INFECTION AND IMMUNITY, July 2003, p. 3690–3698 Vol. 71, No. 7
0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.7.3690–3698.2003
Copy igh © 2003, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Selec ion o Small-Colony Va ian s o Salmonella en e ica Se o a
Typhimu ium in Nonphagocy ic Euca yo ic Cells
Da id A. Cano,
1
† M. G aciela Puccia elli,
2
Ma ina Ma ı´nez-Moya,
3
Josep Casadesu´s,
1
and F ancisco Ga cı´a-del Po illo
2
*
Depa amen o de Gene´ ica, Facul ad de Biologı´a, Uni e sidad de Se illa, 41080 Se ille,
1
and Depa amen o de Bio ecnologı´a
Mic obiana, Cen o Nacional de Bio ecnologı´a, Consejo Supe io de In es igaciones Cien ı´ icas (CSIC), Campus de
Can oblanco,
2
and Cen o de Biologı´a Molecula Se e o Ochoa, Uni e sidad
Au o´noma de Mad id,
3
28049 Mad id, Spain
Recei ed 16 Janua y 2003/Re u ned o modi ica ion 19 Ma ch 2003/Accep ed 31 Ma ch 2003
Salmonella en e ica s ains a e en e opa hogenic bac e ia ha su i e and p oli e a e wi hin acuola com-
pa men s o epi helial and phagocy ic cells. Recen ly, i has been epo ed ha ib oblas cells a e capable o
es ic ing S. en e ica se o a Typhimu ium in acellula g ow h. He e, we show ha p olonged esidence o
bac e ia in he in acellula en i onmen o ib oblas s esul s in he appea ance o gene ically s able small-
colony a ian s (SCV). A o al o 103 SCV isola es, ob ained om ou independen in ec ions, we e subjec ed
o pheno ypic analysis. The ollowing pheno ypes we e obse ed: (i) ␦-aminole ulinic acid auxo ophy; (ii)
equi emen o ace a e o succina e o g ow h in glucose minimal medium; (iii) auxo ophy o a oma ic
amino acids; and (i ) educed g ow h a e unde ae obic condi ions no linked o nu ien auxo ophy. The
exac mu a ions esponsible o he SCV pheno ype in h ee ep esen a i e isola es we e mapped in he lpd,
hemL, and a oD genes, which code o dihyd olipoamide dehyd ogenase, glu ama e-1-semyaldehyde amino-
ans e ase, and 3-dehyd oquina e dehyd a ase, espec i ely. The lpd,hemL, and a oD mu an s had in acel-
lula pe sis ence a es in ib oblas s ha we e 3 o 4 logs highe han ha o he pa en al s ain and dec eased
suscep ibili y o aminoglycoside an ibio ics. All h ee o hese SCV isola es we e a enua ed in he BALB/c
mu ine yphoid model. Complemen a ion wi h lpd
ⴙ
,hem
ⴙ
, and a oD
ⴙ
genes es o ed he le els o in acellula
pe sis ence and an ibio ic suscep ibili y o le els o he wild- ype s ain. Howe e , i ulence was no exhibi ed
by any o he complemen ed s ains. Al oge he , ou da a demons a e ha simila o wha i has been epo ed
o SCV isola es o o he pa hogens, S. en e ica SCV display enhanced in acellula pe sis ence in euca yo ic
cells and a e impai ed in he abili y o cause o e disease. In addi ion, hey also sugges ha S. en e ica SCV
may be a o ed in i o.
The species Salmonella en e ica comp ises a se ies o acul-
a i e in acellula pa hogens ha cause gas oen e i is and
sys emic in ec ions in humans and animals (17, 38). Di e se
hos -adap ed S. en e ica se o a s a e also capable o igge ing
asymp oma ic and pe sis en in ec ions a ela i ely high a es
(12, 15). The majo hallma ks o Salmonella pa hogenesis in-
clude he capaci y o he bac e ia o in ade nonphagocy ic cells
and hei capaci y o su i e wi hin phagocy ic cells. These wo
i ulence ai s we e in e ed in ini ial s udies in ol ing in ec-
ion o issue cul u e cells ( e iewed in e e ences 18, 19, 20,
and 38). Subsequen wo k wi h epi helial cells and mac o-
phages ocused on cha ac e iza ion o he acuola compa -
men in which he bac e ia eside (2, 10, 21, 23, 25, 36, 42,
48–51, 53). The mu ine in ec ion model has also been e y
aluable o iden i ica ion o o he i ulence unc ions, such as
hose encoded in Salmonella pa hogenici y island 2, which a e
c i ical o in acellula su i al and sys emic disease (38).
Despi e he ele ance o he mu ine model, in ec ion o
issue cul u e cells has been e y use ul o de e mining di e -
ences in he in acellula li es yles o S. en e ica wi hin dis inc
hos cell ypes (9). In mos cases, massi e in acellula bac e ial
g ow h is obse ed ega dless o he epi helial o mac ophage
cell line ha is used. The si ua ion is di e en in mac ophages
isola ed om animals, which a e capable o a es ing bac e ial
g ow h (11). Rema kably, in i o s udies ha e demons a ed
ha S. en e ica p oli e a es p edominan ly wi hin li e and
spleen mac ophages du ing sys emic in ec ion o mice (44, 47).
This inding indica es ha in con as o he in o ma ion ob-
ained wi h he in i o in ec ion models, mac ophages a e he
p e e ed niche used by S. en e ica o p oli e a e in i o. In
ecen epo s wo ke s ha e also desc ibed he capaci y o S.
en e ica o colonize o he ypes o an igen-p esen ing cells,
such as dend i ic cells. Howe e , bo h in i o and in i o
s udies ha e demons a ed ha bac e ia loca ed wi hin hese
specialized cells do no exhibi massi e in acellula g ow h
(22, 37).
Recen s udies ha e shown ha cul u ed melanocy es and
ib oblas s es ic in acellula p oli e a ion o S. en e ica (13,
32, 33). Upon in ec ion o hese cell ypes, ei he S. en e ica
emains in a la en nong owing s age (13, 33) o he mass
inc eases bu he cell di ision p ocess does no culmina e (32).
The la e de ec leads o o ma ion o long ilamen ous bac-
e ia wi hin he in ec ed cells (32, 33), a phenomenon also
ecen ly obse ed in mac ophages (46). Wo ke s in ou labo-
a o y ha e demons a ed ha S. en e ica se o a Typhimu-
* Co esponding au ho . Mailing add ess: Depa amen o de Bio ec-
nologı´a Mic obiana, Cen o Nacional de Bio ecnologı´a—CSIC, Cam-
pus de Can oblanco, 28049 Mad id, Spain. Phone: (34) 91 5854923.
