Download Genes encoding putative effector proteins of the type III secretion

Molecular Microbiology (1998) 30(1), 163–174
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
Michael Hensel,1 Jacqueline E. Shea,2 Scott R.
Waterman,2 Rosanna Mundy,2 Thomas Nikolaus,1
Geoff Banks,2 Andrés Vazquez-Torres,3 Colin
Gleeson,2 Ferric C. Fang3 and David W. Holden2 *
1
Lehrstuhl für Bakteriologie, Max von Pettenkofer-Institut
für Hygiene und Medizinische Mikrobiologie, Munich,
Germany.
2
Department of Infectious Diseases, Imperial College
School of Medicine, Du Cane Road, London W12 ONN,
UK.
3
Department of Medicine, Division of Infectious Diseases,
University of Colorado Health Sciences Center, 4200 East
Ninth Avenue, Denver, CO 80262, USA.
Summary
The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of
this pathogen in mice. Cloning and sequencing of a
central region of SPI-2 revealed the presence of genes
encoding putative chaperones and effector proteins
of the secretion system. The predicted products of
the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA,
EspD and EspB, which are secreted by the type III
secretion system encoded by the locus of enterocyte
effacement of enteropathogenic Escherichia coli. The
transcriptional activity of an sseA ::luc fusion gene
was shown to be dependent on ssrA, which is required
for the expression of genes encoding components of
the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely
attenuated in virulence, strains carrying mutations in
sseF or sseG were weakly attenuated, and a strain
with a mutation in sseE had no detectable virulence
defect. These phenotypes were reflected in the ability
of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA,
sseB or sseC failed to accumulate, whereas the growth
rates of strains carrying mutations in sseE, sseF or
Received 7 May, 1998; revised 27 June, 1998; accepted 2 July, 1998.
*For correspondence. E-mail [email protected]; Tel. (0181) 383
3487; Fax (0181) 383 2078.
Q 1998 Blackwell Science Ltd
sseG were only modestly reduced. These data suggest
that, in vivo, one of the functions of the SPI-2 secretion
system is to enable intracellular bacterial proliferation.
Introduction
Several Gram-negative bacterial pathogens secrete virulence proteins via specialized type III secretion systems
(Mecsas and Strauss, 1996). These secretion systems
comprise a large number of proteins required to transfer
specific effector proteins into eukaryotic host cells in a
contact-dependent manner (Rosqvist et al., 1994; Zierler
and Galan, 1995; Collazo and Galan, 1997). Although several components of the secretion system apparatus show
evolutionary and functional conservation across bacterial
species (Salmond and Reeves, 1993), the effector proteins are less well conserved and have different functions.
The Yersinia effectors YpkA and YopH have threonine/
serine kinase and tyrosine phosphatase activities respectively (Galyov et al., 1993; Persson et al., 1995). The actions
of these and other Yops inhibit bacterial phagocytosis by
host cells, which is thought to enable extracellular bacterial proliferation (for review see Cornelis and Wolf-Watz,
1997). The Shigella Ipa proteins, secreted by the mxi/spa
type III secretion system, promote entry of this bacterium
into epithelial cells (Menard et al., 1996). The proteins
EspA, EspB and EspD, encoded by the locus of enterocyte effacement (LEE) of enteropathogenic Escherichia
coli (EPEC) are secreted by a type III secretion system
and cause cytoskeletal rearrangements of host epithelial
cells resulting in the formation of pedestal-like structures
on the host cell surface (for a review see Donnenberg et
al., 1997).
Salmonella typhimurium is unusual in that it contains
two type III secretion systems for virulence determinants.
The first controls bacterial invasion of epithelial cells (Galan
and Curtiss, 1989; Galan, 1996) and is encoded by genes
within a 40 kb pathogenicity island (SPI-1) located at 63
centisomes on the chromosome (Mills et al., 1995). Evidence for the second type III secretion system is based
on a set of corresponding genes within a second pathogenicity island (SPI-2) at 30 centisomes, which are required
for systemic growth of this pathogen in its host (Hensel et
164 M. Hensel et al.
al., 1995; Ochman et al., 1996; Shea et al., 1996). We proposed that the SPI-2 secretion system genes should be
designated as follows: ssa for genes encoding the secretion system apparatus, ssr for genes encoding secretion
system regulators, ssc for genes encoding secretion system chaperones and sse for genes encoding secretion
system effectors (Hensel et al., 1997a).
Many of the genes encoding components of the SPI-2
secretion system are located in a 25 kb segment beginning
at the 31 centisomes boundary of SPI-2 (Hensel et al.,
1997b). On the basis of similarities with genes of other
bacterial pathogens, the first 13 genes from this boundary
(the ssaK /U operon and ssaJ ) encode components of the
secretion system apparatus (Hensel et al., 1997a). A number of additional genes including ssaC (orf 11 in Shea et
al., 1996; spiA in Ochman et al., 1996) and ssrA (orf 12
in Shea et al., 1996; spiR in Ochman et al., 1996), which
encode a secretion system apparatus protein and a two
component regulatory protein, respectively, are found in
a region <8 kb upstream of ssaJ (Shea et al., 1996).
