IRG e ec o p o eins no mally oocu s be ween
di e en membe s o he g oup has no ye been examined.
The coope a i e hyd olysis o GTP by I ga6 has been assumed o be
gene ally ue o he GKS IRG p o eins. In ac , Pawlowski showed ha
al hough pu i ied I gd and I gb6 a e bo h slow GTPases, he hyd olysis
a es a e independen o p o ein concen a ion, aising he possibili y
ha he coope a i e ac i i y o pu i ied I ga6 is an in i o a e ac 42.
I ga6 ca ies an amino- e minal my is oyla ion si e 38 a glycine 2, which
is ac i e and has been shown o pa ially a ou binding o he p o ein
o memb anes 45,46. The ou andem IRG p o eins, I gb2b1, I gb9b8
I gb5b4 and I gb5*b3 also ca y canonical amino- e minal
my is oyla ion sequences, hough only I gb2b1 is known o be
signi ican ly exp essed in C57BL6 mice.
IRG p o eins associa e wi h memb anes o a ying deg ees. Regula o y
11
p o eins bind o memb anes h ough C- e minal helical sequences 47,48
Wi h he excep ion o he plasma memb ane, egula o IRG p o eins
I gm1, I gm2 and I gm3 a e dis ibu ed in dis inc compa men s
including all he memb ane o ganelles: I gm1 is associa ed wi h Golgi
complex 48,49 endosomes, lysosomes 47,50 mi ochond ia 47,50,51 and lipid
d ople s 52,53; I gm2 is associa ed wi h Golgi 29,54. I gm3 localises o he
ER 54,55 and lipid d ople s 52,53. In all h ee cases he di e en
compa men al speci ici ies o he IRGM p o eins can be ep oduced by
sho pep ides de i ed om he C- e minal helical egions o he ull-
leng h p o eins.
In IFN-s imula ed cells, e ec o IRGs (I ga6, I gb6, I gb10 and I gd) a e
ound in GDP-bound o m mainly in he cy osol, and only I ga6 can be
ound associa ed mainly wi h he endoplasmic e iculum memb ane 49,58
1.3.1.2. IRG genes in mouse esis ance immuni y
The i s e idence ha IRGs ha e some ole in esis ance o in ec ions
came om a s udy in I gm3 ( o me ly IGTP) knockou (KO) mice, in
which he e was 100% mo ali y ollowing in ec ion wi h an a i ulen
s ain o T. gondii 58. Then, a se ies o s udies in ol ing mo e gene ic
dele ions o IRG genes in mice ha e suppo ed he no ion o a ole o
he IRG sys em in esis ance agains ce ain pa hogens.
Single knock-ou mouse models a e a ailable o I gm1, I gm3, I gd,
I ga6, I gb10 and I gb6, as well as an I gm1/I gm3 double knock-ou
mouse s ain. E idence om in i o and in i o s udies indica ing
suscep ibili y o esis ance o ce ain o ganisms in compa ison wi h wild
ype C57BL/6 mice is summa ized in Table 1.

12
Table 1. 1. Suscep ibili ies o I g-de icien mice and cells o in acellula
pa hogens
M, mouse; C, cells; R, esis an ; S, sensi i e; Sm, knockou mouse sensi i e; Rm,
knockou mouse esis an ; Sc, knockou cells sensi i e; Rc, knockou cells esis an . m,
mouse, c, cells. whe e no speci ied, he esul s a e om knockou mice. nd, no
de e mined.
Table 1 is adap ed om wo k in he Howa d lab 29 and summa izes a ious s udies on
IRGm149,56,59–64 on IRGm3 56,58,60–62,64, on IRGd59,60,62,64 on I ga660, on I gb1056,65,66, and
on I gb667.
The esis ance sys em linked o he IRG p o eins has been
demons a ed in he knock-ou mouse models o a numbe o IRG
genes, showing g ea e suscep ibili y o ce ain pa hogens including
p o ozoa, unicellula ungi and in acellula bac e ia, illus a ed in Table
1. The loss o pa hogen esis ance o he I gm1 -/- mice has been
de e mined o almos all he pa hogens enume a ed. Loss o I gm1
esul s in loss o esis ance o e e y o ganism ha has been es ed o
which IFNγ-induc ion plays a ole in esis ance. The only es ed
o ganism whe e esis ance was no los in I gm1-de icien mice was he
ema ode Schis osoma mansoni whe e esis ance is comple ely
independen o IFNγ 68. Mice de icien in I gm1 in ec ed wi h a numbe
o di e en o ganisms (Toxoplasma gondii, Mycobac e ium a ium,
T ypanosoma c uzi and Salmonella yphimu ium) su e an imp essi e
collapse o he lymphoyeloid sys em 61,63,69. Despi e ea lie claims 62,
I gm1 does no seem o colocalize wi h pa hogens 51 and i is clea ha
13
suscep ibili y o I gm1-de icien animals is due o he IFN-dependen
lymphomyeloid collapse 61 and no o an e ec o unc ion on hese
pa hogens. Since I gm1 is a nega i e egula o o IRG p o ein
ac i a ion, he cy o oxic e ec o in e e on in he I gm1-de icien mouse
may be a consequence o he induc ion and in acellula ac i a ion o
IRG e ec o p o eins causing damage o o ganelles, pa icula ly
lysosomes du ing lymphomyeloid de elopmen 51,54.
Rega ding he e ec o IRGs, i is e iden ha I gd -/- and I ga6 -/- mice
show signi ican suscep ibili y o a i ulen s ains o T. gondii 59,70, while
he p esence o I gb10 seems o be impo an in he esis ance agains
he bac e ia Chlamydia achoma is 65, Chlamydia psi aci and
F ansisella no icida 66. The meaning o hese di e en esis ance
speci ici ies o di e en e ec o IRG p o eins is comple ely unknown,
and p esumably implies unexpec edly di e en mechanisms o ac ion
om di e en membe s o his well-de ined s uc u al amily.
The di e en ial ole o IRG p o eins in he mouse esis ance o
pa hogens a he le el o cell au onomous immuni y (CAI) is illus a ed
in he T. gondii sys em and de ailed in 1.6. IFNγ is he main media o o
immuni y o oxoplasmosis 67,70. In mice de icien in egula o y IRGs
I gm1, I gm3 o I gm1/I gm3, in ec ion wi h T. gondii leads o he apid
dea h o 100% o mice in he acu e phase o he disease, abou 9-11
days a e an in ape i oneal in ec ion, an in ec ion ou come simila o
ha p e iously desc ibed o IFNγ-de icien mice 58,59,67,71. Howe e , he
suscep ibili y o I gm1-de icien mice p obably has a di e en o igin, as
se ou abo e. In e es ingly, he I gm1/I gm3 double de icien mouse no
longe shows he uni e sal suscep ibili y o he I gm1 de icien animal
and is suscep ible only o T. gondii 72. The absence o I ga6 and I gd
causes a less se e e de ec in esis ance han loss o he IRGM
p o eins. Abou 50% o I ga6- and I gd-de icien mice die om in ec ion
wi h T. gondii in he acu e phase and in he ch onic phase 59,70.
14
Recen ly published da a in I gb6/I gb6* KO show ha 100% o animals
succumb o in ec ion wi h he a i ulen T. gondii s ain, PRU 67. An
independen dele ion o I gb6 and I gb6* has con i med he high
suscep ibili y o PRU in ec ion, bu also showed ha he animals a e
la gely esis an o ano he a i ulen T. gondii s ain, ME49 a he same
le el o I ga6 and I gd esis ance (Claudia Campos, unpublished da a).
IRG p o eins ac as e ec o s agains pa hogens. E idence o suppo
his no ion includes he ac ha IRGs colocalize wi h di e en
pa hogens. I ga6, I gb6, I gd, I gb10 colocalize wi h he Toxoplasma
gondii PVM 45,49,73,74, I ga6, I gb6 and I gb10 a e somewha ec ui ed o
Chlamydia achoma is inclusions 53,75. I gb10 con ols g ow h o
Chlamydia in mice 65; he oles o I ga6 and I gb6 emain unclea .
I gb10 colocalizes wi h F ancisella no icida 66, and I ga6, I gb6, I gd
and I gm2 colocalize wi h he mic ospo idian ungus Encephali ozoon
cuniculi 60. The na u e o he esis ance mechanisms is discussed
below.
1.3.2. Guanyla e-binding p o eins (GBPs)
GBP p o eins, composed o 65-73 kDa uni s, a e s ongly induced by
IFNγ, accoun ing o as much as 20% o IFNγ-s imula ed p o eins 29,32.
In ec ions by he p o ozoan T. gondii o he bac e ium Lis e ia
monocy ogenes, ha induce s ong IFNγ esponses, also s imula e
exp ession o GBPs 76.
GBPs a e p esen in mos e eb a es, wi h 11 genes iden i ied in mice
in clus e s on ch omosomes 3 (mGbp1, mGbp2, mGbp3, mGbp5, and
mGbp7) and 5 (mGbp4, mGbp6, mGbp8, mGbp9, mGbp10, and
mGbp11) 76,77 and se en genes (hGBP1–7) in human ch omosome 1 78
whe e i was i s iden i ied, due o i s obus IFNγ induc ion 79.
15
1.3.2.1. Biochemical P ope ies o GBPs
The GTPase-domain o GBPs binds guanine nucleo ides wi h low
a ini ies, and binds bo h GTP and GDP simila ly o o he GTPases.
Human GBP1 is able o hyd olyze GTP in wo s eps o GMP80. GBPs
a e able o mul ime ize and o ganize in o esicle-like s uc u es (VLS)
in he cy oplasm81, as has been p oposed o mGBP2 mul ime izing
wi h mGBP1 and mGBP3 82.
Human and mouse GBP1, GBP2, and GBP5 con ain a mo i in he C-
e minus, he CaaX sequence, ha is pos ansla ionally modi ied by
isop enyla ion, which is equi ed o memb ane in e ac ion 83,84 eg wi h
he Golgi complex memb ane 81,85 while o he s a e p edominan ly
cy osolic 76,83.
1.3.2.2. Role o GBP agains pa hogens
GBPs play an impo an ole in de ense o he o ganism agains a
b oad a ay o pa hogens such as i uses, bac e ia and pa asi es 86.
The an i-pa hogen ac ions o GBPs 87 a e well cha ac e ized in bac e ial
in ec ions o medical signi icance ac oss he human popula ion, such as
Chlamydia, Salmonella, Lis e ia, F ancisella no icida, and Legionella
pneumophila in ec ions 83,88–90.
Rec ui men o GBPs o se e al bac e ia ha e an e ec on bac e ial
su i al 86, a good example o which can be seen in he ec ui men o
hGBP1 and hGBP2 and mGBP1, mGBP2, mGBP3, mGBP6, mGBP7,
mGBP9, and mGBP10 in he con ol o Chlamydia 91.
To ul il hei an i-mic obial unc ions, GBPs use mul iple mechanisms.
P edominan ly cy osolic GBPs o GBPs p esen in esicle like
s uc u es (VLS) ac as an e ec o , by associa ion wi h memb anous
compa men s 31,33,84 and as a con ibu o o o he mechanisms, such
as ec ui men o o he e ec o complexes o p oduc ion o eac i e
22
DNA sensing leads o in lammasome ac i a ion and hos cell dea h
118,119. Recen s udies 118,120 in he mechanisms o ac ion o GBP
p o eins in esis ance agains in acellula bac e ia eleased in o he
cy osol sugges ha binding o GBPs o he pa hogen su ace ia a
di ec in e ac ion wi h bac e ial su ace lipopolysaccha ides is essen ial
o he ac i a ion o downs eam e ec o mechanisms. I is possible o
imagine simila e en s in he ole o GBPs in esis ance o Toxoplasma.
1.4. Ubiqui ina ion as a mechanism o cellula immuni y
agains in acellula pa hogens.
The ac i a ion o cellula immune esponse, om pa hogen ecogni ion
o pa hogen elimina ion in ol es complex signaling pa hways, including
s ingen y egula ed pos ansla ional modi ica ion by ubiqui ina ion.
Ubiqui ina ion is he mos common, complex, conse ed and e sa ile
pos - ansla ional modi ica ion in euka yo es121. Unde s anding he oles
o ubiqui ina ion in egula ing cellula unc ions equi es an
unde s anding o he componen s o he ubiqui ina ion mechanism.
1.4.1. S uc u e o ubiqui ina ion p ocess
Ubiqui ina ion is a co alen a achmen o he 76 aa p o ein, ubiqui in
(Ub), by an exposed C- e minal ail o lysines om o he p o eins o
modi y i s ac i i ies. Ubiqui in uses se en in e nal lysines (K6, K11,
K27, K29, K33, K48, K63) and me hionine a posi ion 1 (Me 1), o bind
o lysine esidues o a p o ein subs a e, including ubiqui in i sel o o m
polyubiqui in chains 122.

23
The ubiqui in molecule including i s se en lysine esidues (K6, K11, K27, K29, K33,
K48, and K63), he N- e minus amino g oup (Me 1) and he ca boxyl e minus 123.
Ubiqui ina ion is he esul o a h ee-s ep enzyma ic cascade. The
eac ion s a s wi h he Ub-ac i a ing E1 ac i a ing a ubiqui in molecule
in i s C- e minal glycine esidue (Gly76), o bind an E1 speci ic cys eine
esidue. Then he ac i a ed Ub is ans e ed o o m an E2-Ub
hioes e complex (Figu e 8). In he inal s ep o he eac ion, one o
hund eds o ubiqui in ligase E3 p o eins ans e s Ub o he amino
g oup o a lysine esidue on he subs a e p o ein 121,124,125 (Figu e 8).
