Identification of a PEST-like motif in listeriolysin O ... · Lety et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1124–1139. EGDDhly expressing LLOwt under the

Molecular Microbiology (2001) 39(5), 1124±1139

Identification of a PEST-like motif in listeriolysin Orequired for phagosomal escape and for virulence inListeria monocytogenes

Marie-Annick Lety, Claude Frehel, Iharilalao Dubail,

Jean-Luc Beretti, Samer Kayal, Patrick Berche and

Alain Charbit*

INSERM U-411, CHU Necker-Enfants Malades,

156 rue de Vaugirard, 75730 Paris Cedex 15, France.

Summary

The hly-encoded listeriolysin O (LLO) is a major

virulence factor secreted by the intracellular patho-

gen Listeria monocytogenes, which plays a crucial

role in the escape of bacteria from the phagosomal

compartment. Here, we identify a putative PEST

sequence close to the N-terminus of LLO and focus

on the role of this motif in the biological activities of

LLO. Two LLO variants were constructed: a deletion

mutant protein, lacking the 19 residues comprising

this sequence (residues 32±50), and a recombinant

protein of wild-type size, in which all the P, E, S or T

residues within this motif have been substituted. The

two mutant proteins were fully haemolytic and were

secreted in culture supernatants of L. monocyto-

genes in quantities comparable with that of the wild-

type protein. Strikingly, both mutants failed to restore

virulence to a hly-negative strain in vivo. In vitro

assays showed that L. monocytogenes expressing

the LLO deletion mutant was strongly impaired in its

ability to escape from the phagosomal vacuole and,

subsequently, to divide in the cytosol of infected

cells. This work reveals for the first time that the

N-terminal portion of LLO plays an important role

in the development of the infectious process of

L. monocytogenes.

Introduction

Listeria monocytogenes is a Gram-positive bacterium

widespread in nature and responsible for sporadic severe

infections in humans and other animal species (Berche,

1995 and references therein). This pathogen is a facul-

tative intracellular microorganism, capable of invading a

wide variety of eukaryotic cells (Gaillard et al., 1987;

1996; Kuhn and Goebel, 1989; Dramsi et al., 1995),

including endothelial cells (Drevets et al., 1995) and

macrophages (Mackaness, 1962). Each step of the intra-

cellular parasitism by L. monocytogenes is dependent

upon the production of virulence factors (Sheehan et al.,

1994). The major virulence genes (hly, plcA, plcB, mpl,

actA, inlA and inlB) are clustered into two distinct loci on

the chromosome and are controlled by a single pleiotropic

regulatory activator, PrfA, which is required for virulence

(Leimeister-Wachter et al., 1990; Chakraborty et al.,

1992; Renzoni et al., 1999).

Among these virulence factors, 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 at its

N-terminus a 25-residue-long typical signal sequence

(Mengaud et al., 1988). The protein is secreted in the

culture supernatant as a monomer (Geoffroy et al., 1989).

A highly conserved undecapeptide sequence motif near

the C-terminus of LLO has been shown to be essential for

haemolytic activity and is thought to be required for

membrane binding (Michel et al., 1990), leading to the

idea that this region binds cholesterol. However, the

distinction between the membrane binding site of LLO and

the cholesterol binding site remained unclear, as pre-

incubation of LLO with cholesterol inhibits haemolytic

activity but does not alter membrane binding (Jacobs

et al., 1998). LLO belongs to the family of thiol-activated

pore-forming cytolysins (for a review, see Bayley, 1997).

Models have been proposed to explain the mechanisms

of pore formation by cytolysins, based essentially on

data from toxins secreted by extracellular bacteria

(Rossjohn et al., 1997; Palmer et al., 1998; Gilbert et al.,

1999; Shatursky et al., 1999). However, the molecular

mechanism of LLO-dependent phagosomal escape of

intracellular L. monocytogenes remains unknown.

Earlier studies by Portnoy and coworkers (Jones

and Portnoy, 1994; Jones et al., 1996) have shown that,

when PFO was expressed in L. monocytogenes in a

hly-negative mutant, it conferred a haemolytic pheno-

type to the strain, allowed bacterial escape from the host

Q 2001 Blackwell Science Ltd

Accepted 9 November, 2000. *For correspondence. E-mail [email protected]; Tel. (133) 1 40 61 53 76; Fax (133) 1 40 61 55 92.

cell vacuole and intracytoplasmic growth in vitro, but failed

to restore virulence in vivo. Two main reasons were

evoked to account for the lack of functional comple-

mentation and for the cytotoxicity observed upon expres-

sion of PFO in place of LLO: the longer half-life of PFO in

the host cell cytosol and its pH-independent cytolytic

activity (Geoffroy et al., 1989; Portnoy et al., 1992; Jones

et al., 1996). The intracytosolic half-life of LLO could not

be determined accurately because the host cells are killed

during infection. However, indirect studies have indicated

that LLO was probably rapidly degraded in the cytosol

of infected cells in a proteosome-dependent manner

(Villanueva et al., 1995), suggesting that the amount of

LLO present in the cytosol was controlled, at least in part,

by host cell-mediated degradation. The fact that an

internal peptide of LLO (LLO 91±99) has been found

in association with major histocompatibility class I

molecules (Pamer et al., 1997 and references therein)

and represents a dominant CTL epitope of L. monocyto-

genes (Vijh et al., 1999) further supports this notion of

cytosolic LLO degradation.

Proteins with short intracellular half-lives have been

shown to contain one or more regions rich in proline (P),

glutamic acid (E), serine (S) and threonine (T), thus called

PEST (Rogers et al., 1986; Rechsteiner and Rogers,

1996; Meraro et al., 1999). These observations prompted

us to search for the presence of a PEST sequence in LLO.

Using the PEST-FIND program developed by Rechsteiner

and Rogers (1996), a PEST-like motif was identified close

to the N-terminus of mature LLO protein. We address

here the role of this region in the biological activities of

LLO by creating a deletion and a substitution. The

expression and activity of the two mutated LLO proteins

and the effects of the mutations on the virulence of

L. monocytogenes were studied in vitro and in vivo. Our

data demonstrate that this proximal region of LLO,

although not necessary for haemolytic activity, is essential

for bacterial virulence.

