Cellular Microbiology (2000) 2(6), 465±476

The invasion protein InlB from Listeria monocytogenesactivates PLC-g1 downstream from PI 3-kinase

HeÂleÁne Bierne,1² Shaynoor Dramsi,1² Marie-Pierre

Gratacap,2 Clotilde Randriamampita,3 Graham

Carpenter,4 Bernard Payrastre2 and Pascale

Cossart1*1Unite des Interactions BacteÂries-Cellules, 28 rue du Dr

Roux, Institut Pasteur. 75724 Paris, Cedex 15. France.2INSERM Unite 326, HoÃpital Purpan, 31059 Toulouse,

France.3Laboratoire d'Immunologie Cellulaire, CNRS URA 625,

83 Bvd de l'HoÃpital, 75013 Paris, France.4Department of Biochemistry, Vanderbilt University

School of Medecine, Nashville, Tenessee 37232-0146,

USA.

Summary

Entry of the bacterial pathogen Listeria monocyto-

genes into non-phagocytic mammalian cells is mainly

mediated by the InlB protein. Here we show that in the

human epithelial cell line HEp-2, the invasion protein

InlB activates sequentially a p85b-p110 class IA PI 3-

kinase and the phospholipase C-g1 (PLC-g1) without

detectable tyrosine phosphorylation of PLC-g1. Pur-

ified InlB stimulates association of PLC-g1 with one

or more tyrosine-phosphorylated proteins, followed

by a transient increase in intracellular inositol 1,4,5-

trisphosphate (IP3) levels and a release of intracellular

Ca21 in a PI 3-kinase-dependent manner. Infection of

HEp-2 cells with wild-type L. monocytogenes bacteria

also induces association of PLC-g1 with phosphotyr-

osyl proteins. This interaction is undetectable upon

infection with a DinlB mutant revealing an InlB

specific signal. Interestingly, pharmacological or

genetic inactivation of PLC-g1 does not significantly

affect InlB-mediated bacterial uptake, suggesting

that InlB-mediated PLC-g1 activation and calcium

mobilization are involved in post-internalization

steps.

Introduction

Listeria monocytogenes is a Gram-positive pathogenic

bacterium responsible for sporadic severe food-borne

infections in humans, especially in immunocompromised

individuals and pregnant women. The clinical features of

listeriosis ± septicaemias, meningitis and abortions ± are

due to the ability of this organism to cross the intestinal,

blood±brain and placental barriers, and to reside intra-

cellularly in most infected tissues (Cossart and Lecuit,

1998). As with several other invasive bacteria, L.

monocytogenes is able to induce its own phagocytosis

into cells that are normally non-phagocytic, where it can

grow and multiply protected from host defences (reviewed

in Dramsi and Cossart, 1998). L. monocytogenes-induced

phagocytosis occurs by a zipper-like mechanism reminis-

cent of phagocytosis in macrophages (Swanson and

Baer, 1995), a process that is initiated by intimate contact

between bacterial surface-associated proteins and host

cell receptors (Ireton and Cossart, 1997).

The invasion protein InlB mediates entry of L. mono-

cytogenes into various human cell types such as

hepatocyte-like cell lines and some fibroblasts, epithelial,

and endothelial cells lines (Dramsi et al., 1995; Ireton

et al., 1996; Greiffenberg et al., 1998; Parida et al., 1998).

The part of the protein sufficient to promote internalization

is the amino-terminal region containing leucine-rich

repeats (LRR) (Braun et al., 1999), the structure of

which has been determined recently (Marino et al., 1999).

Both purified InlB and the LRR domain can trigger zipper-

like uptake when present on the surface of the non-

invasive bacterium Listeria innocua or of latex beads

(Braun et al., 1999, 1998). InlB-mediated entry requires

actin cytoskeleton rearrangement, tyrosine phosphoryla-

tion and, in some cell types, activation of the mammalian

phosphoinositide (PI) 3-kinase p85a-p110, through asso-

ciation of the regulatory subunit p85a to several tyrosine-

phosphorylated proteins (Ireton et al., 1999).

Understanding how InlB-mediated activation of p85-

p110 induces internalization of L. monocytogenes and/or

activates other cellular processes requires the identifica-

tion of signalling molecules that act downstream of this

lipid kinase during bacterial entry. Recent reports have

established that PLC-g isozymes, either PLC-g1 (Bae

et al., 1998; Falasca et al., 1998) or PLC-g2 (Bourette

et al., 1997; Gratacap et al., 1998), can be activated

downstream from PI 3-kinase, through interactions

between their SH2 and/or PH domains with phosphatidy-

linositol-3,4,5-trisphosphate (PIP3), linking the PI 3-kinase

Q 2000 Blackwell Science Ltd

Received 3 April, 2000; revised 5 May, 2000; accepted 29 May, 2000.*For correspondence. E-mail p.cossart@pasteur.fr; Tel. (133) 1 4568 88 41; Fax (133) 1 45 68 87 06. ²The first two authors contributedequally to this work.

and the PLC-g/calcium signalling pathways (Rameh et al.,

1998; Scharenberg and Kinet, 1998). PLCs play critical

roles in receptor-mediated signal transduction via the

generation of inositol 1,4,5-trisphosphate (IP3) and

diacylglycerol (DAG) after hydrolysis of phosphatidylino-

sitol-4,5-bisphosphate (PIP2) (reviewed in Rhee and Bae,

1997). IP3 interacts with a receptor on the endoplasmic

reticulum to release Ca21 from internal stores. DAG

activates a large family of Ca21/phospholipid-dependent

protein kinase C (PKC) isoenzymes. The PLC-g subfamily

is unique among PLCs in that, in contrast to other

isoforms, it is activated downstream of tyrosine kinases

and contains SH2 and SH3 domains which have the

potential to interact, respectively, with tyrosine-phos-

phorylated proteins and proline-rich proteins, including

some cytoskeletal proteins. PLC-g1 is widely expressed

and can be activated by a variety of receptor and non-

receptor tyrosine kinases, classically through tyrosine

phosphorylation (reviewed in Carpenter and Ji, 1999).

In this work, we have analysed the InlB-mediated signal

transduction cascade in the human epithelial cell line

HEp-2. We have first shown that in these cells the

invasion protein InlB stimulates a p85b-p110 PI 3-kinase.