In this paper we describe an analysis of genes that
encode other components of the secretion system, located
in the region between ssaJ and ssaC of SPI-2. DNA and
protein database searches with these genes identified
three whose products display weak but significant similarity to proteins secreted by the type III secretion systems of
EPEC and Yersinia, and two that are potential chaperones
for these proteins. We show that transcriptional activity of
sseA is dependent on ssrAB, which encodes a two-component regulatory system required for the expression of
ssa genes of SPI-2 (Valdivia and Falkow, 1997). Virulence
tests and in vitro assays with strains containing non-polar
mutations show that several sse genes are critical for Salmonella virulence in mice and are required for bacterial
accumulation within macrophages.
Results
Organization of sse and ssc genes
As part of our effort to characterize SPI-2 genetically and
functionally, we cloned and sequenced a central region of
the pathogenicity island (Fig. 1A). DNA fragments covering the region between ssaC and ssaJ were subcloned
in plasmids p5-2 and p5-4 as indicated in Fig. 1C. The
arrangement and designation of genes in the 8 kb region
between ssaC and ssaK is shown in Fig. 1B. This sequence is available from the EMBL database under accession number AJ224892. The sequenced region extends
the open reading frame (ORF) of a gene encoding a putative subunit of the type III secretion apparatus referred
to as spiB (Ochman et al., 1996). For consistency with
the universal nomenclature for type III secretion system
Fig. 1. Genetic organization of the effector gene region of SPI-2 and position of mutations.
A. The position of this region in relation to other genes of the secretion system is shown.
B. Genes encoding proteins with sequence similarity to effector proteins of other type III secretion systems (shaded arrows), putative
chaperones of SPI-2 (hatched arrows), components of the type III secretion system (filled arrows) and without sequence similarity (open
arrows) are indicated. Positions of a mTn5 insertion in mutant strain P10E11 and the aphT gene cassette in mutant strains HH100, HH102,
HH104, HH107 and HH108 are marked.
C. A restriction map is shown for Bam HI (B), Eco RI (E), Hin dIII (H), Eco RV(V), Sma I (S) and Cla I (C). The positions of subclones p5-2,
p5-4, p5-5, p5-7 and p5-8 are also indicated.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
SPI-2 sse genes 165
Table 1. Features of predicted proteins.
Protein
M r ( kDa)
pI
Tm predictions
Predicted coils
SseA
SseB
SseC
SseD
SseE
SseF
SseG
SscA
SscB
12.5
21.5
52.8
20.6
16.3
26.9
24.4
18.1
16.4
9.3
4.7
6.3
4.8
9.7
4.4
9.2
8.8
4.7
Hydrophilic
One transmembrane helix
Three transmembrane helices
Three transmembrane helices
One transmembrane helix
Four transmembrane helices
Three transmembrane helices
Hydrophilic
Hydrophilic
At least one (trimer)
None
At least two (trimers)
At least one (trimer)
None
None
None
None
None
subunits (Bogdanove et al., 1996) and the nomenclature of
other SPI-2 genes (Hensel et al., 1997a), we propose that
this gene be designated ssaD. The deduced amino acid
sequence of ssaD is 24% identical to YscD of Y. enterocolitica. This is followed by an ORF with coding capacity
for a 9.3 kDa protein, 34% identical to YscE of Y. enterocolitica. Therefore, this gene is designated ssaE. A sequence of 263 bp separates ssaE and a set of nine genes,
several of which encode proteins with sequence similarity
to secreted proteins or their chaperones from other pathogens. These genes are separated by short intergenic
regions or have overlapping reading frames and it is likely
that some are co-transcribed and translationally coupled.
Therefore, the genes with similarity to those encoding
chaperones were designated sscA and sscB, and the
others sseA-G. The amino acid sequence deduced from
sscA shows 26% identity/49% similarity over 158 amino
acid residues to SycD, the product of lcrH of Y. pseudotuberculosis that acts as a secretion-specific chaperone
for YopB and YopD (Wattiau et al., 1994). The amino acid
sequence deduced from sscB shows 23% identity/36%
similarity over 98 amino acid residues to IppI of Shigella
flexneri. IppI is a chaperone for S. flexneri invasion proteins (Ipas) (Baundry et al., 1988). As is the case for the
secretion chaperones SycD, IppI and SicA (Kaniga et al.,
1995), SscB has an acidic pI (Table 1), whereas SscA has
an unusually high pI of 8.8.
