Download Modification of the signal sequence cleavage site of

Microbiology (2003), 149, 1249–1255
DOI 10.1099/mic.0.26072-0
Modification of the signal sequence cleavage site
of listeriolysin O does not affect protein secretion
but impairs the virulence of Listeria
monocytogenes
Marie-Annick Lety,3 Claude Frehel,3 Jean-luc Beretti, Patrick Berche
and Alain Charbit
Correspondence
Alain Charbit
Laboratoire de Microbiologie, INSERM U-570, Faculté de Médecine Necker, 156 rue de
Vaugirard, 75730 Paris Cedex 15, France
[email protected]
Received 24 October 2002
Revised
6 January 2003
Accepted 24 January 2003
Listeriolysin O (LLO, hly-encoded), a major virulence factor secreted by the bacterial pathogen
Listeria monocytogenes, is synthesized as a precursor of 529 residues. To impair LLO secretion,
the four residues of the predicted signal sequence cleavage site (EA-KD) were deleted and the
mutant LLO protein was expressed in a hly-negative derivative of L. monocytogenes. Unexpectedly,
the mutant protein was secreted in normal amounts in the culture supernatant and was fully
haemolytic. N-terminal sequencing of the secreted LLO molecule revealed that N-terminal
processing of the preprotein occurred three residues downstream of the natural cleavage site.
L. monocytogenes expressing this truncated LLO showed a reduced capacity to disrupt the
phagosomal membranes of bone marrow macrophages and of hepatocytes; and the mutant strain
showed a 100-fold decrease in virulence in the mouse model. These results suggest that the first Nterminal residues of mature LLO participate directly in phagosomal escape and bacterial infection.
INTRODUCTION
Proteins to be exported are typically synthesized as preproteins with an amino-terminal peptide that addresses the
protein to the secretion pathway. This signal peptide is
needed to target the protein and to initiate translocation
across the membrane, in both Gram-negative and Grampositive bacteria (von Heijne, 1990; Pugsley, 1993; van Wely
et al., 2001; Antelmann et al., 2001; and references therein).
Upon translocation, the signal peptide is removed by the
action of signal peptidases (Bron et al., 1998; Paetzel et al.,
2000). Although signal peptides do not show a great deal
of sequence homology, they do contain three recognizable
domains: (i) a positively charged amino-terminus (1–5
residues in length); (ii) a central hydrophobic domain (7–15
residues in length); and (iii) a neutral but polar C-terminal
domain. Statistical analyses of the amino acid sequences
surrounding signal sequence (SS) cleavage sites have led to
the definition of a so-called ‘23, 21’ rule, alanine being the
residue most commonly found at these two positions (see
Paetzel et al., 2000 for a recent review). The substrate
specificity of signal peptidases (Spases) has also been
extensively studied in vitro and in vivo. For example, Shen
et al. (1991) demonstrated that Escherichia coli type I Spase
Abbreviations: LLO, listeriolysin O; Spase, signal peptidase; SS, signal
sequence.
3These authors contributed equally to this work.
0002-6072 G 2003 SGM
tolerated only the residues A, S, G or P at position 21, and S,
G, T, V or L at position 23, while almost any residue was
allowed at positions +1, 22, 24 and 25.
Listeria monocytogenes is a Gram-positive bacterium widespread in nature and responsible for sporadic severe infections in humans and other animal species (Berche, 1995).
This pathogen is a facultative intracellular micro-organism,
capable of invading a wide variety of eukaryotic cells
(Dramsi et al., 1995; Gaillard et al., 1987, 1996; Kuhn &
Goebel, 1989), including endothelial cells (Drevets et al.,
1995) and macrophages (Mackaness, 1962). Among the
major virulence factors identified, listeriolysin O (LLO,
encoded by the hly gene) plays a crucial role in the escape of
bacteria from the phagosomal compartment. LLO-negative
mutants remain trapped in the vacuole, do not grow
intracellularly, and are avirulent in the mouse model of
infection (Gaillard et al., 1986; Kathariou et al., 1987;
Portnoy et al., 1988). LLO of L. monocytogenes is composed
of 529 residues and possesses a typical SS at its N-terminus
(Mengaud et al., 1988). After cleavage of the SS, the mature
protein is secreted in the culture supernatant as a monomer
(Geoffroy et al., 1989).