This activation is only partially required for L. monocyto-

genes internalization, as previously observed (Ireton and

Cossart, 1997). In addition, we report that in InlB-

stimulated cells PLC-g1 is activated in the absence of

any detectable tyrosine phosphorylation, through associa-

tion with tyrosine-phosphorylated proteins and down-

stream from PI 3-kinase activation. The resulting

transient increases in IP3 levels and mobilization of

intracellular Ca21 are not required for L. monocyto-

genes-induced phagocytosis in mammalian cells, reveal-

ing additional role(s) for InlB in post-internalization events.

Results

L. monocytogenes entry into HEp-2 cells requires PI 3-

kinase activity

The invasion protein InlB mediates entry of L. monocyto-

genes into many different mammalian cell types. Our

laboratory has demonstrated that this phenomenon requires

PI 3-kinase activation in the monkey kidney cell line Veroand

the chinese hamster ovary (CHO) cell line, as cell treatment

with the PI 3-kinase inhibitor wortmannin or transfection with

the dominant negative form of p85a completely inhibit entry

(Ireton et al., 1996). However, we and others have observed

that wortmannin has a much weaker effect on L. mono-

cytogenes uptake in most human cell lines (Ireton and

Cossart, 1997; Braun et al., 1998; Greiffenberg et al., 1998;

H. Bierne and P. Cossart, unpublished results). To analyse

these differences further, we compared the effects of the two

alternative PI 3-kinase inhibitors, wortmannin and

LY294002, on L. monocytogenes invasion in Vero cells

and in the human epithelial HEp-2 cells. As shown in Fig. 1,

pretreating cells with 50 nM wortmannin decreased entry of

L. monocytogenes into HEp-2 cells to 30% of the control

values, while entry into Vero cells was completely abolished,

i.e. 1% of the control values. In contrast, 50 mM LY294002

reduced invasion to more comparable levels in the two cell

lines, i.e. 30% and 15% of the control value in HEp-2 and

Vero cells respectively (Fig. 1). These results established

that a PI 3-kinase activity is also required for InlB-mediated

uptake in HEp-2 cells.

InlB stimulates PI 3-kinase activity and association of

p85b with tyrosine-phosphorylated proteins in HEp-2 cells

The ability of InlB to stimulate PI 3-kinase activity by

measurement of the levels of lipid products has so far only

been demonstrated in the Vero cell line (Ireton et al.,

1999). Stimulation of PI 3-kinase activity was therefore

also tested in InlB-treated HEp-2 cells. Treatment of cells

for 1 min with 4.5 nM InlB (a time and concentration

previously shown to fully activate PI 3-kinase in Vero

cells) clearly stimulated synthesis of both PI(3,4)P2 and

PI(3,4,5)P3 (Fig. 2A). In contrast, the amount of PI(4,5)P2,

which is not a product of PI 3-kinase, was unaffected. This

result indicated that InlB also activates PI 3-kinase in

HEp-2 cells. Pretreatment of cells with 50 nM wortmannin

Fig. 1. Effects of PI 3-kinase inhibitors on entry of L.monocytogenes in Vero and HEp-2 cells. Vero and HEp-2 cellswere infected with L. monocytogenes strain EGD in the presence ofthe PI 3-kinase inhibitors wortmannin (1Wm) or LY294002 (1LY)or in the presence of the solvent dimethylsulphoxide (control,DMSO 0.1%). Concentration are in nM for wortmannin and mM forLY294002. The level of uptake of bacteria in the absence ofinhibitors was similar in both cell types (approximately 0.5% ofbacteria internalized at a multiplicity of infection of 50:1) and hasbeen assigned a value of 100. The level of entry in the presence ofinhibitors is given as a relative value. Values are the mean ^ SD ofthree independent experiments, repeated in duplicate.

466 H. Bierne et al.

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and 50 mM LY294002 fully inhibited the InlB-mediated

PI 3-kinase activation (Fig. 2A), suggesting that the

residual entry observed in HEp-2 cells in the presence

of these PI 3-kinase inhibitors occurred independently of

PI 3-kinase activity (see above and Discussion).

In Vero cells, InlB stimulates tyrosine phosphorylations

and activates a class IA PI 3-kinase p85a-p110 after

association of the regulatory subunit p85a with phospho-

tyrosyl proteins. These interactions can be demonstrated

by coimmunoprecipitation of p85 with antibodies to

phosphotyrosine (Ireton et al., 1996; 1999). InlB treatment

of Vero and HEp-2 cells led to the appearance of

comparable amounts of phosphotyrosyl proteins

(Fig. 2B). Interestingly, the p85 isoform detected in HEp-

2 cells phosphotyrosine immunoprecipitates was not p85a

but p85b. Experiments to detect both isoforms in total cell

extracts revealed that HEp-2 cells express only the p85b

isoform while Vero cells express both p85a and p85b

proteins (Fig. 2B and C). These findings indicate a cell-type

specific recruitment of p85 isoforms by the InlB stimulus. In

HEp-2 cells InlB is an agonist of a p85b-p110 PI 3-kinase

while in Vero cells, it stimulates the p85a-p110 isoform.