This modi ica ion is a con inuously e e sible p ocess ca ied ou by
deubiqui inases (DUBs), which emo e ubiqui in om he a ge ed
p o eins126.
Figu e 1. 6. Enzyma ic machine y ha leads o subs a e o assembly o ubiqui in
(Ub) chains on a subs a e
Ac i a ion
Conjuga ion
Liga ion
Figu e 1. 5. Ubiqui in and i s lysine esidues
24
The coo dina ed ac i i y o h ee classes o enzymes is equi ed o ubiqui ina ion. The
ubiqui in-ac i a ing (E1) conjuga es wi h ubiqui in o ans e i o a ubiqui in-conjuga ing
(E2), ha wo ks in conce wi h E3 ubiqui in-ligase enzyme (E3) o o m a bond
be ween ubiqui in and on he subs a e p o ein. The subs a e can be he a ge o
single ubiqui in moie ies (monoubiqui ina ion) o by mul iple ubiqui in molecules, which
a ach o he p e iously a ached ubiqui in (polyubiqui ina ion)127.
The di e en ways ha di e en linkages bind o he subs a e, which
can be jus one ubiqui in molecule o se e al molecules
(polyubiqui ina ion) h ough he same link (homo ypic) o h ough
di e en linkages (mixed and b anched) (Figu e 8), allow o a g ea
di e si y o ubiqui ina ion opologies ha o m he “ubiqui in code”,
which ansla es in o di e en and gene alized epe oi e o biological
ou comes 128.
1.4.2. Role o ubiqui in in immuni y agains pa hogens
The ansla ion o he “ubiqui in code”, de ines p o ein unc ion, p o ein
localiza ion and p o ein-p o ein in e ac ions ha may be included in
pa hways used by he cell in he immune esponse o pa hogens 129,130.
Ubiqui ina ion has eme ged as an immune de ence mechanism agains
pa hogenic o ganisms 131, no only by egula ing key and p ac ical
aspec s o he au onomous cell esponse bu also o pa hways
ac i a ed by ecogni ion o PAMPs 132,133. Howe e , he ubiqui in signals
in he con ex o in ec ion a e no ully unde s ood.
The ansla ion o he ubiqui in code h ough ecogni ion o he
ubiqui in-binding domain (UBD) d i e deg ada i e and non-deg ada i e
unc ions.
The unc ions o he wo mos abundan linkages in mammalian cells,
K48-linked and K63-linked chains 128 a e he bes cha ac e ized.
Howe e , knowledge abou he so-called "a ypical" chains o med by
connec ions h ough K6, K11, K27, K29, K33 and Me 1, also in ol ed in
25
he immune esponse is con inuously imp o ing 122. K48, K11 and K29
poly-Ub chains and all he e o ypic Ub chains which include K48
b anched Ub chains, can signal o p o easome deg ada ion 122,129,
whe eas K63 Ub chains a e associa ed wi h lysosomal deg ada ion
126,128,134, and K11 and M1 a e in ol ed in he c i ical s ep o NF-κB
ac i a ion 122,135.
1.4.2.1. Ubiqui in-dependen an imic obial au ophagy
Ubiqui ina ion egula es one o he mos ele an cell au onomous
mechanisms o he elimina ion o in acellula pa hogens, namely
selec i e au ophagy o xenophagy. Ubiqui ina ion is hus bo h
implica ed in au ophagosome biogenesis, h ough modi ica ion o
au ophagy-inducing ac o s which ac a di e en s ages 129,132,136,137, as
well as in he binding o he pa hogen o he au ophagosome o
lysosomal deg ada ion o o o he unknown pu pose.
P o ein ubiqui ina ion ma ks a pa hogen o xenophagy-dependen
deg ada ion ia ecogni ion by he au ophagy adap e s - p o eins ha
possess a ubiqui in binding domain (UBD) as well as an LC3-
in e ac ing egion (LIR) mo i – ollowed by ca go deli e y o au ophagy
pa hways 138.
Ubiqui ina ion is in ol ed in he elimina ion o many bac e ia including
Mycobac e ium ube culosis, Lis e ia monocy ogenes 139,140 and
Chlamydia achoma is 141, bu he bes -s udied example o xenophagy-
dependen con ol is in Salmonella in ec ion142, which sha es some
aspec s o he p ocess wi h o he acuola bac e ia. Exposu e o
Salmonella yphimu ium o he cy oplasm allows PAMP ecogni ion,
which immedia ely igge s assembly o di e en ubiqui in chains on he
su ace o cy osolic bac e ia o wi hin he damaged Salmonella-
con aining acuole 93,132,139,142.
26
Au ophagy ecep o s deli e he bac e ia o he au ophagy machine y o
be ul ima ely deg aded by lysosomes 138. In addi ion o inducing
au ophagy, hese associa ed ubiqui in chains lead o he ac i a ion o
NF-Kß signalling and cy okine p oduc ion 132.
Following damage o Salmonella-con aining acuole, se e al hos E3 ligases assemble
poly-Ub chains on he su ace o Salmonella, which a e ecognized by he p62, NDP52
and OPTN au ophagy adap e s ha deli e he ubiqui ina ed Salmonella o ma u ing
au ophagosomes o deg ada ion143.
Mo e ecen ly ubiqui in has also been associa ed wi h clea ance o
Toxoplasma gondii 141. Al hough no as de ailed as he s udies o
ubiqui ina ion in Salmonella, some epo s ha e eme ged sugges ing
ha ubiqui in and au ophagic componen s, such as LC3, GABARAP
amily p o eins, p62 and NDP52 assemble a ound T. gondii PVs 131.
No wi hs anding he ac ha ubiqui in is en iched in he PV o T. gondii
in IFNγ-ac i a ed mouse cells, and he con ol o pa asi e in ec ion
depends on au ophagy-p o eins, con ol o T. gondii occu s h ough a
mechanism independen o he o ma ion o au ophagosomes 144–146.
The e is also he mys e ious spon aneous ac i a ion and agg ega ion o
e ec o IRG p o eins in IFNγ-induced and in ec ed o unin ec ed cells
de icien in A g5, A g16L o A g12, 75,113,147, beha iou eminiscen o
Figu e 1. 7. In ol emen o ubiqui in in Salmonella clea ance
27
lack o IRG egula o p o eins, bu occu ing in hei p esence,
sugges ing a ole o hese au ophagic p o eins in IRG p o ein unc ion
ha has no been elucida ed 109,113 (Khamine s hesis).
In human cells ubiqui ina ion seems o media e he elimina ion o he
pa asi e by di e en mechanisms, ei he wi hou he in ol emen o
lysosomal usion in HeLa cells 148,149, o wi h ubiqui in- and P62-
dependen lysosomal usion in HUVECs 149,150, and pe haps wi h
ec ui men o TRAF6, p62, LC3B and GABARAPs 104. The di e en
oles o ubiqui in in downs eam signaling emain o be cla i ied. The
same is ue o he ole ha ubiqui in ec ui ed o he T. gondii PV
plays in he con ol o he pa asi e.
1.5. Toxoplasma e ec o s an agonis s o hos IFNγ- Inducible
gene exp ession.
The success o he obliga e in acellula pa asi e T. gondii is
gua an eed by i s su i al o he immune esponse du ing acu e
in ec ion and con e sion o achyzoi es o b adyzoi es and cys s, hus
allowing he ch onici y and ansmissibili y o he disease wi hou killing
he hos . A he hea o his duali y o in e es s – he pa asi e in e es ed
in being es ablished in he hos and he hos ’s in e es in elimina ion o
he pa asi e - is he p oduc ion o IFNγ. As desc ibed in de ail ea lie ,
IFNγ signaling leads o he induc ion o a se o ISGs ia he JAK-STAT
pa hway, ha media e T. gondii elimina ion 151.
Fo example, IRGs and GBPs colonize and dis up he PVM, leading o
subsequen killing o he eleased pa asi e, esul ing in he con ol o
he apid o acu e phase o pa asi e p opaga ion.
Howe e , o coun e ac he immune sys em's esponse, Toxoplasma
discha ges a se ies o p o eins du ing cell in asion ha modula e a
a ie y o hos p ocesses, including he exp ession and ac i i y o
GTPases 105,152,153.

28
Some o hese i ulence ac o s, such as ROP kinase amily membe s
like ROP5, ROP17, and ROP18 om ce ain so-called “ i ulen ” s ains
o T. gondii, ha e been shown o block IRG- and GBP-dependen killing
o he pa asi e in ce ain s ains o mice including C57BL/6. In such
s ains, T. gondii ROP18 associa es wi h he PVM 113,154–156 o
phospho yla e conse ed h eonine esidues o a leas wo e ec o
IRG p o eins, I ga6 and I gb6 157–160 p e en ing hei oligome iza ion
and in e ac ion wi h he PVM and hus p e en ing PVM dis up ion and
killing o he pa asi e 161. The comple e inac i a ion o GTPases is
gua an eed by he join ac ion o ROP5/ROP18 p o eins and he dense
g anule p o ein GRA7 complex 153,157. Such mouse s ains a e highly
ulne able o i ulen T. gondii s ains, o he disad an age o bo h
mouse and pa asi e, since he ea ly dea h o he hos d as ically
educes he p obabili y o ansmission o he pa asi e. The
polymo phic i ulence ac o s de ine di e en clonal lineages; ype I
pa asi es ca y a combina ion o ROP5 and ROP18 alleles ha con e s
high i ulence in mos labo a o y mouse s ains, while ype II and III
pa asi es ca y di e en ROP allele combina ions ha allow long- e m
su i al om in ec ion in labo a o y mice in labo a o y mice 153.
Some wild-de i ed mouse s ains incuding he Mus musculus
cas aneus mouse s ain CIM om Sou h India, and Mus musculus
musculus s ains PWD and PWK om he Czech Republic a e able o
su i e in ec ion wi h highly i ulen Type I Toxoplasma s ains due o
he exis ence o a speci ic allele o he andem IRG decoy p o ein I gb1-
b2, ha is highly exp essed and in e ac s di ec ly wi h he
ROP5/ROP18 kinase complex, p e en ing he phospho yla ion o IRG
e ec o s, and hus limi ing pa asi e g ow h wi h subsequen su i al o
he in ec ed animal 35 ca ying in ec ious cys s in he b ain a ou ing
pa asi e ansmission.
29
In addi ion, T. gondii in ec ion has been shown o in e e e wi h IFNγ-
STAT1/STAT2 dependen gene exp ession, when T. gondii in ec s cells
p io o encoun e ing IFN 162–164. The Toxoplasma sec e ed p o ein
TgIST (inhibi o o STAT1-dependen ansc ip ion) was shown o block
he STAT1 ansc ip ion ac o in human and mouse cells 116 . The
molecula mechanisms a he nucleus le el, whe e STAT1 is a ec ed,
ha e been explo ed. TgIST is sec e ed in o he hos cy oplasm
immedia ely a e in ec ion and a ics o he hos cell nucleus by MYR
166,167 whe e i ec ui s he Mi-2/NuRD complex o he ac i a ed STAT1
and STAT2 o block he subsequen ansc ip ion o genes ha a e
impo an o con ol o Toxoplasma in ec ion, such as IRGs and GBPs,
IRF1 and iNOS 165,168 and IDO1169. In his way, T. gondii achyzoi es
p oli e a e eely in he impo en hos cell 105. This is p esumably he
si ua ion when he pa asi e i s en e s a hos . Wi hin a day o wo,
howe e , IFNγ p oduc ion will ha e been ini ia ed. La e ounds o
cellula in ec ion by he pa asi e will he e o e con on IFNγ-induced
cells, IRG p o ein media ed esis ance, esul ing in subsequen
di e en ia ion o he non- i ulen slow- eplica ing b adyzoi e s a e and
he o ma ion o essen ially silen b ain and muscle cys s.
1.6. Thesis aims.
The ecogni ion and des uc ion o he T. gondii PV is no well
unde s ood. In i o expe imen s ha e demons a ed ha he pa asi e
con on s in e e on-dependen esis an mechanisms o ches a ed by
IRGs and GBPs. A he single-cell le el immedia ely a e in ec ion IRG
p o eins accumula e on he PVM and pa icipa e in i s dis up ion
leading o elimina ion o he pa asi e and nec o ic dea h o he hos cell
45,73,111,145. I has been shown ha bo h ubiqui in and componen s o an
au ophagic mechanism also assemble a he pa asi opho ous acuole
30
and i has been sugges ed ha GTPases a e ubiqui ina ed on he PVM.
Some pa asi es su i e he in e e on-dependen esponse ini ia ed by
IRGs and he mechanism behind his phenomenon is unknown.
We he e o e aimed o in es iga e:
1. The ac o s ha de e mine he dynamics o he IRG p o ein
ec ui men p ocess on o he PVM, o unde s and i he hos cell
o he pa asi e i sel de e mines he di e ence in IRG loading.
2. The ole o ubiqui in in he esis ance o T.gondii, i.e., whe he
Ub is pa o he IRG-media ed mechanism o no .
31
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Toxoplasma gondii Vi ulence and Hos GBP2 Loading . mSphe e
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& S ein eld , T. The Toxoplasma gondii hop y p o ein ROP18 is
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p o ein GRA7. Cell. Mic obiol. 18, 244–259 (2016).
158. Fen ess, S. J. e al. Phospho yla ion o immuni y- ela ed
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mac ophage su i al and i ulence. Cell Hos Mic obe 8, 484–
54
C- e minal isop enyla ion o he CaaX-box mo i ha acili a es
in e ac ion wi h endomemb anes and T. gondii PVs and pa asi e
memb anes 9,25–30.
