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Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, München, Germany;
Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, Würzburg, Germany; and
Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing cells reactive with listeriolysin O 91–99 or p60
217–225, respectively. This CD8 T cell response protected mice against
a challenge with L. monocytogenes. In conclusion, these
findings suggest that YopE is a versatile carrier molecule for type
III-mediated foreign Ag delivery by Salmonella vaccine
strains. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an attempt to circumvent this problem, we have reported the use of the type III protein secretion system of S. typhimurium to target viral T cell epitopes directly to the cytosol of eukaryotic cells (7). Type III secretion systems are currently discovered in an increasing number of taxonomically diverse Gram-negative animal and plant pathogens (8). These systems are specialized for the export of bacterial virulence factors delivered directly into the cytosol of target cells to modulate host cellular functions (9).
In our previous study (7), mice were orally vaccinated with live replicating Salmonellae expressing the translocated type III effector protein Salmonella protein tyrosine phosphatase (SptP)4 fused to an immunodominant CD8 epitope of the murine lymphocytic choriomeningitis virus. In a subsequent challenge experiment, all vaccinated mice were protected against a lethal lymphocytic choriomeningitis virus infection. However, the use of SptP as a carrier protein for heterologous Ags was limited to deliver small protein fragments of 45–55 aa inserted in frame between two functional domains of the Salmonella type III protein. Because a versatile Ag delivery system used by attenuated S. typhimurium strains should be also capable of targeting large protein fragments derived from diverse microorganisms, we were interested in identifying a type III effector protein that could be used in Salmonella for this purpose.
Among different bacterial species, many components of type III
secretion systems reveal functional conservation probably due to the
fact that shared type III genes were recruited by horizontal transfer
during evolution (8). Probably one of the best-studied
type III effector proteins is the Yersinia outer protein E
(YopE). During the interaction of Yersinia enterocolitica
with professional phagocytes, YopE translocation mediates the ability
of the bacteria to resist phagocytosis and to survive at extracellular
sites (10, 11). Cytosolic delivery of YopE into eukaryotic
cells was investigated by techniques based on a variety of reporter
proteins fused to the type III effector molecule (12, 13, 14, 15).
Based on results of these studies, transport signals of YopE for type
III-dependent secretion and translocation have been well defined. The
23-kDa YopE molecule contains an N-terminal secretion domain of
11–15 aa and a translocation domain of at least 50 aa. The latter
domain provides the binding site for the YopE-specific chaperone (SycE)
that is required for YopE translocation. Recently, the versatility of
YopE for heterologous protein delivery into the cytosol of target cells
was expanded. Chaux et al. (16) used Y.
enterocolitica expressing hybrid YopE/MAGE-A1 proteins for Ag
translocation into dendritic cells, resulting in the induction of a
proliferative cytotoxic T cell response. Moreover, for vaccination
purposes, our laboratory made use of Yersinia to inject YopE
fused to a heterologous immunodominant bacterial protein into the
cytosol of infected host cells and to deliver foreign antigenic
peptides to the MHC class I Ag presentation pathway
(17).
In a previous study, Rosqvist et al. (18) have shown that full-length wild-type YopE can be secreted and translocated by S. typhimurium in a type III-dependent manner. This observation on one hand, and the promiscuity of YopE secretion and translocation domains to deliver foreign proteins to the cytosol of target cells in contrast, prompted us to investigate the possible employment of YopE for the delivery of heterologous Ags by attenuated Salmonella. In this study, we demonstrate that S. typhimurium allows secretion and translocation of chimeric YopE fused to large antigenic protein fragments of Listeria monocytogenes, which results in the induction of Ag-specific CD8 T cell responses in orally vaccinated mice and animal protection against a virulent L. monocytogenes challenge.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli 
6060 was used as an intermediate
host for cloning procedures. The M45 epitope tag (MDRSRDRLPPFETETRIL)
is derived from the E4-6/7 protein of adenovirus (19).
Plasmid pSB1187 bearing the genetic information for M45
(20) was used as template DNA to amplify the epitope tag
by PCR (forward primer, M45 XhoI
5'-ATACTCGAGAACATTGCTAATAAA-3'; reverse primer, M45 KpnI
5'-TATGGTACCGCTTCTGCGTTCTGA-3'). The resulting DNA fragment was
cloned into the XhoI and KpnI sites of the
low-copy-number vector pWSK29 (21) to obtain pHR225.
Translational fusions between different lengths of YopE and
listeriolysin O (LLO) or p60 were constructed by PCR cloning
procedures. All resulting protein fusions were checked by DNA
sequencing. Site-specific mutagenesis of the iap gene
(cysteine 396
alanine) coding for p60 was performed according to the
protocol of Nelson and Long (22). Briefly, plasmid pSK50
carrying the wild-type iap gene of L.
monocytogenes strain sv1/2a EGD (23, 24) was used as
template DNA for the first round of PCR amplification (forward primer,
SSM1-iap 5'-CCAGATGCATCAAATGTAGTTGG-3'; reverse primer,
SSM2-iap
5'-GACTGACTGACTGACTGACTTTGGAATTCTTATCTCATCATTT-3'). In primer
SSM1-iap, the mutated triplet (TGCGCA) was introduced.