SseA, SseE, SseF and SseG showed no significant
similarities to DNA or protein database entries. SseB is
25% identical/47% similar to EspA of EPEC over the entire
length of the 192-amino-acid-residue protein. SseD is 27%
identical/51% similar to EspB of EPEC over 166 amino
acid residues. SseC has sequence similarity to a class of
effector proteins involved in the translocation of other
effectors into the target host cell. These include YopB of
Y. enterocolitica, EspD of EPEC and PepB of Pseudomonas aerugunosa. SseC is <24% identical/48% similar
to both EspD of EPEC and YopB of Y. enterocolitica
(Fig. 2). EspD and YopB have two hydrophobic domains
that are predicted to insert into target cell membranes (Pallen et al., 1997). SseC contains three hydrophobic regions
that could represent membrane-spanning domains. Other
features of these predicted effector proteins are shown
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
in Table 1. Using the TMPREDICT program (Hofmann and
Stoffel, 1993), transmembrane helices are predicted for
all the effector proteins apart from SseA, which is very
hydrophilic. Alignments of SseC to homologues in other
pathogens are shown in Fig. 2. Conserved amino acids
are mainly clustered in the central, more hydrophobic portion of the protein, but unlike YopB, there is no significant
similarity to the RTX family of toxins. The conserved residues in SseD are present mainly in the N-terminal half
of the protein. Comparison of the deduced amino acid
sequences of sseABCDEF with entries in the PROSITE
database did not reveal the presence of any characteristic
protein motifs. We subjected the predicted amino acid
sequences of the sse genes to searches using the programs COIL and MULTICOIL (Lupas, 1997) as described by
Pallen et al. (1997). SseA and SseD are predicted to
have one trimeric coil each, and SseC is predicted to have
two trimeric coils (Table 1). As EspB and EspD are predicted to have one and two trimeric coils, respectively
(Pallen et al., 1997), this provides further evidence that
these proteins are functionally related.
Between sseG and ssaJ [a gene encoding a component
of the type III secretion apparatus (Hensel et al., 1997a)],
three short ORFs were identified, designated ssaG, ssaH
and ssaI, encoding proteins of 7.9 kDa, 8.1 kDa and 9.0 kDa
respectively. SsaG has sequence similarity to YscF of Y.
enterocolitica (25% identity over 71-amino-acid residues)
and MxiH of S. flexneri (34% identity over 46 amino acid
residues). No significant sequence similarities were obtained
with SsaH and SsaI.
Expression of sseA is dependent on ssrAB
To establish if the sse genes are part of the SPI-2 secretion system, we investigated the expression of an sseA ::luc
reporter gene fusion, integrated by homologous recombination into the chromosome of different SPI-2 mutant
strains. In accordance with results from a previous study
(Valdivia and Falkow, 1997), no significant transcription
was detected during growth in rich medium. However,
transcriptional activity of sseA in a wild-type background
was detected during growth in minimal medium (Fig. 3).
Transposon insertions in ssrA and ssrB, encoding the
166 M. Hensel et al.
Fig. 2. Alignment of the deduced SseC amino acid sequence to EspD of EPEC, YopB of Yersinia enterocolitica (Hakansson et al., 1993) and
PepB of Pseudomonas aeroginosa (Hauser et al., 1998). The CLUSTALW algorithm of the MACVECTOR 6.0 program was used to construct the
alignments. Similar amino acid residues are boxed, identical residues are boxed and shaded.
sensor component and the transcriptional activator, respectively, resulted in 250- to 300-fold reduced expression
of sseA . Inactivation of hilA , the transcriptional activator of
SPI-1 (Bajaj et al., 1996), had no effect on sseA gene
expression. Transposon insertions in two genes encoding
components of the SPI-2 type III secretion apparatus
(ssaJ ::mTn5; ssaT ::mTn5 ; Shea et al., 1996) also had no
significant effect on the expression of sseA . These data
show that ssrAB is required for the expression of sseA ,
but that hilA is not.
Virulence tests with strains carrying non-polar mutations
DNA sequence analysis suggested that the sse genes
might encode effector proteins of the secretion system,
but apart from a possible polar effect from a transposon
insertion in sscA (strain P10E11, Fig. 1) no strains carrying mutations in these genes were recovered in the
original STM screen for S. typhimurium virulence genes
using mTn5 mutagenesis (Hensel et al., 1995), and their
role in virulence was unclear. To address this question,
we constructed strains carrying non-polar mutations in
sseA , sseB, sseC, sseE, sseF and sseG (Fig. 1) and subjected these strains to virulence tests by inoculating groups
of BALB/c mice intraperitoneally with each strain, as this
was the method by which the original SPI-2 mutants were
identified. Table 2 shows that all mice inoculated with
strains carrying mutations in sseA , sseB and sseC survived a dose of 1 × 104 cfu, three orders of magnitude
greater than the LD50 of the wild-type strain, which is
less than 10 cfu when the inoculum is administered by the
i.p. route (Buchmeier et al., 1993; Shea et al., 1996). The
same strains containing a plasmid carrying the corresponding wild-type allele were also inoculated into mice
at a dose of 1 × 104 cfu. No mice survived these infections,
which shows that each mutation can be complemented at
least partially by the presence of a functional copy of each
gene, and that each of these genes plays an important role
in Salmonella virulence. Strains carrying non-polar mutations in sseE, sseF and sseG caused lethal infections
when <1 × 104 cells of each strain were inoculated into
mice by the i.p. route (Table 2) and were analysed in
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
SPI-2 sse genes 167
Intramacrophage replication of mutant strains
Fig. 3. Expression of an sseA ::luc fusion in wild-type and mTn5
mutant strains of S. typhimurium. Expression in each strain was
determined in triplicate and the standard errors from the means are
shown.
more detail by a competition assay with the wild-type
strain in mixed infections (three mice per test) to determine whether they were attenuated in virulence. The
competitive index, defined as the output ratio of mutant
to wild-type bacteria, divided by the input ratio of mutant
to wild-type bacteria, shows that sseF and sseG do contribute to virulence, but their absence in mutant strains is not
detectable by the relatively insensitive LD50 test (Table 2).