In IFNγ-induced, Toxoplasma-in ec ed mu ine cells he e ec o IRG
p o eins (I ga6, I gb6, I gb10 and I gd) accumula e on he PVM 18,26,31,32
and so do p o eins in ol ed in au ophagy 33, ubiqui in machine y
componen s 34,35 and GBPs 26,28,36, leading o i s “ u ling and
esicula ion” ollowed by dis up ion, hen culmina ing in damage o he
pa asi e’s in acellula niche 37–39. Exposed o he cy oplasm, he
pa asi e dies and e en ually, he hos cell unde goes nec o ic cell dea h
39. The mechanisms by which pa asi e and hos cell dea h occu a e no
well unde s ood.
The i ulen s ains o T. gondii use ROP5 and ROP18 o block he
deposi ion o GKS p o eins on hei PVMs, h ough a ge ed
phospho yla ion o he nucleo ide-binding domain 40,41.
T. gondii has he abili y o in e e e wi h he exp ession o IFN-
esponsi e genes. I T. gondii en e s a cell ha is no induced wi h
IFNγ, subsequen induc ion o esis ance by IFNγ is inhibi ed 42,43.
Al hough IRGs ha e been in ensi ely s udied and much da a has
es ablished he impo ance o GTPase ec ui men o PVs in he con ol
o T.gondii, i is no known how he IRGs selec PVs o des uc ion and
elimina ion. He e we explo e IRG ec ui men o he PVM o unde s and
wha ac o s de e mine he selec ion o pa icula PVs o be co e ed by
he IRGs and hus elimina ed, o no co e ed by he IRG and o pe sis
in he cell. We ask whe he hese a e pa asi e-de i ed o cell in insic
ac o s.

55
2.3. Ma e ial and Me hods
2.3.1. Ma e ial
2.3.1.1. Mammalian cells and media
Mouse emb yonic ib oblas s (MEFs) and diaph agm-de i ed cells
DDCs om C57/BL6 we e cul u ed in Dulbecco’s modi ied Eagle’s
medium (DMEM), high glucose w/o L-glu amine w/o sodium py u a e
(DMEM) supplemen ed wi h 10% hea -inac i a ed e al bo ine se um o
HI-FBS, 2mM L-glu amine, 1mM sodium py u a e, 1x non-essen ial
amino acids, and 1x penicillin/s ep omycin. Human Fo eskin ib oblas s
(Hs27) ob ained om ATCC we e main ained in Isco e's Modi ied
Dulbecco's medium, high glucose (IMDM) supplemen ed wi h 5% hea -
inac i a ed e al bo ine se um, 2mM L-glu amine, 1mM sodium
py u a e and 1x non-essen ial amino acids wi hou an ibio ic.
Fo expe imen s wi h immo alized MEFs, MEFs we e p epa ed om
mice a day 12-14 pos coi um and immo alized wi h DNA: pSV3-neo
plasmid using FuGENE 6 44 o Sc een ec A.
Immo alized MEFs we e also p epa ed om a new C57BL/6 mouse
s ain in which bo h isomo phs o I gb6 we e emo ed by C ispR-Cas9
(Claudia Campos, unpublished esul s)
MEFs we e used in he ollowing condi ions:
1. Fo mic oscopy expe imen s:
• 20000 cells/well pla ed on 13mm co e glass in o 24 well pla es
• 20000 cells/well pla ed on Lab-TekII chambe slide 4 well
• 10000 cells/well pla ed on Lab-TekII chambe slide 8 well
56
2. Fo Wes e n blo expe imen s:
• 250000 cells/well pla ed on i6 well pla es
Fo T. gondii main enance Hs27 (ATCC (CRL-1634)) we e used.
2.3.1.2. Toxoplasma gondii s ains
All he expe imen s we e pe o med using he ype II Me4945, PRUku80,
PRUku80∆IST, PRUku80∆IST+IST-HA 46 (kind gi s om D . Hakimi)
and ype I RH 47 T. gondii s ains.
2.3.1.3. P ima y Imuno eagen s:
Ra monoclonal an i-GRA7 (2.1.2) used a 1/500, abbi se um an i-
I ga6 (165/4) used a 1/4000, abbi se um an i-I gb6 (141/3 ) used a
1/2000, abbi se um an i-I gb10 (940/6 ) used a 1/2000, abbi se um
an i-I gd (081/3 ) used a 1/3000, abbi se um an i-I gb1 (954/1 ) used
a 1/4000, mouse monoclonals an i-I ga6 (10D7 /1000) ) used a
1/1000, mouse monoclonal an i-I gb6 (B34)48 used a 1/2000, abbi
se um an i-GBP149 (kindly p o ided by D . E a F ickel) used a 1/500.
An ibody e e ences ollowed by slash-numbe indica e he numbe o
he abbi bleed.
2.3.1.4. Seconda y an ibodies
Goa an i- abbi Alexa 488, donkey an i- abbi Alexa 488, goa an i- a
Alexa 647, donkey an i-mouse Alexa 555 and donkey an i-mouse Alexa
488, (Molecula P obes) we e used a 1:1000 o immuno luo escence.
57
2.3.2. Cell-biological me hods
2.3.2.1. Passaging, eezing and hawing o mammalian
cells
The C57/BL6 MEFs we e main ained in cul u e by egula passages o
48 hou s, no exceeding a maximum o 15 passages. Regula ly he
cells wen h ough he hawing and eezing p ocesses. Aliquo s o cells
om ea lie passages we e ozen, o allow s o age o lowe passaged
cells.
Passaging:
Cells we e washed wi h s e ile PBS 1X a RT and hen de ached wi h
3ml o a solu ion o 1X ypsin-EDTA o 3-5 min a 37ºC, 10%CO2.
T ypsin ac ion was s opped by adding 7ml o wa m comple e DMEM
(DMEM supplemen ed wi h 10% hea -inac i a ed e al bo ine se um o
HI-FBS, 2mM L-glu amine, 1mM sodium py u a e, 1x non-essen ial
amino acids, and 1x penicillin/s ep omycin). Cells we e cen i uged a
20-25ºC a 290g o 5 min and he pelle was esuspended in DMEM
and spli o a new lask.
F eezing:
Cells we e washed wi h s e ile PBS1X, de ached wi h 1X ypsin-EDTA
o 3-5 min a 37ºC, 10%CO2, hen cen i uged a 290g o 5 min a
4ºC. The pelle was esuspended in cold s e ile FBS-10%DMSO and
hen ans e ed in o c yo ubes in he cold (M . F os y) o -80º C.
Thawing:
Cells we e apidly esuspended in 15ml o comple e DMEM a 37ºC
hen placed in a T75 lask. A e a achmen , he medium was
eplaced.
58
2.3.2.2. P opaga ion o T. gondii
Tachyzoi es we e main ained by se ial passages o 48-72 hou s in 25
cm2 lasks con aining con luen monolaye s o Hs27 cul u ed in
comple e IMDM wi hou an ibio ic. Fo collec ion o achyzoi es, he
suspension om lysed and sc aped HFFs was ha es ed and passed
h ough a 25G sy inge needle se e al imes. The pa asi e suspension
was exposed o wo ounds o di e en ial cen i uga ion o emo e cell
deb is, he i s a 100 g o 5 min RT, ollowed by cen i uga ion o he
supe na an a 700g o 15 min RT. Tachyzoi es we e esuspended in
2ml o medium, and iable pa asi es we e coun ed in a Neubaue
chambe using ypan blue, and immedia ely used o in ec ion
expe imen s and o p opaga ion.
2.3.2.3. Cell induc ion wi h IFN-
γ
and In ec ion wi h T.gondii
A e 24 hou s o g ow h ei he on UV-s e ilized 13mm diame e co e
glass o o he cul u e su aces, o he egula expe imen s, mu ine
ib oblas s we e induced wi h mouse IFNγ (Ca . 315-05 Pep o ech) a
40 ng/ml (200 U/ml) o 18 o 24 hou s o we e le un ea ed. The
cul u es we e hen inocula ed wi h a small olume o a esh
suspension o T. gondii ME49 achyzoi es a a equi ed mul iplici y o
in ec ion (MOI) o 1,5 o 10 and kep a 37°C, 10% CO2 o he equi ed
ime o he essay.
Fo he expe imen s o s udy pa asi e in e e ence wi h he exp ession
and ec ui men o IRG p o eins in he PVM, cells we e exposed o IFN-
γ o dec easing ime pe iods (24, 12, 10.5, 6, 4.5, 3, 2, 1 and 0 hou s
be o e in ec ion) un il hey eached ime 0 when hey we e inocula ed
wi h achyzoi es a a equi ed mul iplici y o in ec ion (MOI) o 1,5 o 10
and kep a 37°C, 10% o CO2 o he equi ed ime o he assay.
59
2.3.3. Immuno luo escence
Fo isualiza ion o he loading o IRG p o eins and GBP1 on o he
PVM by immuno luo escence, MEFs we e allowed o adhe e on a UV
s e ilized co e slip o in s e ile Lab-Tek chambe s o 24 hou s. 24, 12,
6, and 0 h s IFNγ induced and uninduced cells in ec ed wi h Me49
achyzoi es we e washed wice wi h PBS1X supplemen ed wi h 1,8mM
o CaCl2 and 0,8mM o MgSO4 and ixed in 4% pa a o maldehyde
(PFA) o 30 min a oom empe a u e (RT). Be o e
immunos aining, he cells we e washed wice wi h PBS, pe meabilized
in 0.1% saponin o 10min and blocked in blocking bu e (3% bo ine
se um albumin (BSA) and 0,1% saponin) o 1h a oom empe a u e
(RT). These cells we e hen s ained wi h indica ed an ibody eagen s
speci ic o T. gondii GRA7, o one o mo e IRG p o eins (165, 141,
940, 81) and o GBP1, all dilu ed in blocking bu e , o a leas 1 hou
a RT.
Subsequen ly, a e h ee washes wi h blocking bu e , he cells we e
s ained wi h Alexa Fluo -conjuga ed seconda y an ibodies: AF488
donkey an i- abbi , AF647 goa an i- a , and AF555 donkey an i-mouse
dilu ed in blocking bu e , o a leas 45 min a RT. Cell nuclei/DNA
we e labelled by 4’,6-diamidino-2-phenylindole (DAPI D3571— 889 Li e
Technologies) The s ained cells we e washed and moun ed on glass
mic oscope slides wi h P olong gold an i ade eagen . Fo Lab-Tek
chambe s he whole immuno luo escence p ocedu e ook place in he
small chambe s whe e he cells g ow on a s anda d mic oscope slide.
When he p ocedu es we e comple ed, he uppe s uc u e was
emo ed, and he samples we e co e ed wi h co e slips in p esence o
P olong gold an i ade eagen .

60
2.3.4. Analyses o cellula p o eins
2.3.4.1. Wes e n blo analysis
Lysa e p epa a ion om in ec ed cells.
2x105 MEFs we e seeded on day -2, s imula ed wi h 200U/ml o IFNγ
du ing day-1 o 24 hou s (con ol) and o he equi ed imes (12, 6, 3, 2,
1 and 0 hou s) be o e in ec ion, and hen in ec ed wi h Me49
achyzoi es o he equi ed imes. F om hese and con ol non-in ec ed
cells he supe na an was emo ed and he cells washed wice wi h
PBS 1X, hen lysed in 500µl o cold lysis bu e (20 mM T is/HCl (pH
7.6), 140 mM NaCl, 5 mM MgCl2, 0.5% NP-40, supplemen ed wi h
Comple e P o ease Inhibi o s able s (Roche)) o 30min on ice. The
lysa e supe na an was collec ed a e 30 minu es o 14000 pm
(>16000 c ) cen i uga ion a 4ºC. The concen a ion o he lysa e was
de e mined by BCA assay (below) hen dilu ed wi h lysis bu e o
equalize sample concen a ion.
BCA assay
The p o ein concen a ions o he samples we e de e mined based on
colo ime ic de ec ion using he The mo Scien i ic™ Pie ce™ BCA
P o ein Assay, acco ding o he manu ac u e ’s ins uc ions.
The concen a ion was de e mined in compa ison wi h a bo ine se um
albumin (BSA) S anda d. A se ial dilu ion o BSA and BCA wo king
Reagen was p epa ed. The samples (BSA s anda ds and unknown)
we e mixed wi h BCA in a 1:20 a io and a e 30 minu es incuba ion a
37ºC, he abso bance was measu ed a 562nm.
Immunoblo ing Analysis
Samples in 1X p o ein sample bu e (375 mM T is.HCl, 9% SDS, 50%
Glyce ol, 0.03% B omophenol blue and 20% β-me cap oe hanol) we e
61
dena u ed a 90ºC o 3min. 10 o 20 µg o p o ein we e sepa a ed on
10% Sodium dodecyl sulpha e polyac ylamide gel elec opho esis
(SDS-PAGE) wi h a PageRule TM P es ained P o ein Ladde . The
SDS-PAGE was un in unning bu e (250mMT is, 192mM glycine,
1%SDS in wa e ) a 120V o 150V un il he desi ed p o ein sepa a ion
had been achie ed.
The p o eins we e elec opho e ically ans e ed in memb ane
elec opho esis bu e (25mM T is base, 193,7mM Glycine and 20%
MeOH in wa e ) in a semi-d y ans e appa a us (Bio-Rad T ans-Blo
SD Cell) a 5,5mA/cm2 a a maximum o 25V. Gel and memb ane we e
be ween ou shee s o we Wha man pape .