Fax: (34) 91 5854506. E-mail: [email p o ec ed].
† P esen add ess: Diabe es Cen e , Depa men o Medicine, Uni-
e si y o Cali o nia, San F ancisco, San F ancisco, CA 94143-0540.
3690
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ium is able o a enua e in acellula g ow h upon in ec ion o
ib oblas s. Thus, mu a ions ha cause loss o unc ion in i -
ulence- ela ed genes, such as phoP-phoQ,slyA,sp R, and poS,
esul in exace ba ed in acellula bac e ial g ow h (13). This
obse a ion indica es ha he e is delica e in e ac ion be ween
he pa hogen and he in ec ed ib oblas , esul ing in es ablish-
men o a la en s a e o he bac e ia wi hin he hos cell.
Recen ly, he ole o ib oblas s in bac e ial in ec ions was
suppo ed by a s udy ha e ealed he p esence o Mycobac-
e ium ube culosis, a bac e ium ha emains in a la en s a e in
a high pe cen age o he human popula ion, in his speci ic hos
cell ype (28). In e es ingly, i has also been shown ecen ly
ha S. en e ica se o a Pullo um pe sis s in acellula ly in i o
wi hin splenic chicken mac ophages (60). Whe he adap a ion
o he in acellula pe sis ence s age is accompanied by s able
changes in he physiology o S. en e ica is no known.
I has been epo ed ha S aphylococcus au eus a ian s ha
display physiological changes, such as a low g ow h a e unde
ae obic condi ions, a ise a a high equency in he in acellula
en i onmen o cul u ed endo helial cells (54). These a ian s,
known gene ically as small-colony a ian s (SCV), a e e-
quen ly isola ed om cases o pe sis en and elapsing in ec-
ion and ha e an enhanced capaci y o pe sis in acellula ly
when hey a e es ed in issue cul u e in ec ion models (1, 30,
40, 56, 57). A pheno ypic ai sha ed by mos S. au eus SCV is
a de ec in syn hesis o ei he menadione o hemin (34, 58).
Lack o ei he o hese wo compounds likely al e s he unc-
ionali y o he elec on anspo chain, leading o a educed
g ow h a e unde ae obic condi ions and pleio opic e ec s,
such as dec eased alpha- oxin exp ession, dec eased up ake o
aminoglycoside an ibio ics, and inc eased exp ession o clump-
ing ac o and ib onec in-binding p o eins (52, 58). Di e en
au ho s ha e claimed ha S. au eus SCV migh be selec ed by
exposu e o subinhibi o y concen a ions o an imic obial
agen s, al hough SCV ha e also been isola ed a e an ibio ic-
ee in e als ( e iewed in e e ence 58). SCV linked o
ch onic and ecu en in ec ions ha e been desc ibed o o he
pa hogens, such as Esche ichia coli,Pseudomonas ae uginosa,
and Bu kholde ia pseudomallei (26, 27, 45).
SCV isola es o S. en e ica ha e no been cha ac e ized p e-
iously. Howe e , salmonellae a e pa hogens ha a e p one o
igge ch onic and pe sis en in ec ions (5, 62). Since his
pa hogen adap s o es ablish a la en , nong owing s a e in he
in acellula en i onmen o ib oblas s (13, 33), we s udied he
a e o in acellula bac e ia wi hin his speci ic cell ype. Ou
esul s sugges ha , as obse ed o S. au eus, p olonged in-
acellula esidence o S. en e ica se o a Typhimu ium wi hin
euca yo ic cells leads o selec ion o SCV.
MATERIALS AND METHODS
Bac e ial s ains, plasmids, and g ow h condi ions. The S. en e ica se o a
Typhimu ium s ains and plasmids used in his s udy a e lis ed in Table 1. The
genomic lib a y o S. en e ica se o a Typhimu ium s ain LT2/TN1379
(leuBCD485) was p o ided by G. C. Mille (Depa men o Mic obiology, Uni-
e si y o Illinois, U bana). I was cons uc ed in ec o pBR328 by Sau3AI
pa ial diges ion o ch omosomal DNA and liga ion o 8- o 12-kb agmen s o
he BamHI-diges ed ec o . The lib a y was s o ed in P22 HT 105/1 in 201 phage
( e e ed o below as P22 HT). Allele numbe s o S. en e ica SCV isola es
desc ibed in his s udy we e ob ained om he Salmonella Gene ic S ock Cen e ,
Uni e si y o Calga y, Calga y, Albe a, Canada. All s ains we e g own in Lu ia-
Be ani (LB) medium supplemen ed wi h ampicillin (50 ␮gml
⫺1
) o kanamycin
(50 ␮gml
⫺1
) when app op ia e.
