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Global impac o Salmonella ype III sec e ion e ec o S eA on hos cells
Elena Ca denal-Muñoza, Gab iel Gu ié ezb and F ancisco Ramos-Mo ales*
Depa amen o de Gené ica, Facul ad de Biología, Uni e sidad de Se illa,
Apa ado 1095, 41080 Se illa, Spain
a[email p o ec ed]
b[email p o ec ed]
*Co esponding au ho . Mailing add ess: Depa amen o de Gené ica, Facul ad de Biología,
Uni e sidad de Se illa, A da Reina Me cedes, 6, 41012 Se illa, Spain. Phone: 34 95 455
7107. Fax: 34 95 455 7104. E-mail: [email p o ec ed]
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Abs ac
Salmonella en e ica is a G am-nega i e bac e ium ha causes gas oen e i is, bac e emia and
yphoid e e in se e al animal species including humans. I s i ulence is g ea ly dependen
on wo ype III sec e ion sys ems, encoded in pa hogenici y islands 1 and 2. These sys ems
ansloca e p o eins called e ec o s in o euka yo ic hos cell. E ec o s in e e e wi h hos
signal ansduc ion pa hways o allow he in e naliza ion o pa hogens and hei su i al and
p oli e a ion inside acuoles. S eA is one o he ew Salmonella e ec o s ha a e subs a es
o bo h ype III sec e ion sys ems. He e, we used gene a ays and bioin o ma ics analysis o
s udy he gene ic esponse o human epi helial cells o S eA. We ound ha cons i u i e
syn hesis o S eA in HeLa cells leads o induc ion o genes ela ed o ex acellula ma ix
o ganiza ion and egula ion o cell p oli e a ion and se ine/ h eonine kinase signaling
pa hways. S eA also causes ep ession o genes ela ed o immune p ocesses and egula ion
o pu ine nucleo ide syn hesis and pa hway- es ic ed SMAD p o ein phospho yla ion. In
addi ion, a cell biology app oach e ealed ha epi helial cells exp essing s eA show al e ed
cell mo phology, and dec eased cy o oxici y, cell-cell adhesion and mig a ion.
Keywo ds
Salmonella en e ica; ype III sec e ion sys em; S eA; mic oa ay; human epi helial cell; cell-
cell adhesion.
Abb e ia ions
T3SS, ype h ee sec e ion sys em; SPI, Salmonella pa hogenici y island; NCBI, Na ional
Cen e o Bio echnology In o ma ion; LB, Lu ia-Be ani; FDR, alse disco e y a e; LDH,
lac a e dehyd ogenase; qPCR, quan i a i e eal- ime PCR; HEMA, 2-hyd oxye hyl
me hac yla e; GO, gene on ology.
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1. In oduc ion
Many pa hogenic G am-nega i e bac e ia possess ype III sec e ion sys ems (T3SSs) o hei
in e ac ion wi h he hos . These sys ems allow deli e y in o euka yo ic hos cells o e ec o
p o eins ha di ec he di e en s ages o he in ec ion a he cellula le el [1]. Salmonella
en e ica possesses wo dis inc i ulence- ela ed T3SSs, T3SS1 and T3SS2, ha a e encoded
by genes loca ed in Salmonella pa hogenici y islands 1 and 2 (SPI1 and SPI2), espec i ely
[2]. T3SS1 is necessa y o he in asion o non-phagocy ic cells [3], whe eas T3SS2 is
induced a e in asion and is essen ial o su i al and eplica ion wi hin mac ophages [4,5].
S. en e ica injec s mo e han hi y T3SS e ec o s o hei hos cells and some o hem ha e
been shown o manipula e cellula p ocesses such as ac in cy oskele on o ganiza ion, igh
junc ion al e a ions, biogenesis o he Salmonella-con aining acuole and cell dea h [6].
Howe e , he unc ions o many e ec o s a e s ill unknown.