Results

LLO contains a potential PEST sequence at its

N-terminus

The amino acid sequence of LLO was submitted to a

search using the PEST-FIND program (Rechsteiner and

Rogers, 1996). By definition, a score above zero denotes

a possible PEST region, and a value of 5 or higher defines

a probable PEST sequence. We identified one PEST-like

sequence, bracketed by two positively charged residues

(K) presenting a score around 5 (4.71), in the N-terminal

part of the mature LLO protein (Fig. 1). It is worth noting

that the sequence identified contains a short tandemly

duplicated central motif, PPASP, with one P overlap (see

Discussion). As LLO shares significant similarities with the

other members of the thiol-activated cytolysin family, we

also looked for the presence of putative PEST-like motifs

in these proteins. One very high scoring PEST sequence

was identified in the same region of seeligeriolysin,

the 530-residue-long cytolysin from Listeria seeligeri

(with a score of 15.4 for amino acid sequence 32±51).

In streptolysin O, the 571-residue-long cytolysin from

Streptococcus pyogenes, two consecutive potential PEST

sequences were identified in the same region of the

protein (with scores of 12 and 14.7 for amino acid

sequences 35±51 and 51±61 respectively). In contrast,

no PEST-like sequence could be identified in ivanolysin

(the 528-residue-long cytolysin from Listeria ivanovii),

pneumolysin (the 471-residue-long cytolysin from Strepto-

coccus pneumoniae) or perfringolysin (the 500-residue-

long cytolysin from Clostridium perfringens). As illustrated

in Fig. 1A, in the alignment of LLO with PFO (one of the

smallest members of the cytolysin family), the region of

the LLO motif corresponds to the region of the signal

sequence of PFO.

Modifications in the PEST-like region of LLO alter

bacterial virulence by preventing phagosomal escape

Two LLO variants were constructed: a deletion mutant

protein lacking the putative PEST sequence (named LLO-

DPest for simplification), and a recombinant protein of

wild-type size, in which all the P, E, S or T residues within

this motif have been substituted (named LLO-P-like). Two

complementary approaches were used to study the in vivo

and in vitro effects of the mutations created: we first used a

plasmid-based expression system and then confirmed the

results obtained using a chromosomally based system.

These two approaches will be presented successively

below.

Properties of the LLO mutants (plasmid-encoded con-

structs). We first constructed an LLO deletion mutant by

site-directed mutagenesis lacking 19 residues in the

proximal portion of the molecule (deletion of residues

32±50, preprotein numbering; Fig. 1B). The deleted

sequence was substituted with an XbaI restriction site

encoding a serine and a threonine residue. Then, from

this construct, a recombinant gene encoding a protein

of wild-type size (i.e. 529 residues) was created by

subcloning a double-stranded oligonucleotide encoding a

17-residue-long peptide into the XbaI site (for details, see

Experimental procedures). In the recombinant sequence,

all the P, E, S and T residues were substituted by A, G, N

or R residues (Fig. 1B). The two recombinant genes were

carried on pAT28, a Gram-positive/Gram-negative shuttle

`vector (Trieu-Cuot et al., 1990), and the recombinant

plasmids were transferred into EGDDhly by electroporation.

Role of the PEST motif in LLO 1125

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1124±1139

Fig. 1. Constructions.A. Alignment of LLO with PFO. Similar residues are shaded in light grey, identical residues are boxed. The predicted signal sequencecleavage sites of the two proteins are indicated by triangles. The PEST sequence in LLO is boxed and shaded in dark grey (PEST-FIND score of4.71).B. Sequence of the mutated proteins. Top: DNA and amino acid sequences of the LLO-D-Pest mutant. The created XbaI site is underlined,and the corresponding residues are in bold. The numbers above the sequence indicate the amino acid position). Bottom: DNA and amino acidsequences of the LLO-P-like mutant. The XbaI cohesive ends at each extremity of the inserted oligonucleotide are indicated (XbaI and cos/XbaI respectively). A unique NdeI site was also introduced in the middle of the inserted sequence (underlined) in order to facilitate thescreening of the recombinant clones. The alignment of the amino acid sequence of the initial PEST region with the mutated sequence isboxed below; the conserved residues are shaded in light grey.

1126 M.-A. Lety et al.


EGDDhly expressing LLOwt under the same conditions

was used as a positive control (Dubail et al., 2000).

The two plasmid-encoded LLO mutant proteins were

expressed and efficiently secreted into the culture super-

natant of EGDDhly, as shown by Western blot using

polyclonal and monoclonal (Erdenlig et al., 1999) anti-LLO

antibodies (Fig. 2A±C). The two mutant proteins were

normally recognized by the polyclonal serum (Fig. 2A),

and no major degradation product could be detected after

preincubation of the proteins for 4 h at 378C, indicating

that the mutations in the PEST region had not drastically

altered the stability of the protein. Using monoclonal

antibody (mAb) SE1, several additional bands of lower

molecular weight were clearly detected with LLOwt but not

with the two mutant proteins (Fig. 2A). The intensity of

these bands increased slightly upon preincubation of the

protein at 378C, suggesting that LLOwt might be more

unstable than the two mutated proteins. However, mAb

SE1 appeared to react more strongly with LLOwt than

with the two mutant proteins, and the absence of visible

degradation product in the mutant preparations might thus

only reflect a poorer recognition by the antibody. With the

second monoclonal antibody (mAb SE 2; Fig. 2C), no

major difference in detection and stability could be

observed between LLOwt and the two mutant proteins.

The amount of LLOwt, LLO-DPest and LLO-P-like

secreted by EGDDhly was measured by enzyme-linked

immunosorbent assay (ELISA) on concentrated super-

natants using mAb SE2. The total protein concentration of

each preparation, estimated by the Bradford colorimetric

method, was comparable for the three preparations (see

Experimental procedures). The concentration of LLO in

these preparations was determined by direct coating of

serial dilutions onto microtitration plates. Anti-LLO mAb

SE2 was chosen for detection in this assay in order

to minimize non-specific cross-reactions and because it

reacted normally with the LLO mutant proteins in Western

blotting. The assay showed that the three proteins were

present in almost identical amounts in the culture super-

natant of EGDDhly (Fig. 3), indicating that the modifi-

cations in the proximal portion of LLO had not affected

protein secretion.