Stimulation of HEp-2 cells with InlB induces association of

PLC-g1 with phosphotyrosyl proteins but no tyrosine

phosphorylation of PLC-g1

InlB-dependent entry of L. monocytogenes into most non-

phagocytic human cell lines displays a sensitivity to

wortmannin more comparable to that observed in HEp-2

cells than in Vero cells (data not shown). Therefore, the

signal transduction events associated with InlB stimula-

tion were characterized further in HEp-2 cells. We tested

a possible involvement of PLC-g1, which has recently

been described as a downstream target of PI 3-kinase

after growth factor stimulation. Because activation of

PLC-g1 is mostly induced by tyrosine phosphorylation, the

presence of PLC-g1 was first analysed in anti-phospho-

Fig. 2. InlB stimulates PI 3-kinase activity andassociation of p85b with tyrosine-phosphorylated proteins in HEp-2 cells.A. Stimulation of PI 3-kinase activity in HEp-2cells. Cells were left untreated (U) or treatedwith 4.5 nM (300 ng ml21) purified InlB for1 min (InlB). Reactions were then stopped andthe concentrations of the phosphoinositidesPI(3,4)P2, PI(3,4,5)P3 and PI(4,5)P2 weredetermined as described in Experimentalprocedures. When indicated, cells werepretreated by the PI 3-kinase inhibitorswortmaninn 50 nM (InlB 1 Wm) or LY29400250 mM (InlB 1 lY). Values are the average oftwo to three experiments and are expressed asthe relative amount of the particularphosphoinositide, using the counts per minute(c.p.m.) in untreated control cells as thereference (value of 1).B. Recruitment of p85 isoforms with tyrosine-phosphorylated proteins in InlB-stimulatedcells. Vero (V) or HEp-2 (H) cells were leftuntreated (±) or treated with 3 nM InlB (1) for1 min. Cells were then lysed. Tyrosine-phosphorylated proteins wereimmunoprecipitated with anti-pTyr antibodies,and tyrosine-phosphorylated proteins and p85isoforms were detected using anti-pTyrantibodies (pTyr) and anti-p85 antibodies (h-p85a, polyclonal antibodies to human p85a;mab-p85b, monoclonal antibody to humanp85b isoform).C. Cell-type expression of p85 isoforms. Then,20 mg of Vero and HEp-2 cell lysates wereimmunoblotted with antibodies to different p85isoforms. (P, red ponceau staining; h-p85a,polyclonal antibodies to human p85a; mab-p85a and mab-p85b, monoclonal antibodies tohuman p85a and b isoforms).

InlB activates PLC-g1 467

Q 2000 Blackwell Science Ltd, Cellular Microbiology, 2, 465±476

tyrosine (anti-pTyr) immunoprecipitates after a time

course stimulation of HEp-2 cells with 4.5 nM InlB

(300 ng ml21). Detection of the p85b subunit of PI 3-

kinase was also performed. PLC-g1 and p85b were

rapidly and transiently detectable, their levels being

maximal at the earliest time point examined (1 min) and

declining thereafter (Fig. 3A). Maximal detection of PLC-

g1 in anti-pTyr immunoprecipitates occurred after 1 min

stimulation with 9 nM InlB (600 ng ml21; data not shown).

The presence of PLC-g1 in anti-pTyr immunoprecipitates

could result from interactions of the enzyme with

phosphotyrosine residues of key membrane-associated

proteins and/or of PLC-g1 direct tyrosine phosphorylation.

To discriminate between these two possibilities, phos-

photyrosyl proteins and PLC-g1 were immunoprecipitated

from the same amount of cellular extracts, prepared from

resting cells or cells stimulated for 1 min with InlB (9 nM)

or with EGF (17 nM; 100 ng ml21), a known agonist of

PLC-g1 (Meisenhelder et al., 1989). Immunoprecipitated

proteins were then probed with anti-pTyr and anti-PLC-g1.

The amount of PLC-g1 coimmunoprecipitated with phos-

photyrosyl proteins was equivalent in anti-pTyr immuno-

Fig. 3. InlB does not stimulate tyrosine phosphorylation of PLC-g1but induces its association with tyrosine-phosphorylated proteins.A. HEp-2 cells were left untreated (U) or stimulated with InlB (4.5 nM300 ng ml21) for various times. Cells were lysed, tyrosine-phosphorylated proteins were immunoprecipitated with anti-pTyrantibodies and PLC-g1 was detected by immunoblotting with amixture of monoclonal antibodies against PLC-g1. The PI 3-kinaseregulatory subunit p85b was detected in parallel by probing the sameamount of anti-pTyr immunoprecipitated material with a monoclonalanti-p85b antibody. The results shown are representative of threeexperiments.B. HEp-2 cells were left unstimulated (U) or stimulated with100 ng ml21 EGF (17 nM; EGF) or 600 ng ml21 InlB (9 nM; InlB) for1 min pTyr-containing proteins (left panel) and PLC-g1 (right panel)were immunoprecipitated from the same amount of cell extract withagarose-conjugated antiphosphotyrosine antibodies and polyclonalantibodies to PLC-g1 respectively. Blots were probed withmonoclonal anti-pTyr antibodies coupled to peroxidase (anti-pTyrblot) or polyclonal anti-PLC-g1 antibodies (anti-PLC-g1 blot). Theresults shown are representative of three experiments.

Fig. 4. PI 3-kinase inhibition abolish InlB-mediated association ofPLC-g1 with phosphotyrosyl proteins.A. HEp-2 cells were left untreated (±) in presence of the solventDMSO (0.1%), or pretreated with several inhibitors dissolved inDMSO and stimulated by 3 nM InlB (1) for 1 min in the presence orabsence of inhibitors. pTyr-containing proteins wereimmunoprecipitated and probed with polyclonal antibodies to PLC-g1.Inhibitors used were 50 nM wortmannin (Wm), 250 mM genistein(Gen.), 5 mM U73122 (U73122), 5 mg ml21 Cytochalasin D (Cyt.).B. HEp-2 cells were stimulated with 4.5 nM InlB for 1 min in thepresence or absence of several concentrations of wortmannin(1Wm) or LY294002 (1LY). pTyr-containing proteins wereimmunoprecipitated and blots were probed with anti-PLC-g1, anti-p85b or anti-pTyr antibodies. The results shown are representative ofthree experiments.

468 H. Bierne et al.

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precipitates of EGF and InlB-stimulated cells (Fig. 3B, left

panel). However, only stimulation with EGF led to the

detection of the tyrosine-phosphorylated PLC-g1 at

150 kDa, in anti-PLC-g1 immunoprecipitates (Fig. 3B,

right panel). Tyrosine phosphorylation of PLC-g1 was

undetectable in anti-PLC-g1 immunoprecipitates from

InlB-stimulated cells, even after shorter (30 s) or longer

(5 min and 15 min) stimulations with InlB (data not shown).

Phosphotyrosyl proteins that are likely to interact with

PLC-g1 should be detected in anti-PLC-g1 immunopreci-

pitates followed by anti-pTyr immunoblot. As expected,

EGF stimulation induced the appearance of a 180 kDa

pTyr-containing protein corresponding to the EGF recep-

tor (Fig. 3B, right panel) to which PLC-g is known to bind

(Margolis et al., 1989). In contrast, InlB stimulation lead to

the detection of phosphotyrosyl proteins in anti-PLC-g1

immunoprecipitates only after overexposure of the Wes-

tern blots. Two very faint bands of , 180 and 200 kDa

were then detected and may correspond to proteins

interacting with PLC-g1 (data not shown). Taken together

these results indicate that the presence of PLC-g1 in anti-

pTyr immunoprecipitates did not result from tyrosine

phosphorylation of PLC-g1 but from weak interaction(s)

with phosphotyrosyl proteins in response to InlB stimulation.