It has been previously shown that alteration of the SS
cleavage site of a normally exported protein could prevent
SS cleavage, thus resulting in the production of a protein
tethered to the cytoplasmic membrane through its Nterminal hydrophobic SS anchor (Cosma et al., 1998; Raivio
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M.-A. Lety and others
et al., 2000; and references therein). In an attempt to tether
the LLO to the envelope of L. monocytogenes and test how
such LLO-coated bacteria would interact with the phagosomal membrane in infected cells, we deleted residues 23 to
26 (EAKD) of the cleavage site of LLO from EGD-e (whose
genome has been recently sequenced: Glaser et al., 2001).
Unexpectedly, the DSS mutation did not impair protein
secretion but altered bacterial virulence. Implications for
the role of the N-terminus of mature LLO in bacterial
virulence are discussed.
METHODS
Bacterial strains and culture conditions. EGDDhly is a deriva-
tive of EGD (serotype 1/2a), which contains an in-frame chromosomal deletion of 1080 bp in the hly gene (Guzman et al., 1995).
Bacteria were grown in Brain Heart Infusion (BHI) broth (Difco) at
37 ˚C without antibiotics, except for the pAT28-transformed strains,
which were grown on BHI broth containing 60 mg spectinomycin
(Spc) ml21.
Construction of the mutant. Chromosomal DNA, plasmid isola-
tion, restriction enzyme analyses and amplification by PCR were
performed according to standard protocols (Sambrook et al., 1989;
Ausubel et al., 1990). The DSS mutation was generated by in vitro
site-directed mutagenesis on M13mp18-phly-hly as described previously (Lety et al., 2001), using the mutagenic oligonucleotide 59ATTGAATGCAGATGCTCTAGAAGTTTGTTGCGCAATT-39. Upon
mutagenesis, residues 23 to 26 (EAKD) were deleted and substituted
by residues SR (encoded by the underlined XbaI linker introduced)
(Fig. 1). The construction was checked by DNA sequence analysis.
The mutant hly gene, carried on pAT28 shuttle vector (Trieu-Cuot
et al., 1990), was transferred into EGDDhly by electroporation.
EGDDhly expressing wild-type LLO under the same conditions was
used as a positive control (Dubail et al., 2000; Lety et al., 2001) and
EGDDhly as a negative control.
Protein preparation and analyses. Concentrated culture super-
natants from bacteria grown in BHI were prepared as described
previously (Lety et al., 2001). Cell-free supernatants were filtered
through a 0?22 mm pore size Millipore filter. The filtered supernatants were concentrated by centrifugation through ultrafree Biomax
units (cut-off 30 kDa). LLO was identified by Western blotting
with polyclonal anti-LLO antibodies and quantified by ELISA with
the anti-LLO mAb SE2 (kindly provided by Dr A. J. Ainsworth,
Veterinary Medicine, Mississippi State University, USA; Erdenlig
et al., 1999).
For Western blots, identical volumes of each concentrated culture
supernatant were loaded per well. The fraction of LLO still associated
with the envelope was also evaluated. For this, bacterial pellets were
disrupted using a Fastprep FP120 apparatus (Ozyme; BIO101) with
three pulses of 30 s at a speed of 6?5. After centrifugation for 3 min at
10 000 r.p.m., lysates were collected and suspended in 16 SDS-PAGE
sample buffer, as described previously (Garandeau et al., 2002).