Although EGDDhly alone is non-haemolytic on horse

blood agar plates, EGDDhly expressing LLOwt, LLO-DPest

or LLO-P-like showed clear and comparable haemolytic

phenotypes (not shown). The haemolytic activities of culture

supernatants were measured on horse erythrocytes at

different pHs (see Experimental procedures). As shown in

Fig. 4, at pH 6.6, the haemolytic activity in the super-

natant from the plasmid-encoded LLO-DPest mutant was

fourfold higher than that of the plasmid-encoded LLOwt

control (see Experimental procedures). In agreement with

previous observations (Mengaud et al., 1988), a higher

haemolytic activity was recorded for the LLOwt-expressing

Fig. 2. Western blot analyses. Identical amounts of each proteinpreparation (expressed from EGDDhly) were loaded onto 10%SDS±polyacrylamide gel (3 mg of concentrated supernatant in10 ml). WT, LLOwt; DPest, LLO-DPest; P-like, LLO-P-like. Proteinswere transferred electrophoretically onto nitrocellulose and detectedwith anti-LLO antibodies. Membranes were incubated with eitherrabbit polyclonal antibody (A) or mouse monoclonal SE1 (B) andSE2 (C). The numbers to the left of the figures correspond toapparent molecular weights (in kDa). T0, without preincubation; 4 h,after 4 h of preincubation at 378C.



strain at pH 5.6 than at pH 7.4. We found that the activity

of the LLO-DPest-expressing strain was also higher at

pH 5.6 than at pH 7.4 and remained two- to fourfold

higher than that of LLOwt at both pHs. The activity

detected in the supernatant of EGDDhly expressing LLO-

P-like was almost identical to that of EGDDhly expressing

LLO-DPest (not shown).

As noted above, a common property of all thiol-

activated toxins is that preincubation with small amounts

of cholesterol inhibits haemolytic activity (Jacobs et al.,

1998). We therefore tested whether the haemolytic

activity of concentrated supernatant containing the two

mutant LLO proteins could also be inhibited after

preincubation with purified cholesterol. Identical amounts

of each protein preparation (from 5 mg to 1 mg) were

preincubated with 1 mg of cholesterol at 378C for 30 min.

As shown in Fig. 5, the cytolytic activity of the three

concentrated supernatants tested (LLOwt, LLO-DPest,

LLO-P-like) was completely inhibited upon preincubation

with cholesterol, indicating a similar sensitivity of the three

proteins to cholesterol.

We compared the virulence of EGDDhly transformants

expressing the two LLO mutant proteins by following the

kinetics of bacterial growth in the organs of intravenously

infected mice, compared with that of EGDDhly expressing

PFOwt in the same conditions (pAT28 encoded, under

phly promoter control). As shown previously, EGDDhly is

totally avirulent, and virulence is fully restored in this strain

complemented with plasmid-encoded LLOwt: at a dose

of 107 bacteria per mouse, bacteria rapidly grow in the

spleen of infected mice, ultimately resulting in death of

the mice within 2±3 days (Dubail et al., 2000). As shown

in Fig. 6, at a dose of 107 bacteria per mouse, EGDDhly

expressing either LLO-DPest or LLO-P-like were totally

avirulent and were cleared from the spleen of the infected

animals 2 days after inoculation. Unexpectedly, with both

constructs, when injected with a 10-fold higher dose (108

bacteria per mouse), all mice died within 15±30 min after

500

400

300

200

100

0

OD

492

0

µl LLO

LLOwt

LLO-∆Pest

LLO-P-Like

0.1 1 10

500

400

300

200

100

0

OD

492

0.10µg LLOwt1 10

Fig. 3. LLO detection by ELISA. The amounts of LLO proteinexpressed by L. monocytogenes (EGDDhly) were determined in theconcentrated culture supernatants by ELISA. Serial twofold dilutionsof each preparation (starting from 50 ml) were coated directly ontomicrotitration plaques. The coated proteins were detected usinganti-LLO mAb SE2 (final dilution of 1:1000). The ELISA wasrevealed with anti-mouse immunoglobulin peroxidase conjugate(final dilution of 1:1000). The standard curve obtained with purifiedLLOwt is shown in the lower part of the figure. ml in abscissa,amounts (in ml) of extract coated; OD492 in ordinate, the opticaldensity recorded at 492 nm.

Fig. 4. Haemolytic activities of the mutated proteins. Haemolyticactivity of culture supernatants from exponentially grown bacteria at378C in BHI medium (resuspended at a final OD600 of 0.6). Serialtwofold dilutions of each supernatant (starting from 40 ml of non-diluted sample) were tested on HRBCs, essentially as describedpreviously (Jones and Portnoy, 1994), at pH 6.6. Abscissa,reciprocal of the dilution of supernatant; ordinate, percentage ofhaemolysis (the maximal OD450 value recorded was taken as100%). ch/LLOwt, EGDwt; ch/LLO-DPest, chromosomally encodedLLO-DPest (EGDDhly background); pl/LLO-DPest, plasmid-encodedLLO-DPest; pl/LLOwt, plasmid-encoded LLOwt. The haemolyticactivity of chromosomally encoded LLO-DPest was almostindistinguishable from that of EGDwt. In contrast, the plasmid-encoded LLO-DPest mutant was approximately fourfold morehaemolytic than the plasmid-encoded LLOwt.



inoculation with convulsions, revealing a toxic effect of the

recombinant bacterium. The same toxicity was observed

when bacteria were washed before injection. In contrast,

injection of the corresponding supernatant had no effect,

indicating that toxicity was most probably caused by the

presence of the mutated protein at the surface of the

bacteria. With EGDDhly expressing LLOwt, at a dose of

108 bacteria (washed cells), 10% of the mice died within

24 h and the remaining 90% within 48 h, indicating the

development of an infectious process. However, at a

dose of 109 bacteria per mouse, all the mice died within

15±30 min after inoculation with convulsions (at this dose,

EGDDhly is not toxic). These results therefore indicate

that the LLO-DPest protein is < 10-fold more toxic than

LLOwt. The physiological mechanism underlying this

higher toxicity is unknown.

In agreement with previous observations (Jones and

Portnoy, 1994), we found that EGDDhly expressing PFO

from a plasmid-borne gene was highly haemolytic (the

activity recorded in the supernatant from the PFO-

expressing strain was fourfold higher than that recorded

in the LLO-DPest-expressing strain; not shown) but still

totally avirulent. Like the LLO-DPest-expressing strain, at

a dose of 107 bacteria per mouse, the PFO-expressing

strain was also completely eliminated from the spleens by

2 days after infection (Fig. 6) and, at higher doses, the

strain was also toxic to the mice. Toxicity was less

pronounced than with the LLO-DPest construct: at a dose

of 108 bacteria per mouse, 65% of the mice died within

24 h after infection and, at a dose of 109 bacteria per

mouse, 100% of the mice died 2±3 h after inoculation.

These results indicate that toxicity is not strictly correlated

with the haemolytic activity of the protein.

As no major difference was observed between the

two LLO variants (LLO-DPest and -P-like) in vivo, we

focused on the LLO-DPest mutant in the following in vitro

analyses. Bacterial infection and multiplication was

studied in bone marrow-derived macrophages.

