Download Survival in Animal Cells Requires ppGpp for Internalization and

Salmonella enterica Serovar Gallinarum
Requires ppGpp for Internalization and
Survival in Animal Cells
Jae-Ho Jeong, Miryoung Song, Sang-Ik Park, Kyoung-Oh
Cho, Joon Haeng Rhee and Hyon E. Choy
J. Bacteriol. 2008, 190(19):6340. DOI: 10.1128/JB.00385-08.
Published Ahead of Print 11 July 2008.
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JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6340–6350
0021-9193/08/$08.00⫹0 doi:10.1128/JB.00385-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 19
Salmonella enterica Serovar Gallinarum Requires ppGpp for
Internalization and Survival in Animal Cells䌤
Jae-Ho Jeong,1,2 Miryoung Song,1,2 Sang-Ik Park,1,3 Kyoung-Oh Cho,1,3
Joon Haeng Rhee,1,2 and Hyon E. Choy1,2*
Received 17 March 2008/Accepted 30 June 2008
To elucidate the pathogenic mechanism of Salmonella enterica serovar Gallinarum, we examined the expression of the genes encoded primarily in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. These genes were
found to be induced as cultures entered stationary phase under high- and low-oxygen growth conditions, as also
observed for Salmonella serovar Typhimurium. In contrast, Salmonella serovar Gallinarum in the exponential
growth phase most efficiently internalized cultured animal cells. Analysis of mutants defective in SPI-1 genes,
SPI-2 genes, and others implicated in early stages of infection revealed that SPI-1 genes were not involved in
the internalization of animal cells by Salmonella serovar Gallinarum. Following entry, however, Salmonella
serovar Gallinarum was found to reside in LAMP1-positive vacuoles in both phagocytic and nonphagocytic
cells, although internalization was independent of SPI-1. A mutation that conferred defects in ppGpp synthesis
was the only one found to affect animal cell internalization by Salmonella serovar Gallinarum. It was concluded
that Salmonella serovar Gallinarum internalizes animal cells by a mechanism independent of SPI-1 genes but
dependent on ppGpp. Intracellular growth also required ppGpp for the transcription of genes encoded in
SPI-2.
Salmonella enterica serovar Gallinarum is a fowl-adapted
pathogen causing typhoid fever in chickens, often with a high
mortality rate. (59). Most western countries have succeeded in
the elimination of the diseases by improving the surveillance
and slaughter practices. However, in South America and Asia,
outbreaks of fowl typhoid have seriously threatened the poultry industry (60).
In contrast to other Salmonella serotypes, little is known
about the genetic basis of Salmonella serovar Gallinarum virulence and the molecular mechanisms involved in systemic
infection and the development of fowl typhoid. The 85-kb
serovar Gallinarum plasmid is known to be essential for virulence (6). Two pathogenicity islands, Salmonella pathogenicity
island 1 (SPI-1) and SPI-2, which play key roles in mediating
disease by Salmonella enterica through their respective type III
secretion systems (TTSS) (36), have been described. The TTSS
mediates the translocation of various virulence-associated effector proteins from bacteria into the host cells (33). Based on
studies of the cellular and molecular mechanisms of typhoidlike disease in S. enterica serovar Typhimurium-infected mice,
SPI-1 is considered to be essential for the invasion of animal
cells by Salmonella, and SPI-2 is required for intracellular
proliferation and survival (16, 29). Several of the other SPIs
have also been identified in serovar Gallinarum, and these SPIs
share significant homology with those from other serovars,
although their functional role has yet to be confirmed (56, 67).
In the case of serovar Typhimurium, the SPI-1-encoded
TTSS translocates bacterial effector proteins into the host cell
cytosol to reorganize the cytoskeleton during invasion, resulting in membrane ruffling and eventual bacterial uptake, in
which bacteria are enclosed in a membrane-bound vacuole, the
Salmonella-containing vacuole (SCV) (26). The survival of bacteria within the SCV has been attributed to the incomplete
fusion of this phagosome with the prelysosomal and lysosomal
compartments (27, 52). Phagocytes normally incorporate microorganisms into a membrane-bound compartment or phagosome, which matures by sequential fusion with a series of
endomembrane compartments (21). The resulting changes in
its composition and luminal pH confer its characteristic bactericidal properties to the phagosome. The survival of Salmonella can be attributed to its seclusion within SCVs, rendering
it inaccessible to most host defense mechanisms (21, 27,
52, 68).
It has been well documented that the expression of the SPI-1
secretion system and the expression of its secreted effectors in
serovar Typhimurium are coordinately regulated by SPI-1-encoded HilA, a member of the OmpR/ToxR family of transcriptional regulators (42). Other SPI-1 genes regulated by HilA
include invF and sicA (15, 17, 18). InvF, a member of the
AraC/XylS family of transcriptional regulators, in conjunction
with SicA, a TTSS chaperone, takes part in the coordinated
regulation of all SPI-1-encoded genes. It was previously shown
that SPI-1-encoded genes in serovar Typhimurium, including
hilA, are induced at the onset of stationary phase (late exponential phase) under physiologically well-defined standard
* Corresponding author. Mailing address: Department of Microbiology, Chonnam National University Medical College, Gwangju 501746, South Korea. Phone: 82 62 220 4137. Fax: 82 62 228 7294. E-mail
[email protected].
䌤
Published ahead of print on 11 July 2008.
6340
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Center for Host Defense against Enteropathogenic Bacteria Infection and Research Institute of Vibrio Infection,1 and Department of
Microbiology,2 Chonnam National University Medical College, Gwangju 501-746, South Korea, and Biotherapy Human Resources
Center,
College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, South Korea3
ppGpp IN SALMONELLA SEROVAR GALLINARUM
VOL. 190, 2008
TABLE 1. Salmonella strains used in this study
Description
SG3001
SG3003
SG3004
SG3005
SG3015
SG3016
SG3017
SG3018
SG3021
SG3022
SG3023
SG3024
SG3025
Sch2005
SMR2063
Wild-type isolate
SG3001 ⌬spoT::cat ⌬relA::kan
SG3001 ⌬hilA::kan
SG3001 ⌬ssrAB::Kan
SG3001 putA::hilAp::lacZY::Amp
SG3003 putA::hilAp::lacZY::Amp
SG3001 putA::ssrABp::lacZY::Amp
SG3003 putA::ssrABp::lacZY::Amp
SG3001 ⌬sipADCB::kan
SG3001 ⌬invF::tet
SG3001 ⌬siiE::kan
SG3001 ⌬pilV::kan
SG3001 ⌬sefABCDR::kan
Serovar Typhimurium 14028s
Sch2005 hilA080::Tn5 lacZY
Reference
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growth conditions (LB with vigorous aeration) (63) and that
stationary-phase induction requires the stringent signal molecule ppGpp (7). A recent study revealed that the expression of
SPI-2 genes is also ppGpp dependent (66). ppGpp is synthesized by two synthetases, PSI and PSII, which are encoded by
the relA and spoT genes, respectively. Enteric bacteria exert a
stringent control over ribosome production that is mediated by
ppGpp during the transition from exponential growth to stationary phase (54, 65). The accumulation of ppGpp at the end
of the exponential phase has been considered to result in a
reduction of stable RNA synthesis and the activation of those
genes involved in the maintenance of growth-arrested physiology and the survival of environmental stresses (7, 11).
In an attempt to elucidate the pathogenic mechanism of
serovar Gallinarum, we have examined various serovar Gallinarum mutants defective in SPI-1- or SPI-2-encoded genes and
found that SPI-1-encoded genes are not involved in the uptake
of bacteria into animal cells, although they were induced and
secreted in a manner similar to that seen for serovar Typhi-
murium at the onset of stationary phase during growth under
standard conditions. Microscopic observation revealed that
serovar Gallinarum resides in SCVs following entry into both
phagocytic and nonphagocytic cells. We thus conjectured that
an alternative route independent of SPI-1 is responsible for the
uptake of serovar Gallinarum into animal cells. Here, we demonstrate that among various serovar Gallinarum mutants, only
ppGpp-defective serovar Gallinarum showed significantly reduced entry into several types of animal cells, suggesting that
the factor(s) involved in animal cell entry is under the control
of ppGpp.