By comparison, very low competitive indices were obtained
using strains carrying mutations in either ssrA or sseB.
The competitive index for the sseE mutant was not significantly different to that of a fully virulent strain carrying an
antibiotic resistance marker, which implies that this gene
does not play a significant role in systemic Salmonella
infection of the mouse.
Table 2. Virulence of S. typhimurium strains in
mice.
Strain
Genotype
12023
HH100
HH102
HH104
HH106
HH107
HH108
P3F4
Wild-type
sseA D: :aphT
sseBD ::aphT
sseC ::aphT
sseED
sseFD ::aphT
sseG ::aphT
ssrA ::mTn5
We tested several mutant strains for their ability to grow
inside macrophages and macrophage-like cell lines, as
macrophage survival and replication are thought to represent an important aspect of Salmonella pathogenesis in
vivo (Fields et al., 1986), and because Ochman et al.
(1996) reported that an S. typhimurium strain carrying a
mutation in a SPI-2 gene was unable to survive in macrophages. We have reported previously that a number of
SPI-2 mutant strains were not defective for survival or replication within RAW macrophages (Hensel et al., 1997a), but
subsequent experiments have revealed that some SPI-2
mutants can be shown to have a defect if aerated stationary-phase bacterial cultures opsonized with normal mouse
serum are used (see also accompanying paper: Cirillo et
al., 1998). The increase in cfu for different strains in RAW
macrophages over a 16 h period is shown in Fig. 4. Growth
defects were observed for strains carrying mutations in
ssaV (encoding a component of the secretion apparatus),
sseA, sseB, sseC and to a lesser extent for strains carrying
mutations in sseE, sseF and sseG. Partial complementation
of this defect was achieved with strains harbouring plasmids
carrying functional copies of sseC and sseB, and very slight
complementation was observed for sseA. We also investigated the ability of SPI-2 mutant strains to accumulate
inside the J774.1 macrophage cell line (Fig. 5A) and in
periodate-elicited peritoneal macrophages from C3H/HeN
mice (Fig. 5B). Similar defects of S. typhimurium carrying
transposon or non-polar mutations in SPI-2 genes were
observed, regardless of the phagocyte cell-type examined,
although the peritoneal elicited cells had superior antimicrobial activity than either cell line.
Discussion
We have identified a group of genes encoding putative
effector proteins and chaperones of the SPI-2 type III
Mouse survival
after inoculationa
with bacterial
strain
0/ 5
5/ 5
5/ 5
5/ 5
0/ 5
0/ 5
0/ 5
Mouse survival after
inoculationa with
mutant þ complementing
plasmid
Competitive
index in vivo
0.98 b
0/ 5
0/ 5
0/ 5
0.013
0.79
0.222
0.158
0.01
a. Mice were inoculated intraperitoneally with 1 × 104 cells of each strain and survival was
determined after 14 days.
b. Result of competition between wild-type strain 12023 and a virulent mTn5 mutant identified
in the STM screen.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
168 M. Hensel et al.
1994; Reed et al., 1998). The SPI-1 secretion system
forms a needle-like structure that spans the bacterial cell
envelope (Kubori et al., 1998). Both SseC and EspD are
similar to YopB, which is a pore-forming protein required
for translocation of effector proteins of the Yersinia type
III secretion system across the eukaryotic cell plasma
Fig. 4. Intracellular survival and replication of SPI-2 mutant S.
typhimurium in RAW 264.7 macrophages. After opsonization and
infection, macrophages were lysed and cultured for enumeration of
intracellular bacteria (gentamicin protected) at 2 h and 16 h after
infection. The values shown represent the fold increase calculated
as a ratio of the intracellular bacteria between 2 h and 16 h after
infection. Each strain was infected in triplicate and standard error
from the mean is shown.
secretion system. These genes are located between two
clusters of genes encoding components of the secretion
system apparatus. In terms of their overall arrangement
and deduced amino acid sequences, the SPI-2 ssa and
sse genes appear to share greatest similarity to the esc
and esp genes of the EPEC LEE (Elliot et al., 1998).
Although sseBCD are in the same order as the genes to
which they show similarity (espADB ), there is no gene
between espA and espD corresponding to sscA , which
encodes a putative chaperone. The corresponding gene
products of sseCD and espDB are also predicted to
have transmembrane helices and to form trimeric coiled
coils which suggests that the two sets of genes have similar functions.