S eA was iden i ied as a S. en e ica se o a Typhimu ium T3SS e ec o [7] ha can be
sec e ed o cul u e media and ansloca ed in o epi helial cells and mac ophages h ough
T3SS1 and T3SS2, depending on cul u e condi ions, in ec ed cell ypes and in ec ion imes.
The i s 10 amino acids o S eA ac as a signal sequence o i s ansloca ion in o he
euka yo ic cell [8]. The gene s eA is loca ed ou side SPI1 and SPI2, and i s low GC con en
(43%) sugges s ho izon al acquisi ion, common in i ulence-associa ed genes. We ha e
p e iously shown ha i s exp ession is ansc ip ionally con olled by he bac e ial edox
s a us in a PhoQ/PhoP-dependen manne [9]. A s eA null mu an is h ee- old a enua ed o
BALB/c mice i ulence a e in ape i oneal in ec ion [7] and S eA seems o be in ol ed in
he bac e ial pe sis ence du ing long ime in ec ion in 129X1/S J mice [10]. In he hos cell,
S eA localizes o he ans-Golgi ne wo k (TGN) and o Salmonella-induced memb ane
ubules con aining he ans-Golgi ma ke GalT-mChe y [7,11]. A e y ecen epo has
shown ha S eA pa icipa es in he con ol o Salmonella-con aining acuole memb ane
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dynamics![12]. This s udy also sugges s ha S eA should ha e addi ional oles, which emain
o be elucida ed, a ea lie imes o hos cell in ec ion.
Simila i y o p o eins wi h known ac i i ies has been use ul in some cases o de e mine he
unc ion o a speci ic e ec o [13], bu in he case o S eA no sequence simila i ies ha e been
de ec ed. Exp ession o indi idual e ec o s, like A A o SopB, in he budding yeas
Saccha omyces ce e isiae, ha se es as a simpli ied he e ologous model [14], has been a
p oduc i e app oach o s udy he e ec s o hese p o eins on hos cells [15,16,17], he eby
helping in he disco e y o hei ac i i ies. He e, we use mammalian cells as a ele an model
o speci ically analyze he e ec on he hos ansc ip ome o he e ec o S eA. We show ha
in epi helial HeLa cells S eA leads o changes in he exp ession o genes ela ed o
ex acellula ma ix o ganiza ion, cell p oli e a ion, se ine/ h eonine kinase signaling
pa hways, immune p ocesses, egula ion o pu ine nucleo ide syn hesis and pa hway-
es ic ed SMAD p o ein phospho yla ion, and p oduces signi ican changes in cell dea h,
adhesion, and mig a ion.
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2. Ma e ials and me hods
2.1. Bac e ial s ains and bac e ial cul u e
Bac e ial s ains used we e Esche ichia coli DH5α [18] and S. en e ica se o a Typhimu ium
SV5846![8], a de i a i e o s ain 14028 ca ying a s eA::3xFLAG ch omosomal usion. The
s anda d bac e ial cul u e medium was Lu ia-Be ani (LB) b o h. Solid LB con ained aga
1.5% inal concen a ion. An ibio ics we e used a he ollowing concen a ions: kanamycin,
50 µg/ml; ampicillin, 100 µg/ml.
2.2 Plasmid cons uc ion, DNA ampli ica ion, and sequencing
Plasmid pIZ1963 is a de i a i e o pBABEpu o [19] coding o S eA wi h a C- e minal
3xFLAG ag. To cons uc his plasmid, s eA::3xFLAG was ampli ied om s ain SV5846
using p ime s s eApcdnadi and s eA lagxho3, diges ed wi h BamHI and XhoI and liga ed
oge he wi h ec o pBABEpu o p e iously diges ed wi h BamHI and SalI. Ampli ica ion
eac ions and con i ma ion by sequencing we e ca ied ou as p e iously desc ibed [8].
P ime s a e lis ed in Table S1.