MATERIALS AND METHODS
Bacterial strains. The serovar Gallinarum strains, all derived from wild-type
SG3001, used in this study are listed in Table 1. All bacterial strains were constructed
according to the method developed by Datsenko and Wanner (19). The gene(s)
carrying either kan or cat in the place of its open reading frame(s) was generated by
PCR amplification using the respective pair of ⬃60-nucleotide (nt) primers that
included 40-nt homology extensions and 20-nt priming sequences with pKD13 as a
template (Table 2). The PCR products were purified and transformed into bacteria
carrying a Red helper plasmid (pKD46) by electroporation. The electrocompetent
cells were grown in LB broth with ampicillin and L-arabinose (1 mM) at 30°C to an
optical density at 600 nm of ⬃0.5. The mutants were confirmed by PCR using
original and common test primers: k1 (CAGTCATAGCCGAATAGCCT) and k2
(CGGCCACAGTCGATGAATCC) for kan or C1 (TTATACGCAAGGCGACA
AGG) and C2 (GATCTTCCGTCACAGGTAGG) for cat. Serovar Typhimurium
strains carrying lacZ genes transcriptionally fused to the hilA or ssrAB promoter on
the chromosome adjacent to putA gene were constructed by the modification of the
method developed previously (19, 61). Briefly, we cloned the hilA (positions ⫺200 to
⬃⫹100) or ssrB (positions ⫺200 to ⬃⫹100) promoter region into a plasmid derived
from pRS415 (61), which carried a lacZ structural gene and a cat gene in the place
of lacYA in pRS415 (data not shown). Test promoter::lacZ::cat was amplified with
primers that contain sequences for the putA site of S. enterica serovar Typhimurium:
5⬘ primer GAAATCGCCTGTTAATGGTACCAATAGCCTTGACGCAATAGA
GTAATGACCGAGGCCCTTTCGTCTTCAAGAATT and 3⬘ primer CGTCAT
TGTCAGTCTCTTACAGAAAGATTACACGATTATTTCATCGGCAGGAG
ACGTGTGTAGGCTGGAGCTGCTTC, which consisted of 50 or 55 nt of putA
(putA sequences are underlined) and 25- or 22-nt sequences flaking promoter::lacZ::cat in the above-described plasmids. The ⬃5-kbp PCR products were
purified and transformed into bacteria carrying a Red helper plasmid (pKD46) by
electroporation. This putA::test promoter::lacZ::cat construct was confirmed by
PCR.
TABLE 2. Oligonucleotide primers used to generate gene knockout strains
Gene
relA
Primer
(direction)a
relA::Km (F)
relA::Km (R)
spoT
spoT::Cm (F)
spoT::Cm (R)
hilA
hilA::Km (F)
hilA::Km (R)
ssrAB
ssrAB::Km (F)
ssrAB::Km (R)
a
b
Sequenceb
GTG GAT CGC AAG CCT GGG AAT TTC CAG CCA GCA GTC GTG TGA GCG CTT AGG TGT AGG
CTG GAG CTG CTT C
GTG CAG TCG CCG TGC ATC AAT CAC ATC CGG CAC CTG GTT CAG CTT ACC GAA TTC CGG
GGA TCC GTC GAC C
TTA AGC GTC TTC GGC AGG CGT ATC TCG TTG CAC GTG ACG CTC ACG AGG GCT GTA GGC
TGG AGC TGC TTC
GCC AGA TGT ACG CGA TCG CGT GCG GTA AGG CGA ATA AAG GTA CTA TAG ACC ATA TGA
ATA TCC TCC TTA G
GAG GAT GATACT GCT CAT AAC CCT CCT GCC TTT TTG ACG CTA TAA CTG AAG GGA GGT
GTA GGC TGG AGC TGC TTC
CCA GGT TTC ATC GCC GAT TCC TGC TGG GCG ATA GCG TAA AGT AGT TCG GAT TCA TTC
CGG GGA TCC GTC GAC C
ATG AAT TTG CTC AAT CTC AAG AAT ACG CTG CAA ACA TCT TGT GTA GGC TGG AGC TGC
TTC
TTA ATA CTC TAT TAA CCT CAT TCT TCG GGC ACA GTT AAG TAT TCC GGG GAT CCG TCG ACC
F, forward primer; R, reverse primer.
The respective gene sequences are underlined.
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Strains
6341
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JEONG ET AL.
Typhimurium were used (Table 2) (1). PCRs were performed in a total volume
of 50 ␮l containing 1⫻ PCR buffer without MgCl2, 2.5 mM MgCl2, 1 ␮g DNA/
ml, 0.3 ␮M each primer, 0.25 mM each deoxynucleoside triphosphate, and 1 U
Taq DNA polymerase (Takara). PCRs were carried out using a Bio-Rad thermocycler. Species-specific amplification consisted of 30 cycles of 1 min at 94°C,
45 s of an annealing step, and 45 s at 72°C. The first cycle of the amplification
program was preceded by incubation for 5 min at 94°C and followed by a final
5-min extension step at 72°C. Negative controls containing no DNA template
were included in parallel. Five-microliter samples of the PCR products were
analyzed by electrophoresis in a 0.7% agarose gel in Tris-acetate-EDTA buffer
and visualized by UV illumination after ethidium bromide staining.
RESULTS
Analysis of SPIs in Salmonella serovar Gallinarum. In an
attempt to elucidate the pathogenic mechanism of serovar
Gallinarum, we carried out comparative studies between serovar Gallinarum and a better-characterized serovar, serovar Typhimurium. The representative serovar Gallinarum used in
this study was isolated from the chicken liver with fowl typhoid
in a South Korean broiler farm and identified by the modified
rapid slide agglutination test in 2001 (53). The natural serovar
Gallinarum isolate used in this study was found to be fully
infectious, suggesting that the strain contains all virulence
components necessary for fowl infection (see below) (31). The
genetic organization of this serovar Gallinarum isolate was
investigated by determining sizes of the genes amplified by
PCR using the primer sets specific to DNA sequences within
SPI-1 to SPI-5, previously employed to analyze the variation
between serovars of S. enterica within SPIs (Table 3) (1). PCR
amplification was performed with chromosomal DNA isolated
from serovar Gallinarum. The PCR products, a total of 14
amplicons, were found to be exactly the same size as those
predicted for serovar Typhimurium (Fig. 1). All the SPI loci
from different serovars share approximately the same distribution of similarities as the genomes as a whole (varying between
97.7% and 98.6% identity), which suggested that they were
acquired soon after the divergence of Salmonella from E. coli
(22, 44, 46, 48). Thus, we speculated that the genetic structure
of the serovar Gallinarum isolate, at least that of the SPI loci,
does not differ significantly from that of serovar Typhimurium,
implying that the roles of these genes in serovar Gallinarum
and serovar Typhimurium may be similar.
Expression from major promoters in SPI-1 and -2 in Salmonella serovar Gallinarum. It was previously shown that SPI1-encoded genes in serovar Typhimurium are expressed at the
onset of stationary phase when bacteria are grown under standard laboratory conditions, in LB with vigorous aeration, and
that early-stationary-phase cultures are therefore most invasive
(63). This was found to be due to the selective expression of the
master regulator hilA upon entry into stationary phase. Similarly, it was previously reported that SPI-2-encoded genes are
also activated at entry into stationary phase under the same
aerobic growth conditions (66). We therefore monitored the
levels of expression of hilAp and ssrABp (located in SPI-2) in
serovar Gallinarum under standard growth conditions. First,
transcription from these promoters was verified in serovar
Gallinarum. Total RNA was extracted from serovar Gallinarum cells grown to early stationary phase, and specific transcripts generated from hilAp and ssrABp were analyzed by the
primer extension method (Fig. 2). This analysis revealed that
the DNA sequences surrounding the two promoters (between
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Growth conditions. Except when indicated otherwise, cultures were grown in
LB medium (Difco Laboratories) containing 1% NaCl with vigorous aeration at
37°C. For solid support medium, 1.5% granulated agar (Difco Laboratories) was
included. Nutrient broth and brain heart infusion medium were purchased from
Difco Laboratories. Antibiotics were obtained from Sigma Chemical. When
present, antibiotics were added at the following concentrations: 50 ␮g/ml ampicillin, 50 ␮g/ml kanamycin, 15 ␮g/ml chloramphenicol, and 15 ␮g/ml tetracycline.