InlB-induced association of PLC-g1 with pTyr-containing

proteins depends on PI 3-kinase activation

Inhibition of tyrosine kinases by the drug genistein

abolished InlB-induced PLC-g1 association with tyro-

sine-containing proteins, confirming that this event

occurred downstream from InlB-mediated activation of a

tyrosine kinase (Fig. 4A). In contrast, U73122, a com-

monly used inhibitor of PLC activity, and cytochalasin D

which blocks actin polymerization, did not prevent InlB-

inducible association of PLC-g1 with tyrosine-phosphory-

lated proteins, indicating that this association occurred

upstream from PLC-g1 own activation and did not require

InlB-mediated rearrangement of the actin cytoskeleton.

Because PLC-g1 can be activated by PIP3 (Bae et al.,

1998; Falasca et al., 1998), we analysed the effect of PI 3-

kinase inhibitors after InlB stimulation. The amount of

PLC-g1 immunoprecipitated with phosphotyrosyl proteins

was greatly reduced by pretreatment of the cells with

wortmannin or LY294002, whereas the amount and

pattern of total pTyr-containing proteins immunoprecipi-

tated or the amount of p85b associated with pTyr residues

was apparently not affected by these drugs (Fig. 4B).

InlB stimulates PLC-g1 activity and induces Ca21

increase downstream from PI 3-kinase activation

InlB-inducible association of PLC-g1 with tyrosine-phos-

phorylated proteins suggested that PLC-g1 could be

activated upon InlB stimulation. PLC activity was therefore

monitored by measurements of one of its product, IP3.

Stimulation of HEp-2 cells by 9 nM InlB for 1 min caused a

2.3-fold increase in IP3 amounts, a stimulationcomparable in

magnitude to that induced by 17 nM EGF. The InlB-

mediated increase in IP3 was transient, optimal at 1 min

and returned to basal levels after 5 min after InlB stimulation.

This is in contrast to EGF, which maintained a more

sustained response (Fig. 5A). Both genistein and

LY294002 blocked InlB-stimulated IP3 production, indicating

that InlB stimulation of PLC activity was dependent on

tyrosine kinase and PI 3-kinase activities, in agreement with

the results described above (Fig. 5B). These findings

strongly suggested that InlB activates exclusively a PLC-

g1 isoform and not a PLCb isoform, as the latter is not

activatedby tyrosinephosphorylation (Rhee and Bae,1997).

We then determined whether the InlB-mediated IP3

generation could mobilize calcium. In HEp-2 resting cells,

Fig. 5. InlB stimulates IP3 production.A. HEp-2 cells were left untreated (U), ortreated 1 min or 5 min with 9 nM (600 ng ml21)purified InlB (1InlB) or with 17 nM(100 ng ml21) EGF (1EGF). Reactions werethen stopped and the cellular concentration ofIP3 was determined as described inExperimental procedures. `1InlB' values arethe average of two to three experiments andare expressed as the relative amount of IP3,using the disintegrations per min (d.p.m.) inuntreated control cells as the reference (valueof 1).B. Cells were pretreated (or not) with 250 mM ofthe tyrosine kinase inhibitor genistein or 50 mMof the PI 3-kinase inhibitor LY294002,stimulated for 1 min (or not) with 9 nM InlB(1InlB) and IP3 levels were determined, asdescribed in Experimental procedures. Theresults are average 1 SD of two to threeexperiments.

InlB activates PLC-g1 469

Q 2000 Blackwell Science Ltd, Cellular Microbiology, 2, 465±476

the intracellular free calcium concentration ([Ca21]i) was

,140 nM. Addition of 1.5 nM InlB resulted in a rapid and

transient increase in [Ca21]i, which peaked at 250 nM,

2 min after stimulation and returned to basal level after

5 min (Fig. 6A). Thus, the kinetics of calcium release

correlated with the kinetics of PLC-g1 association with

phosphotyrosyl proteins and IP3 generation. As expected,

inhibition of PI 3-kinase activity with LY294002 completely

abrogated the InlB-dependent Ca21 mobilization

response (Fig. 6A). The same results were obtained

when the experiment was performed in Ca21-free

medium, suggesting that InlB-mediated variation of

[Ca21]i resulted from a release of calcium intracellular

stores and not from an influx of Ca21 from the

extracellular medium (data not shown).

We compared the InlB- and EGF-induced Ca21

response, as EGF-induced [Ca21]i elevation is known to

result, in part from Ca21 release from internal stores and

in a larger part from Ca21 influx through the plasma

membrane by the opening of voltage-independent Ca21

channels (Moolenaar et al., 1986). Stimulation of HEp-2

cells by the two agonists was performed at concentrations

known to induce comparable increases in IP3 levels (i.e.

InlB 7.5 nM and EGF 17 nM). Significant differences in

the calcium response were observed (Fig. 6B and C). The

EGF stimulation displayed a shorter lag time (, 20 s) to

induce the calcium initial response and more amplitude

and duration of the Ca21 elevations than the InlB-

mediated response. The magnitude of the InlB-mediated

response was lower and the Ca21 oscillations were more

frequent. Taken together these results indicate that InlB

induces a Ca21 rise by mobilizing Ca21 from intracellular

stores, apparently without influx of extracellular Ca21.

PLC-g1 becomes associated with tyrosine-

phosphorylated proteins during infection with L.

monocytogenes but is not with a D inlB mutant

Because the purified InlB protein induced PLC-g1 activa-

Fig. 6. InlB stimulates Ca21 increase. Fura-2 loaded HEp-2 cellswere stimulated by addition to the extracellular medium of theindicated agonist and intracellular calcium levels were determined,as described in Experimental procedures, in the presence orabsence of 25 mM of the PI 3-kinase inhibitor LY294002. In eachexperiment, n � 15 cells. A. 1 1.5 nM InlB; B. 1 17 nM EGF; C.1 7.5 nM InlB.

Fig. 7. L. monocytogenes infection of HEp-2 cells inducesinteraction of PLC-g1 with pTyr-containing proteins in an InlB-dependent manner. Cells were left untreated or infected with wild-type L. monocytogenes EGD strain or a DinlB mutant at aMOI � 200:1, for various time. Tyrosine-phosphorylated proteinswere immunoprecipitated from equivalent amounts of cell extractsand probed with polyclonal antibodies to PLC-g1. The resultsshown are representative of three experiments.