Antibodies were used at a final dilution of 1/1000, as described
previously (Lety et al., 2001). In ELISA, serial twofold dilutions of
concentrated culture supernatant were coated directly onto microtitration plates. The coated proteins were detected using a 1 : 1 mixture
of anti-LLO mAbs SE1 and SE2 at a final dilution of 1/1000. The ELISA
was revealed with anti-mouse immunoglobulin peroxidase conjugate
at a final dilution of 1/1000.
Protein sequencing. EGDDhly expressing wild-type LLO and
EGDDhly expressing LLO-Cliv were grown overnight with agitation
at 37 ˚C in RPMI 140 synthetic medium containing glucose (3 %
final concentration). After centrifugation, cell-free supernatants
were filtered through 0?22 mm pore size Millipore filters and further
concentrated with Ultrafree columns with a cutoff of 30 kDa
(Millipore). Denatured proteins were separated by SDS-PAGE and
then transferred electrophoretically onto PVDF membranes
(Millipore). Protein bands were visualized by staining the membrane
with amido black. To identify the protein bands corresponding to
LLO, a part of the membrane was cut and tested like a classical
Western blot with anti-LLO antibody. For N-terminal sequencing,
the protein bands detected by the antibody were cut from the PVDF
membrane and sequenced on an Applied Biosystems Procise
Sequencer (J. d’Alayer, Laboratoire de Séquençage des Protéines,
Institut Pasteur, Paris, France).
Haemolysis. Haemolytic phenotype was visualized by spreading
bacteria onto horse- or sheep-blood agar plates with Columbia base
medium (BioMérieux). Haemolytic activity of bacterial culture
supernatant was measured by lysis of horse or sheep erythrocytes as
described by Jones & Portnoy (1994). The values corresponding to
the reciprocal of the dilution of culture supernatant required to lyse
50 % of horse erythrocytes were used to compare the haemolytic
activities in the different supernatants.
Infection of mice and virulence assays. The virulence of the
Fig. 1. Amino acid sequences of the N-terminal portion of
mature wild-type (top) and mutant (bottom) LLO. A horizontal
bar indicates the SS and its cleavage site is represented by a
black triangle. The four residues constituting the cleavage site
of LLOwt were replaced, in LLO-Cliv, by residues serine and
arginine (SR, in bold). The numbers above the sequence indicate the amino acid position (preprotein numbering).
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strain expressing LLO-Cliv was estimated by the lethal dose 50 %
(LD50) using the probit method. Specific-pathogen-free, 6–8-weekold female Swiss mice (Janvier, Le Genest St Isle, France) were used.
Bacteria were grown for 18 h in BHI broth, centrifuged, washed
once, appropriately diluted in 0?15 M NaCl, and inoculated intravenously via the lateral tail vein (0?5 ml). Groups of five mice were
challenged with various doses of bacteria (109, 108, 107 or 106 bacteria per mouse), and mortality was observed for 10 days. The assay
was carried out on animals pre-treated with spectinomycin (1 mg
per mouse per day) in order to overcome in vivo instability of the
recombinant plasmids (Dubail et al., 2000).
Culture of cell lines and microscopic analyses
Cell cultures and infections. Bone-marrow-derived macrophages from
BALB/c mice were cultured as described previously (De Chastellier &
Berche, 1994), and then infected as follows. Overnight-grown bacteria
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Microbiology 149
Signal sequence cleavage site of LLO and virulence
were diluted in cell culture medium to give a bacterium : macrophage
ratio of 5 : 1. Bacteria were allowed to adhere onto cells by incubation
on ice for 15 min, then to enter cells by placing cells at 37 ˚C for
15 min. After thorough washings, infected cells were refed with fresh
medium.