Immunoblo s aining
The memb ane was blocked in milk (powde , comme cial supe ma ke
b and) (5% milk, 0.1% Tween20 in PBS1X, PBST) o 1 hou a RT
hen incuba ed wi h p ima y an ibody agains I ga6 (165, 1:25000) and
I gb6 (141, 1:8000) dilu ed in 0,1% PBST o 1 hou . The unbound
p ima y an ibodies we e washed h ee imes wi h 0,1% PBS/T and he
memb anes hen incuba ed wi h an i- abbi 680 o 800 DyLigh TM
(Rockland) (1:10000) in he da k o 1 hou . Memb anes we e washed
3x in PBS/T and 1x in wa e and hen scanned in an Odyssey CLx
Imaging Sys em.
2.3.5. Mic oscopy and image analyses
2.3.5.1. Fluo escence mic oscopy
Wide ield images we e acqui ed on a Zeiss Axio Obse e Z1
luo escence mic oscope, using ei he a 40x 0.94NA o 63x 1.3NA
objec i e, equipped wi h a Hamama su FlashLT came a, a Zeiss Colib i
62
luo escence illumina o , and app op ia e luo escence il e s all
con olled by he Zen 2011 so wa e.
2.3.5.2. Blind and unbiased analysis
To ensu e eliable analysis o isual images, he mic oscope slides
p epa ed and labelled we e blinded wi h adhesi e ape be o e imaging.
When his was no possible, ano he pe son encoded he iles wi h
acqui ed images be o e quan i ica ion. To minimize bias in he
semiau oma ic quan i ica ion, by applying he mac o (desc ibed in de ail
below), he IRG and GBP1 p o eins we e e alua ed only in acuoles
i s selec ed based on GRA7 exp ession, showing ha he o ganism is
in acellula .
2.3.6. Quan i ica ion o IRG p o eins and GBP1 on he PVM
2.3.6.1. P opo ion o coa ed acuoles
Fo each condi ion a leas 100 GRA7 labelled acuoles we e sco ed.
The equency and in ensi y o di e en IRG p o eins and GBP1 on he
PVM on IFNγ-s imula ed and uns imula ed cells was de e mined and
a ed as a posi i e o nega i e. The isual es ima e o "posi i e" o
"nega i e" was u he e ined by quan i ica ion as below.
Mac o design
An ImageJ mac o was c ea ed o au oma e he quan i ica ion o p o ein
in ensi y a he PVM, by measu emen o luo escence in de ined a eas,
based in GRA7 iden i ica ion.
The mac o assumes a composi e image in czi o ma and based on
sc ip s, di ides i in o 3 dis inc egions: PV, PVM and cy oplasm om
which he luo escence in ensi y is quan i ied.
63
The Mac o and i s uses a e desc ibed in ull in 50.
2.3.6.2. Quan i ica ion o p o ein in ensi y.
The amoun o IRG o GBP1 p o eins p esen on he PVM in IFNγ-
s imula ed and non-s imula ed MEFs was de e mined by measu ing he
luo escence in ensi y o he luo och ome co esponding o each
p o ein using a semi-au oma ed sys em on he Fiji 2.0.0- c69/1,52i
so wa e 51.
Pixel in ensi ies o he luo och omes ha labelled de ined indi idual
a ge an igens, usually wi h a seconda y an i-Ig eagen exci ed a 488
nm, we e measu ed in GRA7-posi i e acuoles, de ec ed wi h
luo escen seconda y an i- a Ig eagen exci ed a 647 nm, by using
he c ea ed mac o on ImageJ.
The czi composi e luo escen images we e loaded in he Fiji so wa e,
hen he mac o was un. The GRA7 channel was used o de ec
in acellula PVs wi hou bias. The ole ance was adjus ed o each
case as necessa y. The wand ool h ough he ole ance allows he
adjus men o he PV line o include some ou o ocus and sca e ed
ligh associa ed wi h he acuole.
The backg ound signal in he cy osol is an impo an a iable. Ve y
o en, IRG p o eins and GBP1 we e de ec ed using abbi i s -s age
an i-IRG an ise a ha ecognise cy osolic and inac i e IRG p o eins, as
well as ac i a ed p o eins on he PVM (See Figu e 2.1). In hese cases,
when he cy osolic backg ound was ma kedly oo b igh and o he s
when he cy sosol was no e y b igh , he cy osolic luo escence
in ensi y was de e mined and sub ac ed. The pixel in ensi y a he PVM
was de ined as he o al excess pixel in ensi y on he PVM a e
backg ound sub ac ion.
70
ollowed by he combina ion o I gb6 wi h I ga6 (85%), I ga6 wi h I gb10
(80%). No mally abou 10% o PVs we e no co e ed by any IRG
p o eins (Figu e 2.4. A).
These esul s la gely con i med he conclusion o Khamine s e al, in
which he se o I ga6-posi i e acuoles is included in he la ge se o
I gb6-posi i e acuoles, and he se o I gd-posi i e acuoles is in u n
included in he la ge se o I ga6-posi i e acuoles, like “Russian
Dolls”. Khamine s e al no ed ha I gb10 is loaded on o almos as many
acuoles as I gb6 and assumed ha hese wo se s we e e ec i ely he
same. PVs co e ed wi h IRG p o eins lowe in he hie a chy we e
essen ially comple ely included in PVs loaded wi h he IRG p o eins
highe in he hie a chy: 97% and 98% o PVs wi h I ga6 we e included
in I gb6 and I gb10 espec i ely, and 94% o I gb10 posi i e PVs we e
included in I gb6 co e ed PVs (Figu e 2.4. B). The excep ion is I gb10,
whe e he p opo ions o acuoles co e ed by I gb6 and I gb10 a e
consis en wi h independence.
The mos abundan ly ep esen ed IRG p o eins o he hie a chy o PV
loading include almos comple ely he less abundan ly ep esen ed
I ga6 and I gd 31. This esul s in a he e ogenei y in acuoles, some
co e ed by 3 IRGs (b6+a6+d), o he s by 2 (b6+a6) and 1 (b6 only) and
no IRGs, which may esul in di e en ou comes o he pa asi e. We
show in Chap e 3 ha he subse o acuoles wi h I gb6 alone is less
likely o be dis up ed ha hose wi h 2 o 3 IRG p o eins. The posi ion o
I gb10 in his dis ibu ion is no en i ely clea . I is, howe e , a well-
ep esen ed IRG p o ein on acuoles, pe haps e y sligh ly lowe han
I gb6, and is he e o e p esen on mos acuoles ca ying IRG p o eins
lowe in he hie a chy, bu seems o load independen ly o I gb6,
sugges ing he e may be a small numbe o acuoles loaded wi h
I gb10 alone.

71
Figu e 2.4. Di e en e ec o IRG co-localize o he same PVM.
WT C56BL/6 MEFs we e ea ed o no wi h IFNγ (200 U/ml) o 24 hou s and in ec ed
wi h T. gondii Me49 o 90 minu es. Cells we e washed, ixed and s ained o GRA7 (JH
2.1.2 mAb) wi h Alexa Fluo 647-conjuga ed seconda y an ibody (magen a) o de ine
in a acuola o ganisms, and o he ollowing pai s o indi idual IRGs: I ga6 (se um
165) and I gb6 (se um 141/1); I ga6 (10D7) and I gb10 (se um 940/6); I gb6 (B34) and
I gb10 (se um 940/6) in each case wi h an i- abbi Alexa Fluo 488-conjuga ed
seconda y an ibody (g een) o an i-mouse Alexa Fluo 555-conjuga ed seconda y
an ibody ( ed). The nuclei we e labelled wi h DAPI (cyan). The quan i ica ion o
luo escen images shows A) he pe cen age o GRA7+ PVs wi hou IRG p o ein
labeling (in g ay) and PVs labelled o one (in ed and g een) o bo h (in o ange) IRG
p o eins; B) he pe cen age o he o al PVs labelled o one o bo h IRG p o eins. A)
and B) he ange be ween h ee (1s and 2nd columns) and wo ( hi d columm)
independen expe imen s was shown.
2.4.3. The ec ui men o I ga6 o PVs is independen o he
ec ui men o I gb6
P e ious wo k 31 showed ha I gb6 loads ea lie on o he PVM han
I ga6 and I gd. This has been in e p e ed o mean ha I gb6 ac s as a
"pionee ", coming i s o he PVM and acili a ing he subsequen a i al
o I ga6 and I gd. One possible iew o his "pionee " ac ion could be
ha he in e ac ion o I ga6 wi h he PVM also equi es a ini y be ween
I ga6 and bound I gb6, and likewise o I ga6 p o iding assis ance o
he subsequen binding o I gd.
AB
0
20
40
60
80
100
120
Copy o 136 and 136B
Posi i e PV (%)
Only IRG below
Bo h
Only IRG abo e
I ga6 I ga6 I gb6
I gb6 I gb10 I gb10
n= 278 292 255
0
20
40
60
80
100
120
Posi i e PV (%)
136, 136B and E24.06.21
516 784 285n=
I gb6
I ga6
+
I gb6
I ga6
I gb10
I ga6
+
I gb10
I ga6
I gb10
I gb6
+
I gb10
I gb6
GRA7 GRA7 GRA7
0
20
40
60
80
100
120
%136,136B E.24.06.21_wi hou GRA7
Posi i e PV (%)
I gb6 I gb10 I gb10
n= 474 508 255
I ga6 I ga6 I gb6
72
I such a model is co ec , he numbe o binding si es o I ga6 should
be posi i ely co ela ed wi h he numbe o I gb6 molecules bound o
he PVM. To de e mine i he pionee I gb6 se es as a pla o m o
ancho I ga6, he indi idual loading o he I ga6 and I gb6 in ensi ies in
he PVM a ea we e measu ed and he da a we e subjec ed o
co ela ion analysis. The su p ising esul was ha he loading
in ensi ies o I ga6 and I gb6 a e no co ela ed (Figu e 2.5. A). Abou
20% o acuoles we e loaded wi h nei he I gb6 no I ga6 ( he IRG-
nega i e subse ). The p esence o hese double-nega i e acuoles
con ibu ed somewha o he co ela ion coe icien , and as expec ed,
emo al o double nega i es educes he co ela ion coe icien e en
u he .
Many acuoles showed I gb6 and I ga6 wi h opposi e in ensi ies
(Figu e 2.5. B), con a y o expec a ion i I gb6 is ac ing as a o m o an
ancho o binding si e o I ga6. This esul was so unexpec ed ha we
sough o alida e he app oach in co ela ion expe imen s by assaying
as a posi i e con ol wo an ibody eagen s, he abbi an i I ga6 se um
165 and he mouse an i I ga6-speci ic monoclonal an ibody 10E7
agains a single IRG p o ein, I ga6. The in ensi ies o hese wo
eagen s on indi idual acuoles we e s ongly posi i ely co ela ed as
expec ed (Figu e 2.5. C).
The esul s e ealed no dependence be ween ec ui men o I gb6 and
I ga6, al hough all I ga6-posi i e acuoles a e included in he se o
I gb6 posi i e acuoles, sugges ing ha I ga6 may bind o a di e en
a ge in he PVM and no o I gb6 di ec ly. Ne e heless, he loading
hie a chy would sugges ha , quali a i ely bu no quan i a i ely, he
p esence o a ge s o I ga6 binding is co ela ed wi h he p esence o
a ge s o I gb6 binding.!
73
Figu e 2.5. I gb6 and I ga6 on PVM a e no co ela ed.
WT C56BL/6 MEFs we e ea ed wi h IFNγ (200 U/ml) o 24 hou s and in ec ed wi h T.
gondii Me49 o 90 minu es. Cells we e washed, ixed and s ained o GRA7 (JH 2.1.2
mAb) wi h Alexa Fluo 647-conjuga ed seconda y an ibody, and o he ollowing pai s
o indi idual IRGs: I ga6 (10D7) and I gb6 (se um 141/1); I ga6 (se um 165) and
(monoclonal ab 10E7), wi h an i- abbi Alexa Fluo 488-conjuga ed seconda y an ibody
(g een) and an i-mouse Alexa Fluo 555-conjuga ed seconda y an ibody ( ed). The
nuclei we e labelled wi h DAPI (cyan). The quan i ica ion o luo escen images shows
he in ensi y o I ga6 and I gb6 on he PVM a ea o e e y GRA7 posi i e PVs. A)
Pea son co ela ion analysis was pe o med P alue > 0,05. B) The in ensi y o he
IRGs is o ganized on wo Y-axes ep esen ing I gb6 and I ga6 in ensi y, wi h each line
ep esen ing a PVM and he symbols a he ends I gb6 (le ) and I ga6 ( igh ). C) The
in ensi y o I ga6 labelled wi h abbi (165) and mouse (10E7) an i-I ga6 an ibodies was
measu ed and submi ed o Pea son co ela ion analysis P alue < 0,0001.
2.4.4. The binding o I gb6 is also no c ucial o ec ui men
o o he GTPases o he PVs.
Khamine s sugges ed ha he ec ui men o GKS p o eins o he PVM
occu s in a consis en hie a chy, wi h I gb6 a i ing ea lie and la e
s abilizing wi h he a i al o I ga6 and I gd. Acco ding o his model,
loading o I gb6 would be essen ial o he loading o I ga6 and I gd. We
he e o e analyzed whe he he absence o I gb6 a ec s he loading o
he o he GTPases on o he PVM in WT and I gb6 KO (Claudia
Campos, unpublished) DDCs a e 24 hou s o IFNγ induc ion and 90
min o in ec ion wi h Me49. The samples we e s ained o I gb6 wi h a
74
mAb B34, o I gb10 wi h se um 940, o I ga6 wi h se um 165 and o
GBP1 wi h se um an i-GBP1 55 and o GRA7 wi h JH 2.1.2 mAb.