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tion and calcium mobilization, it was important to

determine whether it could elicit a similar signal when

present on the bacterial surface during infection. The

presence of PLC-g1 in anti-pTyr immunoprecipitates was

examined after infection of HEp-2 with L. monocytogenes

or an isogenic mutant strain lacking the inlB gene (DinlB,

Dramsi et al., 1995). This mutant is still adherent but does

not enter into cells, as it does not express InlB. As shown

in Fig. 7, infection of HEp-2 cells with wild-type bacteria

allowed the detection of PLC-g1 in anti-pTyr immunopre-

cipitates after 5 min, 15 min and 30 min, whereas it was

undetectable in cells infected with the DinlB mutant

(Fig. 7B). This result strongly suggested that activation

of PLC-g1 occurs during L. monocytogenes infection and

is mediated by InlB.

InlB-mediated entry is dependent on PI 3-kinase activity

but not on PLC-g1 activity

InlB is necessary and sufficient to promote internalization

of L. monocytogenes into HEp-2 cells and several other

cell lines. Therefore, it was tempting to speculate that

InlB-triggered PLC-g1 activation and calcium signalling

played a role in InlB-mediated uptake. To directly test this

hypothesis in the absence of other virulence factors, we

made use of the non-virulent strain L. innocua expressing

the LRR domain of InlB, a region shown to be sufficient to

activate PI 3-kinase and to promote entry (Braun et al.,

1999). We also used InlB-coated latex beads (Braun et al.,

1998). As expected, PI 3-kinase inhibitors blocked the

entry of L. innocua expressing LRR region of InlB as

efficiently as that of the wild-type L. monocytogenes strain

(compare Fig. 1 and Table 1). They also inhibited uptake

of InlB-coated beads. These results indicated that

activation of PI 3-kinase is directly involved in the InlB-

mediated internalization process, in agreement with

previous results (Ireton et al., 1996; Braun et al., 1998).

The effects of the PLC inhibitor U73122, of the intracel-

lular calcium chelator BAPTA/AM (Dieter et al., 1993) and

that of calphostin C, an inhibitor of proteins which have a

DAG-binding domain (Kobayashi et al., 1989), were then

tested on InlB-mediated entry. U73122, BAPTA/AM and

calphostin C did not significantly inhibit entry of the InlB-

coated bacteria or beads (Table 1). These results

suggested that InlB-mediated PLC-g1 activation and

calcium signalling did not play a significant role in

internalization

Finally, we used a genetic approach and tested L.

monocytogenes uptake in immortalized mouse embryo

fibroblasts (MEF) derived from Plcg1 (±/±) embryos

compared with that in wild-type cells (Ji et al., 1997). In

the Plcg1 (1/1) MEF cells, entry was InlB dependent

because the DinlB mutant was not internalized in contrast

to the isogenic wild-type strain EGD (i.e. percentage of

entry , 0.001% for DinlB, compared with 0.17% for EGD).

Entry of two unrelated wild-type L. monocytogenes strains

and that of InlB-coated beads were similar in Plcg1 (±/±)

MEF and in Plcg1 (1/1) MEF (Table 2). Taken together

these results indicate that PLC-g1 is not required for the

internalization process mediated by InlB and is probably

implicated in downstream cellular events.

Discussion

In the present study, we have analysed the signalling

Table 1. Effects of PI 3-kinase/PLC/Ca21 signalling inhibitors on InlB-mediated uptake.

L. innocua(LRR-IR-spa)

InlB-coatedbeads

Control (DMSO 0.1%) 100a 100b

Wortmannin 10 nM 61 ^ 15 ±Wortmannin 50 nM 21 ^ 2 53 ^ 13LY294002 10 mM 37 ^ 5 ±LY294002 50 mM 27 ^ 5 58 ^ 14U73122 2.5 mM 95 ^ 21 ±U73122 5 mM 82 ^ 5 89 ^ 10Calphostin C 1 mM 96 ^ 17 102 ^ 15BAPTA/AM 25 mM ± 95BAPTA/AM 250 mM 80 ^ 18 80 ^ 8BAPTA/AM 250 mM 1 EGTA 10 mM ± 79 ^ 10

HEp-2 cells were preincubated for 20 min with each drug at theindicated concentrations followed by infection with L. innocuaexpressing the LLR region of InlB or with InlB-coated latex beads,for 1 h. Invasion frequencies were determined using the gentamicinsurvival assay when infected with bacteria or using a quantificationassay of intracellular beads by immunofluorescence when using InlB-coated beads, as described in Experimental procedures. The level ofentry in absence of inhibitor and presence of the solvent DMSO 0.1%(control) has been artificially reported as 100. The level of entry in thepresence of inhibitors is given as a relative value.Bold letters indicatesignificant drops in invasion frequencies. Results are the mean ^ SDof three to five independent experiments, done in duplicate.a. The value of 100 corresponds to 0.5±2% of bacteria internalizedfor a multiplicity of infection of 50 bacteria per cell.b. The value of 100 corresponds to 60±70% of beads internalized for5±10 beads associated per cell, in 100±200 cells analysed.

Table 2 Invasion efficiency of L. monocytogenes and InlB-coatedbeads in MEF cell lines.

Plcg1 (1/1) Plcg1 (±/±)

L. monocytogenes (EGD) 0.17 ^ 0.03 0.29 ^ 0.05L. monocytogenes (L028) 0.48 ^ 0.10 0.56 ^ 0.10InlB-coated beadsa 92 ^ 19 93 ^ 9

Invasion assays in MEF cells were performed in 24-well plates usingthe gentamicin survival assay when infected with wild-type L.monocytogenes strains EGD or L028 strains, or quantifying intracel-lular beads by immunofluorescence when using InlB-coated beads,as described in Experimental procedures. Plcg1 (±/±) and Plcg1 (1/1) MEF are described in Ji et al., (1997).Per cent invasion values are expressed as the mean 1 SD of threeindependent experiments, carried out in duplicate.a. For 5±10 beads associated per cells in 100±160 cells analysed.

InlB activates PLC-g1 471

Q 2000 Blackwell Science Ltd, Cellular Microbiology, 2, 465±476

events mediated by the invasion protein InlB from L.

monocytogenes in the human epithelial cell line HEp-2.