Human hepatocellular carcinoma cell line HepG-2 (ATCC HB 8065)
was obtained from S. Dramsi and P. Cossart, Institut Pasteur, Paris,
France. HepG-2 cells were cultured using RPMI 1640 medium
supplemented with 10 % fetal calf serum at 37 ˚C under a 5 % CO2
atmosphere, seeded at 26104 cells per culture dish. Cells were infected
48–72 h after seeding with overnight-grown bacteria, at a ratio of
50 : 1 for 1 h at 37 ˚C. As for macrophages, non-ingested bacteria
were removed by washing, and infected cells were fed with fresh
culture medium.
Processing for confocal microscopy. At selected intervals after
infection, cells were fixed and double fluorescence staining for
F-actin and bacteria was performed as described previously (Lety
et al., 2001), using phalloidin coupled to Alexa 488 (Molecular
Probes) and a rabbit anti-Listeria polyclonal antibody revealed with
anti-IgG coupled to Alexa 546 (Molecular Probes). Images were
scanned on a Zeiss LSM 510 confocal microscope.
Processing for electron microscopy. At selected intervals follow-
ing infection (between time zero and 8 h post-incubation), cells
were fixed and processed as described previously (De Chastellier &
Berche, 1994). Thin sections were stained with 2 % uranyl acetate
and lead citrate.
RESULTS AND DISCUSSION
Secretion and activity of the mutant protein
LLO-Cliv
The deletion of residues EAKD does not alter LLO
secretion and haemolytic activity. Residues 23 to 26
(EAKD) of LLO were deleted by site-directed mutagenesis
and the mutant protein was expressed in EGDDhly (see
Methods for details). As shown by Western blotting with
a polyclonal anti-LLO antibody (Fig. 2a), the mutant protein, designated LLO-Cliv, was secreted in comparable
amounts in the culture supernatant of recipient EGDDhly.
We quantified by ELISA with anti-LLO mAb SE2 that the
amount of secreted LLO-Cliv corresponded to 90 % that
of wild-type LLO (LLOwt) (Fig. 2b). The amounts of
LLO still associated in the envelope fraction from sonicated bacteria were also estimated by Western blotting:
they were identical for the two LLO proteins (Fig. 2c),
confirming the normal secretion of LLO-Cliv.
The mutant strain showed a clear haemolytic phenotype
on horse blood agar plates, similar to that of the LLOexpressing strain (not shown). Moreover, the haemolytic
activity recorded in the culture supernatant of the mutant
was identical to that of the wild-type construct (Fig. 2d).
The SS of LLO-Cliv is processed between residues
A27 and S28. N-terminal sequence analysis of the wild-
type LLO protein secreted by L. monocytogenes strain
EGD-e (serovar 1/2a) showed that the first five residues
of the mature protein were KDASA, indicating that the
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cleavage of the SS occurred between A24 and K25. This
result is in perfect agreement with the specificity of type 1
Spases (with A at position 21) and corresponds to the SS
prediction made by the SignalP algorithm trained on
Gram-positive bacteria (accessible at http://www.cbs.dtu.dk/
services/SignalP/), with a cleavage between EA and KD
(Fig. 1). Notably, the N-terminal sequence analysis of
mature LLO secreted by a strain of L. monocytogenes serovar 4b, reported previously (Kreft et al., 1989), indicated
that SS cleavage occurred between residues K25 and D26
of LLO (i.e. one residue downstream of that in LLO
from EGD).
The N-terminal sequence analysis of LLO-Cliv secreted by
EGDDhly indicated that the cleavage occurred between
residues A27 and S28 (wild-type preprotein numbering,
Fig. 1). Thus, a new SS cleavage site was created in the
mutant protein three residues downstream of the natural
cleavage site (deleted). Notably, the SignalP program also
predicted a cleavage between residues RA and SA for the
mutant protein. This alternative cleavage site respects the
rule of specificity of type I Spases, with an A residue at
position 21 and an S at position 23 (see Fig. 1).