Protein analysis of culture supernatants. Cultures were grown in 5 ml LB broth
with antibiotics and vigorous aeration overnight and then harvested. Bacteria were
pelleted at 8,000 ⫻ g for 15 min, and supernatants were immediately transferred into
clean tubes. The supernatants were filtered through a 0.45-␮m-pore-size syringe
filter (Sartorius), and proteins were precipitated with cold trichloroacetic acid at a
final concentration of 10%. The proteins were collected by centrifugation at 8,000 ⫻
g at 4°C for 15 min and resuspended in 1 ml cold acetone. These mixtures were
centrifuged for 15 min at 8,000 rpm at 4°C, and pellets were resuspended in 20 ␮l 1⫻
phosphate-buffered saline (PBS). Protein sample buffer containing ␤-mercaptoethanol was added to the samples, which were boiled for 5 min, and proteins were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (7.5%). Proteins were visualized with silver stain (63). Protein markers were
obtained from Bio-Rad.
␤-Galactosidase assay. ␤-Galactosidase assays were performed essentially as
described previously by Miller (45) except that cells were permeabilized with
Koch’s lysis solution (51). For determinations of ␤-galactosidase levels in bacteria at different stages of growth, bacteria cultured overnight were diluted 1:50
into medium with or without antibiotics as described in the text. Cultures were
incubated further at 37°C to stationary phase. Samples were taken for enzyme
assays at regular time intervals. Each strain was assayed in triplicate, and average
enzyme activities were plotted as a function of time.
Primer extension analysis. Total RNA was obtained from Salmonella serovar
Gallinarum strains using Trizol reagent according to the manufacturer’s instructions (Invitrogen). Approximately 50 ␮g total RNA was isolated from 50 ml cell
culture. The primers used to detect the hilA and ssrB transcripts were SGhilApx
(5⬘-TAATCACAGTTAGTTATAACAATATTATTA-3⬘) and SGssrBpx (5⬘-CG
CGAGGGCAGCAAAATCAAAGAATATAAG-3⬘), respectively. The 32P-labeled primers (50,000 cpm) were coprecipitated with 50 ␮g total RNA. Primer
extension reactions were performed as described previously by Shin et al. (58).
Infection assay. Infection (gentamicin protection) assays were performed essentially as described previously (40). Mouse peritoneal macrophages (MPM)
from specific-pathogen-free BALB/c mice and chicken peritoneal exudate macrophages (CPM) and chicken embryo fibroblasts (CEF) from specific-pathogenfree inbred Salmonella-susceptible White Leghorn chickens (8 to 12 weeks old)
(Hy-Vac, IA) (68, 69) were prepared as previously described (8). RAW264.7 cells
and HEp-2 cells were grown in Dulbecco’s modified Eagle’s medium (Gibco
BRL) with 10% fetal bovine serum (Gibco BRL) at 37°C under 5% CO2.
Bacteria were grown to the early stationary phase and resuspended at the appropriate dilution in cell culture medium for the infection of cell monolayers.
Bacteria were added to MPM, CPM, RAW264.7, CEF, and HEp-2 cells at the
ratios indicated in the text, and the mixtures were incubated at 37°C under 5%
CO2 for 30 min. Infected cells were washed three times with PBS (pH 7.4),
Dulbecco’s modified Eagle’s medium containing gentamicin (20 ␮g/ml; Sigma)
was added, and the mixtures were incubated for the indicated time period.
Intracellular bacteria were harvested by extraction with lysis buffer (0.05% Triton
X-100 in PBS [pH 7.4]) and replica plated for colony counting on brain heart
infusion agar plates.
Immunostaining and fluorescence microscopy. HEp-2 and RAW264.7 cells
were plated onto eight-well LabTec Chamber slides (Nunc). These cells were
washed twice with PBS and placed into Dulbecco’s modified Eagle’s medium
(with 5% fetal bovine serum). A bacterial culture grown overnight was subcultured into LB broth, grown to early stationary phase, and used for invasion. The
animal cells were infected with bacteria at a multiplicity of infection (MOI) of 10
and incubated for 8 h in the presence of 20 ␮g/ml gentamicin. After incubation,
the animal cells infected with serovar Gallinarum were fixed with formaldehyde
(3.7%) and stained with anti-LAMP1 mouse monoclonal antibody (H4A3; Santa
Cruz Biotech), Texas Red-conjugated goat anti-mouse immunoglobulin G (IgG)
(Molecular Probes), and DAPI (4⬘,6⬘-diamidino-2-phenylindole) according to
the protocol described previously by Scott et al. (55). Salmonella serovar Gallinarum was detected by rabbit anti-salmonella IgG and fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (Serotec). Colocalization of intracellular
serovar Gallinarum with SCV markers (LAMP1) was determined using an Olympus BX51 microscope and an imaging program (analySIS LS starter) (55).
PCR amplification and analysis of SPI-encoded genes. For PCR analysis of
serovar Gallinarum isolate used in this study, oligonucleotide primers (Genotech, Daejon, South Korea) targeting the genes in SPI-1 to SPI-5 of serotype
J. BACTERIOL.
ppGpp IN SALMONELLA SEROVAR GALLINARUM
VOL. 190, 2008
6343
TABLE 3. Oligonucleotide primers for PCR amplification of SPI-encoded gene fragmentsa
SPI
Primer
SPI-1
1
2
3
4
5
6
7
SPI3
8
9
10
11
Sequence
Expected
size (bp)
sitD (F)
sprB (R)
sprB (F)
orgC (R)
orgC (F)
prgK (R)
spaS (F)
spaO (R)
GCC ATC GTG TTT ATT GCG GCA TT
GAG ACG CCG GAA GAG GTA TTG TTT
GGT AGA TTC CAC CAG GCG TAT TGA
TGC TGG CGA TTT GTG ATA AGG
CGC TCA TAC TAA GCG TGA TTG
CCT AAT CTG GTG ACG GAT CCT
CCG ACG GTG GTT AGT GAA CAT T
CGT TGC GCT TTG TAA TCG GTA G
ssaV (F)
sscA (R)
ssrA (F)
ssrB (R)
sseF (F)
ssaE (R)
CGG TAC GTC GAA CAA GGT GGC TTC TG
GGC GTG GCG GCT CGC TGC GTA TGT
CAC GTT TAC CAG CAC AAC GAC TGG A
ATA CGA CAT GGT AAA GCC CGT
CGC CAA TAA CAC ACT GGG ATC GGC A
GCT TCG GCG TCA GCA ACG CTG ATA C
sugR (F)
rhuM (R)
rhuM (F)
rmbA (R)
selC (F)
misL (R)
misL (F)
fidL (R)
GAG AAT CGA CAA ACT TTC ACT GC
TTC TGT TTC CCA TTG TAG CAA ACC
GCG CAG GTA AAG TCA CTA A
GTC TTC CAC ATC GCA ATA ATA TGG
AGG TTC GAC TCC TGT GAT CTT CC
GGC AGA CTA TCA ACG TCA ACC CA
CGG TAT CAG CAG CAT TAG CCT CTA
GTC TGG GTG TAG GGC TTG GAC ATC
2,747
1,634
2,092
3,137
3,326
1,1032
2,600
4,684
2,171
6,283
3,364
SPI4
12
4K (F)
4A (R)
AGC CAG GAA GTG ACA CTA ACC CAG G
GCG GTA ACA ATA CTA AAC TGC GGC TG
2,347
SPI5
13
sopB (F)
sopB (R)
pipC (F)
pipB (R)
CGC TAT GCA AAT ACA GAG CTT C
TAC CTC AAG ACT CAA GAT GTG A
ATC GCC AGA GGT GCT CAA TCT TTC
CTG GGG ACG CGT TAG TTA TTG GCA
1,700
14
a
b
1,026
See reference 1.