Mapping o mu a ions linked o he SCV pheno ype. The ollowing wo map-
ping s a egies we e used o iden i y he mu a ions p esen in SCV isola es: (i)
Tn10dTe mu agenesis and de ec ion o inse ions linked o he small-colony
pheno ype upon P22 HT ansduc ion; and (ii) complemen a ion wi h a pBR328-
de i ed lib a y o se o a Typhimu ium s ain LT2. Tn10dTe mu agenesis was
pe o med by ansducing an F⬘-bo ne Tn10dTe inse ion in o an LT2 de i a i e
ca ying pNK2880, a plasmid ha ha bo s a gene encoding he al e ed a ge
speci ici y (ATS) ansposase (31). Indi idual Tn10-ca ying isola es we e hen
pooled and lysed wi h P22 HT. The lysa e was used o selec Tn10dTe inse ions
TABLE 1. S. en e ica se o a Typhimu ium s ains and plasmids used in his s udy
S ain o plasmid Rele an cha ac e is ic(s) Sou ce o e e ence
S ains
SL1344 hisG psL, mouse i ulen 29
SV4042 SL1344 hemL-801 This s udy (SCV isola e)
SV4043 SL1344 lpd-201 This s udy (SCV isola e)
SV4175 SL1344 a oD This s udy (SCV isola e)
SV4139 SV4042(hemL-801)/pIZ919(hemL
⫹
)This s udy
SV4137 SV4043(lpd-201)/pIZ918(lpd
⫹
)This s udy
SV4601 SV4175(a oD)/pIZ961(a oD
⫹
)This s udy
SV4384 SL1344 a oA551::Tn10 This s udy
Plasmids
pBR328 Cloning ec o , Te
Amp
Used o cons uc ion o S. en e ica se o a Typhimu ium LT2 ch omosomal
DNA lib a y
pBluesc ip II SK(⫹) Cloning ec o , lac
⫹
, Amp
S a agene
PNK2880 Amp
31
PIZ916 pBR328, hemL
⫹
Lib a y clone ha complemen ed SCV pheno ype in SV4042 (hemL)
pIZ917 pBR328, hemL
⫹
Religa ion o 6.5-kb SalI agmen om pIZ916
pIZ919 pBluesc ip II, hemL
⫹
NheI-SalI agmen om pIZ917 cloned in pBluesc ip II SK(⫹)
pIZ912 pBR328, lpd
⫹
Lib a y clone ha complemen ed SCV pheno ype in SV4043 (lpd)
pIZ913 pBR328, lpd
⫹
Religa ion o 8.5-kb HindIII agmen om pIZ912
pIZ915 pBR328, lpd
⫹
Religa ion o 6.0-kb A aI agmen om pIZ913
PIZ918 pBluesc ip II, lpd
⫹
HindIII-A aI agmen om pIZ915, con e ed in blun agmen (A aI si e)
and cloned in HindIII-SmaI si es o pBluesc ip II SK(⫹)
pIZ956 pBR328, a oD
⫹
Lib a y clone ha complemen ed SCV pheno ype in SV4175 (a oD)
PIZ961 pBluesc ip II, a oD
⫹
5.5-kb EcoRV agmen om pIZ956 cloned in pBluesc ip II SK(⫹)
VOL. 71, 2003 SCV OF S.ENTERICA SEROVAR TYPHIMURIUM 3691
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ha changed he SCV pheno ype o he no mal-colony-size pheno ype. The
inse ions we e mapped by using he locked-in Mud-P22 p ophage p ocedu e
(7). The inse ions linked o he SCV pheno ype we e also used o clone he
co esponding mu an alleles and o iden i y he exac mu a ion esponsible o
he slow g ow h on pla es. Fo complemen a ion analysis, he pBR328-de i ed
lib a y o s ain LT2 s o ed in P22 HT was used o ansduce an SCV isola e.
Plasmid DNA om no mal-size colonies was pu i ied and used o e i y he
complemen a ion esul s. The inse s p esen in he complemen ing plasmid
we e subcloned by s anda d gene ic p ocedu es o de e mine he minimal inse
size ha complemen ed he SCV pheno ype. A his s age, he lanking egions
o he inse we e sequenced wi h p ime s designed o ampli y om ec o
egions, and he gene(s) p esen was iden i ied.
Bac e ial in ec ion o ib oblas cells. In ec ion o NRK-49F ib oblas s
(ATCC CRL1570) wi h S. en e ica se o a Typhimui um has been desc ibed
elsewhe e (13). B ie ly, ib oblas s we e g own in Dulbecco modi ied Eagle
medium con aining 5% e al cal se um o 30% con luence a he ime o
in ec ion. Bac e ia we e g own o e nigh in LB medium a 37°C unde s a ic
condi ions wi hou shaking. The mul iplici y o in ec ion was 10 bac e ia pe
ib oblas cell, and he in ec ion ime was 20 min. In ec ed cells we e washed
h ee imes wi h phospha e-bu e ed saline (PBS) (pH 7.4) and incuba ed in esh
issue cul u e medium con aining 100 ␮g o gen amicin pe ml. The an ibio ic
concen a ion was dec eased o 10 ␮gml
⫺1
a 2 h pos in ec ion. A di e en
imes pos in ec ion, in ec ed ib oblas s we e lysed in 1% T i on X-100 o 5 min,
and he numbe o in acellula iable bac e ia was de e mined by pla ing se ial
dilu ions o he cell ex ac on o LB aga pla es. To a oid side e ec s o cell
cul u e sa u a ion in he long- e m in ec ion expe imen s, he in ec ed ib oblas s
we e de ached wi h a ypsin-EDTA solu ion a 48 o 72 h pos in ec ion. The
en i e cell popula ion was ans e ed o la ge cul u e dishes con aining esh
Dulbecco modi ied Eagle medium supplemen ed wi h 5% e al cal se um and
gen amicin (10 ␮gml
⫺1
). Cau ion was aken o a oid mo e han 80% con luence
a any ime pos in ec ion. Cy o oxici y in he in ec ed cell cul u es was negligible
a all imes pos in ec ion, as indica ed by he absence o ib oblas s in he cul u es
pe meable o 1% p opidium iodine. The possibili y ha bac e ia we e p esen in
he ex acellula medium was examined by di ec pla ing o issue cul u e supe -
na an s. This con ol consis en ly showed ha he numbe o iable ex acellula
bac e ia was e y low a any ime pos in ec ion (ⱕ0.1% o he numbe o iable
in acellula bac e ia). The low numbe o iable ex acellula bac e ia was con-
side ed i ele an since h ee washes wi h PBS we e pe o med be o e T i on
X-100-media ed cell lysis.
Pheno ypic analysis o SCV isola es. Pheno ypic cha ac e iza ion o SCV
isola es was pe o med by using nonci a e E (NCE) medium (8). Ca bon
sou ces, including glucose, glyce ol, ace a e, and succina e, we e used a a con-
cen a ion o 0.2% (w / ol). ␦-Aminole ulinic acid (ALA) (50 ␮gml
⫺1
) was used
o es whe he he e was a de iciency in heme g oup syn hesis. To es whe he
he e was auxo ophy o a oma ic compounds, a mix u e con aining 30 mM
phenyalanine, 10 mM yp ophan, 10 mM y osine, 20 mM pa a-aminobenzoa e,
and 20 mM dihyd oxinbenzoa e was used.
An ibio ic suscep ibili y assays. The MIC was de e mined by he E es (AB
Biodisk, Solna, Sweden) (61) by ollowing he manu ac u e ’s ins uc ions.
Vi ulence assays in BALB/c mice. Bac e ia we e g own o e nigh in LB me-
dium a 37°C unde s a ic condi ions wi hou shaking, cen i uged a 10,000 ⫻g
o 15 min, washed wice wi h s e ile PBS (pH 7.4), and inally suspended in he
same bu e . Se ial dilu ions we e used o in ec o ally (25 ␮l) o in ape i oneally
(200 ␮l) g oups o i e BALB/c emale mice ha we e 7 o 8 weeks old. Fo he
o al challenges he bac e ium-con aining suspension was mixed wi h an equal
olume o 2.5% sodium bica bona e–0.2% lac ose o bu e he acidic pH o he
s omach. Su i al o he mice was eco ded o up o 3 weeks pos in ec ion, and
50% le hal doses we e calcula ed by he me hod o Reed and Muench (43).