This led to the generation of an NsiI restriction site,
which was used for restriction analysis. In primer SSM2-iap,
an EcoRI site and the sequence (GACT)5
were inserted to allow cloning into pSK50 and amplification of the
mutated strand, respectively. The resulting 1.8-kb PCR fragment was
annealed with the wild-type iap gene from pSK50 in a second
round of amplification to allow elongation of the mutated strand via
two cycles. In a third PCR step, forward primer SSM3-iap
(5'-GACTGACTGACTGACTGACT-3') and reverse primer SSM4-iap
(5'-TTACTGAATTCTTCATCATAC-3') were added to the reaction mix of the
second PCR, and 35 cycles were run. Subsequently, the resulting PCR
product was separated by agarose gel electrophoresis, excised, and
purified. The DNA fragment was then cleaved with
PstI/EcoRI and inserted into plasmid pTZ18R for
multiplication. The 1.09-kb mutated fragment was finally excised from
pTZ18R by PstI/EcoRI digestion and used to
replace the corresponding wild-type PstI/EcoRI
fragment within the iap gene of pSK50. The resulting plasmid
coding for p60 (CysAla) was used as template DNA to amplify p60
(CysAla) 130–484 (forward primer, p60–130 BamHI
5'-GGAAAAACTGGATCCGTTAACGGT-3'; reverse primer, p60–484
SalI 5'-CGAACTGCTTGGTCGACAGGTTAC-3'). Plasmids pHR220 and
pHR222, expressing YopE 1–18/p60 130–484 and YopE 1–138/p60
130–484, respectively (17), were digested with
BamHI and SalI to remove the iap gene
fragment coding for p60 130–484, and subsequently religated by
insertion of the iap gene fragment coding for p60
(CysAla) 130–484 to obtain plasmids pHR220-Cys and pHR222-Cys.
These latter two vectors were used as templates to amplify DNA
fragments encoding YopE 1–18/p60 (CysAla) 130–477 and YopE
1–138/p60 (CysAla) 130–477, respectively (forward primer, YopE
SacII 5'-CACCCGCGGCAGACCATCAATTTG-3'; reverse primer,
p60 XhoI 5'-GCCCTCGAGATATTTACCCCAGCC-3'). Amplicons were
cloned into SacII and XhoI sites of pHR225 to
receive in-frame fusions of YopE/p60 (CysAla) with M45 epitope tag.
The resulting plasmid pHR226 encodes YopE 1–18/p60 (CysAla)
130–477/M45, whereas pHR242 expresses YopE 1–138/p60 (CysAla)
130–477/M45. In a next cloning step, the sycE gene was
amplified using pHR220 (17) as template DNA. The PCR
product was cloned into SacI and SacII sites of
either pHR226 to obtain pHR240 or of pHR242 to construct pHR241.
Genomic DNA from L. monocytogenes strain sv1/2a EGD was used
to amplify a lisA gene fragment encoding LLO 51–363
(forward primer, LLO BamHI 5'-CCTAAGACGCGGATCCCAAAG-3';
reverse primer, LLO XhoI
5'-GTCCTCGAGGTTACCGTCGATGAT-3'). Plasmids pHR240, pHR241, and
pHR242 were digested with BamHI and XhoI to
remove the gene fragment encoding p60 130–477, and subsequently
re-ligated by insertion of the lisA-PCR fragment. Thus,
plasmids pHR230, pHR231, and pHR232 bear the genetic information for
chimeric YopE/LLO/M45 fusion proteins. Plasmids constructed in this
study were transformed into S. typhimurium SB824
(7) by electroporation. Strain SB824 was engineered by
introducing sptP::kan mutant allele from strain
SB237 (25) into the 
aroA strain SL3261
(26) by P22HTint transduction. In some
experiments, wild-type S. typhimurium strain SL1344
(26) and S. typhimurium strain SB161 were used.
The latter strain carries a nonpolar insertion mutation in
invG (27). S. typhimurium strains
were grown in Luria Bertani (LB) medium supplemented with 0.3 M NaCl,
pH 7, to allow expression of components and targets of the type III
secretion system encoded by Salmonella Pathogenicity Island
1 (SPI1) (28). When required, the antibiotics ampicillin
(100 µg/ml) and kanamycin (30 µg/ml) were added. L.
monocytogenes strain sv1/2a EGD was used for challenge experiments
in Salmonella-vacci- nated mice.
