Action et contrôle des leucotoxines de Staphylococcus aureus sur les cellules cibles Mira Tawk
To cite this version:
Mira Tawk. Action et contrôle des leucotoxines de Staphylococcus aureus sur les cellules cibles. Bactériologie. Université de Strasbourg, 2014. Français. NNT : 2014STRAJ111. tel-01234206
HAL Id: tel-01234206 https://tel.archives-ouvertes.fr/tel-01234206 Submitted on 26 Nov 2015
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UNIVERSITÉ DE STRASBOURG
ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE EA-7290 Virulence bactérienne précoce : fonctions cellulaires et contrôle de l'infection aiguë et subaiguë
THÈSE présentée par : Mira TAWK
soutenue le : 07 juillet 2014
pour obtenir le grade de : !"#$%&'($')R%+,-$&.,# '($'0#&1.2!%&3 Discipline/ Spécialité : V'# #2 S ,2M A1.#!21 M-*M!3* '0#1 #2 C#**3* '0#1 "# * B'-*-%'#
Action et contrôle des leucotoxines de Staphylococcus aureus sur les cellules cibles
THÈSE dirigée par : Dr. PRÉVOST Gilles MCU-PH, Université de Strasbourg Pr. BOURCIER Tristan Professeur, Université de Strasbourg
RAPPORTEURS : Pr. FREY Joachim PR-PUPH, Université de Berne Dr. LADANT Daniel DR, Institut Pasteur-Paris
AUTRES MEMBRES DU JURY : Dr. FAFI-KREMER Samira MCU-PH, Université de Strasbourg Dr. JOUBERT Olivier MCU, Université de Lorraine
TAWK Mira · [email protected]
Université de Strasbourg · EA-7290 Virulence bactérienne précoce : fonctions cellulaires et contrôle de l'infection aiguë et subaiguë
Institut de Bactériologie de la Faculté de Médecine · 3 rue Koeberlé · 67000 Strasbourg · FRANCE
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Remerciements
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Toute ma gratitude Gilles PRVOST $1@&# %>1*&# <*&*A # ,!((!# (2 "!# !(# S%>1*&# permis de la r aliser dans les meilleures conditions. Merci pour tous les efforts qui
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(1@"#-!"#<1,@ !'("#%< *'*"(&%(*C"#E@S*-#%#<@# !#C%*&!/# 0S!"$ &!#%>1*&# t digne de la
,1'C*%',!# E@S*-# S%# %,,1&< !# !(# E@!# ,!# (&%>%*-# soit finalement la hauteur de ses esp rances.
Merci au Pr. Tristan BOURCIER pour avoir co-encadr ma th se et pour le financement travers la Bourse Berthe FOUASSIER.
Je remercie norm ment Emmanuel JOVER pour sa disponibilit . Je le remercie pour mon initiation la microscopie confocale et ses nouvelles id es de manipes. Je le
&! !&,*!# A%-! !'(#$1@&#'1"#<*",@""*1'"#E@*# S1'(#G!%@,1@$#!'&*,2* et pour tous les conseils E@S*-# S%#$&1<*A@ /
Je remercie Cristina POTRICH et Mauro DALLA SERRA pour leur gentillesse et
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Je remercie Zouhair ASFARI pour la synth se des calixar nes et sa disponibilit . Je remercie galement nos collaborateurs de -SJ.KI : Sarah CIANFERANI et Fran ois
DEBAENE pour leur travail sur les calixar nes.
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(IPBS) de Toulouse : Lionel Mourey et Laurent Maveyraud, qui ont contribu ce travail.