2.3 Mammalian cell cul u e, ans ec ion and lysis
HeLa cells (ECAC no. 93021013) we e cul u ed, ans ec ed, and lysed as p e iously
desc ibed [20]. Fo s able ans ec ion, HeLa cells we e elec opo a ed wi h pBABEpu o o
i s de i a i e and selec ion was s a ed 24 h a e elec opo a ion in medium con aining 1
µg/ml pu omycin (In i oGen).
2.4 Bac e ial in ec ion o cul u ed cells and analysis o S eA ansloca ion
HeLa cells we e pla ed in 6-well pla es a 6x105 cells pe well and incuba ed o 24 h.
Salmonella in ec ions we e ca ied ou as p e iously desc ibed![8]. The cell cul u e was

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washed wice wi h PBS 1 h pos -in ec ion and o e laid wi h DMEM con aining 100 µg/ml
gen amicin. One hou la e he concen a ion o gen amicin was lowe ed o 16 µg/ml a e an
addi ional wash wi h PBS. In ec ed mammalian cells we e lysed 6 h pos -in ec ion wi h
Nonide P-40 bu e as desc ibed![8]. The ex ac was cen i uged a 13000 pm o 20 min
and he supe na an was il e ed and analyzed by immunoblo .
2.5 Elec opho esis, immunoblo , and an ibodies
P o eins in cell ex ac s we e esol ed by SDS-PAGE. The gel was blo ed on o a
ni ocellulose memb ane (Ame sham) and p obed wi h mouse monoclonal an i-FLAG M2
p ima y an ibodies (1:5000; Sigma) and mouse monoclonal an i-β-ac in C4 (1:5000; San a
C uz Bio ech) as loading con ol. Goa an i-mouse ho se adish pe oxidase-conjuga ed
an ibodies (1:5000; BioRad) we e used as seconda y an ibodies. De ec ion was ia
chemiluminescence p ocedu es (Pie ce).
2.6 RNA p epa a ion, gene a ay p ocessing, and s a is ical analysis
To al RNA om HeLa cells s ably ans ec ed wi h pBABEpu o o i s de i a i e encoding
he S eA-3xFLAG usion was isola ed in iplica e using 1 ml o TRIzol eagen (In i ogen)
acco ding o he p o ocol supplied by he manu ac u e . An addi ional pu i ica ion s ep was
ca ied ou by using he RNeasy Min Elu e Cleanup Ki (Qiagen). Bio inyla ed single-
s anded cDNA was p epa ed om 100 ng pe sample o o al in ac RNA ex ac ed om 9
independen samples (3 om HeLa pBABEpu o, 3 om HeLa pBABEpu o-S eA-3xFLAG
L2, and 3 om HeLa pBABEpu o-S eA-3xFLAG L4). Labelled cDNA was hyb idized o
GeneChip Human Gene 1.0 ST A ays (A yme ix) ollowing he manu ac u e ’s
ins uc ions. The a ays we e scanned in a 3000 7G Scanne om A yme ix. Image analysis,
luo escen da a quan i ica ion and quali y con ol was ca ied ou wi h A yme ix so wa e.
All p ocedu es and p elimina y da a analysis, including luo escen da a p ocessing,
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no maliza ion using Robus Mul i-a ay A e age (RMA) algo i hms, and anno a ions, we e
pe o med a he Genomics Uni o he Andalusian Cen e o Molecula Biology and
Regene a i e Medicine (CABIMER, Se ille, Spain). Fold change was calcula ed o each cell
line ela i e o he con ol line ans ec ed wi h pBABEpu o. S a is ical signi icance (p alue)
was calcula ed by empi ical Bayes mode a ed - es based on he esul s o h ee a ays pe
condi ion. The alse disco e y a e (FDR) o each p alue was also calcula ed. Genes ha
changed wi h an FDR-adjus ed p alue highe han 0.05 we e emo ed om subsequen
analysis. The mic oa ay da a used in his analysis is a ailable om NCBI’s Gene Exp ession
Omnibus a h p://www.ncbi.nlm.nih.go /geo/ unde accession numbe GSE51043.