EspA, EspB and EspD are necessary to activate epithelial signal transduction pathways leading to host cell
cytoskeletal rearrangements and pedestal-like structures
on intestinal epithelial cell surfaces, to which the bacteria
adhere (Donnenberg et al., 1997). Recent work has shown
that both EspB (Wolff et al., 1998) and EspA (Knutton et
al., 1998) are involved in the translocation of EspB into
both the plasma membrane and cytoplasm of epithelial
cells. EspA is a component of a filamentous surface appendage that forms a contact between the bacterium and the
eukaryotic cell surface (Knutton et al., 1998). These structures resemble specific appendages (invasomes) that can
be detected on the surface of S. typhimurium during epithelial cell invasion, although their relationship to the SPI-1
type III secretion system is uncertain (Ginocchio et al.,
Fig. 5. Intracellular survival and replication of SPI-2 mutant S.
typhimurium in (A) J774.1 cells and (B) periodate elicited peritoneal
macrophages from C3H/ HeN mice. After opsonization and
internalization, phagocytes were lysed and cultured for enumeration
of viable intracellular bacteria at time 0 h. The values shown
represent the proportion of this intracellular inoculum viable at
20 h 6 the standard error of the mean. Samples were processed in
triplicate, and each experiment was performed at least twice.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
SPI-2 sse genes 169
membrane (Hakansson et al., 1996). These similarities
suggest that at least some of the sse genes encode a
SPI-2 translocon that integrates into a target cell membrane and mediates the translocation of other SPI-2 effector proteins. Although intracellular Sse proteins can be
detected in bacterial lysates after growth in laboratory
media using polyclonal antibodies and epitope tags (our
unpublished results), we have not been able to establish
in vitro conditions that induce the secretion of these proteins. We are currently investigating their secretion in
vivo and within cultured host cells. Evidence that the sse
genes encode secreted proteins of the SPI-2 secretion
system is therefore based on their chromosomal location
and order, similarities of predicted sequence and hydrophobicity to Esps of EPEC and Yersinia YopB, and on
the fact that the expression of sseA requires SsrAB, a
two-component regulatory system required for the expression of other genes of the secretion system (Valdivia and
Falkow, 1997; and accompanying paper Cirillo et al., 1998).
The importance of the sse genes was originally unclear
because all the STM-derived transposon insertions in SPI-2
are in genes for structural and regulatory components (Shea
et al., 1996) and sscA , which is predicted to encode a
chaperone. The failure to recover transposon insertions
in the sse genes may have resulted from a low frequency
of mTn5 insertions in this region, because strains carrying
targeted mutations in sseA , sseB and sseC are strongly
attenuated in virulence. These strains also exhibited deficient replication or survival in two different macrophagelike cell lines and elicited mouse peritoneal macrophages.
This phenotype was observed using opsonized bacteria
but does not require exogenous cellular activation by cytokines. This finding prompted us to re-examine the intramacrophage accumulation of strains carrying transposon
insertions in ssrA and ssaV because in an earlier study
using non-opsonized bacterial cells grown under conditions that make them highly invasive, these strains were
not found to have a replication defect (Hensel et al.,
1997a). When ssrA and ssaV mutant strains were opsonized
and grown under non-invasive conditions, their intramacrophage numbers were as low as the strains carrying
mutations in sseA and sseB. These differences may reflect
the different intracellular fate of organisms entering macrophages by pathogen-directed invasion processes (nonopsonized) or by phagocyte-directed uptake (opsonized).
Furthermore, as high invasion leads to a greater degree of
host cell cytotoxicity through SPI-1-mediated apoptosis
(Monack et al., 1996; Chen et al., 1996), cytotoxic effects
of replicating wild-type cells may have released bacteria
from the intracellular environment and resulted in killing
by gentamicin, and therefore an under-estimation of the
number of wild-type cells that had undergone intracellular
replication in our previous studies.
As the phenotypic behaviour of opsonized bacterial
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
strains carrying transposon or non-polar mutations in various SPI-2 genes inside cultured macrophages reflects
their virulence phenotype in vivo, we conclude that the
SPI-2 secretion system is required for bacterial proliferation inside macrophages in vivo. However, this does not
exclude the possibility that the SPI-2 genes have other
functions not apparent from the macrophage assays.
These results confirm and extend the earlier findings of
Ochman et al. (1996), who found that the secretion system
is required for bacterial survival in J774 macrophages.
SPI-2 mutant strains were originally found to be attenuated in virulence by mixed infections of mice with pools
of signature-tagged mutant strains, the majority of which
are virulent (Hensel et al., 1995). This means the SPI-2
mutant strains cannot be rescued in trans by the presence
of virulent strains. The demonstration of SPI-2 gene expression within macrophages (Valdivia and Falkow, 1997), along
with the reduced numbers of SPI-2 mutant cells in cultured
macrophages, suggests that the failure of virulent cells to
rescue the SPI-2 defect may be due to physical separation
of bacteria within phagocytic cells. Their reduced accumulation could be due to either reduced survival (greater susceptibility to macrophage killing) or reduced replication, or
a combination of the two. Ochman et al. (1996) concluded
that the function of the secretion system might be to modify
host factors required for phagosome–lysosome fusion or
phagosome acidification. More recently Valdivia and Falkow
(1997) showed that at least one of the SPI-2 secretion system genes is induced in a variety of host cells including
macrophage-like RAW cells, and speculated that the function of the secretion system might be to translocate bacterial proteins across the vacuolar membrane. Therefore,
the putative translocon encoded by the sse genes reported
here might be inserted in the vacuolar membrane and
influence the intracellular fate of the Salmonella -containing vacuole. Further investigation of Salmonella –macrophage interactions and the functions of the sse gene
products are likely to provide important insights into the
mechanism of SPI-2-mediated virulence.