Detection of secreted proteins and Western blot analysis
Secreted proteins from Salmonella culture supernatants and bacterial cell lysates were prepared and detected, as described previously (29). Briefly, bacterial supernatants were passed through a 0.45-µm-pore-size syringe filter to remove bacteria. Protein in the bacteria-free medium was precipitated by the addition of cold TCA to 10% (v/v) and incubated for 2 h on ice. The protein was collected by centrifugation at 4°C, 10,000 x g for 20 min. Pellets were washed in 0.8 ml cold acetone, dried, and resuspended in PBS buffered with 80 mM Tris-HCl, pH 8. Samples corresponding to 100 µl whole bacterial culture and 200 µl culture supernatant were separated in a 10% discontinuous SDS-PAGE, and transferred to nitrocellulose membranes, as described previously (28). Hybrid YopE/p60/M45 and YopE/LLO/M45 proteins as well as Salmonella invasion proteins B and C (SipB and SipC) were detected by immunoblot analysis. Western blots were treated with a mAb against M45 (kind gift of P. Hearing, State University of New York, Stony Brook, NY) or mAbs directed against SipB and SipC (kind gift of J. E. Galán, Yale School of Medicine, New Haven, CT), followed by incubation with an AP-labeled anti-mouse Ab. Blots were developed by using a chemiluminescent detection kit.
Tissue culture cell invasion assay
The invasion assay was conducted as previously described (30). Briefly, P388D1 cells were grown for 2 days in DMEM supplemented with 5% FBS in 24-well dishes to reach 80% confluence. Next, 1 h before the addition of bacteria, the culture medium was replaced by 500 µl HBSS. Bacteria were grown overnight for 12 h in LB supplemented with 0.3 M NaCl, diluted 1/20 in fresh medium, and grown for another 4 h under mild aeration to reach an OD600 of 0.9. P388D1 cells were infected with S. typhimurium for 2 h with a multiplicity of infection (MOI) of 10 bacteria per cell. To determine the number of intracellular bacteria, cells were washed three times with HBSS and further incubated for 3 h with DMEM containing gentamicin (50 µg/ml) to kill extracellular bacteria before lysis with 0.1% sodium deoxycholate in PBS.
Immunofluorescence analysis of hybrid YopE protein translocation
P388D1 cells were grown on glass coverslips to 60% confluence. One hour before the addition of bacteria, DMEM was replaced by 500 µl HBSS. Bacteria were grown overnight for 12 h in LB supplemented with 0.3 M NaCl, diluted 1/20 in fresh medium, and grown for another 4 h under mild aeration to reach an OD600 of 0.9. P388D1 cells were infected with S. typhimurium at an MOI of 10 for 30 min at 37°C, 5% CO2. Cells were washed three times with HBSS and further incubated for 4.5 h with DMEM containing gentamicin (50 µg/ml) to kill extracellular bacteria, thus reducing extracellular growth of Salmonella. Afterward, cells were washed again three times with HBSS and fixed in PBS, 3.7% formaldehyde. Remaining extracellular bacteria were stained with an anti-Salmonella O-1,4,5,12 polyclonal rabbit antiserum (1:400 in PBS, 3% BSA; Difco, Detroit, MI) and a secondary anti-rabbit tetramethylrhodamine isothiocyanate (TRITC) conjugate (1:100 in PBS, 3% BSA; Sigma, St. Louis, MO). After permeabilization of P388D1 cells (3 min in PBS, 0.1% Triton X-100), extra- and intracellular bacteria were stained with a polyclonal anti-Salmonella antiserum and a secondary anti-rabbit 7-amino-4-methylcoumarin-3-acetic acid (AMCA) conjugate (1:100 in PBS, 3% BSA; Jackson ImmunoResearch, West Grove, PA). Translocated hybrid YopE/LLO/M45 or YopE/p60/M45 proteins were detected using a anti-M45 mAb and an anti-mouse FITC conjugate (1:100 in PBS, 3% BSA; Sigma). Coverslips were mounted on glass slides and analyzed by fluorescence microscopy. Experiments were repeated at least three times.
Mice
Female BALB/c mice, 6–8 wk old, were purchased from Charles River WIGA (Sulzfeld, Germany). All mice were kept under specific pathogen-free conditions (positive pressure cabinet) and were provided with food and water ad libitum.
Oral immunization of mice with recombinant Salmonella and in vivo protection assay
Groups of 10 mice were orally immunized with a single dose of 5 x 108 S. typhimurium SB824 expressing various hybrid YopE proteins or a sublethal i.p. dose of 5 x 103 CFU Listeria 8 wk before the challenge infection. Eight weeks after inoculation, four mice per group were sacrificed, and spleens were used for further ELISPOT analysis. The remaining six mice per group were challenged i.v. with 1 x 103 CFU of log phase L. monocytogenes strain sv1/2a EGD in 0.2 ml PBS. Three days after the challenge, CFU were determined by plating serial dilutions of spleen homogenates on PALCAM Listeria selective agar (Merck, Darmstadt, Germany). Colonies were enumerated after 48 h of incubation. Colony counts were corrected for dilution and averaged to yield CFU/organ. The level of protection was calculated as the log10 difference of the bacterial count from immunized mice and naive control mice. Each experiment was performed at least twice with similar results.