Nucleo ide sequence accession numbe s. The sequences o he hemL and lpd
genes o S. en e ica se o a Typhimu ium s ain SL1344 ha e been deposi ed
in he EMBL da abase unde accession numbe s AJ278741/SEN278741 and
AJ297525/SEN297525, espec i ely. S ain SV4042 (hemL) has an A- o-C poin
mu a ion a posi ion 889 o he hemL nucleo ide sequence, which esul s in a
change om P o o Th . S ain SV4053 (lpd) has a G- o-A poin mu a ion a
posi ion 1084 o he lpd nucleo ide sequence ha esul s in a change om Gly o
Asp.
RESULTS
Selec ion o SCV o S. en e ica se o a Typhimu ium in
cul u ed ib oblas s. The a e o se o a Typhimu ium i ulen
s ain SL1344 in NRK-49F ib oblas s, a cell line ha es ic s
in acellula bac e ial g ow h (13), was in es iga ed. Cells we e
in ec ed o 20 min, and he p og ess o he in ec ion was
moni o ed o 6 days. Unde hese expe imen al condi ions,
he iabili y o he in acellula bac e ia dec eased abou 5 log
uni s by day 6 (Fig. 1A). The main dec ease in iabili y was
obse ed 3 o 6 days pos in ec ion. Consis en wi h p e ious
epo s (13, 33), no phase o ac i e bac e ial g ow h was ob-
se ed a any ime pos in ec ion (Fig. 1A). A ep oducible
phenomenon ha occu ed in hese long- e m in ec ion assays
was he appea ance o SCV when he cellula ex ac p epa ed
a e 6 days was pla ed on o LB aga pla es. SCV did no
appea in he popula ion o in acellula bac e ia a p e ious
imes pos in ec ion (da a no shown). The p opo ion o SCV
in he in acellula popula ion anged om 45 o 100% in ou
independen long- e m in ec ion expe imen s (Fig. 1B). These
esul s p o ided e idence ha long- e m pe sis ence o S. en-
e ica se o a Typhimu ium wi hin ib oblas s a o s he selec-
ion o SCV isola es. Simila o wha has been desc ibed o
o he pa hogens, such as S. au eus and E. coli (4, 45, 54, 57),
FIG. 1. Pe sis ence o S. en e ica se o a Typhimu ium in ib oblas s a o s selec ion o SCV isola es. (A) NRK-49F ib oblas s we e in ec ed
wi h se o a Typhimu ium s ain SL1344 o 20 min. The numbe s o iable in acellula bac e ia su i ing gen amicin ea men we e de e mined
a di e en imes pos in ec ion. The da a a e exp essed ela i e o he numbe o iable bac e ia quan i ied a 2 h pos in ec ion. The alues a e
medians (and s anda d de ia ions) o a ep esen a i e expe imen wi h a o al o six epe i ions. (B) Numbe s o no mal-colony-size and SCV
isola es ob ained in ou di e en expe imen s a 6 days pos in ec ion.
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he S. en e ica SCV may ha e acqui ed speci ic mu a ions ha
p omo e in acellula pe sis ence wi hin euca yo ic cells.
Pheno ypic analysis o S. en e ica SCV ob ained om NRK-
49F ib oblas s. To gain insigh in o he na u e o he mu a-
ions esponsible o he SCV pheno ype, 103 SCV isola es
ob ained om he ou independen ib oblas in ec ion expe -
imen s (Fig. 1B) we e sc eened o pheno ypes desc ibed o
SCV o o he pa hogens ha ha e been isola ed (34, 58).
These pheno ypes included auxo ophy o compounds in he
heme biosyn he ic pa hway and di e en ial g ow h on NCE
minimal medium supplemen ed wi h in e media es o he
K ebs cycle as al e na i e ca bon sou ces. Auxo ophy o a -
oma ic amino acids was also es ed since i is known ha a o
mu an s g ow mo e slowly han he wild ype in LB medium,
which is limi ing o hese compounds. SCV isola es belonging
o each o hese h ee pheno ypic g oups we e iden i ied (Ta-
ble 2). In addi ion, ou SCV isola es g ew in NCE minimal
medium con aining glyce ol and he e o e we e conside ed
p o o ophs. In e es ingly, a single SCV pheno ypic class was
obse ed in mos , i no all, he SCV isola es ob ained om he
same expe imen (Table 2). The pheno ypic homogenei y in
he SCV popula ions is consis en wi h s ong selec ion o he
SCV pheno ype (abou one SCV-linked mu a ion pe in acel-
lula bac e ial popula ion consis ing o ⬃10
4
CFU). Because o
he small size o he sample, he p obabili y o selec ing wo
di e en ypes o SCV mu a ions in he same expe imen could
be expec ed o be ex emely low. Finally, we obse ed ha all
103 SCV isola es examined in his sc eening analysis beha ed
like s able a ian s, wi h a a e o e e sion o no mal colony
size o 10
⫺6
o 10
⫺7
, which is in he ange o spon aneous
mu a ion equencies. Al oge he , ou da a indica e ha he e
a e a a ie y o mu a ions ha can cause he SCV pheno ype in
S. en e ica. Some o he pheno ypes ound closely co espond
o wha has been ound in S. au eus, such as a de iciency in
syn hesis o he heme g oup (4). Ou s udy e ealed ha o he
ypes o mu a ions p omo e selec ion o S. en e ica SCV in
ib oblas s, including mu a ions ha cause ei he a de ec in
p o iding ca abolic compounds o he K ebs cycle o a educed
g ow h a e due o nu ien auxo ophy (a o ype mu a ions).
Gene ic cha ac e iza ion o S. en e ica SCV isola es selec ed
in cul u ed ib oblas s. Th ee SCV isola es collec ed in inde-
penden expe imen s (expe imen s 1, 2, and 4 [Table 2]) we e
designa ed s ains SV4042, SV4053, and SV4175, p opaga ed,
and subjec ed o a gene ic analysis o iden i y he spon aneous
mu a ion esponsible o he SCV pheno ype. Unless indica ed
o he wise, he s a egy in ol ed (i) Tn10dTe mu agenesis and
de ec ion o inse ions linked o he SCV pheno ype upon P22
HT ansduc ion; (ii) mapping o he inse ion wi h he locked-
in Mud-P22 p ophage p ocedu e (7); and (iii) complemen a-
ion wi h a pBR328-de i ed lib a y o se o a Typhimu ium
s ain LT2.