The da a con i med he absence o I gb6 in I gb6KO cells, while he
ec ui men o I gb6 on PVM in WT cells occu ed no mally wi h 80% o
PVs posi i e (Figu e 2.6. A). The p esence o I gb10 on PVM (many
wi h an unusual acuola mo phology (Supplemen al Figu e 2.1.) is
sligh ly a ec ed (in ensi y and p opo ion) in I gb6KO cells wi h
app oxima ely 50% o posi i e acuoles loaded wi h I gb10 agains
app oxima ely 75% in WT cells (Figu e 2.6. B). The ec ui men o I ga6
was ba ely a ec ed by he absence o I gb6 (Figu e 2.6. C), suppo ing
he supposi ion abo e om he lack o a quan i a i e co ela ion
be ween I gb6 and I ga6 loading ha he wo e ec o p o eins bind
essen ially independen ly o di e en a ge s on he PVM. The
ec ui men o GBP1 is also ba ely a ec ed by he absence o I gb6
(Figu e 2.6. D).
75
Figu e 2.6. The ec ui men o o he s GTPases is no g ea ly a ec ed by he
absence o I gb6.
WT and I gb6KO DDCs we e ea ed o no wi h IFNγ (200 U/ml) o 24 hou s and
in ec ed wi h T. gondii Me49 o 90 minu es. Cells we e washed, ixed and s ained o
GRA7 (JH 2.1.2 mAb) wi h Alexa Fluo 647-conjuga ed seconda y an ibody (magen a)
A
B
C
D
WT I gb6KO
I gb6
I ga6
Nuclei
T. gondii
Nuclei
T. gondii
I gb10
Nuclei
T. gondii
WT I gb6KO
WT I gb6KO
Nuclei
T. gondii
GBP1
WT I gb6KO
WT I gb6KO
0
2
4
6
8
10
12
14
16
18
20
Log2 I gb6 pixel in ensi y
I gb6_In ensi y
****
WT I gb6KO
10
12
14
16
18
20
Log2 I ga6 pixel in ensi y
I ga6_In ensi y
ns
WT I gb6KO
6
8
10
12
14
16
18
20
Log2 I gb10 pixel in ensi y
I gb10_In ensi y
***
WT I gb6KO
6
8
10
12
14
16
18
20
Log2 GBP1 pixel in ensi y
GBP1 In ensi y
*
WT I gb6KO
0
20
40
60
80
100
% GBP1_139B&E24.06.21
GBP1 posi i e PV (%)
n=360 311
WT I gb6KO
0
20
40
60
80
100
139C.&E24.06.21. % I ga6
I ga6 posi i e PV (%)
n=458 305
WT I gb6KO
0
20
40
60
80
100
I gb6 posi i e PVs (%)
139C. &E24.06.21.% I gb6
n=481 321
WT I gb6KO
0
20
40
60
80
100
I gb10 Posi i e PVs (%)
139C. &E24.06.21 % I gb10
n=551 285

76
o de ine in a acuola o ganisms, and o he ollowing pai s o indi idual IRGs: I gb6
(mAb B34), I ga6 (se um 165), I gb10 (se um 940/6) and GBP1 se um in each case
wi h Alexa Fluo 488-conjuga ed seconda y an ibody (g een) o Alexa Fluo 555-
conjuga ed seconda y an ibody ( ed). The nuclei we e labelled wi h DAPI (cyan).
Rep esen a i e Images, a e shown. Quan i ica ion o luo escen images shows he
in ensi y o IRG on he PVM and pe cen age o GRA7+ PVs wi h A) I gb6 B) I gb10 C)
I ga6 and GBP1 labeling B) he ange be ween wo (D) and h ee independen
expe imen s was shown (A, B and C). ****, P<0,0001;*** P<0,001; n.s., nonsigni ican .
These da a show ha he p esence o I gb6 may p edic he p esence
o o he GTPases, bu is no equi ed o i . The somewha educed
binding o bo h I ga6 and I gb10 o he I gb6-de icien acuoles is
eminiscen o he educed binding o I gb6 o ME49 PVM in he
absence o I ga6 (Khamine s 2010, Figu e 6.F) 31 a esul in e p e ed a
ha ime in e ms o some kind o in e ac ion be ween he wo e ec o
GTPases esul ing in s abiliza ion on he PVM, and his may s ill be
alid. Ne e heless, i is abundan ly clea ha he binding o a leas
I ga6 is no quali a i ely dependen on bound I gb6 as a “pionee ”.
2.4.5. IRG p o ein accumula ion on PVs s a s ea ly and
inc eases o e ime.
I has been shown ha IFNγ-induced IRG p o eins begin o accumula e
and ac i a e on a leas some acuoles immedia ely a e T. gondii
en y31. I was p e iously shown ha a 90 min pos in ec ion 80% o
90% o PVs we e co e ed by e ec o IRG p o eins (Figu e 2.3). To
unde s and he iming o IRG p o ein ec ui men on o T. gondii PVs
IFNγ s imula ed MEFs we e in ec ed wi h Me49 o 15, 30, 60 and 120
minu es. A e e e y ime poin he samples we e ixed and kep in
blocking bu e un il he las ime poin in ec ion. Samples we e
immunos ained o GRA7 de ec ed wi h seconda y an ibody coupled o
he luo och ome 647, and o I ga6, I gb6, I gb10 and I gd, by using
an ise um 165/4, 141/1, 940/6 and 81/3 espec i ely de ec ed wi h
77
seconda y an ibodies coupled o he same luo och ome.
Subsequen ly, IRG p o ein loading in ensi y was coun ed om o e 100
GRA7 labelled PVs (Ma e ials and Me hods, chap e 2.3.4.3).
The esul s o his expe imen indica e ha IRG p o ein accumula ion a
he PVM s a s soon a e in ec ion. A e 15 minu es o in ec ion a
majo p opo ion o PVs was al eady co e ed by IRGs (abou 60%),
sugges ing, as Khamine s showed, ha he loading o IRG p o eins
on o he PV begins immedia ely a e in ec ion. The p opo ions
inc eased o e ime om 60% o abou 75% hen 81% a 60 minu es.
A 120 minu es pos in ec ion ewe appa en ly in ac PVs (67%) we e
co e ed by IRG p o eins, and mo e IRG-posi i e agg ega es we e
p esen , p obably esul ing om PVM dis up ion (Figu e 2.7. A. and B).
The inc ease in he p opo ion o PVs loaded by IRG p o eins was
accompanied by an inc ease in he p o ein loading as measu ed by
luo escence in ensi y. I was clea ha much o he loading is al eady
comple e 15 minu es a e in ec ion. Subsequen ly, he g ea es
inc ease was egis e ed be ween 15 o 30 minu es, a e which sligh
inc eases in in ensi y we e eco ded. Also, he he e ogenei y o
in ensi y inc eased signi ican ly in his i s 30 minu es o in ec ion
(Figu e 2.7. C). The da a all sugges ed ha he i s minu es pos
in ec ion a e c i ical o IRG ec ui men o he PVM and i s dis up ion,
and con i m he ea lie esul s o Khamine s, who concluded ha
loading o IRG p o eins on o he acuole may begin a he poin and
ime o en y.
78
Figu e 2.7. Accumula ion o IRG p o eins on PVs s a s ea ly and inc eases o e
ime.
WT C56BL/6 MEFs induced wi h IFNγ (200 U/mL) o 24 hou s and in ec ed wi h T.
gondii Me49 o 15, 30, 60 and 120 minu es. Cells we e washed, ixed and s ained
simul aneously o I ga6 (an ise um 165/4, I gb6 (an ise um 141/1), I gb10 (940/6 pAS)
and I gd (an ise um 81/3) wi h he same seconda y an ibody coupled wi h Alexa Fluo
488-conjuga ed seconda y an ibody (g een) and o GRA7 (JH 2.1.2 mAB) wi h Alexa
Fluo 647-conjuga ed seconda y an ibody (magen a). The nuclei we e labelled wi h
DAPI (cyan). A) Images aken in a luo escen mic oscope show me ged GRA7 and
DAPI channels and IRG channels. B) 117, 131, 188 and 222 GRA7 labelled PVs, o
ime poin 15 min o 120min espec i ely, we e classi ied as IRG posi i e (wi h isible
accumula ions o IRG p o eins) o nega i e (wi hou isible accumula ions o IRG
p o eins) and p esen ed in a g aphic as a pe cen age o GRA7 posi i e
acuoles. C) The pixel in ensi y co esponding o he IRG channel o each GRA7-
labelled PVM a each ime poin was quan i ied using Image J Fiji so wa e as
desc ibed in ma e ials and me hods.
15
30
60
120
T.gondii Nuclei I ga6,b6, b10 and d Time o in .
(min)
A
C
B
15 30 60 120
0.0
5.0×104
1.0×105
1.5×105
1.5×105
2.0×105
2.5×105
3.0×105
Time o in ec ion (min)
IRG pixel in ensi y
IRGs 2nd quan i ica ion
15 30 60 120
0
20
40
60
80
100
IRG posi i e PV (%)
IRGs on he PVM
Time o in ec ion (min)
79
2.4.6. The cellula concen a ion o IRGs is no a limi ing
ac o in i s ec ui men o acuoles.
In ag eemen wi h Khamine s da a31 i was demons a ed he e ha 10%
o 20% o he PVs a e ne e co e ed by any IRG p o eins. The inding
opened he in iguing ques ion o wha de e mines whe he PVs a e
co e ed by IRGs o no . A e ac o s in insic o pa asi es o o cells
making his de e mina ion? To ind ou whe he an ob ious po en ial
cellula componen , namely he cy osolic concen a ion o IRG p o eins,
is a key ac o in IRG ec ui men o PVs, MEFs we e s imula ed wi h
IFNγ and in ec ed wi h Me49 a low MOI. The cells we e ixed and
s ained simul aneously wi h an ise a agains I ga6 and I gb6 and
de ec ed oge he wi h AF488 and o GRA7 de ec ed wi h AF555. The
nuclei we e labelled wi h DAPI. Only cells in ec ed wi h a single
pa asi e we e selec ed o classi ica ion o he PVs as a posi i e o
nega i e.!I he cy osolic IRG concen a ion de e mines he in ensi y o
IRG loading, he e should be a clea co ela ion be ween hese wo
a iables. The immuno luo escence da a e ealed he le el o IRGs
exp essed wi hin he cy oplasm, bu con a y o expec a ion he
co e age o PVs by IRGs was no uni e sal. Only abou 75% o he
PVs we e loaded by I ga6 and I gb6. The emaining 25% we e no
co e ed by ei he o hese wo p o eins e en in he case o high IRG
in ensi ies in he cy oplasm, meaning ha IRG p o ein concen a ions
a e, wi hin b oad limi s no a limi ing ac o o PV loading (Figu e 2.8. A
and B). To de e mine whe he he p o ision o cy oplasmic IRGs is
co ela ed wi h he amoun o IRG p esen a he PVM, he p o ein
in ensi ies in he PVM and cy oplasm we e measu ed, and he da a
submi ed o Pea son co ela ion and p esen ed in (Figu e 2.8. C). Mos
su p isingly, he quan i a i e loading o IRG p o ein on o he PVM was
essen ially unco ela ed wi h he IRG concen a ion measu ed in he
cy oplasm.
86
adjacen o indi idual PVs was measu ed. E) Mo e han a hund ed PVs labelled wi h
GRA7 we e e alua ed and he p opo ion o I gb6 and I ga6 posi i e PVs a e shown. F)
A e backg ound in ensi y sub ac ion, he da a was shown as 4 mini-his og ams
showing he equency dis ibu ion o PVM I gb6 in ensi y unde he 4 condi ions. ****,
P<0,0001
Fu he , o de e mine he minimum du a ion o IFNγ s imula ion p io o
in ec ion, o allow an IFNγ dependen esponse agains T. gondii, MEFs
we e p e ea ed wi h IFNγ o 3, 2 o 1 hou s be o e in ec ion,
simul aneously wi h in ec ion and 1 hou a e in ec ion. Cells we e
in ec ed wi h he a i ulen Me49 T. gondii pa asi es o le unin ec ed o
9 hou s. Cell lysa es we e collec ed and analyzed by SDS-PAGE,
ollowed by Wes e n blo ing p obed wi h I gb6 and inculin an ibodies,
as desc ibed p e iously in Ma e ials and Me hods. This p ocedu e
showed a decline o I gb6 exp ession le el wi h he educ ion o IFNγ
p e-s imula ion du a ion in cells wi h Me49 (Figu e 2.11. A).
The examina ion o luo escen images showed ha when cells we e
in ec ed simul aneously wi h IFNγ s imula ion ewe han 30% o cells
exp ess ei he I ga6 o I gb6 (Figu e 2.11. B and C).
The esul s showed a speci ic impai men o IRG p o ein exp ession in
pa asi e-in ec ed cells. Abou 65% o he cells ha do no exp ess IRG
wi hin he cy oplasm con ain di iding pa asi es in hei cy oplasm while
ewe han 20% o cells ha exp ess IRGs con ain pa asi es (Figu e
2.11. B) (Figu e 2.11. B and D). Taken oge he , hese esul s
demons a ed ha , in he absence o a su icien ly long IFNγ p e-
induc ion, T. gondii in e e es wi h subsequen IRG exp ession.