T cell lines and Ag presentation assay
CD8 T cell lines specific for p60 217–225 (31) and
LLO 91–99 (32) were derived from L.
monocytogenes-infected BALB/c mice and propagated by repeated
restimulation in the presence of the appropriate synthetic peptide, as
described previously (33). The detection limit of the CD8
T cell lines used was between 10-11 and
10-12 M peptide. CD8 T cell activation by
infected P388D1 macrophage-like APC was measured
by the detection of IFN- in culture supernatants (34).
Briefly, P388D1 cells were infected with S.
typhimurium in 96-well flat-bottom microwell plates for 10 min by
200 x g centrifugation. After 2 h at 37°C,
infected APC were washed and culture medium supplemented with 25
µg/ml gentamicin was added. CD8 T cells were added to each well, and
after 12–18 h at 37°C, supernatants were harvested and the IFN-
concentration was measured by means of an IFN--specific ELISA that
binds and detects IFN- with a pair of specific mAb. Results were
corrected for dilution of the sample to yield the sample concentration
in ng/ml.
ELISPOT assay
The frequency of T lymphocytes in mice immunized with attenuated
S. typhimurium was determined with an IFN--specific
ELISPOT assay (34, 35). Assays were performed in
nitrocellulose-backed 96-well microtiter plates (Nunc, Wiesbaden,
Germany) coated with rat anti-mouse IFN- mAb (RMMG-1; BioSource
International, Camarillo, CA). Unseparated splenocytes (6 x
105/well) were stimulated for 6 h in
round-bottom microtiter plates in the presence of
10-8 M peptide. Subsequently, activated cells
(4 x 105/well or 4 x
104/well) were transferred to ELISPOT plates and
incubated overnight. ELISPOT plates were developed with biotin-labeled
rat anti-mouse IFN- mAb (clone XMG1.2; PharMingen, San Diego,
CA), HRP streptavidin conjugate (Dianova, Hamburg, Germany), and
aminoethylcarbazole dye solution. The frequency of Ag-specific cells
was calculated as the number of spots per splenocytes seeded. The
specificity and sensitivity of the ELISPOT assay were controlled with
IFN--secreting CD8 T cell lines specific for p60 217–225 and LLO
91–99. Recovery of seeded CD8 T cells was higher than 90% for both
cell lines. CD4 T cells did not contribute to the fraction of
IFN--secreting cells. Recently, we have demonstrated that both MHC
class I-restricted nonamer peptides do not stimulate LLO- or
p60-specific CD4 T cells (36).
Statistical analysis
The statistical analysis of the results of in vitro experiments was performed with the Newman-Keuls multiple comparison test at the 0.05 significance level. The statistical significance of the results of in vivo experiments was checked with the nonparametric Tukey multiple comparison test at the 0.05 significance level. All tests were performed using WINKS statistical analysis software (Texasoft, Cedar Hill, TX).
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Results |
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After invasion of target cells, it is a characteristic feature of L. monocytogenes to escape from the host phagosome into the cytosol (37). Destruction of the phagosomal membrane is mediated by the secretion of the pore-forming virulence factor LLO (38, 39). Another protein that is constitutively secreted by Listeria is a murein hydrolase designated as p60 (40). Bacterial secretion of LLO and p60 into the cytosol of infected cells directs these listerial Ags to the MHC class I Ag-processing pathway, leading to presentation of Ag-derived peptides to MHC class I-restricted CD8 T cells (41). Analysis of CD8 T cell clones from Listeria-infected mice revealed that one such clone recognizes residues 91–99 of LLO in the context of the H2-Kd MHC class I molecule (32). Further investigations indicated that p60 217–225 is presented by H2-Kd as well (31, 42). CD8 T cells specific for these epitopes were shown to transfer protective immunity against L. monocytogenes to naive mice (35).
To assess the possible employment of YopE for the delivery of two
independent heterologous Ags by attenuated Salmonella, large
portions of LLO and p60 consisting of >300 aa were fused to various
amino-terminal parts of YopE (Fig. 1
). It
is important to emphasize that both Ag fragments do not elicit any
membrane- or bacterial cell-lysing activity (data not shown). This was
achieved by deleting the N-terminal 50 and the C-terminal 165 aa of LLO
(43) and by exchanging cysteine 396 of p60, which has been
shown to be directly involved in cell division to alanine
(44). Plasmids pHR230 and pHR240 encode the N-terminal 18
aa of YopE fused to aa 51–363 of LLO or aa 130–477 of p60
(CysAla), respectively. The resulting chimeric proteins are both
tagged at their C terminus with an M45 epitope and contain the
secretion, but lack the translocation domain of YopE. In contrast, both
plasmids pHR231 and pHR 241 bear the genetic information for hybrid
YopE proteins containing the N-terminal secretion and translocation
domains of YopE (Fig. 1). Plasmid pHR231 encodes the N-terminal 138 aa
of YopE fused to LLO 51–363/M45, whereas pHR241 encodes YopE 1–138
fused to p60 (CysAla) 130–477/M45. Plasmids pHR230, pHR231, pHR240,
and pHR241 also bear the sycE gene coding for SycE. To
assess the influence of rSycE produced in direct vicinity to hybrid
YopE proteins on the translocation and Ag presentation efficiency of
LLO and p60, plasmids pHR232 and pHR242 were constructed lacking the
sycE gene (Fig. 1).