F, forward primer; R, reverse primer. The sequences are found in the designated genes.
positions ⫺200 and ⫹100) and the transcription start sites were
the same, suggesting that a similar mechanism governs these
promoters. Next, hilAp and ssrABp activities were determined
in serovar Gallinarum cells carrying these promoters fused to
lacZ. To avoid possible complications due to changes in the
copy number of plasmid-borne test promoters during growth
to stationary phase, we placed the promoter::lacZ constructs
on the serovar Gallinarum chromosome near the putA site
using a modified linear DNA transformation method (19, 24).
We found that both promoters were activated at the onset of
stationary phase when serovar Gallinarum was grown under
standard conditions, as determined by the ␤-galactosidase assay (Fig. 3), further supporting the possibility that at least
SPI-1 and SPI-2 in serovar Gallinarum function similarly as in
serovar Typhimurium.
FIG. 1. Analysis of gene fragments encoded by SPIs (SPI-1 to
SPI-5) in Salmonella serovar Gallinarum. The PCR product in each
lane (lanes 1 to 14) was obtained using the respective primer sets
shown in Table 3. The primer sets specific to sequences flanking the
indicated SPI genes were the same as those previously described (1).
FIG. 2. Identification of the hilA and ssrB transcriptional start sites
by primer extension. The right panel shows the hilA transcript (arrow)
and the left panel shows the ssrB transcript (arrow) next to DNA
sequencing ladders. The circled letter indicates the transcriptional start
site, ⫹1.
Downloaded from http://jb.asm.org/ on February 27, 2014 by PENN STATE UNIV
SPI-2
Gene (direction of
sequence)b
6344
JEONG ET AL.
To further validate the functional expression of SPI-1 genes
in serovar Gallinarum, we determined the presence of secreted
effector proteins (35) in the culture medium at the onset of
stationary phase (Fig. 4). Total supernatant was collected, precipitated, and analyzed by SDS-PAGE. The secreted proteins
SipA (89 kDa), SipB (67 kDa), SipD (62 kDa), and SipC (42
kDa) were clearly detected in the supernatant of the serovar
Gallinarum culture, similar to what was seen for the serovar
Typhimurium culture (63). These proteins were not observed
in mutants lacking the hilA or sipADCB gene block, validating
the positive role of hilA in the expression of SPI-1 genes. It
should be noted that these effector proteins were also undetected in a serovar Gallinarum mutant defective in ppGpp
synthesis (⌬relA ⌬spoT) (see below). Analysis of the DNA
region encompassing SPI-1 in serovar Gallinarum by pulsedfield gel electrophoresis/long-range PCR (71) or comparative
genomic hybridization (10, 50) revealed that it is not markedly
different from that in serovar Typhimurium, suggesting that a
mechanism similar to that in serovar Typhimurium would govern the expression of those SPI-1 genes in serovar Gallinarum.
Entry of Salmonella serovar Gallinarum into cultured animal cells. To monitor the entry of animal cells and intracellular
replication by serovar Gallinarum, we first determined its in-
FIG. 4. Expression of secreted TTSS components by various Salmonella strains. Proteins excreted into the media were analyzed on a
7.5% SDS-PAGE gel. The secreted proteins SipA (89 kDa), SipB (67
kDa), SipD (62 kDa), and SipC (42 kDa) were identified by their sizes
as described previously (63). Protein markers are shown in the far left
lane. MW, molecular weight marker (in thousands).
FIG. 5. Internalization capability of serovar Gallinarum (black
bars) in comparison with that of serovar Typhimurium (gray bars)
using various animal cells including RAW264.7, MPM, HEp-2, CEF,
and CPM cells.
ternalization capability using various animal cells including
MPM, HEp-2, RAW264.7, CPM (peritoneal exudates), and
CEF cells (Fig. 5). Human pharyngeal epithelial HEp-2 cells
and murine macrophage-like RAW264.7 cells are the most
commonly used cell lines to assay for infection by serovar
Typhimurium. MPM and chicken-derived cells (CEM and
CEF) were extracted from BALB/c mice and Salmonella-susceptible White Leghorn chickens, respectively (8, 69). Serovar
Gallinarum has been shown to proliferate in macrophages
from the susceptible line due to the inefficient expression of
proinflammatory chemokines and cytokines (68, 69). Serovar
Gallinarum cells grown under the standard conditions were
mixed with the animal cells at an MOI of 10 for 30 min, and
intracellular bacteria were counted. Serovar Typhimurium was
included as a control. The number of intracellular bacteria
varied depending on the cell type. However, it was clear that
serovar Gallinarum inefficiently entered each of these cell
types, at about 10% of the frequency of that observed for
serovar Typhimurium (ST), which might be due to a different
antigenicity (O9) or a lack of motility (4, 68). This result is
consistent with reports that serovar Gallinarum poorly internalizes and survives in animal cells, even those of chicken
origin (3, 14).
We then analyzed the entry of serovar Gallinarum at different phases of growth cultured under standard conditions (LB
with aeration) into phagocytic (RAW264.7) and nonphagocytic
(HEp-2) cells (Fig. 6, black bars). Most interestingly, the internalization capability of serovar Gallinarum during exponential growth (2 h) was significantly greater than that at early (4
h) or late (8 h) stationary phase and overnight culture in both
cell types. Clearly, this finding is inconsistent with the hilAp
activity measured under standard growth conditions (Fig. 3). It
was previously reported that the expression of SPI-1-encoded
genes including TTSS components is activated during growth
under specific culture conditions (2). Specifically, anaerobiosis
was shown to increase the internalization of serovar Typhimurium by HEp-2 cells (2, 25, 39). We therefore grew serovar
Gallinarum in low oxygen and measured its invasiveness using
the same animal cells (Fig. 6, gray bars). In contrast to what
was seen for serovar Typhimurium (63), serovar Gallinarum
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FIG. 3. Expression of hilAp::lacZ (left) and ssrABp::lacZ (right) in
Salmonella serovar Gallinarum growing under standard conditions.
The curves with open circles represent growth (A600), and the curves
with filled circles represent promoter activities as determined by ␤galactosidase assay (Miller units).
J. BACTERIOL.
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ppGpp IN SALMONELLA SEROVAR GALLINARUM
6345
grown in low oxygen was more invasive than when grown under
standard conditions. Moreover, exponential-phase serovar
Gallinarum grown in low oxygen was consistently most invasive
for both types of animal cells. We therefore determined the
levels of expression from hilAp in serovar Gallinarum and
serovar Typhimurium grown in low oxygen using hilAp::lacZ
fusion strains and found that hilAp was activated at the onset
of stationary phase, similar to what was found under standard
growth conditions (Fig. 7). Note that the degree of induction
was much greater in serovar Gallinarum than in serovar Typhimurium. This set of results revealed a discrepancy: the
early-exponential-phase culture was most readily internalized,
while hilA and, therefore, those SPI-1 genes under its control
were highly expressed later at the onset of stationary phase
irrespective of growth conditions. These results suggested that
for serovar Gallinarum, SPI-1 genes might not be involved in
the uptake into animal cells.
Intracellular localization of Salmonella serovar Gallinarum.
SPI-1-encoded TTSS in serovar Typhimurium are essential for
the internalization of bacteria into both phagocytic and nonphagocytic cells, and they function by actively reorganizing the
host cell actin cytoskeleton. Once inside the host, the bacteria
FIG. 7. Expression of hilAp::lacZ in serovar Typhimurium (circles)
or in serovar Gallinarum (triangles) grown under low-oxygen conditions. The curves with open symbols represent growth (A600), and the
curves with closed symbols represent hilAp activity, as determined by a
␤-galactosidase assay (Miller units).
reside in large SCVs that selectively interact with elements of
the endocytic pathway (64). We therefore examined whether
internalized serovar Gallinarum was associated with the SCV
despite the apparent absence of a functional role for SPI-1encoded genes in internalization, as indicated by the abovedescribed results. LAMP1, a lysosomal glycoprotein known to
be recruited to mature SCVs within 30 min of infection (52),
was visualized together with serovar Gallinarum by microscopy. Phagocytic (RAW264.7) (Fig. 8, top) and nonphagocytic
(HEp-2) (Fig. 8, bottom) cells were infected with serovar Gallinarum at an MOI of 10 and incubated for 8 h before staining
for LAMP1 and bacteria with specific antibodies and nuclei
with DAPI. In both cell types, we could visualize SCV by
staining for LAMP1 in cytosolic compartments following serovar Gallinarum infection. Serovar Gallinarum colocalized with
LAMP1 in SCVs only in the infected cells (Fig. 8A and B).