A e Tn10dTe mu agenesis in SV4042 and u he ans-
duc ion, an inse ion mapping be ween cen isomes 3 and 4,
za -6309::Tn10dTe , was selec ed as an elemen ha exhibi ed
30% linkage o he SCV pheno ype. Complemen a ion analysis
wi h he LT2 genomic lib a y and subsequen subcloning p o-
ided a 2-kb agmen ha es o ed no mal colony size (plas-
mid pIZ919 [Table 1]). Sequencing o his inse e ealed he
p esence o a single open eading ame co esponding o he
hemL gene, which encodes glu ama e-1-semialdehyde amino-
ans e ase. This enzyme ca alyzes he las s ep in he biosyn-
he ic pa hway o ALA, a p ecu so o he heme g oup (16).
Cloning and comple e sequencing o he hemL allele ha bo ed
by SV4042 showed ha he e was a poin mu a ion ha caused
a nonconse a i e amino acid change. No mal size colony was
ob ained upon complemen a ion wi h a hemL wild- ype gene,
con i ming ha no addi ional mu a ion esponsible o he
SCV pheno ype was p esen in SV4042. The SCV-linked in-
se ion ob ained in SV4053 a e ansposon mu agenesis,
zad-6310::Tn10dTe , was mapped a cen isome 3, be ween he
nadC and p oA loci. Complemen a ion o no mal colony size
and subcloning yielded a minimal 2.5-kb inse (plasmid
pIZ918 [Table 1]), which ha bo ed a agmen o he aceF gene
and he comple e lpd gene encoding lipoamide dehyd ogenase
o he E3 componen o py u a e and 2-oxoglu a a e dehyd o-
genases (41). The Lpd p o ein has also been shown o be he L
componen o he enzyma ic complex known as he glycine
clea age sys em (41). As in SV4042, cloning and sequencing o
he lpd allele ha bo ed by he SV4053 isola e showed ha he e
was a poin mu a ion ha caused a nonconse a i e amino acid
change. Complemen a ion analysis wi h an lpd
⫹
gene es o ed
wild- ype colony size, elimina ing he possibili y ha he e
we e addi ional mu a ions in SV4053 esponsible o he SCV
pheno ype.
Gene ic analysis o SV4175 was simple since his SCV iso-
la e was a ep esen a i e o he A o
⫺
pheno ypic class (Table
2, expe imen 4). Complemen a ion o he auxo ophy wi h he
LT2 plasmid lib a y, ollowed by subcloning, e ealed ha
he e was a minimal 5.5-kb inse ha es o ed bo h p o o o-
phy and la ge colony size (plasmid pIZ961 [Table 1]). Sequenc-
ing o an inse bounda y e ealed he p esence o an open
eading ame homologous o ydiF,anE. coli gene whose
unc ion is no known. In bo h E. coli and S. en e ica,ydiF lies
nea he a oD gene, which in u n is 14 kb away om a oH.
Since he inse ha es o ed no mal colony size was only 5.5
kb long, we concluded ha he mu a ion ca ied by he SV4175
SCV isola e mapped in a oD, he gene ha encodes 3-dehyd-
oquina e dehyd a ase. This enzyme is in ol ed in he biosyn-
hesis o cho isma e, a common p ecu so o a oma ic amino
acids (35).
S. en e ica se o a Typhimu ium SCV show inc eased in a-
cellula pe sis ence wi hin ib oblas s. I has been shown
ha S. au eus SCV display inc eased in acellula pe sis ence
wi hin cul u ed euca yo ic cells (4, 57). To assess whe he he
S. en e ica SCV selec ed in ou s udy beha ed in a simila
TABLE 2. Pheno ypes o S. en e ica se o a Typhimu ium SCV
isola es selec ed in NRK-49F ib oblas s
Exp
No. o
SCV
isola es
No. o isola es in he ollowing pheno ypic
g oups o SCV isola es:
ALA
auxo ophy
Ace a e-succina e
equi emen o
g ow in NCE-
glucose
A oma ic
compound
auxo ophy
P o o ophy
(g ow h in
NCE-
glyce ol)
113 0 13 0 0
260 57 0 0 3
312 11 0 0 1
418 0 0 18 0
To al 103 68 13 18 4
VOL. 71, 2003 SCV OF S.ENTERICA SEROVAR TYPHIMURIUM 3693
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manne , isola es SV4042 (hemL), SV4053 (lpd), and SV4175
(a oD) we e used indi idually o in ec NRK-49F ib oblas s.
All h ee SCV isola es exhibi ed in acellula pe sis ence a es
highe han he in acellula pe sis ence a e o he wild ype
(Fig. 2A). The di e ences in in acellula iabili y be ween he
wild ype and he SCV isola es inc eased p og essi ely a e 3
days pos in ec ion (Fig. 2A). On day 6, bo h he hemL and
a oD mu an s exhibi ed a iabili y a e ha was close o 5 log
uni s highe han ha o he wild ype, whe eas he iabili y
a e o he lpd mu an was abou 4 log uni s highe (Fig. 2A).
None o he mu an s o e g ew he gen amicin-con aining ex-
acellula medium since di ec pla ing on his medium yielded
low numbe s o bac e ia (ⱕ0.1% o he iable in acellula
bac e ia) a all imes pos in ec ion. In addi ion, he di e ences
in he in acellula pe sis ence a es exhibi ed by he SCV
isola es selec ed in ib oblas s we e ep oduced wi h SCV
s ains econs uc ed wi h he SCV-linked Tn10dTe inse ions
and SCV s ains ca ying knocked-ou hemL,lpd,o a oD
alleles (da a no shown). Complemen a ion o he poin mu-
a ions selec ed in ib oblas s wi h hemL
⫹
,lpd
⫹
, and a oD
⫹
genes esul ed in dec eased in acellula pe sis ence a es ha
we e e en lowe han he a e o he wild- ype s ain (Fig. 2B
o D). Al oge he , hese da a con i med ha he e was clea -
cu linkage be ween he SCV pheno ype and an enhanced
capaci y o S. en e ica se o a Typhimu ium o pe sis wi hin
ib oblas cells.