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To assess the ability of S. typhimurium aroA sptP
mutant strain SB824 to secrete recombinant hybrid YopE/LLO/M45 and
YopE/p60/M45 proteins, strains carrying the respective plasmids were
grown under conditions that allow expression of all components of the
type III secretion system encoded by SPI1 (see Materials and
Methods). Fig. 2A reveals
that all six constructed chimeric YopE proteins were stably expressed
in the cytosol of SB824. Furthermore, similar amounts of these proteins
could be detected in bacterial culture supernatants.
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B, no secretion of chimeric
YopE by SB161 could be detected, even though all fusion proteins were
found in the bacterial cytosol. Taken together, it is demonstrated that the N-terminal 18 aa of YopE are engaged by the SPI1 type III secretion system, resulting in sufficient secretion of large antigenic protein fragments fused to YopE.
Type III-dependent secretion of hybrid YopE proteins does not alter the invasion phenotype of Salmonella
The invasion phenotype of Salmonella is crucial for colonization of the host and is mediated by components and effectors of the invasion-associated SPI1 type III secretion system. It is conceivable that concomitant secretion of hybrid YopE proteins and Salmonella type III effectors could lead to saturation of the export machinery, resulting in reduced secretion of essential Salmonella type III invasion molecules.
To investigate the possible influence of type III-dependent hybrid YopE
protein engagement on secretion of SPI1 effector molecules, the
secretion efficiencies of SipB and SipC by various
Salmonella strains were compared. As shown in Fig. 3, the nontransfected vaccine strain
SB824 secreted both type III proteins into the culture supernatant.
Under these in vitro conditions, it is a characteristic feature of SipC
that a relatively small amount of the protein was detected in the
bacterial whole cell lysate as compared with SipB (28).
SB824 expressing recombinant YopE/LLO/M45 or YopE/p60/M45 produced SipB
and SipC in comparable amounts as nontransfected SB824 (whole cell
lysates). Moreover, coexpression of chimeric YopE did not lead to
differences in secretion of SipB and SipC into culture supernatants. To
further verify this observation, a comparison of the invasion
competence of all SB824 used in this study was conducted. Fig. 4 reveals that no statistically
significant difference among the invasion competence of
Salmonella wild-type SL1344, the attenuated carrier vaccine
strain SB824, and SB824 expressing hybrid YopE proteins was detected.
In contrast, negative control strain SB161 lacking the essential type
III molecule InvG was found to be invasion defective.
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Cytosolic delivery of hybrid YopE proteins into APC
Infections of P388D1 cell monolayers with SB824 were conducted to study the potential of Salmonella to deliver chimeric YopE proteins into the cytosol of infected cells. Five hours after infection, cells were fixed and processed for differential immunofluorescence staining with Abs directed against M45 and Salmonella’s O-Ag, as indicated in Materials and Methods.
Fig. 5, upper panel, shows
typical images obtained by an overlay of the fluorescence signals from
TRITC (extracellular bacteria) and AMCA (intra- and extracellular
bacteria). Thus, internalized bacteria (AMCA+ and
TRITC-) appear blue, whereas extracellular
salmonellae (AMCA+ and
TRITC+) exhibit a purple fluorescent color
(mixture of blue and red). No fluorescent anti-M45 FITC signal
(green color) was detected in P388D1 cells
infected with Salmonella SB824 expressing YopE 1–18/LLO/M45
or YopE 1–18/p60/M45 encoded by plasmids pHR230 or pHR240,
respectively (Fig. 5, middle panel). In contrast, both YopE
1–138/LLO/M45 and YopE 1–138/p60/M45 gave strong fluorescent FITC
signals in samples infected with SB824 (pHR231) or SB824 (pHR241).
Optical sectioning and confocal microscopy revealed that these hybrid
proteins were equally distributed throughout the cytoplasm of
Salmonella-infected P388D1 cells (data
not shown). Thus, chimeric YopE containing the secretion and
translocation domain of YopE was efficiently delivered into the cytosol
of host cells by internalized bacteria. Besides coding for hybrid YopE
proteins, plasmids pHR231 and pHR241 also bear the genetic information
for SycE (Fig. 1). Translocation of hybrid YopE 1–138 was dependent on
the presence of rSycE, because no visible fluorescent FITC signals were
observed in cells infected with SB824 that were transformed with
plasmids pHR232 or pHR242, which lack the sycE gene (Fig. 5, middle panel).