Merci 7%'*!--%# L5449# 1*&# *'*(* # # -%# %'*$@-%(*1'# %(*1'#!(##-%#,1@$!/#N!&,*# A%-! !'(##-S E@*$!#*<#KJIOP#
E@*# S%#$!& *"# Je remercie Jean-Michel SCHEFFTEL !(# 3& < &*,# PIK49NN# E@*# S1'(# <1'' # -S1$$1&(@'*( #%@M#.&%(*E@!"# Un g &%'<# !&,*# # -S Unit Inserm U1110 (Institut de Virologie, Strasbourg) : M lanie, Sylvie, Thomas, Bin et les autres qui nous ont c d les PMNs de leurs E Buffy coats F. Cette th se a t soutenue par la fondation de France N Bourse Berthe FOUASSIER, par @'!#G1@&"!#!&"*( # 0!#&! !&,*!#-S!'"! G-!#<@#$!&"1''!-# Un grand merci Daniel KELLER, pour sa disponibilit , pour son savoir faire technique et pour son aide dans la recherche des solutions tous les probl mes. 0!# (*!'"# # !M$&* !&# %# A&%(*(@!&"# 4%H 1' 2@ %*'!"/#N!&,*#1*&#C%*(#< ,1@>&*&#-S9-"%,!#!(# -S%-"%,*!'# Z !# "*# 1'# 'S%# $%"# !@# -!# (! $"# %',!&\/# N!&,*# $1@&# "%# G1''!# humeur et les gteaux lors des pauses quotidiennes avant son d part, et le E ravitaillement F lors de ses visites actuelles. Je remercie les th sards qui taient au labo mon arriv e, Benot-Joseph, Wardi, P1$2*!#!(#O2%-<1@'#E@*# S1'(#$!& *"#@'!#(& "#&%$**!#<@#-%G1/# Je remercie Galle pour sa serviabilit , sa gentillesse et surtout pour sa bonne humeur et les moments de rigolade. Je la remercie galement pour sa contribution aux manipes. Je remercie aussi les nouveaux th sards, lodie et Xavier pour -S% G*%',!#%@#-%G1#!(#-!"#$%@"!"# 4' Merci tous mes amis qui S1'(# %*< # # $%""!&# ,!"# "!$(# %'"# !# !'# France et E@*# S1'(#$!& *"# % *D# %>!,# E@*# FS%*# $%"" # -%# $-@$%&(# !,# E@*# FS%*# $%&(%A # ,!((!# exp rience. Patrick, Ranine, Omar, Marianne, Ahmad, Ricardos pour leur pr sence, les sorties, les repas, les voyages que nous avons effectu ensemble... Laurent D., 8%@&!'(#./D#0 & HD#N%( #!(#-!"# %@(&!"# %>!,#E@*#FS%*#< ,1@>!&(#@'#%@(&!# 1' Enfin, je remercie ma grande famille pour son soutien et son amour. Farid et N1'*E@!#$1@&# S%>1* G!&A !#$!'<%'(# !"#$&! * &!"#%'' !"##P(&%"G1@&A/#P% mira et Samia pour leur soutien inconditionnel. X!1&A!"D# 9<<*"" # !(# (1@"# -!"# %@(&!"G# 0!# remercie surtout mon p re et ma m re, autant pour leur soutien financier que pour leur soutien moral et leurs encouragements pendant les moments les plus durs. 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SIGNALISATION CALCIQE 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 EJRQH7 QsV 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 GXJX`:C1 Xs 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 EJRQH7 QsV RX]VJR:J V RV C: HC: .`1JV ^CDE_ 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 EJRQH7 QsV RX]VJR:J V RVs H:0XQCVs 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 EJRQH7 QsV 1JRX]VJR:J V RV C: HC: .`1JV V RV C: H:0XQC1JV8888888888888888888888888888888888888888888888888888888888888888888888888888888888888 EJRQH7 QsV RX]VJR:J V RVs FCQ 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88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 EPRESSION ET PRIFICATION DES LECOTOINES 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 COPLAGE À N FLOROCHROME 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 DQs:$V ]`Q X1_%V ]:` C: IX .QRV RV B`:R`Q`R 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 TEST D ;ACTIITÉ DES LECOTOINES 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 S]VH `Q`C%Q`1IX `1V 7 DX V`I1J: 1QJ RV C: 0:`1: 1QJ RV C: HQJHVJ `: 1QJ 1J `:HVCC%C:1`V VJ H:CH1%I 88888888888 C7 QIX `1V VJ `C%6 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 P`1JH1]V RV C: H7 QIX 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HC$CLHC$B V RV C: LP 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 IRVJ 1`1H: 1QJ RVs `Xs1R%s VssVJ 1VCs ; C: `16: 1QJ RV L%@SRP ; C :R 8888888888888888888888888888888888888888888888888888888888888888888888 A` 1HCV J 7 RVs1R%Vs VssVJ 1:C `Q` P:J QJR:CVJ 1JV LV%@QH1R1J S HQI]QJVJ G1JR1J$ Q 1 s HVCC `VHV] Q` s%$$Vs GQ . ]C:s 1H1 7 :JR :R:] :G1C1 7 1J 1 s 1J V`:H 1QJ s%``:HV 8888888888888888888888888888888888888888888888888888888888888888888888 D1sH%ss1QJ 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 CQJHC%s1QJ 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 PV`s]VH 10Vs 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 CHAPITRE I 7!