The intracellular fate of EGDDhly expressing either

LLOwt (Fig. 7A±C) or LLO-DPest (Fig. 7D±F) was

examined by confocal microscopy after double staining

with an anti-Listeria antibody and with beta-phalloidin to

visualize the F-actin (Gaillot et al., 2000). EGDDhly alone

was used as a negative control (Fig. 7G±I). The early

bacterial uptake (up to 4 h; not shown) was similar for

the three strains. In contrast, after 6 h of infection, only

bacteria expressing LLOwt had multiplied, and many

bacteria were visible inside the cytoplasm surrounding the

nucleus of infected cells (Fig. 7B). Bacteria were found

Fig. 5. Inhibition of haemolytic activity bycholesterol. The assays were performed onserial twofold dilutions of concentratedsupernatants. Cytolysis of erythrocytes isvisualized by a uniform grey colour of the well(corresponding to haemoglobin release). Adark spot at the bottom of the wellcorresponds to sedimented intacterythrocytes. The black triangles indicate thelast dilution of protein preparation yieldinghaemolysis. WT, LLOwt expressed inEGDDhly; DPest, LLO-DPest expressedin EGDDhly; P-like, LLO-P-like expressed inEGDDhly. First lane, 1 mg of LLO or LLOmutant protein preincubated with 1 ml of asolution of cholesterol at 1 mg ml21 in ethanolat 378C for 30 min; a complete inhibition ofhaemolysis is observed. Second lane, 1 mg ofLLO or LLO mutant protein preincubated with1 ml of pure ethanol; no inhibition is observed.Third lane, no preincubation.

Fig. 6. In vivo kinetics of infection. The kinetics of bacterial growthwas followed in organs of mice infected with EGDDhly expressingLLO-DPest, LLO-P-like or PFOwt (plasmid-borne genes). EGDDhlyexpressing LLOwt was used as a positive control. Mice wereinoculated with 2 � 107 bacteria. Bacterial survival was followed inthe spleens of infected animals over a 3 day period. Only the miceinfected with EGDDhly expressing LLOwt died 3±4 days afterinfection. ²Death. Bacteria expressing LLO-DPest, LLO-P-like orPFOwt were completely eliminated from the spleens 3 days afterinfection.



associated with tails of polymerized actin (coloured in

green). After 8 h, the number of bacteria expressing

LLOwt had diminished, and cells had started to lyse, but

most bacteria were still surrounded by polymerized actin

(and for a few of them with tails of polymerized actin were

still visible; Fig. 7C). Bacteria expressing LLO-DPest

wereunable to multiply and did not show any actin

polymerization (Fig. 7E and F). After 8 h, a few bacteria

were still visible, but no actin polymerization was

detectable (Fig. 7F). As expected, the EGDDhly control

strain was also unable to multiply and to polymerize actin

(Fig. 7G±I).

In order to determine whether the mutant bacteria

unable to polymerize actin were still trapped in the host

cell phagosomes or were free in the cytosol, electron

microscopic analyses were then carried out. In agreement

Fig. 7. Kinetics of infection of bone marrow-derived macrophages (plasmid-encoded protein). Macrophages were infected (20 bacteria cell21)with EGDDhly expressing LLOwt or LLO-DPest. F-actin was stained with phalloidin (green), and bacteria were labelled with anti-Listeriaantibodies (red). EGDDhly alone was used as a negative control. The kinetics of infection was followed by confocal microscopy over an 8 hperiod. EGDDhly expressing LLOwt at T0 (A), after 6 h (B) and after 8 h (C) of reincubation; EGDDhly expressing LLO-DPest at T0 (D), after6 h (E) and after 8 h (F) of reincubation; EGDDhly alone at T0 (G), after 6 h (H) and after 8 h (I) of reincubation. After 6 h, bacteria expressingLLOwt multiplied rapidly in the cytoplasm of infected cells (B). Intense actin polymerization (bacteria coloured yellow±green) and cometformation (in green) were visible. After 8 h, cells started to lyse (C). In contrast, with bacteria expressing LLO-DPest (D±F) as well as withEGDDhly alone (G±I), bacterial multiplication was strongly impaired (E and F; H and I), and no actin polymerization could be detected after6 h or 8 h (all the bacteria detected are coloured in red). Size bar (bottom right of I) � 10 mm.



with the confocal analyses, after 6 h of infection, only the

bacteria expressing LLOwt had multiplied and were visible

inside the cytoplasm of infected cells (Fig. 8A and B).

Most cells were lysed (Fig. 8A), and the bacteria were

surrounded by polymerized actin (Fig. 8B). In contrast,

bacteria expressing LLO-DPest (Fig. 8C and D) did not

multiply, and only a few of the cells were lysed (Fig. 8C).

The vacuolar membranes were still visible around the

bacteria, and no actin polymerization could be visualized

(Fig. 8D), demonstrating that the bacteria were still

trapped inside phagosomes. We found that a significant

proportion of the vacuolar membranes showed a slightly

altered morphology (in 40% of the phagosomes), but

this partial damage was insufficient to allow bacterial

escape and subsequent actin polymerization (Fig. 8D). As

expected, the negative control strain EGDDhly (Fig. 8E

and F) was also unable to multiply and did not induce cell

lysis (Fig. 8E). Most of the bacteria were found still

trapped within the host cell vacuoles and did not show any

actin polymerization (Fig. 8F).

We then tested whether LLO-DPest produced by

infected macrophages (including the proteins still trapped

inside phagosomes) could be immunoprecipitated from

whole cells. Immunoprecipitations were performed on

metabolically labelled infected bone marrow-derived

macrophages (for details, see Experimental procedures).

Detection of intracellular LLO-DPest was technically

difficult because bacteria expressing the mutated protein

did not replicate intracellularly, which was limiting for the

recovery of the LLO protein produced. In order to optimize

the amount of LLO protein to be detected, monolayers

were infected with a high dose of bacteria (i.e. 75±100

bacteria cell21) and for only a short period of time (1 h)

to avoid cytotoxicity. Under these optimized conditions,

cells infected with EGDDhly expressing either LLOwt or

LLO-DPest contained approximately the same amount of

bacteria (<50), as estimated by immunofluorescence,

and actin polymerization started to be visible around

several bacteria in cells infected with the LLOwt construct

(data not shown). Cells infected with EGDDhly alone were

used as a negative control.

As shown in Fig. 9, wild-type LLO was efficiently and

specifically immunoprecipitated from infected macro-

phages by the anti-LLO mAb (lane WT). In the control

lane (Neg), corresponding to macrophages infected with

EGDDhly, there was no non-specific cross-reaction. In

agreement with previous observations (Moors et al.,

1999), several smaller species (open triangle) likely to

correspond to proteolytic degradation products of LLOwt

were detected below the full-length 58 kDa protein (black

triangle). The LLO-DPest construct (lane DPest) could

also be specifically immunoprecipitated from infected cells.