Note that LAMP1 in uninfected cells was distributed evenly in
the cytosolic compartments (Fig. 8C). It appears that serovar
Gallinarum as well as serovar Typhimurium can generate
SCVs, although its SPI-1-encoded TTSS and effector proteins
may not play an obvious role during the internalization into
animal cells.
A Salmonella serovar Gallinarum mutation affecting entry
and intracellular survival. Finally, to identify the gene or gene
block involved in the internalization of animal cells by serovar
Gallinarum, we created a series of mutations at serovar Gallinarum loci reported to be implicated in early stages of pathogenesis by various Salmonella serotypes. These included hilA,
invF, and the sipADCB gene block in SPI-1; ssrAB in SPI-2; the
sef gene block in SPI-10; siiE in SPI-4; and the pilV gene block
(23, 30, 72). SsrA and/or SsrB, a sensor kinase, in serovar
Typhimurium is required for the expression of SPI-2 genes (12,
20, 34, 47, 57). A gentamicin protection assay (38) was employed to enumerate the intracellular bacteria after incubation
of serovar Gallinarum mutants and nonphagocytic HEp2 cells
for 30 min (Fig. 9). Only a mutant defective in ppGpp synthesis
(⌬relA ⌬spoT) exhibited a strikingly reduced level of internalization (⬎90%).
Subsequently, we determined the entry and intracellular replication capacities of ppGpp-defective mutants in various animal cells including RAW264.7, MPM, CPM, CEF, and HEp-2
cells (Fig. 10). Serovar Gallinarum derivatives with mutations
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FIG. 6. Internalization of animal cells by Salmonella serovar Gallinarum at different growth phases under low-oxygen (gray bars) and
high-oxygen (black bars) conditions. RAW264.7 (right) and HEp-2 (left) cells were infected with serovar Gallinarum harvested at the indicated
time at an MOI of 10, and intracellular gentamicin-resistant bacteria were counted by a standard method (38). The fractions of intracellular
bacteria (percent) are plotted.
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JEONG ET AL.
J. BACTERIOL.
in hilA in SPI-1 or in ssrAB in SPI-2 were also included. Wildtype serovar Gallinarum was internalized by these cells at a
rate of about 0.1% at an MOI of 1 (106 cells and 106 bacteria)
irrespective of cell type. The number of intracellular wild-type
FIG. 9. Internalization of Hep-2 cells by wild-type (SG3001), ⌬hilA
(SG3004), ⌬sipADCB (SG3021), ⌬invF (SG3022), ⌬ssrAB(SG3005),
⌬sefABCDR (SG3025), ⌬siiE (SG3023), ⌬pilV (SG3024), and ⌬relA
⌬spoT (SG3003) strains of Salmonella serovar Gallinarum. Bacteria
were incubated with HEp-2 cells for 30 min at an MOI of 10. After
removing excess bacteria by washing, internalized bacteria were
counted by a standard plating method.
serovar Gallinarum cells increased ⬃103-fold during 24 h of
incubation in all cell types. The ⌬hilA mutant internalized and
replicated like wild-type serovar Gallinarum, and the ⌬ssrAB
mutant invaded as well as the wild type but replicated poorly in
all cell types tested. The ppGpp-defective mutant internalized
much less efficiently than the wild type (⬃10-fold less) and
failed to replicate intracellularly in all cell types. We therefore
determined the expression of SPI-2 genes in serovar Gallinarum using the strain carrying the ssrABp::lacZ construct inserted near the chromosomal putA locus under standard
growth conditions (Fig. 11). ssrABp was found to be activated
in wild-type serovar Gallinarum as the culture entered stationary phase but not in the ppGpp-defective mutant. It was suggested that ppGpp was required for the activation of ssrAB,
which in turn allowed the expression of the SPI-2 genes necessary for the intracellular replication of serovar Gallinarum.
Taken together, we concluded that serovar Gallinarum internalizes animal cells through a route independent of SPI-1 but
dependent on ppGpp and that SPI-2-encoded proteins under
ppGpp control are essential for intracellular replication.
DISCUSSION
In this study, using a clinical strain of Salmonella serovar
Gallinarum isolated from a chicken liver with typical fowl typhoid, we found that (i) serovar Gallinarum was capable of
invading and replicating in animal cells within SCVs (Fig. 8);
(ii) serovar Gallinarum at the exponential phase grown under
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FIG. 8. Colocalization of Salmonella serovar Gallinarum with SCV in animal cells as shown by immunofluorescence staining. RAW264.7 (top) and
HEp-2 (bottom) cells were infected with serovar Gallinarum and incubated for 8 h in the presence of gentamicin (20 ␮g/ml). The cells were then fixed,
permeabilized, and immunostained for LAMP1 (red) (b) and salmonellae (green) (c). Animal cell nuclei were stained with DAPI (blue) (a). (d) Merged
images. Arrows mark the colocalization of intracellular Salmonella serovar Gallinarum and LAMP1. (A and B) Essentially the same Salmonella serovar
Gallinarum-infected cells, except that A is shown at high magnification to show cytosolic details. (C) Uninfected cells for negative control.
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ppGpp IN SALMONELLA SEROVAR GALLINARUM
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low-oxygen conditions was most readily internalized (Fig. 6);
(iii) SPI-1 was not required for internalization, although it was
functionally expressed and excreted similarly as in serovar Typhimurium at the onset of stationary phase in a ppGpp-dependent manner (Fig. 3, 4, 7, and 10); (iv) an unidentified gene(s)
under the control of ppGpp was involved in internalization
(Fig. 9 and 10); and (v) SPI-2, also expressed at the onset of
stationary phase in a ppGpp-dependent manner, was required
for the intracellular replication of serovar Gallinarum (Fig. 10
and 11).
FIG. 11. ppGpp-dependent expression of SPI-2 genes. Shown are
data for the expression of ssrABp::lacZ in wild-type (left) (SG3017)
and ⌬relA ⌬spoT (right) (SG3018) strains of Salmonella serovar Gallinarum cultures grown under standard growth conditions. The curves
with open circles represent growth (A600), and closed circles represent
ssrABp activity as determined by the ␤-galactosidase assay (Miller
units).
It should be noted that serovar Gallinarum internalized animal cells, even those extracted from Salmonella-susceptible
White Leghorn chickens, at a rate about 10% of that of serovar
Typhimurium (Fig. 5). But following entry, the intracellular
serovar Gallinarum level increased by roughly 1,000- to 10,000fold during a 24-h incubation (⬃103 to ⬃107), as estimated by
the gentamicin protection assay (Fig. 10). It was previously
reported that serovar Gallinarum is phagocytosed by macrophages of both avian and murine hosts at a rate of ⬍10% of
that of serovar Typhimurium (9, 49). The reduced level of
invasion by serovar Gallinarum, especially in vitro, has been
ascribed to its poor motility (4, 68) and the absence of mannose-sensitive hemagglutination type 1 fimbriae (70). Type 2
fimbriae expressed by serovar Gallinarum play no role in adherence and invasion of bacteria in animal cells (32). Based on
the lack of evidence of membrane ruffling or macropinocytosis
during serovar Gallinarum entry, as examined by transmission
electron microscopy, it has been suggested that serovar Gallinarum is taken up by murine phagocytic cells by a mechanism
different from that for serovar Typhimurium (49). It is, however, not the scope of this study to elucidate the difference
between serovar Gallinarum and serovar Typhimurium for the
invasion of animal cells. Nevertheless, we could localize serovar Gallinarum in LAMP1-positive vacuoles in both phagocytic
(RAW264.7) and nonphagocytic (HEp2) cells (Fig. 8). Irrespective of the mechanism of invasion, the serovar Gallinarum
employed in this study seems to be as capable as serovar
Typhimurium in proliferating in SCVs.