2.7 Func ion p edic ion o gene se s
To p edic he unc ional associa ion ne wo ks o he ac i a ed and ep essed genes om he
mic oa ay, we used he web-based so wa e applica ion ool GeneMANIA [21]. GO e ms
associa ed o genes in Table 1 we e sea ched using AmiGO e sion 1.8 [22].
2.8 Quan i a i e eal- ime PCR (qPCR)
The p o ocol o qPCR was p e iously desc ibed [9]. DNA p ime s a e indica ed in Table S1.
Gene exp ession le els we e no malized o ansc ip s o BCAT1, gene ha appea ed no
a ec ed by he p esence o S eA in he mic oa ay.
2.9 P oli e a ion assays
HeLa cells we e seeded in 6-well pla es a a densi y o 6x104 cells pe well and g own a
37ºC, 5% CO2 in DMEM. A 24 h in e als, he cells om a well pe cell line we e
ypsinized and coun ed on a haemocy ome e
2.10 Cell dea h, cell adhesion, and cell mig a ion assays
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Cell dea h was measu ed using a p e iously desc ibed p o ocol based on lac a e
dehyd ogenase (LDH) elease in he cul u es [20]. Cell adhesion was de e mined as
p e iously desc ibed [23]. Fo cell-cell adhesion assays, 6x104 cells/ml we e seeded on o 6-
well pla es coa ed wi h poly-(2-hyd oxye hyl me hac yla e) (poly-HEMA) (Sigma) in o de
o p e en cell-ma ix in e ac ions. A e 48 h o cul u e in suspension, cells we e
pho og aphed on an in e ed mic oscope. Fo he mig a ion assay (wound closu e assay), we
ollowed he p o ocol p e iously desc ibed [24].
2.11 S a is ical analysis
S uden ’s es was used o analyze di e ences in g ow h, cy o oxici y pe cen age, and
ela i e mig a ion. p alues o 0.05 o less we e conside ed signi ican .
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3. Resul s and Discussion
3.1$cDNA$mic oa ay$analysis$o $gene$exp ession$in$HeLa$cells$in$ esponse$ o$S eA$
To in es iga e he consequences o he p esence o he Salmonella T3SS e ec o S eA in
human epi helial cells, we gene a ed h ee s ably ans ec ed HeLa cell lines, one wi h he
emp y ec o pBABEpu o and wo wi h a de i a i e o he same ec o encoding a S eA-
3xFLAG usion (lines S eA L2 and and S eA L4). The exp ession le el o agged S eA in
hese cell clones was de e mined by Wes e n blo analysis and compa ed o he le el o S eA
ansloca ed in o HeLa cells du ing in i o in ec ions wi h S. en e ica se o a Typhimu ium
s ain SV5846. As seen in Figs. 1A and 1B, he le el o S eA was highe in L4 han in L2.
Impo an ly, in bo h cell lines he le els we e lowe han a e ansloca ion om Salmonella.
This esul sugges s ha he e ec s o S eA s udied in his wo k a e no due o a non-
physiological le el o exp ession o he e ec o . Nex , we pe o med mic oa ay
hyb idiza ion wi h RNA om s ably ans ec ed cells. Bio in-labelled cDNAs p epa ed om
o al RNA we e hyb idized o mic oa ay chips con aining 28,869 sequenced human genes.
The numbe o genes ha changed in esponse o S eA exp ession in a s a is ically signi ican
manne (FDR-adjus ed p alue < 0.05) is ep esen ed in Fig. 1C (up egula ed genes) and Fig.