Experimental procedures
Bacterial strains, phages and plasmids
The bacterial strains, phages and plasmids used in this study
are listed in Table 3. Unless otherwise indicated, bacteria
were grown in LB broth or on LB agar plates with the addition,
when appropriate, of ampicillin (50 mg ml¹1 ), kanamycin (50 mg
ml¹1 ) or chloramphenicol (50 mg ml¹1 ).
DNA cloning and sequencing
Clones harbouring fragments of SPI-2 were identified from a
library of genomic DNA of S. typhimurium in l 1059, which
has been described previously (Shea et al., 1996). The sse
170 M. Hensel et al.
and ssc genes were subcloned from clone l 5 on a 5.7 kb
Eco RI fragment and a 5.8 kb Hin dIII fragment in pKSþ as
indicated in Fig. 1 and Table 3. DNA sequencing was performed using a primer walking strategy. The dideoxy method
(Sanger et al., 1977) was applied using the Pharmacia T7
sequencing system for manual sequencing and the dye terminator chemistry for automatic analysis on a ABI377 sequencing instrument. Assembly of contigs from DNA sequences
was performed by means of ASSEMBLYLIGN and MACVECTOR software (Oxford Molecular). For further sequence analyses, programs of the GCG package version 8 (Devereux et al., 1984)
were used on the HGMP network.
Construction of non-polar mutations
The construction of non-polar mutations in sseA , sseB, sseC,
sseE and sseF are described below. All chromosomal modifications were confirmed by PCR and Southern hybridization
analysis.
SseA
Plasmid p5-5 was cut at a unique Hpa I site within sseA . The
linear product was then used as template for a PCR with
primers sseA1 (58-GAAGGCCTTTTTCTTTATCATCATTCCCC-38) and sseA2 (58-GAAGGCCTGAAACAACTTAATGCTCAAGCC-38) to delete an internal region of 246 bp from
sseA , corresponding to amino acids 8–90. The linear product
contains Stu I sites at each terminus and was circularized by
ligation after Stu I digestion and transferred into E. coli DH5a.
A 900 bp Hin cII fragment of pSB315 containing an aminoglycoside 38-phosphotransferase gene (aphT ) from which the
transcriptional terminator had been removed (Galan et al.,
1992) was ligated in the same orientation into the unique
Stu I site created in the sseA deletion plasmid. An Nhe I fragment containing the DsseA ::aphT gene was inserted into the
Xba I site of the plasmid pCVD442 (Donnenberg and Kaper,
1991) and transferred by conjugation from E. coli S17-1 l pir
to S. typhimurium 12023. Exconjugants were selected for kanamycin resistance and screened for ampicillin sensitivity.
Table 3. Phages, plasmids and bacterial strains used in this study.
Phage, plasmid or strain
Description
Reference
Phages
l5
Clone from a library of S. typhimurium genomic DNA in l1059
Shea et al. (1996)
Plasmids
pKSþ, pSKþ
pUC18
pACYC184
pGP704
pCVD442
pBL02
pGPL01
pSB315
p5-2
p5-4
p5-5
p5-7
p5-8
psseA
psseB
psseC
Ampr ; high-copy-number cloning vectors
Ampr ; high-copy-number cloning vector
Cmr,Tet r ; low-copy-number cloning vector
R6K ori, Ampr ; lpir -dependent suicide vector
R6K ori, Ampr ; lpir -dependent sucrose selection suicide vector
R6K ori, Ampr ; luc fusion suicide vector
R6K ori, Ampr ; luc fusion suicide vector
Kanr, Ampr
Ampr ; 5.7 kb Eco RI fragment of l5 in pKSþ
Ampr ; 5.8 kb Hin dIII fragment of l5 in pSKþ
Ampr ; Pst I digestion of p5-4 and religation of the larger fragment
Ampr ; 1.9 kb fragment of p5-4 in pKSþ
Ampr ; 2.2 kb Pst I /Hin dIII fragment of p5–2 in pSKþ
Cmr ; sseA in pACYC184
Cmr ; sseB in pACYC184
Cmr ; sseC in pACYC184
Stratagene, Heidelberg
Gibco-BRL, Eggenstein
Chang and Cohen (1978)
Miller and Mekalanos (1988)
Donnenberg and Kaper (1991)
Gunn and Miller (1996)
Gunn and Miller (1996)
Galan et al. (1992)
This study
This study
This study
E. coli strains
DH5a
S17-1 lpir
CC118 lpir
See reference
lpir phage lysogen (see reference)
lpir phage lysogen (see reference)
Gibco-BRL
Miller and Mekalanos (1988)
Herrero et al. (1990)
S. typhimurium strains
12023
CS015
P3F4
P2D6
P4H2
P11D10
HH100
HH101
HH102
HH103
HH104
HH105
HH106
HH107
HH108
Wild-type
phoP -102::Tn10d -Cm
ssrA ::mTn5
ssaV ::mTn5
hilA ::mTn5
ssaJ ::mTn5
sseAD ::aphT, Kmr ; non-polar mutation
HH100 containing psseA
sseBD ::aphT, Kmr ; non-polar mutation
HH102 containing psseB
sseC ::aphT, Kmr ; non-polar mutation
HH104 containing psseC
sseED ; non-polar mutation
sseFD ::aphT, Kmr ; non-polar mutation
sseG ::aphT, Kmr ; non-polar mutation
NCTC, Colindale, UK
Miller et al. (1989)
Shea et al. (1996)
Shea et al. (1996)
Monack et al. (1996)
Shea et al. (1996)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This
This
This
This
study
study
study
study
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
SPI-2 sse genes 171
SseB
Plasmid p5-5 was cut at the unique Cla I site within sseB.