We found that serovar Gallinarum carries all SPI loci found
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FIG. 10. Internalization and intracellular replication of wild-type and mutant Salmonella serovar Gallinarum strains in various animal cells
(A) RAW264.7 cells. (B) MPM. (C) CPM. (D) CEF. (E) HEp-2 cells. Bacteria were incubated with the animal cells for 30 min at an MOI of 10.
After removing excess bacteria by washing, the cells were incubated in the presence of gentamicin (20 ␮g/ml) for the duration of experiment, and
the intracellular bacteria were counted by the standard plating method. Open circles represent wild-type (SG3001), closed circles represent ⌬hilA
(SG3004), open triangles represent ⌬ssrAB (SG3005), and closed triangles represent ⌬relA ⌬spoT (SG3003) strains of Salmonella serovar
Gallinarum.
6348
JEONG ET AL.
play roles during early stages of Salmonella pathogenesis (28).
Among many candidates identified from the literature, three
gene blocks on pathogenicity islands were identified in serovar
Gallinarum by PCR amplification using the primer sets specific
to DNA sequences within their respective SPIs (data not
shown). These include the sef operon in SPI-10, implicated in
uptake and survival in macrophages (23); siiE in SPI-4, implicated in adhesion to eukaryotic cells (30); and the pilV gene
block, implicated in type IV pilus-mediated intestinal cell attachment by Salmonella enterica serovar Typhi (72). However,
none of these genes was found to be involved in the internalization of animal cells by serovar Gallinarum (Fig. 8). Instead,
we observed that a ppGpp-defective serovar Gallinarum strain
entered animal cells at ⬃10% or less of the wild-type invasion
frequency and did not persist within vacuoles as judged by
bacterial survival and direct microscopic visualization (data not
shown). A simple explanation is that ppGpp is required for the
expression of the protein(s) involved in the internalization of
animal cells by serovar Gallinarum. Under standard laboratory
growth conditions, the transition from the exponential phase to
the stationary phase presumably represents a stress condition
that is sensed and translated to the intracellular signal ppGpp.
Based on a microarray analysis of serovar Typhimurium gene
expression, we found that roughly 7.1% of open reading frames
were increased upon entry into the stationary phase (data not
shown). Interestingly, most SPI genes, if not all, were induced
at entry into the stationary phase. All this stationary-phase
gene induction required ppGpp. Thus, the protein(s) responsible for the internalization of serovar Gallinarum might also
be induced at the stationary phase in a ppGpp-dependent
manner. Confirmation of this idea awaits the identification of
the protein(s). Nevertheless, our findings suggest that virulence-associated genes are induced under the stress conditions
that Salmonella encounters in the intestinal lumen and inside
various host cells during the course of animal infection (43).
ACKNOWLEDGMENTS
This work was supported by Korea Health 21 R&D (01-PJ10-PG601GM02-002), the Ministry of Health and Welfare, a Korea Science
and Engineering Foundation grant funded by MOST (no. 2007-04213),
On-Site Cooperative Agriculture Research Project (20070401080077),
RDA, a CNU specialization grant funded by Chonnam National University, the Brain Korea 21 Project, Center for Biomedical Human
Resources, and the Biotherapy Human Recourses Center at Chonnam
National University, Republic of Korea, in 2007. J.H.R. was supported
by a grant (no. RT105-01-01) from the Regional Technology Innovation Program of the MOCIE, Republic of Korea.
REFERENCES
1. Amavisit, P., D. Lightfoot, G. F. Browning, and P. F. Markham. 2003.
Variation between pathogenic serovars within Salmonella pathogenicity islands. J. Bacteriol. 185:3624–3635.
2. Bajaj, V., R. L. Lucas, C. Hwang, and C. A. Lee. 1996. Co-ordinate regulation
of Salmonella typhimurium invasion genes by environmental and regulatory
factors is mediated by control of hilA expression. Mol. Microbiol. 22:703–
714.
3. Barrow, P. A., M. B. Huggins, and M. A. Lovell. 1994. Host specificity of
Salmonella infection in chickens and mice is expressed in vivo primarily at
the level of the reticuloendothelial system. Infect. Immun. 62:4602–4610.
4. Barrow, P. A., and M. A. Lovell. 1989. Invasion of Vero cells by Salmonella
species. J. Med. Microbiol. 28:59–67.
5. Barrow, P. A., M. A. Lovell, and B. A. D. Stocker. 2000. Protection against
experimental fowl typhoid by parenteral administration of live SL5828, an
aroA-serC (aromatic dependent) mutant of a wild-type Salmonella gallinarum strain made lysogenic for P22 sie. Avian Pathol. 29:423–431.
6. Barrow, P. A., J. M. Simpson, M. A. Lovell, and M. M. Binns. 1987. Con-
Downloaded from http://jb.asm.org/ on February 27, 2014 by PENN STATE UNIV
in serovar Typhimurium, as determined by analysis of the gene
fragments in SPIs and partial DNA sequencing (hilA and ssrAB
promoter regions) (Fig. 1 and 2). It was previously reported
that all the SPI loci from different serovars are not significantly
different enough to account for genomic changes that contribute to a serovar’s degree of host adaptation (22, 44, 46, 48). In
the case of serovar Gallinarum, no obvious alternation has
been reported, especially in the SPI-1 region in serovar Gallinarum, as assessed by multilocus enzyme electrophoresis or
whole-genome microarray. These methods, however, would
not allow one to monitor the differences on a minor scale, like
transcriptional changes due to point mutations, silencing of
genes, and small deletions (10, 22, 44, 46, 48, 50, 71). Note that
we could generate various deletion mutants of serovar Gallinarum based on serovar Typhimurium sequences by homologous recombination using the lambda Red system (19). In
addition, some of the mutants (⌬hilA and ⌬sipADCB) displayed the expected defects of the secreted protein profiles
(Fig. 4). This suggests that there are no major genetic rearrangements between serovar Typhimurium and serovar Gallinarum, at least in these regions of the genome.
It was interesting that SPI-1 genes in serovar Gallinarum
seemed to play no role in animal cell internalization, as examined using MPM, CPM, CEF, HEp-2, and RAW264.7 cells
(Fig. 9 and 10). No obvious defect in the internalization of
these cells was noted with serovar Gallinarum derivatives carrying mutations in hilA or invF, which are responsible for the
coordinated expression of SPI-1 genes (15, 17, 18, 42) and in
the sipADCB gene block, which encodes the proteins involved
in effector translocation mediated by SPI-1-encoded TTSS
(Fig. 4) (13). It was previously reported that the internalization
frequency of serovar Typhimurium was severely impaired by
the mutation in hilA, invF, or sipADCB genes (15, 16, 73).
Taken together, it was perceived that the pathogenesis of fowl
typhoid by serovar Gallinarum would be different from that of
murine typhoid by serovar Typhimurium, as suggested previously (59, 60). It was previously shown that serovar Gallinarum
preferentially invaded the cecal tonsil and Peyer’s patch in
chicken lacking peripheral or mesenteric lymph (5, 41). It is
therefore possible that the uptake of serovar Gallinarum may
not require SPI-1 genes even though these were clearly expressed (Fig. 3, 4, and 7). However, this interpretation would
be inconsistent with a previous study that reported the role of
the SPI-1 and SPI-2 TTSS on the virulence, uptake, distribution, and pathology of serovar Gallinarum infections in the
chicken through experimental infections with functional
knockout mutations in either the SPI-1 TTSS (spaS) or the
SPI-2 TTSS (ssaU) (37). The SpaS⫺ mutant was found to be
less invasive, as determined in vitro using primary chick kidney
cells while fully persistent within chicken macrophage-like
cells. In contrast, the SsaU⫺ mutant was fully invasive in chick
kidney cells but failed to persist in macrophages. This discrepancy may also be due to differences in our clinical strain isolated from a South Korean broiler farm and the previously
described strain SG9 (62). However, we also found with the
inbred Salmonella-susceptible White Leghorn chicken that the
SPI-1 mutant was fully virulent, while the SPI-2 mutant was not
(data not shown).