In contrast to LLOwt, only a single band was detected,

indicating the absence of major degradation products. The

intensity of the band of LLO-DPest was, however, sig-

nificantly lower than that of LLOwt, suggesting a lower

amount of protein produced (the fact that the mutated

protein comprises three methionine residues instead of

four in LLOwt prevents direct comparison).

Chromosomal integration of the gene encoding LLO-

DPest. The above results were further supported using a

construction in which LLO-DPest was expressed from a

gene integrated into the chromosome of EGDDhly.

The gene encoding the mutated protein was integrated

in the chromosome of EGDDhly, using the integrative

vector pAT113 (Trieu-Cuot et al., 1991). The insertion site

of the recombinant plasmid in the chromosome of Listeria

was determined by DNA sequencing (for details, see

Experimental procedures). One insertion that occurred in

an open reading frame (ORF) encoding a hypothetical

protein of unknown function, which did not share any

significant similarity (, 25% identity) with other proteins in

the databases, was chosen for further analyses. The

growth curve of this mutant in brain±heart infusion (BHI)

medium (at 378C with agitation) was identical to that of

EGDwt, confirming that the insertion had occurred in a

silent portion of the Listeria chromosome.

The haemolytic activity of the mutant strain was

determined and compared with that of EGDwt. In contrast

to the plasmid-encoded construct, the haemolytic activity

of the chromosomally encoded LLO-DPest mutant was

almost identical to that of EGDwt at pH 6.6 (Fig. 4), as well

as at pH 5.6 and 7.4 (not shown). This result confirmed that

the deletion of residues 32±50 of LLO had no major

deleterious effect on the overall structure of the protein.

We compared the amounts of LLO-DPest produced

from the chromosome or plasmid-encoded gene by ELISA

on concentrated culture supernatant with anti-LLO mAb

SE2 (not shown). We found that the plasmid-encoded

construct produced < three- to fourfold more LLO protein

than the chromosomal one. This difference of expression

is in good agreement with the higher haemolytic activity of

the plasmid-encoded construct and is likely to account for

its higher toxicity in vivo.

The intracellular growth of the chromosomally encoded

LLO-DPest was followed in bone marrow-derived macro-

phages by confocal (Fig. 10) and electron microscopy

(Fig. 10). The results obtained supported those obtained

with the plasmid-encoded construct. Confocal microscopy

showed that the mutant strain was strongly impaired in its

ability to multiply within infected cells (Fig. 10C and D).

After 8 h of reinfection, actin polymerization was still

visible in the wild-type strain (EGDwt, Fig. 10B), whereas

with the LLO-DPest mutant, bacteria had not multiplied

(Fig. 10D) and, in most cases, no actin polymerization

was visible. Electron microscopy (Fig. 10) confirmed that,

after 6 h, the wild-type strain (EGDwt) did multiply;





bacteria were found surrounded by polymerized actin

(Fig. 10A), and infected host cells were partially lysed. In

contrast, the mutant strain did not multiply (Fig. 10B), and

most of the bacteria remained trapped within unlysed

vacuoles (Fig. 10C).

Finally, we tested the attenuation of the LLO-DPest-

expressing strain in vivo. With EGDwt, at a dose of

106 bacteria per mouse, all the mice died within

4 days after infection (and at a 10-fold higher dose,

mice died 24±48 h after infection). The chromosomally

encoded LLO-DPest mutant was avirulent at doses of

106 and 107 bacteria per mouse (100% survival after

6 days of infection). However, in contrast to the

plasmid-encoded construction, the administration of a

dose of 108 bacteria per mouse was lethal within 2±

3 days after infection. The chromosomally encoded

mutant thus appeared to be significantly attenuated

but not totally avirulent (the LD50 of the mutant was

< 107.5, approximately two logs higher than the

parental strain).

Discussion

In the present work, we have identified a putative PEST

sequence in the N-terminal portion of the protein and

focused on the role of this motif in the biological activities

of LLO. Our data demonstrated that this region of LLO,

although not required for haemolytic activity, was involved

in phagosomal escape of bacteria in infected cells and

was critical for bacterial virulence.

The PEST-like region of LLO is important for bacterial

virulence

The two LLO variants, LLO-DPest and LLO-P-like,

expressed from plasmid-borne genes in EGDDhly, were

secreted in amounts comparable with that of the wild-type

protein by L. monocytogenes and were highly haemolytic.

However, in vivo, these strains were totally avirulent, and

bacteria were very rapidly eliminated from the organs of

infectedanimals. Inoculation ofhigher doses did not promote

virulence but, instead, had a toxic effect. It is worth recalling

that the toxicity of purified LLO has already been documen-

ted, and it has been shown that a dose of 1 mg of toxin

provokes death within 1±2 min with convulsions and

opisthotonos (Geoffroy et al., 1987). The precise physiolo-

gical mechanism by which these cytolysins are toxic remains

tobe elucidated. Invitro assaysdemonstrated that the loss of

virulence of the strain expressing LLO-DPest was caused by

a defect in phagosomal escape, preventing bacterial

replication in the cytoplasm of infected cells.

The data obtained with LLO-DPest expressed from a

single chromosomally integrated gene fully supported

those obtained with the plasmid-encoded construct. The

haemolytic activity of the chromosomally encoded LLO-

DPest mutant was fourfold lower than that of the plasmid-

encoded construct, in good agreement with a three- to

fourfold lower level of protein expression. This activity was

identical to that of wild-type L. monocytogenes at all pHs

tested, indicating that the modifications in the proximal

portion of LLO were perfectly tolerated and had no

deleterious effects on the overall structure of the protein.

In vitro assays confirmed that escape from the host cell

Fig. 9. Immunoprecipitation of LLO from L. monocytogenes-infectedbone marrow-derived macrophages. Bacterial proteins weremetabolically labelled with [35S]-methionine during growth in bonemarrow macrophages and immunoprecipitated with monoclonalanti-LLO antibody (see Experimental procedures). Each lanecorresponds to a monolayer (<3 � 106 cells). Cells were infectedwith EGDDhly expressing either LLO-DPest (DPest) or LLOwt (WT)(at a ratio of < 75 bacteria cell21). As a negative control, cells wereinfected in the same conditions with EGDDhly alone (Neg.). The gelwas scanned with a Molecular Dynamics PhosphorImager, and theresulting image was analysed with IMAGEQUANT software (MolecularDynamics). The autoradiograph shown corresponds to a 72 hexposure. The black triangle points to the full-length protein; theopen triangle points to one of the degradation products. The numbersto the left correspond to molecular weight markers (in kDa).