The modification of the SS cleavage site of LLO
alters phagosomal escape and impairs bacterial
virulence
Phagosomal escape is moderately reduced. Bacterial
infection and intracellular multiplication was studied
in bone-marrow-derived macrophages from BALB/c mice
as described previously (Lety et al., 2001), at a bacterium : macrophage ratio of 5 : 1 for confocal and electron
microscopy analyses. The intracellular fate of EGDDhly
expressing the LLO mutant was examined by confocal
microscopy, after double staining with an anti-Listeria
antibody and with beta-phalloidin to visualize the F-actin
(Lety et al., 2001). Bacterial uptake was monitored up to
5 h after reinfection (Fig. 3). Images were scanned on a
Zeiss LSM 510 confocal microscope. Immunofluorescence
microscopy analyses showed that phagosomal escape and
actin polymerization were affected in the mutant. After
4 h, many actin comets were visible with the strain
expressing LLOwt while almost none were detected with
LLO-Cliv (Fig. 3). We monitored quantitatively the capacity of the mutant strain to promote the disruption of the
phagosomal membrane and to multiply intracellularly, by
calculating the percentage of phagosomal escape (i.e. the
number of bacteria surrounded by polymerized actin/total
number of bacteria in cells), and the number of infected
cells containing bacteria surrounded by polymerized actin
(determined on an average of at least 50 cells). After 2 h,
phagosomal escape of the mutant LLO-Cliv represented
about 65 % that of LLOwt and the percentage of cells in
which actin polymerization was visible was also reduced,
to only about 40 % of that observed with LLOwt (values
are the mean of two independent experiments). Electron
microscopic analyses confirmed that, after 4–5 h of
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M.-A. Lety and others
Fig. 2. Expression and activity of LLO-Cliv. (a) Western blot of culture supernatants. Identical amounts of each culture
supernatant were loaded onto 10 % SDS-polyacrylamide gel. Proteins were transferred electrophoretically onto nitrocellulose.
Membranes were incubated with rabbit anti-LLO polyclonal antibody (at a final dilution of 1/1000). (b) ELISA. Serial twofold
dilutions of concentrated culture supernatant were coated directly onto microtitration plates. The coated proteins were
detected using anti-LLO mAb SE2 at a final dilution of 1/1000. Dil on the abscissa represents the reciprocal of the dilution of
concentrated supernatant coated (1=non-diluted sample). %, LLO-Cliv; e, LLOwt. A standard curve was obtained with a
preparation of purified LLO of known concentration coated in the same conditions (inset). (c) Western blot of envelope
fraction. The bacterial pellets were resuspended in cold water and disrupted using a Fastprep apparatus. Loading in each well
corresponds to 100 ml of bacterial culture adjusted to an OD600 of 1. (d) Haemolysis. The haemolytic activity of culture
supernatant from exponentially grown bacteria at 37 ˚C in BHI medium was measured as described in Methods. Serial twofold
dilutions were tested on horse erythrocytes at pH 6?6. Abscissa, reciprocal of the dilution of supernatant; ordinate, percentage
haemolysis (the maximal OD450 value was taken as 100 %). %, LLO-Cliv; e, LLOwt.
infection, most cells were lysed and the bacteria were
surrounded by a meshwork of polymerized actin, with
bacteria expressing LLOwt as well as with those expressing
LLO-Cliv (not shown).
Comparable results were obtained with the human hepatocellular carcinoma cell line HepG-2 (ATCC HB 8065).
After 4 h, while 70 % of bacteria expressing LLOwt had
escaped from the phagosomes, only 28 % of bacteria
expressing the LLO-Cliv mutant had escaped. Thus, in
both professional phagocytic and non-phagocytic cells, the
LLO-Cliv mutant showed a reduced capacity to escape from
phagosomes.
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Intracytosolic protein stability is not affected. Although
the intracytosolic half-life of LLO can not be determined
accurately because the host cells are killed during infection, it is generally believed that the amount of LLO
present in the cytosol is, at least in part, controlled
by host-cell-mediated degradation (Pamer et al., 1997;
Villanueva et al., 1995; and references therein). Different
types of motifs have been identified which mark proteins
as short-half-life molecules for proteolytic degradation.