An ibio ic suscep ibili y analysis o he S. en e ica se o a
Typhimu ium SCV isola es. SCV isola es o S. au eus,P. ae u-
ginosa, and E. coli ha e been shown o sha e de ec s in he
elec on anspo espi a o y chain, which explains he low
g ow h a es o hese a ian s unde ae obic condi ions (4, 45,
57). I has been p oposed ha hese de ec s in he espi a o y
chain educe he memb ane po en ial and impai ene gy-de-
penden p ocesses, such as anspo o molecules ac oss he
memb ane. Up ake o an ibio ics such as aminoglycosides,
which a e dependen on a no mal ene gized s a e o he mem-
b ane, is no ably educed. Acco dingly, some wo ke s ha e
epo ed diminished aminoglycoside suscep ibili y o SCV o S.
au eus and P. ae uginosa eco e ed om cys ic ib osis pa ien s
(27, 30, 58). To de e mine whe he he S. en e ica se o a
Typhimu ium SCV isola es beha e in a simila manne , hei
suscep ibili ies o dis inc classes o an ibio ics, including ␤-lac-
am an ibio ics, aminoglycosides, quinonoles, chlo ampheni-
col, and ime hop im-sul ame hoxazole, we e examined. The
MICs o gen amicin and amikacin o he SCV isola es we e
inc eased and we e app oxima ely 10- old highe o he hemL
FIG. 2. In acellula pe sis ence o ep esen a i e S. en e ica se o a Typhimu ium SCV isola es. (A) NRK-49F ib oblas s we e in ec ed wi h
wild- ype s ain SL1344 (E) and he SCV de i a i es SV5053 (lpd)(䊐), SV4042 (hemL)(‚), and SV4175 (a oD)({). The numbe s o iable
in acellula bac e ia we e calcula ed o di e en imes pos in ec ion and a e exp essed ela i e o he alue ob ained a 2 h pos in ec ion. (B o
D) Beha io o he SV4137 (lpd/lpd
⫹
), SV4139 (hemL/hemL
⫹
), and SV4601 (a oD/a oD
⫹
) complemen ed s ains (■) compa ed o he beha io
o he SL1344 wild- ype s ain (E) and he co esponding SCV isola es (䊐). No e ha complemen a ion wi h he wild- ype genes caused a ma ked
dec ease in he in acellula pe sis ence.
TABLE 3. An ibio ic suscep ibili ies o ep esen a i e S. en e ica se o a Typhimu ium SCV isola es
An ibio ic
MIC (␮g/ml)
SL1344
(wild ype)
SV4042
(hemL)
SV4053
(lpd)
SV4175
(a oD)
SV4139
(SV4042 hemL
⫹
)
SV4137
(SV4053 lpd
⫹
)
SV4601
(SV4175 a oD
⫹
)
Amoxicillin-cla ulanic acid 0.75 0.38 0.38 0.75 NA
a
NA NA
Ce o axime 0.064 0.032 0.032 0.064 NA NA NA
Amikacin 1.5 16 3 3 1 1 1
Gen amicin 0.75 8 3 1.5 0.75 0.75 0.75
Nalidixic acid 6 1.5 4 2 3 3 4
O loxacin 0.094 0.094 0.094 0.094 0.094 0.047 0.064
Chlo amphenicol 1.5 1 0.75 2 1 1 1.5
T ime hop im-sul ame hoxazole 0.125 0.125 0.125 0.125 0.125 0.125 0.125
a
NA, no applicable since he s ain ha bo s he co esponding complemen ing gene cloned in he pBluesc ip II SK(⫹) plasmid, which ca ies an ampicillin
esis ance de e minan (Table 1).
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mu an (Table 3). A ou old inc ease in he MIC o gen ami-
cin was also no ed o he lpd mu an (Table 3). No majo
di e ences we e obse ed be ween he wild ype and he SCV
in e ms o suscep ibili y o ␤-lac am an ibio ics, chlo amphen-
icol, ime hop im-sul ame hoxazole, and he quinonoles nali-
dixic acid and o loxacin (Table 3). These esul s sugges ha S.
en e ica SCV isola es a e less sensi i e o aminoglycosides, an
expec ed inding conside ing ha hei mu a ions may po en-
ially lead o de ec s in espi a o y me abolism. Complemen-
a ion o he hemL,lpd, and a oD mu an s wi h he co e-
sponding wild- ype genes inc eased an ibio ic suscep ibili y
(Table 3), con i ming ha he e is a di ec ela ionship be-
ween he poin mu a ions esponsible o he SCV pheno ype
and he obse ed changes in sensi i i y o aminoglycoside an-
ibio ics.
S. en e ica se o a Typhimu ium SCV isola es a e a enu-
a ed in he BALB/c mouse i ulence model. P e ious epo s
p o ided e idence ha he e is educed p oduc ion o i u-
lence ac o s in SCV. Fo ins ance, S. au eus SCV isola es ha e
dec eased alpha- oxin p oduc ion and low coagulase ac i i y
(58, 59), which may p e en o e disease and hos cell damage.
Likewise, SCV o P. ae uginosa ha e been shown o be less
i ulen in no mal and mode a ely leukopenic mice (24). The
hemL,lpd, and a oD S. en e ica SCV isola es showed ma ked
a enua ion in BALB/c mice when hey we e es ed by using
he o al o in ape i oneal ou e (Table 4). The da a s ongly
sugges ha con e sion o S. en e ica o SCV no ably educes
he capaci y o his pa hogen o cause disease. In e es ingly, no
appa en e e sion o a i ulen , no mal-colony-size o m was
obse ed in he animals since bac e ia isola ed om o gans
e ained he SCV pheno ype on pla es (da a no shown). We
also es ed whe he complemen a ion o he SCV pheno ype
wi h he wild- ype hemL
⫹
,lpd
⫹
, and a oD
⫹
genes could esul
in he eappea ance o i ulence. Among he complemen ed
s ains, only he lpd/lpd
⫹
s ain displayed a sligh e lowe me-
dian le hal dose, al hough he le hal dose was only on he o de
o 1 log lowe han he le hal dose o he lpd SCV mu an
(da a no shown). In con as , bo h he hemL/hemL
⫹
and
a oD/a oD
⫹
s ains emained as a enua ed as he pa en al
SCV isola es. The la e esul s led us o examine whe he he
complemen ing plasmid was uns able in i o. Li e and spleen
ex ac s we e p epa ed om mice ha su i ed a e 20 days o
bac e ial challenge wi h he hemL/hemL
⫹
and a oD/a oD
⫹
s ains. In e es ingly, SCV appea ed in some cases, whe eas
plasmid-bea ing bac e ia we e no de ec ed in any o gan ex-
ac a his ime pos in ec ion (da a no shown). These esul s
we e consis en wi h apid loss o he complemen ing plasmid
du ing in ec ion.