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MHC class I-restricted Ag presentation of cytosolic hybrid YopE proteins
The ability of S. typhimurium expressing chimeric
YopE/LLO/M45 or YopE/p60/M45 to deliver listerial nonamer peptides to
the MHC class I-restricted Ag-presenting pathway was examined. Murine
P388D1 cells were infected with various
Salmonella strains and used as APC. The ability of infected
cells to present the immunodominant epitopes LLO 91–99 and p60
217–225 to CD8 T cells with corresponding specificities was assessed
in an in vitro Ag presentation assay by measuring secreted IFN- in
the culture supernatant (Fig. 6).
P388D1 cells infected with SB824 (pHR231)
expressing and translocating YopE 1–138/LLO/M45 were efficiently
recognized by LLO 91–99-specific T cells (Fig. 6A), whereas
APC infected with SB824 (pHR230) or SB824 (pHR232) expressing and
secreting hybrid YopE proteins stimulated a much weaker LLO
91–99-specific T cell response. In contrast, the negative control
strain SB824 did not induce any measurable level of IFN- secretion
into the culture supernatant. Similar results were obtained when APC
infected with p60-expressing Salmonella strains were tested
with the p60 217–225-specific T cell line (Fig. 6B).
P388D1 cells infected with SB824 (pHR240) or
SB824 (pHR242) expressing and secreting, but not translocating chimeric
YopE/p60/M45 proteins were less efficiently recognized by p60
217–225-specific T cells than APC infected with SB824 (pHR241), which
translocates p60 into the cytosol of the host cell. Thus, Ag
presentation and CD8 T cell stimulation were significantly enhanced by
cytosolic delivery of the respective listerial nonamer peptide by the
Salmonella type III secretion/translocation system.
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production at higher MOI was observed. This phenomenon is probably due
to the fact that the infection of macrophages with higher numbers of
Salmonella can lead to the death of phagocytes by apoptosis
(46, 47). In vivo induction of LLO- and p60-specific T cell responses by Salmonella translocating hybrid YopE proteins
The potential of attenuated S. typhimurium expressing
chimeric YopE proteins to induce cytotoxic CD8 T cells in vivo was
investigated. For this purpose, BALB/c mice were orally inoculated with
a single dose of 5 x 108 S.
typhimurium SB824 harboring the indicated plasmids. Control groups
received a sublethal i.p. dose of 5 x 103
Listeria (Fig. 7). Eight weeks
after inoculation, ELISPOT assays were performed to determine the
frequency of LLO- and p60-specific T cells in vivo. The frequency of
LLO 91–99- and p60 217–225-specific CD8 T cells was calculated as the
number of IFN- spots generated per 1 x
105 spleen cells in the presence of the
corresponding synthetic peptide. Mice immunized with SB824 (pHR241)
translocating YopE 1–138/p60/M45 revealed similar numbers of
IFN--producing cells reactive with p60 217–225 as mice infected
with L. monocytogenes (Fig. 7). In contrast, immunization of
animals with SB824 (pHR240) secreting YopE 1–18/p60/M45 resulted in
induction of a significantly lower number of p60-reactive CD8 T cells.
The difference between the potential of secreted Ags on one hand and
translocated Ags in contrast to elicit peptide-specific CD8 T cell
priming was even more pronounced, when mice were immunized with the
LLO-expressing strains SB824 (pHR231) or SB824 (pHR230). The frequency
of IFN--producing cells reactive with LLO 91–99 in the former group
of animals indicated a prominent Ag-specific T cell response, whereas
the number of LLO-specific T cells in mice of the latter group was
below the detection limit of the ELISPOT assay. In summary, these
experiments demonstrate that in vivo type III-mediated cytosolic
delivery of chimeric YopE proteins by Salmonella results in
superior efficacy of MHC class I-restricted Ag presentation as compared
with secreted Ag display.
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Eight weeks after oral immunization with Salmonella
expressing hybrid YopE proteins, mice were i.v. challenged with 1
x 103 L. monocytogenes. To compare
the contribution of translocated vs secreted LLO and p60 on
vaccine-induced protection, CFU were determined in spleens 3 days after
the challenge. Spleens of uninfected mice and of animals infected with
the nontransfected vaccine strain SB824 were colonized with 1.6 ±
1.2 x 105 and 1 ± 0.6 x
105 CFU of Listeria, respectively
(Fig. 8). In contrast, no bacteria were
detected in spleens of mice that had received a sublethal i.p. dose of
5 x 103 CFU of Listeria 8 wk
before the challenge infection. Mice immunized with SB824 (pHR240) or
SB824 (pHR230) secreting but not translocating p60 or LLO,
respectively, showed a significant (p < 0.05)