For example, PEST motifs (Rechsteiner & Rogers, 1996)
are thought to target eukaryotic proteins for phosphorylation and/or degradation by the proteasome. Although a
putative PEST-like motif has been identified close to the
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Signal sequence cleavage site of LLO and virulence
Fig. 3. Kinetics of infection of bone-marrowderived macrophages. Bone-marrow-derived
macrophages from BALB/c mice were infected
(five bacteria per cell) with EGDDhly expressing LLOwt (WT) or LLO-Cliv (Cliv) at t0 and
after 2, 4 and 5 h of re-incubation. F-actin was
stained with phalloidin (green) and bacteria
were labelled with anti-Listeria antibodies
(red). Bar, 10 mm.
N-terminus of mature LLO (Decatur & Portnoy, 2000;
Lety et al., 2001), we very recently demonstrated that the
susceptibility of LLO to intracellular proteolytic degradation was not related to the presence of a high PEST score
sequence (Lety et al., 2002). Furthermore, we showed that
sequence alterations immediately downstream of this
region strongly impaired phagosomal escape and bacterial
virulence. Another degradation signal often mentioned in
the literature as conferring metabolic instability is the
presence of a destabilizing N-terminal residue (the N-end
rule: Varshavsy, 1996).
The fact that the N-terminus of mature LLO-Cliv was different from that of LLOwt (SAF instead of KDA) prompted
us to compare the intracytosolic half-life of LLO-Cliv with
that of the wild-type protein. Immunoprecipitations were
performed on metabolically labelled infected bone-marrowderived macrophages with anti-LLO mAbs, as described
previously (Lety et al., 2001). Briefly, monolayers of bonemarrow-derived macrophages from BALB/c mice were
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infected at a bacterium : macrophage ratio of 10 : 1. After
30 min, monolayers were washed and re-incubated for 1 h
or 4 h (under these conditions, infected cells contained 5–10
bacteria per cell, as estimated by immunofluorescence; not
shown). The medium was then replaced with methioninefree RPMI minimal medium containing 10 % dialysed fetal
calf serum and 225 mg cycloheximide ml21. After 30 min,
monolayers were pulse-labelled for 1 h with 100 mCi
(3?7 MBq) [35S]methionine. Cells were lysed and the lysates,
prepared as described, were incubated with anti-LLO
mAbs (a 1 : 1 mixture of mAbs SE1 and SE2). As shown
in Fig. 4, LLOwt was specifically immunoprecipitated by
the anti-LLO mAbs. The amounts of LLO detected by the
PhosphorImager were quantified with the ImageQuant
software (Molecular Dynamics). LLOwt was still detected,
although in lower amounts, after 4 h of infection (35 % of
the value measured at 1 h). With LLO-Cliv, one major band
was also detected after 1 h, and the mutant protein was still
clearly detected after 4 h of infection (67 % of the value
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M.-A. Lety and others
(Faculté Necker-Enfants Malades, Paris, France). This work was
supported by CNRS, INSERM, Université Paris V and the EEC (BMH4 CT 960659).
REFERENCES
Fig. 4. Immunoprecipitation of LLO from L. monocytogenesinfected macrophages. Proteins were metabolically labelled with
[35S]methionine during growth in bone marrow macrophages
and LLO was immunoprecipitated with anti-LLO mAb. Each
lane corresponds to a monolayer (approx. 36106 cells). LLOwt
and LLO-Cliv were immunoprecipitated after 1 h or 4 h of
infection. The gel was scanned with a Molecular Dynamics
PhosphorImager. The autoradiograph shown corresponds to a
72 h exposure.
measured at 1 h). As compared to LLOwt, the intensity of
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LLOwt were identical (LLO-Cliv 109 % that of LLOwt) after
4 h. This assay thus demonstrated that the nature of the
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