We have demonstrated that InlB stimulates sequentially

host protein tyrosine phosphorylation, PI 3-kinase and

PLC-g1 activities, resulting in the generation of IP3 and

Ca21 release from intracellular stores. The mechanism of

PLC-g1 activation by this bacterial agonist appears to be

original because it does not require PLC-g1 tyrosine

phosphorylation.

Our laboratory has recently established that gC1qR

acts as a host cell receptor for InlB but that other

receptors may be involved in transducing signals from the

cell surface to the cell machinery (Braun et al., 2000).

How InlB triggers the signal transduction cascade

described here is presently unknown, but it is clear that

these signals are reminiscent of those induced by growth

factors, hormones and Fcg receptors (reviewed in Van

der Geer et al., 1994). We have previously reported that

the p85-p110 PI 3-kinase becomes associated with

tyrosine-phosphorylated adaptor proteins upon InlB sti-

mulation, via the p85a subunit (Ireton et al., 1999). Here

we show that the nature of the p85 isoform recruited is

dependent on the cell type, being a p85a in Vero cells and

a p85b in HEp-2 cells. Several reports suggest that

different p85 isoforms may have non-redundant functions,

may be regulated differently and may associate with

different subsets of intracellular proteins (Reif et al., 1993;

Hartley et al., 1995; Shepherd et al., 1997). The relevance

of these differences is unclear, but understanding their

nature and their significance may highlight cell specific

differences in the InlB signalling pathway.

In any case, our results suggest that activation of p85-

p110 PI 3-kinase by the InlB protein is a general

phenomenon. This activation is only partially required for

InlB-mediated induced phagocytosis in HEp-2 cells, as

30% of bacteria are still internalized in conditions where

PI 3-kinase activity is totally inhibited. The relative

importance of PI 3-kinase activation in Listeria uptake by

the InlB-mediated pathway has previously been ques-

tioned. While the PI 3-kinase inhibitor wortmannin strongly

inhibits Listeria uptake in Vero and CHO cells (Ireton et al.,

1996), it has weaker inhibitory effects on InlB-mediated

entry in several other cell lines, such as HEp-2, HeLa

(Ireton and Cossart, 1997; Braun et al., 1998), HBMEC

(Greiffenberg et al., 1998) and HepG2 (H. Bierne and P.

Cossart, unpublished results). Such differences may be

linked to variations in cell sensitivity to inhibitors and/or to

bypass of the PI 3-kinase pathway. Activation of PI 3-

kinase by the InlB protein could also be involved in

functions other than phagocytosis. Identification of the

possible downstream targets of PI 3-kinase in InlB

signalling should help to elucidate such phenomenons.

In this work we identify PLC-g1 as one of these targets.

Interestingly, InlB activates PLC-g1 by a mechanism which

does not depend on PLC-g1 tyrosine phosphorylation but

on PLC-g1 association with tyrosine-phosphorylated

Fig. 8. Model of InlB-mediated activation of PI 3-kinase, PLC-g1 and Ca21 mobilization. PI 3-kinase is recruited to activated InlB-receptors bybinding to specific phosphotyrosine residues on adaptor proteins via the SH2 domain of the p85 subunit. The p110 catalytic subunit of PI 3-kinase generates PIP3, which recruits PLC-g1 to the plasma membrane via binding to the SH2 and/or PH domain(s) of PLC-g1. Theseinteractions facilitate the association of SH2 domains of PLC-g1 to phosphotyrosine residues of either activated InlB receptors or adaptorproteins. Binding of PLC-g1 to PIP3 and to phosphotyrosyl proteins stabilizes the enzyme in close proximity to its substrate, PIP2, andenhances hydrolysis of PIP2 to IP3 and DAG, which lead to the release of Ca21 from intracellular stores and activation of PKC.

472 H. Bierne et al.

Q 2000 Blackwell Science Ltd, Cellular Microbiology, 2, 465±476

proteins in a PI 3-kinase manner. This observation is in

agreement with two recent findings which suggest that

even although PLC-g1 is phosphorylated extensively after

growth factors stimulation, activation of this enzyme can

occur in the absence of its tyrosine phosphorylation (Yeo

et al., 1997; Kayali et al., 1998). InlB-mediated PLC-g1

activation seems to be entirely dependent on PI 3-kinase

activity, as shown by full inhibition of IP3 release and

calcium mobilization by the PI 3-kinase inhibitor

LY294002, in contrast to other situations in which PI 3-

kinase only partially regulates PLC-g1 activity (Rameh

et al., 1998; Scharenberg and Kinet, 1998). We propose

that InlB-mediated activation of p85b-p110 generates

PIP3, which recruits non-phosphorylated PLC-g1 at the

plasma membrane. This interaction facilitates association

of PLC-g1 SH2 domains with pTyr residues generated by

the InlB stimulus, through activation of as yet unidentified

tyrosine kinase(s). These phosphotyrosine containing

proteins, either the activated InlB receptor itself or adaptor

proteins remain to be characterized. Interaction of PLC-g1

with PIP3 and tyrosine-phosphorylated proteins would

target the enzyme to its membrane substrates, leading to

PLC-g1 catalysed hydrolysis of PIP2 to IP3 and DAG,

calcium mobilization and probably PKC activation (Fig. 8).

InlB stimulation induces very transient increases in

intracellular IP3 and Ca21 levels and does not lead to a

sustained response, in contrast to growth factors-

mediated calcium signalling, for which Ca21 release

from intracellular stores is accompanied by an enhanced

influx of external Ca21 through voltage-independent Ca21

channels in the plasma membrane (Moolenaar et al.,

1986). The InlB-induced Ca21 release from internal stores

is likely to activate highly localized cellular processes,

which would be able to respond to slight changes in the

concentration of this potent signalling ion (Berridge et al.,

1998). In agreement with this idea, it has been reported

that the magnitude and/or the duration of intracellular

calcium elevations differentially activate transcription

factors (Dolmetsch et al., 1997).

Involvement of phosphoinositide metabolism and cal-

cium signalling during interaction of bacterial pathogens

with eukaryotic cells have been previously reported upon

adherence of enteropathogenic Escherichia coli (Baldwin

et al., 1991; Foubister et al., 1994) and invasion of

Salmonella typhimurium (Ruschkowski et al., 1992).