decrease of the bacterial load in spleens (1.9 ± 1.8 x
104 and 1.1 ± 1.5 x
104 CFU) as compared with nonimmunized mice.
However, animals orally inoculated with SB824 (pHR241) or SB824
(pHR231) translocating p60 or LLO revealed a more pronounced reduction
of bacterial colonization in their spleens (0.9 ± 1.1 x
102 and 0.6 ± 0.8 x
102 CFU). Thus, in comparison with secreted Ag
display, MHC class I-restricted Ag presentation and CD8 T cell
stimulation of translocated listerial Ags resulted in a significantly
better protection against L. monocytogenes in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study describes the efficient protection against the intracellular pathogen L. monocytogenes by oral vaccination with attenuated S. typhimurium translocating two defined listerial Ags, LLO and p60, via the SPI1 type III secretion system. The first major finding of our experiments is that the N-terminal translocation domain of the Yersinia type III effector molecule YopE is engaged by Salmonella’s type III secretion system, resulting in excellent secretion and translocation of large heterologous antigenic proteins. In Yersinia, N-terminal YopE transport domains were shown to be sufficient for secretion and translocation of fused reporter proteins. Sory et al. (12) used recombinant Y. enterocolitica expressing truncated YopE proteins fused to a calmodulin-dependent adenylate cyclase of Bordetella pertussis, resulting in an increase in cAMP in the eukaryotic cytosol. In another approach, Lee et al. (14) constructed hybrid YopE-neomycin phosphotransferase proteins to study type III effector protein targeting. Furthermore, Jacobi et al. (15) used the green fluorescent and the firefly luciferase proteins fused to various residues of YopE to investigate translocation. However, this is the first report that describes the use of YopE for heterologous Ag delivery in Salmonella. Use of YopE by S. typhimurium’s type III secretion system allows secretion and translocation of large antigenic proteins, thus avoiding limitations to single epitopes, which might occur using Salmonella’s type III effector molecule SptP for vaccination purposes (7). Remarkably, the concomitant secretion of hybrid YopE proteins and Salmonella type III effectors did not lead to saturation of the export machinery that would result in reduced secretion of essential Salmonella type III invasion molecules. Impaired invasion and persistence of Salmonella is not a desirable feature of a carrier vaccine strain because these phenotype alterations are clearly associated with reduced immunogenic properties.
By using differential immunofluorescence staining of Salmonella-infected APC, we demonstrate that LLO and p60 fused to the N-terminal translocation domain of YopE comprised of 138 aa were efficiently delivered into the cytosol of P388D1 cells. Protein translocation resulted in efficient listerial Ag presentation and peptide-specific CD8 T cell stimulation in vitro. In contrast, no cytosolic delivery of these hybrid YopE proteins was observed in the absence of recombinant, plasmid-borne SycE. SycE is a small homodimeric protein that interacts with YopE in the Yersinia cytoplasm (53), and this interaction is absolutely required for YopE translocation (14), whereas YopE secretion is not affected. Obviously, SycE serves a similar essential purpose when expressed in Salmonella. Hybrid YopE proteins containing the N-terminal secretion domain (aa 1–18), but lacking the translocation domain, could not be detected in the cytosolic compartment of APC as well. The presence of secreted but nontranslocated chimeric YopE proteins in the Salmonella-containing endosomal compartment could not be visualized by immunofluorescence staining. It is tempting to speculate that after secretion, a rapid degradation of these proteins occurs. As compared with translocation, hybrid YopE protein secretion resulted in significantly less efficient, but measurable listerial Ag presentation and weak peptide-specific CD8 T cell stimulation in vitro. Obviously, a minimal portion of secreted but nontranslocated chimeric YopE was introduced from the endosome to the MHC class I processing pathway. Whether this is due to endosomal loading of empty MHC class I molecules (54) with listerial peptides after rapid degradation of hybrid YopE proteins, or release of these peptides into the cytosol of APC with subsequent classical MHC class I Ag processing and presentation (55) remains to be elucidated.
The second major finding of our study is that in accordance with the in
vitro results, translocated rather than secreted Ags elicited a much
more pronounced peptide-specific CD8 T cell priming in immunized mice,
resulting in the induction of stronger protective immunity. As
determined by ELISPOT assays, animals that were orally inoculated with
SB824 (pHR241) expressing translocated hybrid YopE/p60/M45 protein
revealed a significantly higher number of IFN--producing splenocytes
reactive with p60 217–225 than mice immunized with SB824 (pHR240)
expressing secreted but nontranslocated hybrid YopE/p60/M45 protein.
The effect that a nontranslocated listerial Ag induces a weak but
measurable induction of peptide-specific CD8 T cells in vivo was
observed for p60, but not for LLO. However, in vivo protection assays
showed that oral vaccination of mice with either Salmonella
strain SB824 (pHR230) or SB824 (pHR240) led to a slight but significant
decrease of the bacterial load in spleens after challenge with
Listeria as compared with control mice. It remains to be
elucidated whether this low-level protection could be also due to the
induction of LLO- or p60-specific CD4 T cells by secreted hybrid YopE
comprised of >300 aa of the respective listerial Ag. In fact, adoptive
transfer experiments revealed that p60-specific Th1 clones mediate
significant protection against L. monocytogenes infection
(56). In contrast, Salmonella species have been
reported to inhibit activated macrophages in their ability to present
peptides from homologous or heterologous Ags in the context of MHC
class II molecules (57). In further studies, we will focus
on aspects of MHC class II-restricted immune responses and CD4 T cell
priming elicited by type III-mediated Ag display by recombinant
Salmonella.