*+-*.*)*#+!$%!/;'( TION DES LECOTOINES DE S8 ARES 8888888888888888888888888888888888888888888888888 LES CALIARÈNES 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 IJ `QR%H 1QJ 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 RXs%C : s 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 GJ.1G1 1QJ%RV%C;:H 1QJ%RV"%CV%HQ Q61JV"%"%`%CV"%.D F" 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 HC$CLHC$B V C: LP 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 LVs :% `Vs CV%HQ Q61JVs 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 H0:C%: 1QJ%RV%C;V``V %.XIQC7 1_%V%RV"%H:C16:`WJV" 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 H0:C%: 1QJ%RV%C;V``V %CV%HQH7 Q Q61_%V%RV"%H:C16:`WJV" 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 IXX0:C%: 1QJ%RV%C;V``V %1J.1G1 V%`%RV"%H:C16:`WJV" 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 IVH.V`H.V%R;%J%V``V %:J 1G1Q 1_%V%RV%HV"%H:C16:`WJV" 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 CQJHC%s1QJ 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 PV`s]VH 10Vs 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 CHAPITRE 7 CONCLSION 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 PATHOLOGIES ASSOCIÉES A HLG CLH LG B ET A LA LP 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 RÔLE DES LECOTOINES DANS LES PATHOLOGIES 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 CONCLSION GÉNÉRALE 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 CHAPITRE I 7 BIBLIOGRAPHIE 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 6' LISTE DES TABLEA TABLEA II8 7 LES DIFFÉRENTS CALIARÈNES TILISÉS 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 TABLEA II8 7 RÉFÉRENCES ET DOSES DES DROGES TILISÉES POR L ;EPÉRIMENTATION ANIMALE 888888888888888888888888888888888888888888 T& TABLEA III8 7 TABLEA RÉCAPITLATIF DES EFFETS DES DIFFÉRENTS INHIBITERS SR L ;AGMENTATION DE LA C A I PAR RAPPORT À L _EFFET DE LA TOXINE T 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 TABLEA I8 7 RÉSMÉ DES IC DES DIFFÉRENTS CALIARÈNES SR HLG CLH LG B ET LA LP 8888888888888888888888888888888888888888888888 TABLEA I8 S ICWR DES CALIXARÈNES SUR LA FIXATION DE S N M DE LUK S#VPV SUR LES H PMN S AVANT ET APRÈS NEUTRALISATION DES CALIXARÈNES T 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 TABLEA I8 7 EFFET DES CALIARÈNES SR LA PERMÉABILISATION DE LA MEMBRANE DE HPMN S A BET888888888888888888888888888888 TABLEA 8 7 EFFETS CELLLAIRES DES DIFFÉRENTS COPLES DE LECOTOINES 8 8888888888888888888888888888888888888888888888888888888888888888 6'' LISTE DES FIGRES FIGRE I8 7 STAPHLOCOCCS ARES 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 EASION D SSTÈME D COMPLÉMENT 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 SCHÉMA SIMPLIFIÉ D CCLE D ;N BIOFILM 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 LES PRINCIPA FACTERS DE IRLENCE DE S8 ARES 88888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 MODE D ;ACTION DES PFT S8 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 STRCTRE TRIDIMENSIONELLE DE SOS RNITÉS DE LECOTOINES BIPARTITES 8 888888888888888888888888888888888888888888888888888 FIGRE I8 7 STRCTRE TRIDIMENSIONNELLE D PORE FORMÉ PAR HLG ARH LG B 888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 ADAM8 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 PHAGOCTOSE