Fig. 8. Thin section of macrophages 6 h after infection (plasmid-encoded protein). Thin sections of macrophages infected with EGDDhlyexpressing LLOwt (A and B), LLO-DPest (C and D) or EGDDhly alone (E and F). In the overviews (A, C and E), the size bar (bottom right ofE) � 2.5 mm. Bacteria are indicated by an arrow. In the enlarged views (B, D and F), the size bar (bottom right of F) � 0.2 mm. As shown in(A), the bacteria expressing LLOwt multiplied actively and were found free in the cytoplasm of lysed cells (A), surrounded by a meshwork ofactin filaments (B). In contrast, the bacteria expressing LLO-DPest did not multiply (C) and were still entrapped in the phagosomes (D).EGDDhly alone was also unable to multiply (E) and to escape from the phagosomes (F). The black arrowheads point to the intact phagosomalmembranes entrapping the bacteria (D and F) and the black arrows (D) to slightly damaged portions of a phagosomal membrane. The emptyarrowhead (B) points to the polymerized actin around a cytosolic bacterium expressing LLOwt.



vacuole, although not totally abolished, was strongly

impaired in the LLO-DPest mutant. It is important to

note that the properties of the LLO-DPest mutant were

completely different from those of PFO. Indeed, Portnoy

and coworkers have shown that PFO, expressed in an

LLO-negative strain, promoted escape from the host cell

vacuole and subsequent growth in the cytosol of infected

cells in vitro (Jones and Portnoy, 1994).

Possible roles for the PEST motif

LLO is a virulence factor adapted for the intracellular life

cycle of L. monocytogenes. Its expression has been

shown to be transcriptionally and post-transcriptionally

regulated in infected cells (Villanueva et al., 1995; Bubert

et al., 1999; Moors et al., 1999). As LLO is a pore-forming

toxin, its rapid degradation in the cytosol may ensure that

the host cytoplasmic membrane remains intact during

infection, allowing L. monocytogenes to multiply and

propagate to adjacent cells. Statistical evidence indicates

that PEST regions in proteins serve as proteolytic signals

(Rechsteiner and Rogers, 1996; Meraro et al., 1999),

some of which are constitutive, whereas others are

conditional (Borges and Gomes, 2000 and references

Fig. 10. Infection with the chromosomally encoded LLO-DPest mutant.1. Confocal microscopy. Macrophages were infected (20 bacteria cell21) with either EGDwt (positive control, A and B) or with thechromosomally encoded LLO-DPest mutant (C and D). The infection was followed over a 6 h period. After 6 h, EGDwt multiplied rapidly in thecytoplasm of infected cells, and actin polymerization was visible (B). In contrast, with the bacteria expressing LLO-DPest, multiplication wasseverely impaired, and no actin polymerization could be detected (D). Size bar (bottom right of D) � 5 mm.2. Electron microscopy. Macrophages were infected (50 bacteria cell21) with EGDwt (A) or with the chromosomally encoded LLO-DPestmutant (B and C). With EGDwt, after 6 h, actin polymerization was visible around cytosolic bacteria (A). In contrast, with the bacteriaexpressing LLO-DPest, multiplication was severely impaired (B, overview), and bacteria remained trapped in the phagosomes (C). Sizebars � 0.25 mm (A); 3.5 mm (B); 0.18 mm (C).



therein). Therefore, our initial assumption was that

disruption of the putative PEST motif of LLO, provided

that it had no major effect on the overall structure of LLO,

would generate a mutant protein more resistant to

cytosolic proteolytic degradation. We found that disruption

of the putative PEST motif of LLO was critical for an

earlier step in the infectious process, i.e. phagosomal

escape. One possible explanation to account for these

results is that the PEST motif in LLO constitutes a

recognition site for an as yet unknown ligand and that this

interaction would be required to disrupt the phagosomal

membrane. The fact that the LLO variant lacking the

PEST motif was still able to lyse red blood cells efficiently

also suggests that this hypothetical interaction is specific

for the phagosomal membrane. Supporting this hypo-

thesis, it has been shown recently that PEST motifs can

also serve as binding sites for ligands other than

proteases (Marchal et al., 1998; Perkel and Atchison,

1998; Roth and Davis, 2000). For example, proline-rich

stretches are known to participate in protein±protein

interactions (Kay et al., 2000), and the five proline

residues within the PEST motif of LLO (Fig. 1) could

constitute such a binding site. Of interest, mutants

lacking the phosphatidylinositol-specific phospholipase C

(PI-PLC, plcA encoded) have been shown to escape less

efficiently from the primary vacuoles than wild-type

L. monocytogenes (Camilli et al., 1993), and a co-

operative action between LLO and PI-PLC has been

proposed for lysing the intracellular vacuoles (reviewed by

Songer, 1997). Moreover, it has been reported that LLO

and PI-PLC could act synergistically to induce cell

signalling (Sibelius et al., 1996; Wadsworth and Goldfine,

1999). It is thus tempting to speculate that the PEST

region of LLO could be involved in the interaction with the

PI-PLC. This hypothesis will have to be addressed

experimentally. An alternative explanation would be that

the PEST motif serves as a proteolytic signal inside the

vacuole, thus allowing LLO cleavage and subsequent

interaction with the phagosomal membrane.

The fact that a significant proportion of phagosomal

membranes was altered when macrophages were

infected with the LLO-DPest construct suggested leaki-

ness and a possible cytoplasmic release of the mutated

protein. This observation prompted us to test whether

LLO-DPest produced by infected macrophages could be

immunoprecipitated from metabolically labelled infected

bone marrow-derived macrophages. In our assay, wild-

type LLO was efficiently immunoprecipitated from infected

macrophages, and several smaller species likely to

correspond to proteolytic degradation products were

also detected. The LLO-DPest construct could also be

immunoprecipitated from infected cells as a single band,

suggesting that the removal of the PEST sequence

rendered the protein more resistant to proteolytic

degradation. The increased resistance of the LLO-DPest

construct might indeed account for its increased cytotoxi-

city observed in vivo (bacteria expressing EGDDhly-

expressing LLO-DPest appeared to be < 10-fold more

toxic than wild-type LLO). These data are compatible with

the notion that the PEST sequence would be responsible

for the cytosolic degradation of LLO: after phagosomal

escape, the PEST motif might serve as a proteolytic

signal for cytosolic proteases by an as yet unknown

mechanism. In this respect, it is worth mentioning that

PEST-like sequences have recently been shown to serve

as ubiquitination signals in eukaryotic cells, which serve a

broad spectrum of cellular processes, including protea-

some-dependent and -independent protein degradation,

but also non-proteolytic functions (Roth and Davis, 2000;

Shih et al., 2000).

In conclusion, the present work shows that the PEST

motif close to the N-terminus of LLO plays a critical role in

phagosomal escape. The precise molecular mechanisms

by which this sequence participates in the opening of the

vacuole remains to be elucidated.