1D (down egula ed genes) using Venn diag ams. As a esul o his analysis we decided o
ocus on 58 genes (0.2 % o he whole human gene numbe analyzed) ha showed
di e en ial exp ession in bo h o he cell lines exp essing S eA (L2 and L4) when compa ed
o he con ol cell line. O hese, 13 genes showed highe exp ession while 45 genes showed
lowe exp ession. These genes a e lis ed in Table S2. A gene al conclusion ha can be d awn
om he mic oa ay analysis is ha he p esence o S eA wi hin HeLa cells causes mo e gene
ep ession han ac i a ion. This is ue o bo h s ably ans ec ed cell lines, L2 and L4. The
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[20] J. Be nal-Baya d, F. Ramos-Mo ales, Salmonella ype III sec e ion e ec o Sl P is an E3
ubiqui in ligase o mammalian hio edoxin, J Biol Chem 284 (2009) 27587-27595.
[21] K. Zube i, M. F anz, H. Rod iguez, J. Mon ojo, C.T. Lopes, G.D. Bade , Q. Mo is,
GeneMANIA p edic ion se e 2013 upda e, Nucleic Acids Res 41 (2013) W115-122.
[22] S. Ca bon, A. I eland, C.J. Mungall, S. Shu, B. Ma shall, S. Lewis, AmiGO: online
access o on ology and anno a ion da a, Bioin o ma ics 25 (2009) 288-289.
[23] S. Muelle , Cadenas, E., Schön hal, A. H., p21WAF1 egula es ancho age-independen
g ow h o HCT116 colon ca cinoma cells ia E-cadhe in exp ession., Cance Res 60
(2000) 156-163.
[24] W. Kleebe ge , Bo a, G. S., Nielsen, M. E., He awi, M., Chuang, A. Y., Eps ein, J. I.,
Be man, D. M., Roles o he s em cell associa ed in e media e ilamen Nes in in
p os a e cance mig a ion and me as asis., Cance Res 67 (2007) 9199-9206.
[25] O. S eele-Mo ime , L.A. Knodle , S.L. Ma cus, M.P. Scheid, B. Goh, C.G. P ei e , V.
Du onio, B.B. Finlay, Ac i a ion o Ak /p o ein kinase B in epi helial cells by he
Salmonella yphimu ium e ec o SigD, J Biol Chem 275 (2000) 37718-37724.
[26] H. Wu, R.M. Jones, A.S. Neish, The Salmonella e ec o A A media es bac e ial
in acellula su i al du ing in ec ion in i o, Cell Mic obiol 14 (2012) 28-39.
[27] J.E. Galán, Common hemes in he design and unc ion o bac e ial e ec o s, Cell Hos
Mic obe 5 (2009) 571-579.

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Figu e legends
Fig. 1. Exp ession o S eA-3xFLAG in s ably ans ec ed HeLa cells (A) HeLa cells we e
s ably ans ec ed wi h plasmids pBABEpu o (Vec o ) and pBABEpu o-S eA-3xFLAG (S eA
L2 and S eA L4) o we e in ec ed wi h S. en e ica se o a Typhimu ium s ain SV5846
(In ec ed) and lysa es we e immunoblo ed wi h monoclonal an i-FLAG an ibodies and wi h
an ibodies agains β-ac in as a loading con ol. (B) Quan i ica ion o S eA-3xFLAG wi h
espec o β-ac in and no maliza ion ega ding he S eA L2 quan i ica ion. Means and
s anda d de ia ions om wo independen expe imen s a e ep esen ed. (C and D) Venn
diag ams o genes wi h di e en ial exp ession be ween HeLa cells exp essing S eA-3xFLAG
and con ol HeLa cells. Numbe o signi ican ly (FDR-adjus ed p alue < 0.05) up egula ed
(C) o down egula ed (D) genes in clones exp essing S eA-3xFLAG compa ed o he con ol
(L2/Con ol and L4/Con ol) is shown.