However, the bacterial factors involved and the host

PLCs have not been clearly characterized. In L. mono-

cytogenes, the pore-forming toxin listeriolysin LLO and

the bacterial phospholipase PI-PLC have been shown to

induce phosphoinositide turnover in endothelial cells and

calcium signalling in macrophages (Sibelius et al., 1996;

Wadsworth and Goldfine, 1999), but the PLC isoforms

activated by these factors are still unknown. In HEp-2

cells, we have observed that wild-type L. monocytogenes

and a DinlB mutant induce a comparable and sustained

calcium response, suggesting that listerial factors other

than InlB are able to promote PLC signalling in epithelial

cells (S. Dramsi and P. Cossart, unpublished results).

However, infection of HEp-2 cells with L. monocytogenes,

but not with the DinlB mutant, induces association of PLC-

g1 with phosphotyrosyl proteins, suggesting that activa-

tion of the PLC-g1 isoform is specific to the InlB stimulus.

InlB is thus the first bacterial factor described as an

inducer of PLC-g1 in non-phagocytic cells.

PLC-g activation has been proposed to be involved in

actin rearrangements, as this enzyme modulates the

levels of PIP2 and Ca21, two well-known regulators of

actin-binding proteins (Lee and Rhee, 1995). It was

therefore tempting to speculate that activation of PLC-g1

was required for the reorganization of the actin cytoske-

leton that occurs during InlB-induced phagocytosis.

However, this hypothesis is not sustained by two sets of

data. First, entry of InlB-coated beads and that of InlB-

expressing bacteria are not affected by the PLC inhibitor

U73122, or by the Ca21 chelator BAPTA/AM. Second, L.

monocytogenes internalization is not decreased in Plcg1

knockout cells. Therefore, InlB-mediated PLC-g1 activa-

tion and intracellular calcium rises are apparently not

involved in the internalization process. In contrast, our

very recent results suggest that extracellular calcium is

important for InlB-mediated entry of bacteria (S. Dramsi

and P. Cossart, unpublished results) and could play a role

in the interaction of InlB with one of its coreceptor (Marino

et al., 1999; Braun et al., 2000).

The signalling cascade described here is not a

prerequisite for InlB-induced phagocytosis, as in the

case of professional phagocytosis (Di Virgilio et al.,

1988), a phenomenon also associated with increased

PLC activity, enhanced tyrosine phosphorylation of PLC-

g1 and a rise in [Ca21]i (reviewed in Greenberg, 1995). In

that case, such signals play a role in post-ingestion

phenomenons, such as in subsequent phagolyzosome

fusion (Jaconi et al., 1990) or in killing of bacteria by

regulation of the generation of toxic oxygen metabolites

(Zheng et al., 1992; Wilsson et al., 1996). We do not

know yet in which step of the events downstream to

phagocytosis PLC-g1 activation by InlB is involved. An

attractive hypothesis could be a link between PLC-g1 and

NFkB activation, and therefore in some control of gene

expression. In agreement with this hypothesis, we have

recently demonstrated that InlB can stimulate NFkB in

macrophages in HEp-2 cells (Mansell et al. 2000) and

BAPTA/AM has been shown to block EGF-induced NFkB

activation in A431 cells (Sun and Carpenter, 1998). Such

links are among investigations currently in progress in

order to identify the precise role of PLC-g1 activation and

Ca21 mobilization induced by InlB during dissemination of

L. monocytogenes in cells and tissues.

InlB activates PLC-g1 473

Q 2000 Blackwell Science Ltd, Cellular Microbiology, 2, 465±476

Experimental procedures

Media, bacterial strains and cell lines

The Listeria strains used were the wild-type L. monocytogenesstrains EGD (BUG 600) and L028 (Vicente et al., 1985), aDinlB isogenic mutant of EGD (BUG 1047; Dramsi et al., 1995)and a L. innocua derived strain expressing the LRR-IR regionof InlB (BUG 1642; Braun et al., 1999). Strains were grown at378C in brain±heart infusion (BHI) agar (Difco). The humanlaryngeal epithelial cell line HEp-2, the African green monkeykidney cell line Vero, the Plcg1 (±/±) and Plcg1 (1/1) MEF (Jiet al., 1997) were cultured in Dulbecco's modified Eagle'smedium (DMEM, Gibco) supplemented with 10% fetal calfserum (FCS, Sera-Lab) 2 mM glutamine and 1% non-essential amino acids (Gibco) at 378C in 10% CO2.

Proteins, InlB-coated beads, antibodies and other

materials

The InlB protein was the InlB6xHis protein, purified aspreviously described (Braun et al., 1997). Human recombi-nant EGF was from Upstate Biotechnology (UBI). Latexbeads were from Molecular Probes (1 mM diameter, catalo-gue no. F8817). Covalent coupling of purified InlB protein tolatex beads was carried out as described previously (Braunet al., 1998). Antibodies to PLC-g1 were either anti-bovinephospholipase C-g1 PowerClonalTM mixed monoclonalpreparation (UBI) or a polyclonal rabbit antibody raisedagainst a peptide which is unique to PLC-g1 (sc-81).Monoclonal antibodies against p85a and p85b were kindlyprovided by Drs I. Gout and D. Waterfield. Polyclonal affinity-purified antibody against human p85a was kindly provided byDr M. Thelen. InlB-specific affinity-purified polyclonal anti-bodies were as previously described (Braun et al., 1997).Fluorescein isothiocyanate-labelled goat anti-rabbit IgGswere from Biosys. Monoclonal antibodies against phospho-tyrosine were from UBI (4G10) or Transduction Laboratory(RC20). Protein A-Sepharose CL-4B beads used for immu-noprecipitation were from Pharmacia. U73122 was pur-chased from Alexis corporation, wortmannin and LY294002were obtained from Biomol, genistein and calphostin C fromCalbiochem, cytochalasin D from Sigma, and BAPTA/AMfrom Molecular probes. Inhibitors were dissolved in DMSO.At the concentrations used, these inhibitors had little or noeffect on cell viability, as determined by trypan Blue staining.U73122 at 5 mM and BAPTA/AM at 250 mM induced celldetachment (25% and 50% respectively). These effects weretaken into account in invasion frequency determination.