DISCUSSION
To ou knowledge, his s udy is he i s one in which S.
en e ica se o ype Typhimu ium SCV we e isola ed and cha -
ac e ized. Pheno ypic sc eening pe o med wi h 103 S. en e ica
SCV isola es showed ha a leas some o hem esemble SCV
o o he bac e ial pa hogens, such as S. au eus. Thus, S. en e ica
SCV isola es wi h mu a ions in genes ela ed o espi a o y
me abolism we e iden i ied. Examples include mu an s equi -
ing ALA and mu an s equi ing in e media es o he K ebs
cycle o no mal g ow h (hem and lpd pheno ypic g oups, e-
spec i ely). In e es ingly, a de ec in he hemB gene was he
i s cha ac e ized mu a ion leading o an S. au eus SCV phe-
no ype (57). A hi d pheno ypic class, auxo ophy o a oma ic
compounds, was also obse ed. S ikingly, mos i no all SCV
isola ed om each o he long- e m ib oblas in ec ions be-
longed o he same pheno ypic g oup. This esul was in acco d
wi h he ela i ely low numbe o in acellula bac e ia ha
we e analyzed. In ac , he size o he sample o iable in a-
cellula bac e ia eco e ed om ib oblas s was on he o de o
10
3
o 10
4
cells. Despi e his ela i ely low numbe o bac e ia,
we success ully de ec ed a leas one mu a ion linked o he
SCV pheno ype in each expe imen . A de ailed gene ic anal-
ysis o each o he SCV isola es eco e ed om a single expe -
imen should p o ide clues abou whe he he isola es a e
de i a i es o a single clone. Rega dless o whe he all mu an s
o a pheno ypic class bea he same mu a ion, ou esul s
demons a e ha he equency o o ma ion o S. en e ica SCV
in ib oblas s is on he o de o 10
⫺3
o 10
⫺4
, in close ag ee-
men wi h he s udy o Vesga e al., in which hese au ho s
es ima ed ha he a e o o ma ion o S. au eus SCV in cul-
u ed endo helial cells was 10
⫺3
(54). Howe e , one di e ence
be ween he wo pa hogens is ha eme gence o S. au eus SCV
in endo helial cells occu s a e 72 h o in ec ion, whe eas S.
en e ica SCV a e selec ed in cul u ed ib oblas s a e 6 days o
in ec ion. The easons o his di e ence a e no known, bu
ac o s such as he hos cell ype and/o bac e ial ype may
ce ainly con ibu e o i .
An in e es ing aspec o ou s udy is he di e si y o he
mu a ions ha appa en ly accoun o an inc eased capaci y o
S. en e ica o pe sis in acellula ly wi hin ib oblas s. In he
hem and lpd mu an s he espi a o y me abolism capaci y may
ha e been al e ed, p obably leading o dec eased gene a ion o
oxygen- eac i e subp oduc s. In his sense, S. en e ica SCV
isola es would be less p one o sel -in oxica ion in he in a-
acuola en i onmen wi h hese oxygen-de i ed compounds.
Fu he analysis o he cy och ome con en and measu emen
o he espi a o y a es o hese mu an s should ce ainly p o-
ide new clues conce ning his hypo hesis. Ou s udy also
showed ha mu a ions ha cause a de ec in syn hesis o a o-
ma ic amino acids, as in he case o a oD, a o in acellula
bac e ial pe sis ence in ib oblas s. P eceden s o linkage be-
ween a o mu a ions and S. en e ica pe sis ence exis o a oA
pu A double mu an s, which pe sis o many mon hs in o gans
o BALB/c mice (62). We obse ed ha in ec ion o ib oblas s
wi h a well-cha ac e ized S. en e ica a oA mu an esul ed in an
in acellula pe sis ence a e compa able o ha o he a oD
TABLE 4. Vi ulence a ibu es o ep esen a i e S. en e ica se o a
Typhimu ium SCV isola es in he mu ine yphoid model
S ain Median le hal dose
a
O al In ape i oneal
SL1344 (wild ype) 6 ⫻10
4
10
SV4042 (hemL)4⫻10
7
1.5 ⫻10
3
SV4053 (lpd)⬎5⫻10
9
⬎10
4
SV4175 (a oD)⬎5.5 ⫻10
8
⬎1.1 ⫻10
5
a
Median le hal doses we e calcula ed by he me hod o Reed and Muench
(43).
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mu an (da a no shown). In e es ingly, bo h a oA and a oD
mu an s a e de ec i e o syn hesis o cho isma e, a p ecu so
o no only a oma ic amino acids bu also olic acid, ubiqui-
none, and en e obac in. Lack o en e obac in migh esul in
i on s a a ion and subsequen impai men o cy och ome syn-
hesis. The ac ha all SCV isola es selec ed in ou s udy a e
po en ially de ec i e in espi a o y me abolism led us o pos-
ula e ha swi ching o non espi a o y me abolism may be a
majo mechanism used by S. en e ica o inc ease in acellula
pe sis ence wi hin euca yo ic cells.
The analysis o an ibio ic sensi i i y showed ha , as epo ed
p e iously o SCV isola es o S. au eus and P. ae uginosa (3, 4,
34, 39, 45, 57, 58), he S. en e ica SCV isola es ha e educed
aminoglycoside sensi i i y. This pheno ype could indica e ha
selec ion o S. en e ica SCV wi hin cul u ed ib oblas cells was
me ely caused by p olonged exposu e o in acellula bac e ia
o gen amicin, an an ibio ic main ained in he issue cul u e
medium. Se e al obse a ions a gue agains his possibili y.
Fi s , selec ion o S. au eus SCV by exposu e o suble hal
concen a ions o gen amicin has been epo ed o ake place
in as li le as 30 min (34). In con as , eco e y o S. en e ica
SCV isola es om in ec ed ib oblas s occu s a an app eciable
equency only a e y la e in ec ion imes (6 days pos in ec-
ion). Second, acquisi ion o gen amicin esis ance concomi-
an wi h he SCV pheno ype seems o be a e e sible phe-
nomenon in S. au eus since an ibio ic- esis an SCV e e
apidly o sensi i e o ms upon incuba ion in an ibio ic- ee
media (34). High- equency e e sion o no mal colony size
and an ibio ic-sensi i e o ms was no obse ed in any o he
S. en e ica SCV isola es desc ibed in his s udy, which sugges s
ha he e may be in insic di e ences be ween SCV selec ion
unde an ibio ic exposu e condi ions and in acellula pe sis-
ence wi hin ib oblas cells. Thi d, he di e ences in he e-
e sibili y o he pheno ype a e accompanied by dis inc le els
o an ibio ic esis ance. Whe eas he MICs o gen amicin o S.
en e ica SCV a e 2- o 10- old highe han hose o he wild
ype (Table 3), he MICs o S. au eus SCV a e ⱖ20- old
highe ha hose o he pa en al s ain (34). Fou h, despi e
he inc eased MICs o gen amicin o he h ee S. en e ica SCV
isola es analyzed (1.5, 3, and 8 ␮gml
⫺1
o he a oD,lpd, and
hemL mu an s, espec i ely [Table 3]), none o hese a ian s
o e g ew he issue cul u e medium con aining 10 ␮go
gen amicin pe ml du ing he ib oblas in ec ion expe imen .