87
Figu e 2.11. Sho ime p es imula ion wi h IFN-γ allow T. gondii o educe and
supp ess IRG exp ession.
A) C56BL/6 MEFs we e s imula ed wi h IFNγ o indica ed pe iods (3, 2, 1, hou s p io
in ec ion), simul aneously wi h in ec ion and 1 hou pos in ec ion hen in ec ed
wi h T,gondii Me49 o le unin ec ed o 9 hou s. To al I gb6 exp ession was de ec ed
om cell lysa es wi h 141 an ise um as desc ibed in ma e ials and me hods. Vinculin
le els a e shown as a loading con ol. B) C56BL/6 MEFs simul aneously in ec ed wi h
Me49 and s imula ed wi h IFNγ o 13 hou s we e washed and s ained wi h
immuno eagen s agains I gb6 (141) o I ga6 (165/3) using seconda y an ibodies
coupled o AlexaFluo 488 (g een) and agains GRA7 (JH 2.1.2 mAB) using seconda y
an ibodies coupled o AlexaFluo F555 ( ed). Nuclei we e labelled wi h DAPI (blue). C)
The p opo ion o he wo popula ions o cells: I gb6 o I ga6 posi i e (g ey) and I gb6 o
I ga6 nega i e (black) we e ob ained o a o al o 167 and 232 cells espec i ely. D) The
I gb6 and I ga6 posi i e (+) and nega i e (-) popula ions o he cells in C we e classi ied
as in ec ed (black) o non-in ec ed (g ey) wi h T. gondii based on GRA7 labelled PVs.
****, P<0,0001
88
2.4.8. Toxoplasma gondii educes IRG p o ein exp ession
and loading in he absence o TgIST
Khamine s 31 showed ha he ini ia ion o IRG loading on o PVs has a
s ochas ic ime dependence: some acuoles a e loaded by IRGs wi hin
a couple o minu es a e en y and PV o ma ion, whe eas o he
acuoles a e loaded nea ly hal an hou la e .
We hypo hesized ha an ex ended s ochas ic delay in IRG loading may
p o ide he necessa y ime o modi ica ion o he PVM induced by he
enclosed pa asi e, enabling some kind o “ma u a ion” o he acuole
ha migh ul ima ely comple ely p e en i s loading by IRGs, and
he eby pe haps p o ide some explana ion o he pe sis en
obse a ion o abou 20% o acuoles ca ying no IRG p o eins.
P e ious s udies showed ha when T. gondii in ade cells ha a e no
p imed wi h IFNγ, he pa asi es block signaling downs eam o IFNγ ia
TgIST 46,61 which, as shown abo e, in u n blocks exp ession o IRG
p o eins 46 .
Howe e he p esence o TgIST p e en s a di ec expe imen on any -
hypo he ical acuole ma u a ion due o pa hogen esidence, since e en
a sho p eincuba ion o wild- ype pa asi es supp esses IRG
exp ession. We he e o e exploi ed a TgIST mu an s ain, TgΔIST
gene ously p o ided by D Ali Hakimi. Wi h his s ain we expec ed o
be able o a y in acellula esidence imes a will in ela ion o IFNγ
exposu e wi hou inhibi ing IRG p o ein induc ion and exp ession ia
TgIST. In his way, we we e able o explo e whe he ex ended
esidence o he pa asi e in a acuole could educe he abili y o he
acuola memb ane o accumula e IRG p o eins.
89
2.4.8.1. TgIST is no he only pa asi e-de i ed ac o limi ing
exp ession o IRG p o eins
In he ollowing expe imen , we we e unable o show a sys ema ic,
ime-dependen ma u a ion e ec , bu we did e eal ha in ec ion wi h
TgIST-de icien pa asi es also imposes a limi a ion on IRG p o ein
loading, albei weake han he e ec due o TgIST. In he expe imen
we conside “s anda d condi ions”, ie cells we e in ec ed o 1,5h s only
a e IFNγ p e-induc ion o ei he 4,5h s (4,5+1,5) o 10,5h s
(10,5+1,5), o cells we e in ec ed a he ime o IFNγ induc ion, ie 0 h
p einduc ion, 6 o 12 h s co-incuba ion (0+6, 0+12). In ec ion was wi h
wild- ype PRUku80, PRUku80TgΔIST and by PRUku80TgΔIST
complemen ed wi h TgIST.
In bo h 0+6 and 0+12 p o ocols he in ec ion wi h mu an
PRUku80ΔIST showed signi ican ly highe cy osolic I gb6 in ensi ies
and mo e I gb6 posi i e cells han in ec ion wi h he wild- ype o TgIST-
complemen ed mu an pa asi es (Figu e 2.12. A-D). The nume ical
esul s a e summa ized in (E). As expec ed, no simila e ec was
appa en in he 4,5+1,5 o 10,5+1,5 in ec ion p o ocols. Howe e , i
was appa en ha in all cases cells in ec ed wi h he TgΔIST pa asi es
in he 0+6 o 0+12 p o ocols ailed o de elop he equencies o I gb6-
posi i e cells o he cy osolic I gb6 in ensi ies associa ed wi h he
4,5+1,5 o 10,5+1,5 p o ocols, hough signi ican ly mo e han he wild-
ype o complemen ed s ains. Thus he e is indeed a u he ac o in
he pa asi e ha can con ol he IFNγ-media ed induc ion o IRG
p o eins, albei o a lesse ex en han TgIST.
90
Figu e 2.12. T. gondii impac he cy osolic I gb6 ia TgIST and TgIST independen
ac o , independen ly o ime.
DDCs we e seeded and p e induced wi h IFNγ (200 U/ml) o 4,5, 10,5 (con ol) and 0
hou s. The cells we e hen in ec ed wi h P u ku80 (WT), P uku80ΔIST o P u ku80ΔIST
+ HA-IST a a MOI=10 o 1,5 hou s o simul aneously induced wi h IFNγ and in ec ed
wi h he men ioned T. gondii s ains o 6 and 12 hou s.
The samples we e p ocessed o Immuno luo escence o GRA7 and o I gb6
(condi ions as in Figu e 2.12). Quan i ica ion o cy osolic I gb6 in ensi y in in ec ed cells
was measu ed and plo ed in he g aphics and he mean was p esen ed in he able. A)
Mean o cy osolic I gb6 in ensi y
A
0
20
40
60
80
100
I gb6 posi i e cells(%)
Copy o I gb6 posi i e cells
1,5
6
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
4,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
** **
ns
****
****
****
0
1×106
2×106
3×106
4×106
6×106
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Cy osolic I gb6 in ensi y
0/12_10,5/1,5
1,5
12
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
10,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
**** ****
ns ns
C
B D
E
0
20
40
60
80
100
I gb6 posi i e cells(%)
Copy o I gb6 posi i e cells
1,5
12
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
10,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
****
****
****
********
0.0
5.0×105
1.0×106
1.5×106
2.0×106
4×106
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Cy osolic I gb6 in ensi y
0/6_4,5+1,4
1,5
6
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
4,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
**** **** *ns
91
and B) Fluo escence in ensi ies we e plo ed compa e each expe imen al ime poin wi h
i s con ol. C) and D) compa e he equency o I gb6 posi i e cy osols wi h de ec able
I gb6 p o ein (mo e han 650000au).. E) The I gb6 mean in ensi y o each condi ion
was lis ed in he able. *, P < 0.05; **, P<0,01, **** P<0,0001; n.s., nonsigni ican .
These expe imen s we e designed o sea ch o an e ec on acuole
ma u a ion, no an addi ional pa asi e-speci ic inhibi o o I gb6
exp ession. The nex expe imen s examined he le els o I gb6
exp ession on indi idual acuoles wi hin he same expe imen al
p o ocols.!
A e obse ing he e ec o TgIST in he exp ession o cy osolic I gb6,
we u he analysed his p o ein le el on he PVM, unde he same
in ec ion condi ions depic ed on Figu e 2.12. The esul s (Figu e 2.13)
gene ally ollowed he same end p e iously seen o he cy osolic
le els.
In bo h he 0+6 and 0+12 g oups he TgΔIST showed mo e I gb6-
posi i e acuoles han ei he he wild ype s ain o he TgΔIST s ain
complemen ed wi h TgIST, and in bo h cases he le els we e ma kedly
lowe han he acuoles in he 4.5+1.5 o 10.5+1.5 g oups, bo h in
e ms o pe cen age o posi i e acuoles (Figu e 2.13. A, B) obse ed
and he in ensi y le els o I gb6 on he acuoles (Figu e 2.13. C, D, F
and G).

92
Figu e 2.13. T. gondii a ec he I gb6 ec ui men o he PVM pa ially ia TgIST.
DDCs we e p e- ea ed wi h IFNγ and in ec ed ollowing he same condi ion desc ibed
in Figu e 2.13. The samples we e p ocessed o Immuno luo escence o GRA7 and o
Mean o cy osolic I gb6 in ensi y
A
B D
C
0
20
40
60
80
100
I gb6 posi i e PV (%)
%IRG PVM
1,5
6
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
4,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
****
****
** ns
****
********
P uku80
ΔIST ΔIST+IST
0.0
2.0×104
4.0×104
6.0×104
8.0×104
1.0×105
1.2×105
1.4×105
2×105
4×105
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PVM I gb6 pixel in ensi y
WT_PVM I gb6 In ensi y_ze o
4,5
6
IFNγ&Toxo (h)
0
P e ind. IFNγ(h)
1,5
10,5
12
0
1,5
**** ****
0.0
2.0×104
4.0×104
6.0×104
8.0×104
1.0×105
1.2×105
1.4×105
2×105
4×105
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PVM I gb6 pixel in ensi y
Del a IST_PVM I gb6 In ensi y_ze o
****
****
4,5
6
IFNγ&Toxo (h)
0
P e ind. IFNγ(h)
1,5
10,5
12
0
1,5
0.0
2.0×104
4.0×104
6.0×104
8.0×104
1.0×105
1.2×105
1.4×105
2×105
4×105
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PVM I gb6 pixel in ensi y
dIST+IST_PVM I gb6 In ensi y_ze o
4,5
6
IFNγ&Toxo (h)
0
P e ind. IFNγ(h)
1,5
10,5
12
0
1,5
****
****
0.0
2.0×104
4.0×104
6.0×104
8.0×104
1.0×105
1.2×105
1.4×105
2×105
4×105
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PVM I gb6 pixel in ensi y
PVM I gb6 In ensi y_ze o
1,5
12
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
10,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
**
ns ns
**
**** ****
****
E F G
H
0.0
2.0×104
4.0×104
6.0×104
8.0×104
1.0×105
1.2×105
1.4×105
2×105
4×105
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PVM I gb6 pixel in ensi y
PVM I gb6 in ensi y
1,5
6
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
4,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
** **** ns
****
**** ****
ns
0
20
40
60
80
100
I gb6 posi i e PV (%)
Copy o %IRG PVM
1,5
12
ΔIST
WT ΔIST
+IST
P uku80
IFNγ&Toxo (h)
10,5
0
P e ind. IFNγ(h)
ΔIST
WT ΔIST
+IST
****
****
ns
ns
****
****
****
Mean o cy osolic I gb6 in ensi y
ΔIST
E F
G
0.0
2.0×104
4.0×104
6.0×104
8.0×104
1.0×105
1.2×105
1.4×105
2×105
4×105
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PVM I gb6 pixel in ensi y
Del a IST_PVM I gb6 In ensi y_ze o
****
****
4,5
6
IFNγ&Toxo (h)
0
P e ind. IFNγ(h)
1,5
10,5
12
0
1,5
ns
0
20
40
60
80
100
Copy o %IRG PVM
I gb6 posi i e PV (%)
4,5
6
IFNγ&Toxo (h)
0
P e ind. IFNγ(h)
1,5
10,5
12
0
1,5
ΔIST
93
I gb6 (de ails in Figu e 2.12). A) and B) Quan i ica ion o I gb6 loading on o di e en T.
gondii s ains PVs was sco ed o mo e han 100 in acellula pa asi es and esul s
we e plo ed as a p opo ion o posi i e PV (in ela ion o he G a7 posi i e PVs). C), D)
F) and G) Fluo escence in ensi ies o I gb6 we e measu ed in a leas 100 PVM as
desc ibed in Ma e ial and Me hods; C) and D) he mean o in ensi y compa e each
expe imen al ime poin wi h i ’s con ol E) The equency o I gb6 posi i e PVs o
TgΔIST and F) he luo escence in ensi ies we e plo ed, compa ing di e en ime
poin s. G) The I gb6 mean in ensi y o each condi ion was lis ed in he able. *, P <
0.05; **, P<0,01, **** P<0,0001; n.s., nonsigni ican .
While his esul seems ob ious, i is impo an o emembe ha when
indi idual acuoles om wild- ype T. gondii we e sco ed o in ensi y o
loading and cy osolic le els he e was i ually no co ela ion. I is no
immedia ely ob ious how hese disco dan esul s a e o be econciled.
One possibili y o be explo ed is ha he induc ion o IRG p o eins is
delayed by esidence o he TgIST mu an pa asi es in he cy oplasm.
Ne e heless, i is clea ha he e is no addi ional e ec o acuole-
speci ic ma u a ion beyond a gene alised educ ion in IRG loading le el
ha is con incingly co ela ed wi h he educed cy osolic le els (Figu e
2.13. E and F).
2.4.9. GBP1 is ec ui ed o T. gondii PVs and colocalizes o
some ex en wi h IRG p o eins.