In an attempt to identify the protein(s) involved in animal
cell internalization, if any, we examined those genes known to
J. BACTERIOL.
VOL. 190, 2008
7.
8.
9.
10.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
6349
33. Hensel, M. 2000. Salmonella pathogenicity island 2. Mol. Microbiol. 36:
1015–1023.
34. Hensel, M., J. E. Shea, S. R. Waterman, R. Mundy, T. Nikolaus, G. Banks,
A. Vazquez-Torres, C. Gleeson, F. C. Fang, and D. W. Holden. 1998. Genes
encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Mol. Microbiol. 30:163–174.
35. Hong, K. H., and V. L. Miller. 1998. Identification of a novel Salmonella
invasion locus homologous to Shigella ipgDE. J. Bacteriol. 180:1793–1802.
36. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens
of animals and plants. Microbiol. Mol. Biol. Rev. 62:379–433.
37. Jones, M. A., P. Wigley, K. L. Page, S. D. Hulme, and P. A. Barrow. 2001.
Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity
island 1 type III secretion system for virulence in chickens. Infect. Immun.
69:5471–5476.
38. Kim, H. J., E. Y. Kim, Y. Hong, J. H. Rhee, and H. E. Choy. 2006. Alternative
methods to limit extracellular bacterial activity for enumeration of intracellular bacteria. J. Microbiol. Methods 64:17–26.
39. Lee, C. A., and S. Falkow. 1990. The ability of Salmonella to enter mammalian cells is affected by bacterial growth state. Proc. Natl. Acad. Sci. USA
87:4304–4308.
40. Lee, C. A., B. D. Jones, and S. Falkow. 1992. Identification of a Salmonella
typhimurium invasion locus by selection for hyperinvasive mutants. Proc.
Natl. Acad. Sci. USA 89:1847–1851.
41. Lowry, V. K., G. I. Tellez, D. J. Nisbet, G. Garcia, O. Urquiza, L. H. Stanker,
and M. H. Kogut. 1999. Efficacy of Salmonella enteritidis-immune lymphokines on horizontal transmission of S. arizonae in turkeys and S. gallinarum
in chickens. Int. J. Food Microbiol. 48:139–148.
42. Lucas, R. L., and C. A. Lee. 2000. Unravelling the mysteries of virulence gene
regulation in Salmonella typhimurium. Mol. Microbiol. 36:1024–1033.
43. Marcus, S. L., J. H. Brumell, C. G. Pfeifer, and B. B. Finlay. 2000. Salmonella pathogenicity islands: big virulence in small packages. Microbes Infect.
2:145–156.
44. McClelland, M., L. Florea, K. Sanderson, S. W. Clifton, J. Parkhill, C.
Churcher, G. Dougan, R. K. Wilson, and W. Miller. 2000. Comparison of the
Escherichia coli K-12 genome with sampled genomes of a Klebsiella pneumoniae and three Salmonella enterica serovars, Typhimurium, Typhi and
Paratyphi. Nucleic Acids Res. 28:4974–4986.
45. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
46. Mirold, S., K. Ehrbar, A. Weissmuller, R. Prager, H. Tschape, H. Russmann,
and W. D. Hardt. 2001. Salmonella host cell invasion emerged by acquisition
of a mosaic of separate genetic elements, including Salmonella pathogenicity
island 1 (SPI1), SPI5, and sopE2. J. Bacteriol. 183:2348–2358.
47. Ochman, H., F. C. Soncini, F. Solomon, and E. A. Groisman. 1996. Identification of a pathogenicity island required for Salmonella survival in host
cells. Proc. Natl. Acad. Sci. USA 93:7800–7804.
48. Parkhill, J., G. Dougan, K. D. James, N. R. Thomson, D. Pickard, J. Wain,
C. Churcher, K. L. Mungall, S. D. Bentley, M. T. Holden, M. Sebaihia, S.
Baker, D. Basham, K. Brooks, T. Chillingworth, P. Connerton, A. Cronin, P.
Davis, R. M. Davies, L. Dowd, N. White, J. Farrar, T. Feltwell, N. Hamlin,
A. Haque, T. T. Hien, S. Holroyd, K. Jagels, A. Krogh, T. S. Larsen, S.
Leather, S. Moule, P. O’Gaora, C. Parry, M. Quail, K. Rutherford, M.
Simmonds, J. Skelton, K. Stevens, S. Whitehead, and B. G. Barrell. 2001.
Complete genome sequence of a multiple drug resistant Salmonella enterica
serovar Typhi CT18. Nature 413:848–852.
49. Pascopella, L., B. Raupach, N. Ghori, D. Monack, S. Falkow, and P. L.
Small. 1995. Host restriction phenotypes of Salmonella typhi and Salmonella
gallinarum. Infect. Immun. 63:4329–4335.
50. Porwollik, S., C. A. Santiviago, P. Cheng, L. Florea, and M. McClelland.
2005. Differences in gene content between Salmonella enterica serovar Enteritidis isolates and comparison to closely related serovars Gallinarum and
Dublin. J. Bacteriol. 187:6545–6555.
51. Putnam, S. L., and A. L. Koch. 1975. Complications in the simplest cellular
enzyme assay: lysis of Escherichia coli for the assay of beta-galactosidase.
Anal. Biochem. 63:350–360.
52. Rathman, M., L. P. Barker, and S. Falkow. 1997. The unique trafficking
pattern of Salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry. Infect. Immun.
65:1475–1485.
53. Runnells, R. A., C. J. Coon, H. Farley, and F. Thorp. 1927. An application of
the rapid-method agglutination test to the diagnosis of bacillary white diarrhoea infection. J. Am. Vet. Med. Assoc. 70:660–662.
54. Sands, M. K., and R. B. Roberts. 1952. The effects of a tryptophan-histidine
deficiency in a mutant of Escherichia coli. J. Bacteriol. 63:505–511.
55. Scott, C. C., P. Cuellar-Mata, T. Matsuo, H. W. Davidson, and S. Grinstein.
2002. Role of 3-phosphoinositides in the maturation of Salmonella-containing vacuoles within host cells. J. Biol. Chem. 277:12770–12776.
56. Shah, D. H., M. J. Lee, J. H. Park, J. H. Lee, S. K. Eo, J. T. Kwon, and J. S.
Chae. 2005. Identification of Salmonella gallinarum virulence genes in a
Downloaded from http://jb.asm.org/ on February 27, 2014 by PENN STATE UNIV
11.
tribution of Salmonella gallinarum large plasmid toward virulence in fowl
typhoid. Infect. Immun. 55:388–392.
Cashel, M., D. R. Gentry, V. J. Hernandez, and D. Vinella. 1996. Escherichia
coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 2. ASM
Press, Washington, DC.
Chadfield, M., and J. Olsen. 2001. Determination of the oxidative burst
chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes. Vet. Immunol. Immunopathol. 80:289–308.
Chadfield, M. S., D. J. Brown, S. Aabo, J. P. Christensen, and J. E. Olsen.
2003. Comparison of intestinal invasion and macrophage response of Salmonella Gallinarum and other host-adapted Salmonella enterica serovars in
the avian host. Vet. Microbiol. 92:49–64.
Chan, K., S. Baker, C. C. Kim, C. S. Detweiler, G. Dougan, and S. Falkow.
2003. Genomic comparison of Salmonella enterica serovars and Salmonella
bongori by use of an S. enterica serovar Typhimurium DNA microarray. J.
Bacteriol. 185:553–563.
Chang, D. E., D. J. Smalley, and T. Conway. 2002. Gene expression profiling
of Escherichia coli growth transitions: an expanded stringent response
model. Mol. Microbiol. 45:289–306.
Cirillo, D. M., R. H. Valdivia, D. M. Monack, and S. Falkow. 1998. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type
III secretion system and its role in intracellular survival. Mol. Microbiol.
30:175–188.
Collazo, C. M., and J. E. Galan. 1997. The invasion-associated type III
system of Salmonella typhimurium directs the translocation of Sip proteins
into the host cell. Mol. Microbiol. 24:747–756.
Collins, F. M., and P. B. Carter. 1978. Growth of salmonellae in orally
infected germfree mice. Infect. Immun. 21:41–47.