Finally, i has been demons a ed p e iously by using a quan-
i a i e enzyme-linked immunoso ben assay ha unlike pha-
gocy ic cells, NRK-49F ib oblas s do no accumula e gen ami-
cin in acellula ly (33). Al oge he , hese indings led us o
conside selec ion o S. en e ica SCV isola es in he in acellu-
la en i onmen o ib oblas s a unique e en un ela ed o he
e en ual exposu e o in acellula bac e ia o gen amicin.
Aside om he same beha io in e ms o pe sis ence wi hin
ib oblas s cells and an ibio ic suscep ibili y, he h ee S. en-
e ica SCV clones cha ac e ized a a gene ic le el (hemL,lpd,
and a oD) we e highly a enua ed in he BALB/c mouse y-
phoid model. S. en e ica a oA mu an s, which pe sis e icien ly
in NRK-49F ib oblas s, we e he i s gene ically de ined
s ains in which ma ked a enua ion was obse ed in he mouse
model (29). In a subsequen s udy he wo ke s also epo ed
a enua ion o a hemA mu an o se o a Typhimu ium (6),
which, in ag eemen wi h he esul s o he p esen s udy,
ein o ces he con ibu ion o he biosyn he ic pa hway in-
ol ed in syn hesis o he heme g oup o S. en e ica pa hogen-
esis. We ound e idence ha when adminis a ed by he o al
ou e, he hemL,lpd, and a oD SCV isola es a e capable o
eaching he li e and spleen (da a no shown). These esul s
a e consis en wi h no mal passage h ough he in es inal ep-
i helium and subsequen ailu e o he bac e ia o su i e o
p oli e a e wi hin phagocy ic cells p esen in a ge o gans.
Addi ional wo k is equi ed o assess whe he hese SCV iso-
la es also pe sis in acellula ly in i o. Globally, ou da a a e
consis en wi h p e ious obse a ions made wi h S. au eus and
P. ae uginosa ha show ha he e is di ec linkage be ween he
SCV pheno ype and an inabili y o cause o e disease (24, 57,
58). In e es ingly, we did no ind e idence o con e sion o he
no mal-colony-size ype du ing in ec ion wi h SCV isola es.
Fu he mo e, ou a emp s o complemen he a enua ion
pheno ype displayed by he S. en e ica SCV isola es wi h plas-
mids ca ying he wild- ype genes hemL
⫹
,lpd
⫹
, and a oD
⫹
we e unsuccess ul. The ac ha he complemen ing plasmid
was los apidly du ing in ec ion may explain hese esul s. In
addi ion, plasmid-bea ing bac e ia a e impai ed in e ms o he
abili y o su i e in acellula ly (Fig. 2), which sugges s ha he
complemen ed bac e ia may be apidly coun e selec ed in i o.
O e all, hese esul s con i m ha he e is a s ic ela ionship
be ween he SCV pheno ype and i ulence a enua ion in S.
en e ica and selec ion occu ing agains no mal-colony-size
pheno ype once he bac e ia ha e swi ched o an SCV.
A inal conside a ion in ou wo k was he possibili y ha
simila o S. au eus, na u al SCV o S. en e ica could be e-
sponsible o igge ing ch onic and pe sis en in ec ions. So
a , i ually no hing is known abou he mechanisms o
S. en e ica pe sis ence. Howe e , as p e iously no ed, an S.
en e ica se o a Typhimu ium a oA pu A double mu an , which
is an SCV, pe sis s in BALB/c mice (62). Al hough he basis o
his phenomenon has no been de ined, i suppo s he hypo h-
esis ha he e is p obably linkage be ween he SCV pheno ype
and he in i o pe sis ence o S. en e ica. This hypo hesis does
no con adic he esul s o epidemiological s udies showing
ha bac e ia eleased in o he en i onmen by asymp oma ic
human and animal ca ie s e ain pa hogenic po en ial. Thus,
no mal-colony-size bac e ia may coexis in i o wi h SCV, as
has been obse ed in long- e m in ec ion o ib oblas s (Table
2, expe imen s 1, 2, and 4). Unde hese condi ions, an e ec i e
hos immune esponse elici ed by he SCV (e.g., by an a o
mu an [29]) may limi he abili y o no mal-colony-size bac e-
ia o cause disease. Reac i a ion o disease due o non-SCV
bac e ia would occu only du ing pe iods when immuni y is
educed. Al e na i ely, eac i a ion o he disease may be as-
socia ed wi h he appea ance o second mu a ions ha sup-
p ess he SCV pheno ype. Such a concep has been pos ula ed
o he eac i a ion o in ec ion in he case o ch onic in ec-
ions caused by an E. coli hemB mu an (45). Finally, i should
be no ed ha in he speci ic case o se o a Typhi, a hos -
adap ed se o a ha igge s ch onic and asymp oma ic in ec-
ions in humans, many clinical isola es a e auxo ophs o
a oma ic amino acids (55). Al oge he , hese obse a ions in-
dica e ha a mo e p o ound molecula analysis o S. en e ica
SCV isola es is necessa y, which may p o ide clues abou
whe he swi ching o a s able SCV pheno ype has conse-
quences o Salmonella pa hogenesis. The ecen disco e y
3696 CANO ET AL. INFECT.IMMUN.
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ha polynucleo ide phospho ylase is a global egula o o i -
ulence and pe sis ence in S. en e ica (14) suppo s he hypo h-
esis ha he e is a delica e balance be ween hese wo p o-
cesses. Addi ional wo k is equi ed o con i m whe he he
eme gence o s able SCV isola es is a consequence o he
in e play be ween such bac e ial egula o y ne wo ks and he
hos immune de enses.
ACKNOWLEDGMENTS
We hank Nu ia Go´mez-Lo´pez and Es he Campana io o assis-
ance wi h sequencing o he hemL and lpd genes and pheno ypic
analysis o SCV collec ions. We also hank Thomas Ellio o sending
s ains.
This wo k was suppo ed by g an s om he Minis e io de Ciencia
y Tecnologia (g an BIO2001-0232-C02) and he Eu opean Union
(g an QLK2-1999-00310). M.G.P. was a ecipien o a pos doc o al
ellowship om he Comunidad de Mad id.
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Edi o : B. B. Finlay
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