Mu ine GBP p o eins belong o a second la ge amily o IFNγ-inducible
GTPases, some membe s o which a ge T. gondii PVs 26,29,63. To
analyze he epo ed associa ion o GBP1 wi h he p esence o IRG
p o eins a PVs, C56BL/6 MEFs and DDCs we e s imula ed wi h IFNγ
o kep uns imula ed o 24 hou s, and hen in ec ed wi h a ype II T.
gondii s ain o 90 min. Samples we e p ocessed by
immuno luo escence as desc ibed in Ma e ials and Me hods.
In acellula pa asi es we e iden i ied wi h an i-GRA7 Ab, I ga6 wi h 10
D7 mAb and GBP1 wi h a abbi an i-GBP1 an ise um.
94
As p e iously shown, GBP1 shows a cy oplasmic dis ibu ion s ongly
enhanced in IFNγ s imula ed cells (Figu e 2.14. A, middle column). In
hese cells, GBP1 is obus ly exp essed and associa ed wi h T. gondii
PVs (Figu e 2.14. A, B). GBP1 is ec ui ed o 65% o he acuoles,
I ga6 o 53%, wi h he expec a ion on he basis o independence o
34% double-posi i e GBP1/I ga6, agains 47% obse ed, a small bu
possibly meaning ul excess (Figu e 2.14. C). Fo I gb6, he da a also
show 62% o in acellula PVs loaded wi h GBP1, 80% loaded wi h
I gb6 and 54% loaded by bo h GBP1/I gb6 agains an expec a ion o
50% (Figu e 2.14. D and G and Supplemen al able 2.2).
These da a a e hus consis en wi h he loading o GBP1 being la gely
o ully independen o I ga6 and I gb6 loading. The in ensi y o labelling
o GBP1 is co ela ed wi h he loading o I gb6 be e han I ga6
(Figu e 2.14. F and G).
On hose acuoles whe e bo h an IRG p o ein and GBP1 a e loaded
he colocaliza ion o GBP1 and I ga6 o I gb6 on he T. gondii PVs, he
loca ion was analyzed by supe - esolu ion mic oscopy. E iden ly GBP1
and I ga6 a e ec ui ed o he same a ea o PVs and a e e y close o
each o he , bu he esolu ion does no allow a con incing
de e mina ion whe he hey a e on di e en memb anes, one on he
PVM, one on he plasma memb ane o he T. gondii (Figu e 2.14. E).
95
!
Figu e 2.14. GBP1 is ec ui ed o PVs independen o I ga6 and I gb6.
WT C56BL/6 MEFs we e induced wi h IFNγ (200 U/mL) o 24 hou s o le uninduced
and in ec ed wi h T. gondii Me49 o 90 minu es. Cells we e washed, ixed and s ained
simul aneously o GRA7 (JH 2.1.2 mAB) using seconda y an ibodies coupled o
AlexaFluo F647 (magen a), GBP1 (pAS) 55 wi h Alexa Fluo 488-conjuga ed seconda y
an ibody (g een) o I ga6 (mAb 10D7) o o I gb6 (mAb B34) wi h Alexa Fluo 555-
conjuga ed seconda y an ibody ( ed). DAPI was used o label he nuclei (cyan). A)
Immuno luo escence images a e shown me ged GRA7 and DAPI, as well as indi idual
GBP1 and I ga6 channels. B) GRA7 labelled PVs o IFNγ non-induced and induced
condi ions we e a ge ed o luo escen signal in ensi y o GBP1 measu emen , using
ImageJ Fiji as desc ibed in ma e ials and me hods. C) and D) GRA7 posi i e PVs we e
classi ied as a GBP1, I ga6 o I gb6 posi i e PVs, espec i ely (Supplemen al able
2.4). The ange be ween wo (C) and 3 (D) independen expe imen s is shown. E) 3D
su ace plo and plo p o ile o a ansec ed PV om he ep esen a i e con ocal Image,
showing GBP1 and I ga6, was ob ained by using ImageJ Fiji. F) and G) The in ensi ies
o each acuole labelled wi h GBP1 and I ga6, and GBP1 and I gb6, we e submi ed o
Pea son co ela ion analysis. F) P alue > 0,002, G) P alue < 0,0001.
I gb6 GBP1 GBP1/I gb6
0
20
40
60
80
100
Posi i e PV (%)
136B&142
01×1052×1053×105
0
1×105
2×105
3×105
4×105
5×105
GBP1
I ga6
Copy o GBP1/I ga6 co ela ion
0.2907
01×1052×1053×105
0
1×105
2×105
3×105
4×105
5×105
GBP1
I gb6
Col: En e ing eplica e da a
0.5853
=0,2907
=0,5853
GBP1 I ga6 GBP1/I ga6
0
20
40
60
80
100
Posi i e PV (%)
combine da a
GBP1
T.gondii Nuclei I ga6
GBP1 I ga6
A B
C
D
E
-IFNγ
+IFNγ
GBP1 I ga6
-IFNγ+IFNγ
0.0
5.0×104
1.0×105
1.5×105
2.0×105
2.5×105
GBP1 pixel in ensi y
GBP1 on PVM
F
G
102
Supplemen al Figu e 2. 2. Toxoplasma gondii in ec ion impac he IFNγ–induced
I gb6 exp ession.
DDCs we e seeded and p e induced wi h IFNγ (200 U/ml) o 4,5 and 10,5 (con ol)
hou s. The cells we e hen in ec ed wi h P u ku80 (WT) and P uku80ΔIST a a MOI=10
o 1,5 hou s. The samples we e p ocessed o Immuno luo escence o GRA7 (JH
2.1.2 mAB) and o I gb6 (se um 141). A and C) Quan i ica ion o cy osolic I gb6
in ensi y in in ec ed and non-in ec ed cells we e measu ed and plo ed in he g aphic
and he mean p esen ed in he able. B and D) The popula ions o in ec ed and o non-
in ec ed cells we e classi ied, acco ding o he in ensi y, as I gb6 posi i e (mo e han
650000) o I gb6 nega i e (less han 650000 au). *, P < 0.05; ****, P<0,0001; n.s.,
nonsigni ican .
0
1×106
2×106
3×106
4×106
5×106
6×106
9×106
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Cy osolic I gb6 in ensi y
Copy o NI_da a
+
-
P uku80
IFNγ&Toxo(h)
P. ind. IFNγ(h)
1,5
10,5
4,5
****
****
+
-
0
1×106
2×106
3×106
4×106
5×106
6×106
9×106
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Cy osolic I gb6 in ensi y
Copy o ISTKO
+
-
ΔIST
IFNγ&Toxo(h)
P. ind. IFNγ(h)
1,5
10,5
4,5
+
-
****
****
A B
D E
Mean o cy osolic I gb6
in ensi y
Mean o cy osolic I gb6
in ensi y
C
F
0
20
40
60
80
100
I gb6 posi i e cells (%)
Copy o con ingency WT In /NI
+
-
P uku80
IFNγ&Toxo(h)
P. ind. IFNγ(h) 1,5
10,5
4,5
+
-
**** *
0
20
40
60
80
100
Copy o con ingency IST
I gb6 posi i e cells (%)
+
-
ΔIST
IFNγ&Toxo(h)
P. ind. IFNγ(h) 1,5
10,5
4,5
+
-
**** ns

103
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Rep ess STAT1 T ansc ip ion and Block IFN-γ-Dependen Gene
Exp ession. Cell Hos Mic obe 20, 72–82 (2016).
62. F anco, M. e al. A no el sec e ed p o ein, MYR1, is cen al o
Toxoplasma’s manipula ion o hos cells. MBio 7, 1–17 (2016).
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63. Vi ei a Win e , S. e al. De e minan s o GBP ec ui men o
Toxoplasma gondii acuoles and he pa asi ic ac o s ha con ol
i . PLoS One 6, (2011).
64. Halda , A. K., Pi o, A. S., Pilla, D. M., Yamamo o, M. & Coe s, J.
The E2-like conjuga ion enzyme A g3 p omo es binding o IRG
and Gbp p o eins o Chlamydia- and Toxoplasma- con aining
acuoles and hos esis ance. PLoS One 9, (2014).
65. Rosenbe g, A. & Sibley, L. D. Toxoplasma gondii sec e ed
e ec o s co-op hos ep esso complexes o inhibi nec op osis.
Cell Hos Mic obe 1–13 (2021). doi:10.1016/j.chom.2021.04.016
111
Chap e 3
3. Ubiqui ina ion as a mechanism o con ol
o Toxoplasma gondii in ec ion.
Ana Rod igues1 Claudia Campos1 and Jona han C. Howa d 1,2
1Fundação Calous e Gulbenkian, Ins i u o Gulbenkian de Ciência,
2780- 156 Oei as, Po ugal,
2Ins i u e o Gene ics, Uni e si y o Cologne, 50674 Cologne, Ge many
118
e al bo ine se um, 2mM L-glu amine, 1mM sodium py u a e and 1x
non-essen ial amino acids wi hou an ibio ic addi ion.
Table 3.1. Cell ypes used o pe o m expe imen s
Cell ype
Re .
No e
F om C57/BL6
Immo alized WT
MEF
28
P epa ed om mice a day 14 pos coi um
and immo alized wi h pSV3-neo plasmid
ans ec ed wi h FuGENE 6 o
X emeGene 9
Immo alized WT
DDC
29
Cells we e p epa ed om diaph agm
issue o adul mouse
Immo alized
I ga6KO MEF
28
Cells we e gene a ed om I ga6-/- mice
and Immo alized as abo e
Immo alized I gdKO
MEFs
30
Kindly p o ided by D . G eg Taylo , cells
we e immo alized wi h pSV3-neo plasmid
using FuGENE 6 o X emeGene 9
Immo alized W c l
and I gb10KO ea
Fib oblas s
31
Kindly p o ided by D . Thi umala-De i
Kannegan i, cells we e immo alized wi h
pSV3-neo plasmid using Sc eenFec ®A
T ans ec ion Reagen ki (InCella)
Immo alized
I gm1/m3KO MEFs
32
Kindly p o ided by D G ego y Taylo ,
cells we e immo alized wi h pSV3-neo
plasmid using FuGENE 6 o X emeGene
9
Immo alized W c l
and TRAF6KO MEF
33
Kindly p o ided by D . Jö n Coe s,
geno ype con i med by PCR
Immo alized W c l
and TRIM21KO MEF
34
Kindly p o ided by D E a F ickel,
geno ype con i med by PCR
Immo alized I gb6
KO DDCs
Cells we e p epa ed om diaph agm
issue o adul mouse Howa d Lab

119
Cell ype
Re e ence
No e
F om CIM
Immo alized WT
DDCs
35
Kindly p o ided by D . Tobias
S ein eld
Immo alized I gb2-
b1CIM KO (T17),
T17+ I gb2-b1CIM,
T17+ I gb2-b1P20,
T17+ I gb2-b1BL6
36
Kindly p o ided by D Tobias
S ein eld
Immo alized WT
DDC
36
Cells we e immo alized wi h pSV3-
neo plasmid using Sc eenFec ®A
T ans ec ion Reagen ki (InCella)
F om Human
Hs27
35
ATCC (CRL-1634)
The ollowing condi ions we e used:
− 20.000 cells/well pla ed on 13mm co e glass in o 24 well pla es
− 20.000 cells/well pla ed on Lab-TekII chambe slides (4 well)
− 10.000 cells/well on Lab-TekII chambe slides (8 well)
− 18.0000 cells/well on 12 well pla es
− 25.000 cells/well pla ed on mul ichannel mic oscope slides
(MMS) (6-channel µ-Slide VI0.4 slides om IBIDI).
3.3.1.2. Toxoplasma gondii (T.gondii) s ains
Table 3.2.T . gondii used o pe o m expe imen s
S ain Name
O igin
Re e ence
No e
Type II
Me49
Sheep
36
PRU YFP
Human
37
T ansgenic RH s ain
exp essing d Toma o
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S ain Name
O igin
Re e ence
No e
Type I
RH
Human
38
!!
RH-YFP
Human
37
RHΔROP5
Human
39
T ansgenic RH s ain,
he ROP5 locus has
been dele ed
RHΔGRA7
Human
40
!!
BK
Human
40
Adap ed om 41
3.3.1.3. Plasmids
I ga6-c ag1-GFP (Howa d Lab) and RFP-Ubiqui in 42 (kindly p o ided
by D Jacques Nee jes) we e used.
3.3.1.4. An ibodies:
Fo all he expe imen s, he ollowing an ibodies we e used:
Ra monoclonal an i-GRA7 (2.1.2) a 1/500, abbi se um an i-I ga6
(165/4) a 1/4000, abbi se um an i-I gb6 (141/3) a 1/2000, abbi
se um an i-I gb10 (940/6 ) a 1/2000, abbi se um an i-I gd (081/3 ) a
1/3000, abbi se um an i-I gb1 (954/1) a 1/4000, mouse monoclonal
an i-I ga6 (10D7) a 1/1000, ( om he Howa d labo a o y) mouse
monoclonal an i-I gb6 (B34) a 1/2000 43, abbi se um an i-GBP1
(Kindly p o ided by D . E a F ickel) a 1/500, mouse monoclonal an i-
ubiqui in (FK2) (BML-PW8810) om Enzo a 1/300, abbi monoclonal
an i-ubiqui in linkage –speci ic K48 (ab140601) om Millipo e a 1/500,
abbi monoclonal an i-ubiqui in linkage –speci ic K63 (ab179434) om
Millipo e a 1/100 and mouse monoclonal an i-SAG1 TP3 a 1/250.