Experimental procedures

Sequence analyses

Similarity searches were performed with BLAST (Altschul et al.,1997). Sequence alignments were produced with CLUSTALW

(available on the internet site http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl). The PEST sequencein LLO was identified with PEST-FIND (available on the inter-net site http://bioweb.pasteur.fr/seqanal/interfaces/pestfind-simple.html; Rogers et al., 1986; Rechsteiner and Rogers,1996).

Bacterial strains and culture conditions

The wild-type virulent strain of L. monocytogenes EGD(denoted EGDwt) belongs to the serovar 1/2a (Gaillard et al.,1986). EGDDhly is a derivative of EGDwt (serotype 1/2a) thatcontains an in frame chromosomal deletion of 1080 bp in thehly gene (Guzman et al., 1995). EGDDhly was transformedwith the different recombinant plasmids by electroporation asdescribed previously (Park and Stewart, 1990). EGDDhlyexpressing LLOwt from a pAT28-borne hly gene (under thephly promoter) has been described previously (Dubail et al.,2000).

Bacteria were grown in BHI broth (Difco Laboratories) at378C without antibiotics, except for the pAT28-transformedstrains, which were grown on BHI broth containing 60 mg ml21

spectinomycin (Spc).

Construction of the recombinant cytolysins

Chromosomal DNA, plasmid isolation, restriction enzymeanalyses and amplification by polymerase chain reaction



(PCR) were performed according to standard protocols(Sambrook et al., 1989; Ausubel et al., 1990).

Deletion of residues 32±50 of LLO (LLO-DPest). Themutation DPest was generated by in vitro site-directedmutagenesis on M13mp18-phly hly. A 1.6 kb DNA fragmentcomprising the hly gene (encoding LLOwt) preceded by itspromoter phly was obtained from plasmid pAT28-phly hly(Dubail et al., 2000) by digestion with restriction enzymesBamH1 and SalI. The 1.6 kb DNA fragment was clonedbetween the BamH1 and SalI sites of the replicative formof M13mp18. Site-directed mutagenesis was performedaccording to standard procedures (Sambrook et al., 1989).The oligonucleotide used for the site-directed deletionmutagenesis was 5 0-CTTTTCGATTGGCGTTCTAGAATTGAATGCAGATGC-3 0. Upon mutagenesis, the deleted portion(19 residues) was substituted by two residues, serine andthreonine (S and R), encoded by a unique XbaI restrictionsite (underlined) (Fig. 1). The mutation was then transferredonto plasmid pAT28 by restriction enzyme excision from thereplicative form (double digestion with BamH1 and SalI) andinsertion into similarly restricted pAT28, yielding plasmidpAT28-phly hlyDPest.

Cloning of a DNA fragment encoding a 17-amino-acid-longpeptide lacking P, E, S or T residues (LLO-P-like). Thetwo complementary synthetic oligonucleotides encoding thepeptide were: coding strand 5 0-CTAGAAATGCAATTGCGGCAATGGCAGGTGGAGCATATGGTGGAGCAAATG-3 0; comple-mentary strand 5 0-CTAGCATTT-GCTCCACCATATGCTCCACCTGCCATTGCCGCAATTGCATTT-3 0. Plasmid DNA frompAT28-phly hlyDPest was linearized by digestion with XbaI.Insertion of the double-stranded oligonucleotide into the XbaIsite of the linearized plasmid was performed as describedpreviously (Charbit et al., 2000). Upon insertion of the double-stranded oligonucleotide in the correct orientation into the XbaIsite of hly DPest, an XbaI site is recreated only at the 5 0 end ofthe inserted sequence (Fig. 1).

Chromosomal insertion. We used the integrative vectorpAT113 (Trieu-Cuot et al., 1991). Integration of pAT113 inthe chromosome of Gram-positive bacteria requires thepresence (in trans) of the transposon-encoded integrase inthe recipient. Therefore, EGDDhly was first transformed withplasmid pAT145, carrying the transposon-encoded integraseInt-Tn. Then, the BamHI±SalI fragment of plasmid pAT28-phly hlyDPest was cloned into the BamHI±SalI sites of vectorpAT113. The resulting plasmid, pAT113-phly hlyDPest, wasfinally transferred into EGDDhly/pAT145 by electroporationand selection on erythromycin (8 mg ml21). Several trans-formants corresponding to single chromosomal insertionswere conserved. For one of them (clone 10), the site ofinsertion in the chromosome of Listeria was mappedprecisely by sequence analysis. For that, we used thestrategy described previously by Trieu-Cuot et al. (1991).Briefly, 5 mg of chromosomal DNA was digested with Bgl II(the pAT113-phly hlyDPest vector is devoid of a BglII site).After self-ligation, DNA was introduced by transformation inEscherichia coli, and clones were selected on kanamycin(50 mg ml21) and erythromycin (8 mg ml21). The sequenceof the chromosomal region adjacent to the att sites of the

recombinant plasmid was determined by PCR sequencingusing primer SeqR (5 0-CGTGAAGTATCTTCCTACAGT-3 0)with the automated ABI-Prism 310 sequencer (Perkin-Elmer,Applied Biosystems). The DNA sequence obtained waslaunched in the complete 2 900 000 bp Listeria genomedatabase (BLASTN search). This analysis revealed that therecombinant transposon had inserted in the upstream portionof a putative ORF (accession number 966.1) encoding ahypothetical protein of unknown function in a region of thechromosome that did not participate in any known biologicalactivity of L. monocytogenes.

Protein preparation and analyses

The proteins were prepared from supernatants of theEGDDhly strain transformed with the different pAT28derivatives. For each mutant, 25 ml of an 8 h culture at378C in BHI±Spc broth was added to 500 ml of RPMI-1640minimal medium containing glucose (3% final concentration).The suspension was grown overnight with agitation at 378C(under these conditions, the titre of the cultures was of2±2.5 � 108 ml21). After centrifugation, cell-free super-natants were filtered through a 0.22 mm pore size Milliporefilter. The filtered supernatants were first concentrated into15 ml by tangential flux through miniplate YM30 (Millipore)with a cut-off of 30 kDa and then into a final volume of 1.5 mlby centrifugation through ultrafree Biomax units (with cut-offsof 30 kDa). The total protein concentration of the preparationwas estimated by the Bradford colorimetric method. Com-parable values were recorded for the three preparations(<0.6 mg ml 21 ^ 10%). The concentration of LLO in thesepreparations was then determined in ELISA by direct coatingof serial dilutions of each preparation and detection with anti-LLO mAb SE2 (at a final dilution of 1:1000). Purified LLOwtprotein, prepared as described previously (Kayal et al.,1999), was used as a standard in the assay. The concen-tration of LLO was almost identical in the three proteinpreparations (LLOwt, LLODPest and LLO-P-like). The LLOmutant proteins were also identified by Western blot analysis,as described by Charbit et al. (2000). Five microlitres of eachconcentrated culture supernatant was loaded per well in10 ml of sample buffer.