Fig. 2 Mic oa ay and qPCR esul s o selec ed di e en ially exp essed genes. (A)
Mic oa ay esul s o 7 di e en ially exp essed genes in HeLa pBABEpu o-S eA-3xFLAG
(S eA L2 and S eA L4) compa ed o HeLa pBABEpu o. (B) qPCR esul s showing mRNA
ela i e amoun o he same genes in he same s ains. Means and s anda d de ia ions om
h ee expe imen s pe o med in iplica e a e ep esen ed. Gene names a e indica ed. M (log)
is he log2 o he a io L2/con ol o L4/con ol o each gene
Fig. 3. E ec o S eA on cell dea h. (A) P oli e a ion o HeLa cells exp essing S eA-
3xFLAG (S eA L4) compa ed o con ol cells (Vec o ) du ing a pe iod o 5 days. (B) HeLa
cells s ably ans ec ed wi h pBABEpu o (Vec o ) and pBABEpu o-S eA-3xFLAG (S eA L4)
we e cul u ed in se um- ee medium o 24 h and cell dea h was de e mined by LDH elease.
Means om ep esen a i e expe imen s ca ied ou in iplica e a e ep esen ed. E o ba s
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ep esen he s anda d de ia ions. The as e isk indica es a signi ican di e ence (p < 0.01) by
he S uden ’s es .
Fig. 4. HeLa cells exp essing S eA ha e a de ec in cell-cell adhesion and cell mig a ion. (A)
HeLa cells s ably ans ec ed wi h pBABEpu o (Vec o ) and pBABEpu o-S eA-3xFLAG
(S eA L4) we e obse ed unde an in e ed mic oscope o de ec di e ences in cellula shape
(uppe panels) and in cell colony shape (lowe panels). (B) HeLa cells we e ans e ed o
poly-HEMA-coa ed pla es. A e 48 h o cul u e in suspension, cells we e pho og aphed
unde an in e ed mic oscope. Depic ed a e con ol cells HeLa pBABEpu o (Vec o ), which
o m dense agg ega es, and HeLa pBABEpu o-S eA-3xFLAG (S eA L4), which adhe e o
each o he only e y weakly. (C) Rep esen a i e images o wound-healing assays showing
ha HeLa pBABEpu o (Vec o ) closed he wound 4 days a e wounding, whe eas HeLa
pBABEpu o-S eA-3xFLAG (S eA L4) did no . Wounds a e ou lined. (D) Wound wid hs
measu ed 48 h a e wounding and no malized o he alue ob ained o HeLa pBABEpu o
a e g aphed, showing means and s anda d de ia ions om six expe imen s. The as e isk
indica es a signi ican di e ence (p < 0.01). Scale ba s, 200 µm.
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Table 1
Selec ed biological unc ions associa ed o a subse o di e en ially exp essed genes in s eA-
ans ec ed HeLa cells
Up egula ed
genes
Biological p ocess o molecula unc ion
P oli e a ion
Dea h/apop osis
ECMa
Adhesion
Mig a ion
Endocy osis
CAV1
X
X
X
CCND1
X
CTGF
X
X
X
X
X
C5AR1
X
X
C5o 23 (NPR3)
X
EFEMP1
X
LUM
X
MAP2K6
X
SDC2
X
Down egula ed
genes
Biological p ocess o molecula unc ion
P oli e a ion
Dea h/apop osis
ECMa
Adhesion
Mig a ion
Endocy osis
AKR1C2
X
ALDH3A1
X
CNTNAP3
X
COL15A1
X
X
X
CYFIP2
X
X
FURIN
X
X
IER3
X
MPDZ
X
MYH10
X
X
NFE2L2
X
NOG
X
X
RARRES1
X
SLC7A11
X
TGFB1
X
X
X
X
X
TNS1
X
UNC13B
X
aECM: ex acellula ma ix egula ion. X indica es ha his GO e m appea s associa ed o his gene.
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Figu e 1
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Figu e 2

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Figu e 3
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Figu e 4
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Figu e S1