This linear DNA was used as the template for PCR using
primers sseB1 (58-ATTGGATCCGGTGGAGATACCGTC-38)
and sseB2 (58-TATGGATCCTGTTGTTAGGGTCGGG-38).
The product, with terminal Stu I sites, was digested with Stu I
and self-ligated to generate an sseB gene containing an internal
deletion of 333 bp corresponding to amino acids 55–166. The
blunt-ended aphT cassette (see above) was ligated into the
Stu I site in the sseB deletion. An Nhe I fragment containing
the sseB deletion was ligated into the unique Xba I site of plasmid pCVD442 and transferred to the S. typhimurium chromosome as described above.
SseC
A 2.6 kb fragment was recovered after Bam HI and Cla I digestion of p5-2 and subcloned in Bam HI/Cla I-digested pKSþ.
The resulting construct was digested by Hin dIII, blunt ended
using the Klenow fragment of DNA polymerase and ligated
to the aphT cassette as indicated above. The resulting plasmid was digested with Sal I and Xba I and the insert was
ligated to Sal I/Xba I-digested pGP704. This plasmid was electroporated into E. coli S17-1 l pir and transferred into S. typhimurium 12023 by conjugation. Exconjugants in which the sseC
gene had been replaced by the cloned gene disrupted by insertion of the aphT cassette were selected by resistance to kanamycin and screened for sensitivity to carbenicillin.
SseE
Plasmid p5-8 was used as template for a PCR with primers
sseE1 (58-ATTATGCATGCATGGGAGCGACCTTTACACAGCTT-38) and sseE2 (58-ATTTAGCATGCGGCGGTCTCCCCTAAATATGCAGG-38). The PCR product, containing terminal
Sph I sites, was digested with Sph I and self-ligated to create
an internal deletion of 261 bp in sseE corresponding to amino
acids 26–112. The plasmid was digested with Sal I and Sst I
and a 2.0 kb fragment containing the deleted sseE gene
was ligated into pCVD442. The resulting plasmid was used
to transfer the mutated gene onto the Salmonella chromosome as described above, using sucrose selection to obtain
cells from which the suicide vector had been lost (Donnenberg
and Kaper, 1991).
SseF
The N-terminal and C-terminal regions of sseF were isolated
from plasmid p5–2 on a 2.3 kb Pst I/Hin dIII fragment and a
2.7 kb Eco RI/Sst II fragment respectively. The Pst I/Hin dIII
fragment was subcloned in pBluescript SK, the resulting construct linearized at the polylinker Xba I site and the cohesive
ends made flush by treatment with T4 DNA polymerase.
The Eco RI/Sst II fragment was treated with T4 DNA polymerase and ligated to the linearized vector to form plasmid
psseF2, which contains sseF with a central deletion corresponding to amino acids 81–178. Plasmid psseF2 was linearized at a Bam HI site adjacent to the destroyed Xba I site and
ligated with the aphT gene (see above). A plasmid was identified carrying the aphT gene in the required orientation.
Q 1998 Blackwell Science Ltd, Molecular Microbiology, 30, 163–174
SseF ::aphT was isolated on a Eco RI/Sst II fragment, bluntended as before, ligated into the Sma I site of pCVD422 and
the mutation transferred to the Salmonella chromosome as
described above.
SseG
A 1.0 kb Sau IIIA fragment containing sseG was subcloned
from p5-7 into pUC18. The resulting construct was digested
with Asc I, blunt-ended using Klenow fragment of DNA polymerase and ligated to the aph T cassette as indicated above.
The resulting plasmid was digested with Eco RI and Xba I and
the insert was ligated into Eco RI/Xba I-digested pGP704.
This plasmid was introduced into S. typhimurium 12023 and
exconjugants carrying the aph T cassette in sseG were isolated as described above.
For complementation of non-polar mutations in sseA , sseB
and sseC, the corresponding genes were amplified by PCR
from genomic DNA using a series of primers corresponding
to the region 58 of the putative start codons and to the 38
ends of the genes. These primers introduced Bam HI restriction sites at the termini of the amplified genes. After digestion
by Bam HI, the genes were ligated to Bam HI-digested
pACYC184 (Chang and Cohen, 1978) and transferred into
E. coli DH5a. The orientation of the inserts was determined
by PCR, and, in addition, DNA sequencing was performed
to confirm the orientation and the correct DNA sequence of
the inserts. Plasmids with inserts in the same transcriptional
orientation as the Tet r gene of pACYC184 were selected for
complementation studies and electroporated into the S. typhimurium strains harbouring corresponding non-polar mutations.