Darwin, K. H., and V. L. Miller. 1999. InvF is required for expression of
genes encoding proteins secreted by the SPI1 type III secretion apparatus in
Salmonella typhimurium. J. Bacteriol. 181:4949–4954.
Darwin, K. H., and V. L. Miller. 1999. Molecular basis of the interaction of
Salmonella with the intestinal mucosa. Clin. Microbiol. Rev. 12:405–428.
Darwin, K. H., and V. L. Miller. 2000. The putative invasion protein chaperone SicA acts together with InvF to activate the expression of Salmonella
typhimurium virulence genes. Mol. Microbiol. 35:949–960.
Darwin, K. H., and V. L. Miller. 2001. Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella
typhimurium. EMBO J. 20:1850–1862.
Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad.
Sci. USA 97:6640–6645.
Deiwick, J., T. Nikolaus, S. Erdogan, and M. Hensel. 1999. Environmental
regulation of Salmonella pathogenicity island 2 gene expression. Mol. Microbiol. 31:1759–1773.
Desjardins, M., L. A. Huber, R. G. Parton, and G. Griffiths. 1994. Biogenesis
of phagolysosomes proceeds through a sequential series of interactions with
the endocytic apparatus. J. Cell Biol. 124:677–688.
Edwards, R. A., G. J. Olsen, and S. R. Maloy. 2002. Comparative genomics
of closely related salmonellae. Trends Microbiol. 10:94–99.
Edwards, R. A., D. M. Schifferli, and S. R. Maloy. 2000. A role for Salmonella fimbriae in intraperitoneal infections. Proc. Natl. Acad. Sci. USA 97:
1258–1262.
Elliott, T. 1992. A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon. J. Bacteriol.
174:245–253.
Ernst, R. K., D. M. Dombroski, and J. M. Merrick. 1990. Anaerobiosis, type
1 fimbriae, and growth phase are factors that affect invasion of HEp-2 cells
by Salmonella typhimurium. Infect. Immun. 58:2014–2016.
Galan, J. E. 2001. Salmonella interactions with host cells: type III secretion
at work. Annu. Rev. Cell Dev. Biol. 17:53–86.
Garcia-del Portillo, F., and B. B. Finlay. 1995. Targeting of Salmonella
typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors. J. Cell Biol.
129:81–97.
Gerlach, R. G., and M. Hensel. 2007. Protein secretion systems and adhesins:
the molecular armory of gram-negative pathogens. Int. J. Med. Microbiol.
297:401–415.
Gerlach, R. G., and M. Hensel. 2007. Salmonella pathogenicity islands in
host specificity, host pathogen-interactions and antibiotics resistance of Salmonella enterica. Berl. Munch. Tierarztl. Wochenschr. 120:317–327.
Gerlach, R. G., D. Jackel, B. Stecher, C. Wagner, A. Lupas, W. D. Hardt, and
M. Hensel. 2007. Salmonella pathogenicity island 4 encodes a giant nonfimbrial adhesin and the cognate type 1 secretion system. Cell. Microbiol.
9:1834–1850.
Guiney, D. G., F. C. Fang, M. Krause, S. Libby, N. A. Buchmeier, and J.
Fierer. 1995. Biology and clinical significance of virulence plasmids in Salmonella serovars. Clin. Infect. Dis. 21(Suppl. 2):S146–S151.
Hancox, L. S., K. S. Yeh, and S. Clegg. 1997. Construction and characterization of type 1 non-fimbriate and non-adhesive mutants of Salmonella
typhimurium. FEMS Immunol. Med. Microbiol. 19:289–296.
ppGpp IN SALMONELLA SEROVAR GALLINARUM
6350
57.
58.
59.
60.
61.
63.
64.
65.
66.
chicken infection model using PCR-based signature-tagged mutagenesis.
Microbiology 151:3957–3968.
Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification of
a virulence locus encoding a second type III secretion system in Salmonella
typhimurium. Proc. Natl. Acad. Sci. USA 93:2593–2597.
Shin, D., S. Lim, Y. J. Seok, and S. Ryu. 2001. Heat shock RNA polymerase (E
sigma(32)) is involved in the transcription of mlc and crucial for induction of the
Mlc regulon by glucose in Escherichia coli. J. Biol. Chem. 276:25871–25875.
Shivaprasad, H. L. 1997. Diseases of poultry, p. 82–96. In B. W. Calnek (ed.),
Pullorum disease and fowl typhoid, 10th ed. Iowa State University Press,
Ames, IA.
Shivaprasad, H. L. 2000. Fowl typhoid and pullorum disease. Rev. Sci. Tech.
19:405–424.
Simons, R. W., F. Houman, and N. Kleckner. 1987. Improved single and
multicopy lac-based cloning vectors for protein and operon fusions. Gene
53:85–96.
Smith, H. W. 1956. The use of live vaccines in experimental Salmonella
gallinarum infection in chickens with observations on their interference
effect. J. Hyg. (London) 54:419–432.
Song, M., H. J. Kim, E. Y. Kim, M. Shin, H. C. Lee, Y. Hong, J. H. Rhee, H.
Yoon, S. Ryu, S. Lim, and H. E. Choy. 2004. ppGpp-dependent stationary
phase induction of genes on Salmonella pathogenicity island 1. J. Biol.
Chem. 279:34183–34190.
Steele-Mortimer, O., S. Meresse, J. P. Gorvel, B. H. Toh, and B. B. Finlay.
1999. Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway. Cell. Microbiol. 1:33–49.
Stent, G. S., and S. Brenner. 1961. A genetic locus for the regulation of
ribonucleic acid synthesis. Proc. Natl. Acad. Sci. USA 47:2005–2014.
Thompson, A., M. D. Rolfe, S. Lucchini, P. Schwerk, J. C. Hinton, and K.
J. BACTERIOL.
67.
68.
69.
70.
71.
72.
73.
Tedin. 2006. The bacterial signal molecule, ppGpp, mediates the environmental regulation of both the invasion and intracellular virulence gene programs of Salmonella. J. Biol. Chem. 281:30112–30121.
Wallis, T. S., M. Wood, P. Watson, S. Paulin, M. Jones, and E. Galyov. 1999.
Sips, Sops, and SPIs but not stn influence Salmonella enteropathogenesis.
Adv. Exp. Med. Biol. 473:275–280.
Wigley, P., S. Hulme, L. Rothwell, N. Bumstead, P. Kaiser, and P. Barrow.
2006. Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential
expression of cytokines and chemokines following Salmonella enterica challenge. Infect. Immun. 74:1425–1430.
Wigley, P., S. D. Hulme, N. Bumstead, and P. A. Barrow. 2002. In vivo and
in vitro studies of genetic resistance to systemic salmonellosis in the chicken
encoded by the SAL1 locus. Microbes Infect. 4:1111–1120.
Wilson, R. L., J. Elthon, S. Clegg, and B. D. Jones. 2000. Salmonella enterica
serovars Gallinarum and Pullorum expressing Salmonella enterica serovar
Typhimurium type 1 fimbriae exhibit increased invasiveness for mammalian
cells. Infect. Immun. 68:4782–4785.
Wu, K. Y., G. R. Liu, W. Q. Liu, A. Q. Wang, S. Zhan, K. E. Sanderson, R. N.
Johnston, and S. L. Liu. 2005. The genome of Salmonella enterica serovar
Gallinarum: distinct insertions/deletions and rare rearrangements. J. Bacteriol. 187:4720–4727.
Zhang, X. L., I. S. Tsui, C. M. Yip, A. W. Fung, D. K. Wong, X. Dai, Y. Yang,
J. Hackett, and C. Morris. 2000. Salmonella enterica serovar Typhi uses type
IVB pili to enter human intestinal epithelial cells. Infect. Immun. 68:3067–
3073.
Zhou, D., M. S. Mooseker, and J. E. Galan. 1999. Role of the S. typhimurium
actin-binding protein SipA in bacterial internalization. Science 283:2092–
2095.
Downloaded from http://jb.asm.org/ on February 27, 2014 by PENN STATE UNIV
62.
JEONG ET AL.