Anti-Listeria and anti-LLO antibodies

The polyclonal anti-LLO serum, raised in rabbits againstdenatured LLO (Geoffroy et al., 1989), was used in Westernblotting at a final dilution of 1:2000. Two monoclonal anti-LLOantibodies (SE1 and SE2, kindly provided by Dr Ainsworth),raised in mice after the injection of concentrated L. mono-cytogenes extracellular proteins (Erdenlig et al., 1999), wereused in Western blotting at final dilutions of 1:500 and 1:2000respectively. The polyclonal rabbit anti-Listeria antibody(J. Rocourt, Institut Pasteur, Paris, France) was used inimmunofluorescence at a final dilution of 1:200.

Detection of LLO produced in macrophages

LLO-DPest or LLOwt produced by EGDDhly (plasmid-encoded constructs) were detected by immunoprecipitation



with anti-LLO mAb (SE2). EGDDhly alone was used as anegative control. The assay was adapted from the method ofdetection of LLO in infected macrophages described pre-viously (Brundage et al., 1993; Moors et al., 1999). Briefly,monolayers of bone marrow-derived macrophages, seededinto 60 mm dishes, were infected at a bacterium±macrophageratio of 75±100:1 (i.e. 2±3 � 108 bacteria per < 3 � 106 cells).After 30 min, monolayers were washed and reincubated for 1 h.Then, the medium was replaced with methionine-free RPMIminimal medium containing 10% dialysed fetal calf serum and225 mg of cycloheximide ml21. After 30 min, monolayers werepulse-labelled for 1 h with 100 mCi of [35S]-methionine (in vivocellular labelling grade at 1000 Ci mmol21; Amersham). Cellswere then lysed with 1 ml of RIPA buffer (150 mM NaCl,50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% NonIdet P-40, 0.5%deoxycholate, 0.1% SDS and containing a protease inhibitorcocktail; Complete protease inhibitor cocktail, BoehringerMannheim). The lysate was further solubilized by passagethrough a needle, subjected to centrifugation in a microcen-trifuge for 10 min at 48C, after which the supernatant wasrecovered.

Ten microlitres of anti-LLO mAb (SE2) were added to eachsupernatant fluid, and the sample was rocked for 30 min at48C. Protein A±agarose (50 ml of a suspension at 3 mg ml21;Boehringer Mannheim) was then added, and the suspensionwas incubated for 2 h at 48C. The immunoprecipitatedmaterial (processed as recommended by the manufacturer)was finally resuspended in 30 ml of electrophoresis samplebuffer and boiled for 5 min before SDS±PAGE.

Haemolysis

Haemolytic phenotypes were visualized by spreadingbacteria onto horse blood agar plates (BioMerieux). Haemo-lytic activity of bacterial culture supernatants was measuredby lysis of horse red blood cells (HRBCs) at various pHs, asdescribed previously (Jones and Portnoy, 1994). The valuescorresponding to the reciprocal of the dilution of culturesupernatant required to lyse 50% of HRBCs were usedto compare the haemolytic activities in the differentsupernatants.

Inhibition of haemolytic activity by cholesterol was per-formed essentially as described previously (Geoffroy et al.,1989; Jacobs et al., 1998) on concentrated protein prepara-tions. Identical amounts of each preparation (from 5 mg to1 mg) were preincubated with 1 ml of a solution of cholesterolat 1 mg ml21 in ethanol at 378C for 30 min in a final volume of200 ml. As negative controls, each preparation was incubatedwith 1 ml of ethanol.

Infection of mice and virulence assays

Six- to 8-week-old pathogen-free ICR female Swiss mice(Janvier) were used for the kinetics of infection. Groups offive mice were inoculated intravenously in the lateral tail vein.Mice were inoculated with 108, 107 or 106 bacteria, andbacterial survival was followed in the spleen over a 3 dayperiod. Organs were removed aseptically and homogenizedseparately in 0.15 M NaCl. Bacterial numbers in organhomogenates were determined at various intervals on BHI

plates containing appropriate antibiotics. The assays werecarried out on animals pretreated with Spc (1 mg of Spc permouse twice a day) in order to overcome in vivo instability ofthe recombinant plasmids.

Infection of macrophages and microscopic analyses

Bone marrow-derived macrophages from six C57/Black micewere cultured and infected as described previously (DeChastellier and Berche, 1994) at a bacterium±macrophageratio of 10±20:1 for confocal microscopy analyses and of50:1 for electron microscopy analyses.

Processing for confocal microscopy. Double fluorescencelabelling of F-actin and bacteria was performed as describedpreviously (Gaillot et al., 2000), using phalloidin coupled toOregon green 488 (Molecular Probes) and a rabbit anti-Listeria polyclonal antibody revealed with anti-IgG antibodycoupled to Alexa 546 (Molecular Probes). Images werescanned on a Zeiss LSM 510 confocal microscope.

Processing for electron microscopy. At selected intervalsafter infection (between time zero and 8 h of reincubation),cells were fixed for 1 h at room temperature and processedas described previously (De Chastellier and Berche, 1994).Thin sections were stained with 2% uranyl acetate and leadcitrate.

Acknowledgements

We thank Dr D. Portnoy for the gift of plasmid pD 1868, andDr T. Chakraborty for the gift of EGDDhly. We are grateful toDr A. J. Ainsworth for providing us with the anti-LLOmonoclonal antibodies SE1 and SE2. The wish to thankYann Goureau (CHU Necker-Enfants Malades, Paris,France) for expert technical work in confocal microscopy,and Colin Tinsley for careful reading of the manuscript. Wealso thank the European Listeria Genome Consortium forgenerous access to the Listeria genome sequence. This workwas supported by CNRS, INSERM, UniversiteÂ Paris V andthe EEC (BMH-4 CT 960659).

Note added in proof

Upon acceptance of this manuscript, which was submitted on27 July 2000, Decatur and Portnoy (Science, November2000, 290: 992) reported similar results on the critical role ofthe PEST motif in LLO on bacterial virulence. However, theauthors did not observe effects on phagosomal escape.Thus, it is possible that the efficiency of LLO-dependentphagosomal escape varies between cell lines and betweenstrains of L. monocytogenes.

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Identification of a PEST-like motif in listeriolysin O ... · Lety et al. Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1124–1139. EGDDhly expressing LLOwt under the