However, a much stronger impact on vaccine-induced protection was achieved by immunization of mice with SB824 (pHR231) or SB824 (pHR241), which deliver chimeric YopE/LLO or YopE/p60 proteins directly to the cytosol of infected host cells, thus mimicking the situation of a L. monocytogenes infection. In additional experiments, we were interested in investigating how long this protection against listeriosis is able to persist. Therefore, 100 days after a single immunization with Salmonella vaccine strains, ELISPOT assays and challenge infections with Listeria were conducted as described above (data not shown). Interestingly, the results of these experiments showed no significant differences than the data obtained from animals 8 wk after immunization. Thus, in contrast to secreted Ag display, translocation of chimeric YopE by recombinant Salmonella induced prolonged protective immunity against listeriosis based on prominent Ag-specific CD8 T cell priming.
Remarkably, in mice orally immunized with a single dose of Salmonella, type III-dependent translocation and cytosolic delivery of hybrid YopE proteins into host cells resulted in the induction of prominent LLO- and p60-specific T cell responses, which were comparable with the levels of peptide-specific CD8 T cell priming in animals immunized with a sublethal dose of wild-type Listeria. This observation emphasizes the efficacy of type III-mediated induction of MHC class I-restricted immune responses. Obviously, recombinant Salmonella translocating a truncated version of a single immunodominant Ag from Listeria did not elicit the same protective ability as an immunizing sublethal dose of wild-type L. monocytogenes, which naturally displays a variety of listerial peptides from different Ags to CD8 T cells of the vaccinated host. Despite that it has been shown that selected or cloned CD8 T cells from Listeria immune mice are able to adoptively transfer antilisterial protection (6), for a potent vaccine candidate against listeriosis it might be necessary to display several pathogen-derived Ags. In future studies, we will investigate whether type III-mediated dual translocation of LLO and p60 by a single Salmonella vaccine strain enhances the protective ability against listeriosis.
In the experimental setup described in this study, mice were immunized with attenuated Salmonella expressing a single immunodominant listerial Ag, either LLO or p60, which led to significant protection of animals against a Listeria challenge infection. Similar results were obtained by Hess et al. (6), who used the E. coli hemolysin export apparatus to secrete LLO or p60 by recombinant Salmonella. In this study, the authors compared the efficacy of secreted vs somatic listerial Ag display. Secretion of LLO or p60 resulted in prominent peptide-specific CD8 T cell priming. This discrepancy to our study is probably due to the fact that we took special care of constructing hybrid YopE/LLO and YopE/p60 proteins that lack any membrane-lysing activity (43, 44). By truncating N- and C-terminal domains of the pore-forming protein LLO and by exchanging cysteine 396 to alanine of the autolysin p60, we were able to reduce secondary leakage from the endosomal compartment to the cytosol of infected cells. In our hands, type III-mediated translocation of antigenic hybrid proteins is mandatory for efficient MHC class I-restricted Ag presentation elicited by Salmonella carrier vaccines.
Taken together, we demonstrate for the first time that a type III effector molecule from Yersinia can be used as a carrier protein for translocation of large Ags by Salmonella’s type III secretion system. Our study shows that efficient stimulation of peptide-specific CD8 T cells and protection against listeriosis require cytosolic delivery of the respective listerial Ag. By engaging well-defined secretion and translocation domains of YopE, targeting of Ags into different subcellular compartments of APC can be orchestrated, which might expand the use of attenuated S. typhimurium strains for future oral vaccination strategies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Holger Rüssmann, Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig Maximilians Universität München, Pettenkoferstrasse 9a, 80336 München, Germany. E-mail address: ruessmann{at}m3401.mpk.med.uni-muenchen.de
3 Current address: Microbiological Analytics, Merck KGaA, 64271 Darmstadt, Germany.
4 Abbreviations used in this paper: SptP, Salmonella protein tyrosine phosphatase; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; LB, Luria Bertani; LLO, listeriolysin O; MOI, multiplicity of infection; Sip, Salmonella invasion protein; SPI1, Salmonella pathogenicity island 1; SycE, YopE-specific chaperone; TRITC, tetramethylrhodamine isothiocyanate; YopE, Yersinia outer protein E.
Received for publication January 8, 2001. Accepted for publication April 18, 2001.
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) or p60 217–225
(
) is shown as the number of reactive cells per 1 x
105 splenocytes. The dotted line at 0.2 x
10-5 indicates the detection limit (DL) of the ELISPOT
assay. The SD of triplicate cultures is indicated.