An ibodies e e ences ollowed by slash-numbe indica e he numbe o
he abbi bleed.
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Fo p ima y an ibody de ec ion by immuno luo escence we used Goa
an i- abbi Alexa 488, donkey an i- abbi Alexa 488, goa an i- a Alexa
647, donkey an i-mouse Alexa 555, donkey an i-mouse Alexa 488,
(Molecula P obes) all a 1:1000.
3.3.2. Cell-biological me hods
3.3.2.1. Cell cul u e main enance
Cells we e kep in cul u e h ough con inuous passage. To passage
cells, he s ock lask was washed wi h s e ile PBS 1X a RT and hen
de ached wi h a solu ion o 1X ypsin-EDTA o 3-5 min a 37ºC,
10%CO2. Cells we e ha es ed and cen i uged a 20-25ºC a 400g o
5 min, and he pelle was esuspended in DMEM and spli o a new
lask o keep in cul u e. Fo eezing, he pelle was esuspended in
cold solu ion o 90%FBS-10%DMSO, which was hen aliquo ed in o
c yo ubes and placed in o a Nalgene M . F os y eezing con aine o
ans e o a slowly cooled down o -80ºC, be o e s o ing in liquid
ni ogen. When necessa y, cells we e hawed by apid esuspension in
15ml o comple e DMEM a 37ºC wa e ba h and placed in a 75 cm2
(T75) lask. A e a achmen , he medium was eplaced.
3.3.2.2. P opaga ion o T. gondii
Tachyzoi es om he s ains o T. gondii enume a ed in Table 2 we e
main ained by se ial passaging in T25 con luen monolaye s o Hs27
s ain human o eskin ib oblas s (HFFs) cul i a ed in Isco e's modi ied
Dulbecco's medium, high glucose (IMDM) supplemen ed wi h 5% FCS,
2 mM L-glu amine, 1mM sodium py u a e, and 1x non-essen ial amino
acids wi hou an ibio ic added a 37ºC in an a mosphe e sa u a ed wi h
10% CO2.
122
E e y 48 hou s, a ime pe iod mo e o less coinciden wi h lysis o Hs27
cells, he achyzoi es in suspension we e collec ed. The emaining
Hs27 cells we e sc aped om he lask, and hen he suspension was
ha es ed and passed h ough a 25G sy inge needle se e al imes.
The pa asi e suspension was subjec ed o wo di e en ial
cen i uga ions: he i s (100 g o 5 min RT) o ge id o cell deb is and
a second one (700g o 15 min RT) o ob ain a small pelle en iched in
achyzoi es. Tachyzoi es we e esuspended in 2ml o medium, a
sample coun ed in a Neubaue chambe , and immedia ely used o
in ec ion o a ge cells.
3.3.2.3. T ans ec ion o mammalian cells
T ansien DNA ans ec ion o cells was conduc ed using
Sc eenFec ®A T ans ec ion Reagen acco ding o he manu ac u e ’s
ins uc ions. App op ia e numbe s o a ge cells we e seeded on a 12
well pla e 24 hou s be o e ans ec ion in o de o ob ain 50-70%
con luen wells on he day o ans ec ion. A complemen a y DNA
(cDNA) solu ion (con aining cDNAs encoding he usion p o eins I ga6-
c ag1-GFP and RFP-Ubiqui in) and a Sc een ec A solu ion we e
p epa ed, mixed and incuba ed o 20min, a e which he mix was
added in a d opwise ashion o he a ge cells. A e 24 hou s he
medium was changed and exp ession o he p o ein obse ed in a
luo escence mic oscope. Cells we e ypsinized, coun ed and
ans e ed o 6-channel µ-Slide VI0.4 slides o expe imen s.
3.3.2.4. Cell induc ion wi h IFN
γ
and In ec ion wi h T.gondii
Mu ine ib oblas s we e seeded on o UV-s e ilized 13mm diame e
co e glasses, 12 well pla es, Lab-Tek chambe s o mul ichannel
mic oscope slides (MMS), hen induced wi h mouse IFNγ (Ca . 315-05
123
Pep o ech) a 40 ng/ml (200 U/ml) o 18 o 24 hou s o we e le
un ea ed. The cells we e hen inocula ed wi h a small olume o he
esh suspension o T. gondii ME49, RH and BK achyzoi es a a
mul iplici y o in ec ion (MOI) o 1,5 o 10 and o he equi ed ime o
he essay, a 37°C, 10% o CO2. Fo expe imen s ha equi e li e-cell
imaging acquisi ion, he achyzoi e suspension was il e ed h ough a
3µm po e size il e (Nucleopo e Ca No. WHA110612) o ge “clean”
pa asi es, i.e., de oid o Hs27 cell deb is.
3.3.3. Immuno luo escence
Fo isualiza ion o IRG p o eins and ubiqui in coa ing on he PVM by
luo escence, cells we e allowed o adhe e on o a co e slip o on o
mul ichannel mic oscope slides (MMS) o 24 hou s. Fo mo e sensi i e
cells, MMS we e p e- ea ed wi h 250µg/ml o Poly-D-Lysine o 1hou ,
ollowing he manu ac u e ’s ins uc ions. The 24 h s IFNγ- s imula ed
as well as he uns imula ed cells we e in ec ed wi h he indica ed s ain
o T.gondii o he indica ed amoun o ime, a e which hey we e
ex ensi ely washed (2x) wi h PBS and ixed in 4% pa a o maldehyde
(PFA) o 30 min a oom empe a u e (RT). Be o e immunos aining, he
cells we e washed wice wi h PBS, pe meabilized in 0.1% saponin o
10min and hen blocked in blocking bu e (3% bo ine se um albumin
(BSA) and 0,1% saponin) o 1h a oom empe a u e (RT). These cells
we e hen s ained wi h he indica ed an ibody eagen s speci ic o T.
gondii GRA7, o one o mo e IRG p o eins (165/4 , 141/3 , 940/6 ,
081/3 ), o GBP1 and o Ubiqui in (FK2) dilu ed in blocking bu e , o
a leas 1 hou a RT.
Subsequen ly, a e h ee washes wi h blocking bu e , he an ibodies
we e de ec ed wi h Alexa Fluo -conjuga ed seconda y an ibodies [647
goa an i- a , 488 donkey an i- abbi , 488 goa an i- abbi , 555 donkey
an i-mouse, all om Molecula P obes/In i ogen] dilu ed in blocking

124
bu e , o a leas 45 min a RT. Cell nuclei/DNA we e labelled by 4’,6-
diamidino-2-phenylindole (DAPI D3571— 889 Li e Technologies). The
s ained cells we e washed and moun ed in o a mic oscope glass slide
wi h P olong Gold an i ade eagen . Expe imen s made on o he
suppo s ollow he same p ocedu es. Lab-Tek chambe s we e
disassembled and co e ed wi h co e glass wi h P olong gold an i ade
eagen .
3.3.4. Nec osis assay using PI
3.3.4.1. P epa a ion o cells
IFNγ-s imula ed and non-s imula ed cells seeded in an MMS we e
in ec ed wi h po e- il e ed ype I and/o ype II (Me49, PRU-YFP, RH) a
he indica ed desi ed MOI ( om 2 o 10). The il e ed achyzoi es we e
added in a suspension con aining comple e DMEM supplemen ed wi h
0.1 mg/ml o P opidium iodide (PI) (P4170—sigma), 1 µg/ml o Hoechs
33342 ihyd ochlo ide (sc-200,908—San a C uz Bio echnology) and 25
mM HEPES 29.
3.3.5. Nec osis and T. gondii assays by Flow Cy ome y
1,8 x105 cells we e pla ed on day -2, s imula ed o uns imula ed wi h
200 U/ml IFNγ o 24 hou s and in ec ed wi h Me49 a MOI om 15 o
20 o 7 hou s. A e ha ime, he cells and supe na an we e
ha es ed wi h Accu ase (BioLegend), pelle ed o 5 min a 500g,
washed wice wi h PBS1X and s ained wi h li e dead LIVE/DEAD® dye
(1:500) in PBS1X. Resuspended cells in 150µl o LIVE/DEAD® dye
we e incuba ed o 30 min a RT in he da k. A e insing he dye o
wi h h ee washes wi h PBS, cells we e ixed in 4% PFA o 20 min. The
PFA was emo ed by 3 washes in PBS1X and he cells esuspended in
125
FACS bu e (PBS + 1% BSA) be o e low cy ome y analysis.
In cases when he aim was also o de e mine he le els o pa asi e
in ec ion, he pelle ed cells ee o PFA we e equilib a ed in ice-cold
pe meabiliza ion/wash (P/W) bu e —BD Biosciences o 20 min and
incuba ed wi h an an i-SAG1 TP3 mouse monoclonal an ibody (1:250)
dissol ed in P/W bu e o 30min a 4º C wi h cons an ocking. A e
wo washes wi h P/W bu e cells we e incuba ed wi h AF488 goa an i-
mouse seconda y an ibody o 30 min a 4ºC wi h cons an ocking. The
seconda y an ibody was washed o wi h P/W bu e and cells we e
esuspended in 250µl FACS bu e (PBS + 1% BSA) o low cy ome y
analysis.
1x104 cells we e acqui ed in wo di e en exci a ion channels, 405nm
gi ing he li e/dead s a us and 488nm gi ing he in ec ed s a us, using
LSR Fo essa X20 and eco ded using BD FACsDi a so wa e. Da a
we e plo ed and quan i ied using FlowJO so wa e e sion 10.5.3.
Based on p ope con ol popula ions (non-in ec ed cells a e SAG1
nega i e and uns ained cells a e Li e/dead nega i e) ou ga es we e
es ablished o which u he analysis was es ic ed: Q1 including ali e
in ec ed cells, Q2 nec o ic in ec ed cells, Q3 nec o ic non-in ec ed cells,
and Q4 nec o ic non-in ec ed cells. The p opo ion o nec o ic cells was
ob ained conside ing only in ec ed cell popula ions29.
3.3.6. Mic oscopy and image analyses
3.3.6.1. Fluo escence mic oscopy
Wide ield images we e acqui ed on a Zeiss Axio Obse e Z1
luo escence mic oscope, using ei he a 40x 0.94NA o 63x 1.3NA
objec i e, equipped wi h a Hamama su FlashLT came a, a Zeiss Colib i
126
luo escence illumina o , and app op ia e luo escence il e s all
con olled by he Zen 2011 so wa e.
Fo each condi ion slides we e blinded and a minimum o 100 acuoles
we e sco ed o quan i ica ion o GRA7, IRG, Ubiqui in, IRG/Ubiqui in,
and GBP1/Ubiqui in posi i e acuoles.
Z-se ies s ack images we e acqui ed on a Leica SP5 con ocal,
equipped wi h 488, 568 and 633nm lase lines, using a 63x 1.3NA Oil
imme sion objec i e, and spec al de ec ion adjus ed o he emission o
he Alexa488, 568 and 633 luo och omes. Images we e decon ol ed
wi h he Huygens so wa e ( 17) o enhance con as and imp o e
esolu ion.
3.3.6.2. S uc u ed Illumina ion Mic oscopy
Images we e acqui ed on a GE Heal hCa e Del a ision OMX
S uc u ed Illumina ion sys em, equipped wi h 2 PCO Edge 5.5 sCMOS
came as, using a 60x 1.42NA Oil imme sion objec i e, GFP + CY5
luo escence il e se s. Images we e decon olu ed/ econs uc ed wi h
Applied P ecision's so Wo x so wa e. Images we e econs uc ed in
3D using Ima is so wa e.
3.3.6.3. Image acquisi ion o nec osis assay
Images we e acqui ed on an epi luo escence mic oscope equipped
wi h a digi al came a, imaging so wa e, and a hea sou ce o main ain
cells a 37ºC. Pic u es wi h 20x magni ica ion o a leas 10 andom
posi ions pe sample we e aken each ~4–5.5 min o no longe han up
o ~8–10 h pos -in ec ion s a ed a e 20min pos -in ec ion. When he
127
expe imen did no equi e all he ime se ies, he cells we e e u ned o
he incuba o un il he las equi ed imepoin imaging.
3.3.7. Blinding and unbiased analysis
Fo all expe imen s, he acquisi ion o images was based on he phase
con as channel. To ensu e eliable analysis o images mic oscopic
slides p epa ed and labeled we e blinded wi h adhesi e ape be o e
imaging. When his was no possible he iles wi h acqui ed images
we e encoded by ano he pe son be o e quan i ica ion. To minimize
bias in he semiau oma ic quan i ica ion by applying he mac o
(desc ibed in de ail below), he IRG, GBP1 and Ubiqui in p o eins we e
sco ed in acuoles selec ed based on GRA7 exp ession. This selec ion
ensu ed ha he obse ed T. gondii pa asi es we e in acellula , and
ha selec ion was no based on exp ession o ma ke s o in e es .
3.3.8. Quan i ica ion o IRG, GBP1 and Ubiqui in on he PVM
3.3.8.1. P opo ion o coa ed acuoles
The equency o PVMs ca ying IRG p o eins, GBP1 o ubiqui in in
IFNγ-s imula ed and uns imula ed cells was de e mined by blind sco ing
o a minimum o 100 in acellula pa asi es, based on he exp ession o
he T. gondii GRA7 p o ein, o each condi ion 18.
3.3.8.2. Mac o design
An ImageJ mac o was c ea ed o au oma e he quan i ica ion o p o ein
in ensi y a PVM, by measu emen o luo escence in a de ined a ea.
The mac o assumes an o iginal czi o ma image loaded in o ImageJ in