Virulence tests and macrophage survival assays
Groups of female BALB/c mice (20–25 g) were inoculated
intraperitoneally with either single or mixed S. typhimurium
strains. The inoculum consisted of either 1 × 105 or 1 × 104 cfu
in 0.2 ml of physiological saline. Bacterial strains were cultured
as described by Shea et al. (1996).
For mixed infections, wild-type and mutant strains were
grown separately and mixed before injection. The cfu of both
strains was checked by plating a dilution series of the inoculum
onto LB and LB supplemented with kanamicin. For mixed
infections involving HH106, strain 12023 was first transformed
with pACYC184 (which does not affect its virulence, unpublished results) and cfu were checked by plating onto LB and
LB supplemented with chloramphenicol. Mice were killed 48 h
after inoculation and bacterial cfu were counted after plating
dilution series of spleen homogenates onto LB and LB supplemented with the appropriate antibiotic.
RAW 264.7 cells (ECACC 91062702), a murine macrophage-like cell line, were grown in Dulbecco’s modified Eagle
medium (DMEM) containing 10% fetal calf serum (FCS) and
2 mM glutamine at 378C in 5% CO2 . S. typhimurium strains
were grown in LB to stationary phase and diluted to an OD600
of 0.1 and opsonized for 20 min in DMEM containing 10%
normal mouse serum. Bacteria were then centrifuged onto
macrophages seeded in 24-well tissue culture plates at a
multiplicity of infection of <1:10 and incubated for 30 min.
After infection, the macrophages were washed twice with
PBS to remove extracellular bacteria and incubated for 90 min
172 M. Hensel et al.
(2 h after infection) or 16 h in medium containing gentamicin
(12 mg ml¹1 ). Infected macrophages were washed twice with
PBS and lysed with 1% Triton X-100 for 10 min and appropriate aliquots and dilutions were plated onto LB agar to enumerate cfu.
Survival of opsonized S. typhimurium strains in J774.1
cells (Ralph et al., 1975) or C3H/HeN murine peritoneal exudate cells (from Charles River Laboratories) was determined
essentially as described by DeGroote et al. (1997), but without the addition of interferon g. Briefly, peritoneal cells harvested in PBS with heat-inactivated 10% fetal calf serum 4
days after intraperitoneal injection of 5 mM sodium periodate
(Sigma) were plated in 96-well flat-bottomed microtitre plates
(Becton-Dickinson) and allowed to adhere for 2 h. Non-adherent cells were flushed out with prewarmed medium containing 10% heat-inactivated fetal calf serum. In previous studies,
we have established that >95% of the cells remaining after
this procedure are macrophages. S. typhimurium from aerated
overnight cultures was opsonized with normal mouse serum
and centrifuged onto adherent cells at an effector to target
ratio of 1:10. The bacteria were allowed to internalize for
15 min and washed with medium containing 6 mg ml¹1 gentamicin to kill extracellular bacteria. At 0 h and 20 h, cells were
lysed with PBS containing 0.5% deoxycholate (Sigma), with
plating of serial dilutions to enumerate cfu.
Macrophages were examined microscopically over the course
of these experiments and did not show significant levels of
cytotoxicity.
Construction and analysis of sseA reporter gene fusion
A 1.1 kb Sma I/Hin cII fragment of p5-4 was subcloned into
pGPL01, a suicide vector for the generation of luc fusions
(Gunn and Miller, 1996). The resulting construct, in which
1.0 kb upstream and 112 bp of sseA is translationally fused
to luc was used to transform E. coli S-17 l pir, and conjugational transfer to S. typhimurium performed as described previously (Gunn and Miller, 1996). Strains that had integrated
the reporter gene fusion into the chromosome by homologous
recombination were confirmed by PCR and Southern hybridization analysis. Subsequently, the fusion was moved by P22
transduction into the wild-type and various mutant strain
backgrounds with mTn5 insertions in SPI-1 or SPI-2 genes
(Maloy et al., 1996). As a control, a strain was constructed
harbouring a chromosomal integration of pLB02, a suicide
plasmid without a promoter fusion to the luc gene (Gunn
and Miller, 1996). For the analysis of gene expression, strains
were grown for 16 h in minimal medium with aeration. Aliquots
of the bacterial cultures were lysed and luciferase activity was
determined using a luciferase assay kit according to the
manufacturer’s protocol (Boehringer Mannheim). Photon
detection was performed on a Microplate scintillation/luminescence counter (Wallac). All assay were carried out in triplicate and replicated on independent occasions.
Acknowledgements
This project was supported by grants from the MRC (UK) to
David Holden, and DFG (Germany) (grant no. 1964/2–1) to
Michael Hensel. A.V.T. and F.C.F. were supported in part
by a grant from the National Institutes of Health (AI39557).
We gratefully acknowledge the use of the network service
at HGMP Resource Centre, Hinxton, UK. We are grateful to
Dr J. S. Gunn for providing pGPL01 and pLB02 and to Dr Carmen Beuzon for help with the sequence analysis.
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