Immunoprecipitation and immunoblotting

Cells were seeded in 75 cm2 or 25 cm2 tissue culture flasks(approximately 2.106 or 1.106 cells respectively) and grown for2 days. Cells were starved for 2 h in serum-free DMEM and,when indicated, were pretreated for 20 min with 0.1% DMSOor inhibitors. Cells were stimulated by addition of InlB or EGFor by infection with L. monocytogenes strains at a multiplicity ofinfection (MOI) of 200 bacteria per cell. Immunoprecipitationswere performed as described (Ireton et al., 1999). Proteinconcentration of the lysates were determined after preclearing

with protein A-sepharose CL-4B beads (Pharmacia) using theBCA system and equivalent amounts of total protein wereused for immunoprecipitation. Immunoprecipitates were sepa-rated on 8% SDS±PAGE and transferred to nitrocellulose(Hybond C, Amersham) or PVDF (Immobilon-P, Millipore)membranes with a semi-dry apparatus. Membranes wereblocked overnight by incubation in Tris-buffered saline (TBS)with 0.1% Tween 20 containing 3% BSA (for immunoblottingwith anti-pTyr IgG) or 5% non-fat milk (for all other antibodies).Proteins were detected using the enhanced chemilumines-cence systems (ECL or ECL Plus, Amersham).

Measurements of in vivo levels of PI(3,4)P2, PI(4,5)P2 and

PI(3,4,5)P3

Measurement of phosphoinositides were carried out asdescribed in Ireton et al., 1999. Approximately 8 � 105 cellswere seeded in 75 cm2 flasks, grown at 378C in 5% CO2 for40±46 h and labelled for 5 h by incubation in serum-freeDMEM without phosphate containing 250 mCi ml21 32Pi.Cells were then stimulated or not by addition of 4.5 nM InlB inDMEM for 1 min. When indicated, cells were pretreated withthe inhibitors LY294002 (50 mM) or wortmannin (50 nM) for15 min prior to stimulation. After stimulation cells werewashed with ice-cold PBS and immediately removed byscrapping. Reactions were stopped by the addition of cold 2.4N HCl. Lipids were extracted and separated by thin-layerchromatography. The spots corresponding to PIP2 and PIP3

were recovered by scrapping. After deacylation the differentphosphoinositides were separated and quantitated by a high-performance liquid chromatography technique as described(Ireton et al., 1999).

Quantification of intracellular IP3

For measurements of IP3, cells were grown to 90%confluency in 75 cm2 culture dishes and were labelledovernight with myo-(2±3H) inositol (2mCi ml21) in inositol-free DMEM. Labelled cells were washed once with DMEMand pretreated with 10 mM LiCl for 15 min, in the presence orabsence of the inhibitors genistein (250 mM) and LY294002(50 mM). Cells were left untreated or treated with purified InlB(9 nM) or EGF (17 nM) for various times. Inositol phosphateswere extracted and separated on a Dowex AG 1-X8 anionexchange resin, as described (Berridge et al., 1983).Samples were processed for liquid scintillation counting.

Intracellular calcium determination

HEp-2 cells were plated on glass coverslips 2 days prior tothe experiment. The day before (or at least 4 h before theexperiment), the culture medium was exchanged for FCS-free medium. Cells were loaded at room temperature with400 nM Fura-2/AM for 30 min. Fura-2 loading and Ca21

experiments were carried out in D-PBS 1 1 mMCa21 1 2 g l21 glucose 1 20 mM HEPES, pH 7.4. Cellswere stimulated by InlB or EGF at the indicated concentra-tion. Ca21 measurements were performed at 378C with anIMSTAR imaging system as previously described (Donnadieuet al., 1994) and are averages of 15 cells in the same field,

474 H. Bierne et al.

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sampled at 5 s interval. For PI 3-kinase inhibition, LY294002(25 mM) was added to the loading medium and to the assaymedia during Ca21 measurements. For experiments in Ca21-free medium, the assay medium was D-PBS 1 140 mMNaCl 1 5 mM KCl 1 1 mM MgCl21 1 20 mM HEPES1 0.5 mM EGTA.

Invasion assays with bacteria and InlB-coated beads

Invasion assays of bacteria or of InlB-coated latex beadswere performed as described previously (Braun et al., 1998).When indicated, cells were pretreated with DMSO orinhibitors 15 min before infection and during the 60 mininfection. Briefly, the Listeria strains were grown toOD � 0.8±1, washed in PBS, and diluted in DMEM suchthat the MOI was about 50 bacteria:1 cell. Bacterialsuspensions were added to mammalian cells and centrifugedat 200 g for 1 min. Cells were washed and non-invasivebacteria were killed by adding gentamicin 10 mg ml21 for 2 h.After washing, cells were lysed in 0.2% Triton and thenumber of viable bacteria released from the cells wasassessed by titering on agar plates.

For invasion analysis of InlB-coated beads, approximately5 � 104 cells were seeded on glass coverslips, grown for2 days and incubated with latex beads (MOI of about 20:1) bycentrifugation at 200 g for 1 min. Cells were incubated at 378Cin 10% CO2 for 1 h, washed once in PBS, and fixed for 20 minwith 3% paraformaldehyde in PBS at room temperature.Extracellular beads were successively labelled with polyclonalantibodies to InlB and with FITC-labelled goat anti-rabbit IgGantibodies. Because the beads were already fluorescent in the650±700 nM range, extracellular beads were labelled bothgreen and red, whereas intracellular beads were labelled onlyin red. To quantify invasion, beads associated with 100±200cells were analysed in each experiment. The efficiency of entryis expressed as the average number of intracellular particlesper cell relative to the total number of beads per cell (bothextracellular and intracellular).

BAPTA/AM (250 mM) and U73122 (5 mM)-induced celldetachment were taken into account in the determination ofinvasion frequencies of bacteria. It does not interfere withdirect counting of cell-associated beads by immunofluores-cence labelling.

Acknowledgements

We gratefully acknowledge K. Ireton for sharing unpublished

results. We thanks Drs I. Gout and D. Waterfield for providing the

p85a and p85b monoclonal antibodies and Dr M. Thelen for thegift of the p85a human affinity-purified antibody. Dr Arpita Maiti is

thanked for critical reading of the manuscript. This work was

supported by European Economic Community (grant BMH4-CT96±0659), the MinisteÁre de la Recherche et de la Technologie

(programme microbiologie 1998) and the Pasteur Institute. G.

Carpenter received support from a NIH grant (CA75195). H.

Bierne is on the INRA staff.

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