INDITE PAR MAC R 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 STRCTRE TRI RDIMENSIONNELLE DE CCR ET CCR 8 88888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 MÉCANISME D ;ACTION DES GPCR S 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 ENDOCTOSE ET TRI INTRACELLLAIRE 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 MÉCANISME D ;ACTION DE LA SHIGA RTOINE 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 MÉCANISME D ;ACTION DE L ;EOTOINE DE BACILLS ANTHRACIS 88888888888888888888888888888888888888888888888888888888888888888888 FIGRE II8 7 PRIFICATION DES PMN S 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE II8 7 IMMNO RMARQAGE EN SANDICH POR DOSAGE BIO RP LE ^M ANEL BIO RAD _ 8888888888888888888888888888888888888888888888 FIGRE III8 7 COPES DE RÉTINE DE LAPIN ALBINOS DE RACE NE EALAND 8888888888888888888888888888888888888888888888888888888888888888888888 T& FIGRE III8 S EFFETS DE HLG C H LG B ET LA LPV SUR LA VARIATION DE LA C A I LIBRE EN PRÉSENCE ET EN ABSENCE DE CAT& EXTRACELLULAIRE T 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE III8 S EFFET DU CAT& EXTRACELLULAIRE SUR LA FIXATION DES LEUCOTOXINES 8888888888888888888888888888888888888888888888888 FIGRE III8 S EFFET DU CAT& EXTRACELLULAIRE SUR L _ENTRÉE DE BET DANS LES H PMN S 888888888888888888888888888888888888888888 FIGRE III8 S COMPARAISON DE L _EFFET DE S =M DE F MLP BLEU ET SQT N M DE CW A ROUGE SUR LES H PMN S 88888888 FIGRE III8 S EFFETS DU F MLP ET DU CW A DANS LES H PMN S EN PRÉSENCE DE THAPSIGARGINE OU D _IONOMYCINE 8888888 FIGRE III8 S EFFETS DE DIVERS INHIBITEURS SUR LA SIGNALISATION CALCIQUE DE HLG C H LG BT 88888888888888888888888888888888 FIGRE III8 S EFFET DE L _IONOMYCINE SUR LA SIGNALISATION DE LA LPV 8888888888888888888888888888888888888888888888888888888888888888888 FIGRE III8 S DOT PLOTS DES H PMN S SOUS L _EFFET DE HLG C H LG B ET LA LPV À UR ET XR MIN 88888888888888888888888888888888 FIGRE III8 S EFFETS DE HLG C H LG B CB ET LA LPV SF SUR LA SÉCRETION DE L _!"Z ET L _!"S VRA PAR LES H PMN S 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 S STRUCTURE TYPE D _UN CALIX N ARÈNE Q 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 STRCTRE DE SC5 SC ET SC ^P ERRET ET AL 85 _8 888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 STRCTRES PLANES ^GACHE _ ET D ^DROITE _ D THIA VSCV 5 D CARBOXYMÉTHOXY VPVSULFONATE PHÉNOL Q DU DCMVSCV ET D MCVSCVT 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 STRCTRES PLANES ^GACHE _ ET D ^DROITE _ D BIS VB ENZO VSCV 5 D BIS VN APHTO VSCVQ DU BIS VD IBENZO V SCV ET D BIS VE SCULETIN VSCVT 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 7 EFFET DES CALIARÈNES SR LA PERMÉABILISATION DES H PMN S A BET DE À HLG CLH LG B ET LA LP8 8888888888888 FIGRE I8 7 EFFETS DES CALIARÈNES SR HLG ALH LG B ET LK ELL K D 8888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 S TEST D _HÉMOLYSE DES CALIXARÈNES T 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 FIGRE I8 S EFFETS DU BIS VD IBENZO VSCV ET DU SCZ ZAS SUR LES H PMN ST 88888888888888888888888888888888888888888888888888888888 6''' A 0M4' 2'-,1 ADN A!'"# DM1-670' -N3!*M'/3# C T& ' !-,!#,20 2'-, "# ! *!'3+ ',20 !#**3* '0# ADAM A D#1',#%0', A," M#2 **-.0-2# 1# %0 !!#11-07 %#,# 0#%3* 2-0 AIP A32-VI,"3!2#30 P#.2'"# AMP! A"M,-1',# M-,-P&-1.& 2# !7!*'/3# APT A" .2-0 P0-2#', !-+.*#6VT APP .0M!301#30 "# .* /3# +7*-_"# ATP A"M,-1',#VWVT0'P&-1.& 2# BAR B',VA+.&'.&71',VR41SXY B 1-31V3,'2M .0-2M 1# "3 $ !2#30 B BET %0-+30#+"_-.&'"'3++ B'1VB#,8-VSCV 2M20 V13*$-, 2# "# N V '1V #,8-V!-30-,,#VXV! *'6V 0L,# B'1VD' #,8-VSCV . 0 V13*$-, 2# "# N V '1V"' #,8-V!-30-,,#VXV! *'6V 0L,# B'1VE1!3*#2',VSCV 2M20 V13*$-, 2# "# N V '1V#1!3*#2',V!-30-,,#VXV! *'6V 0L,# B'1VN .&2-VSCV 2M20 V13*$-, 2# "# N V '1V, .&2-V!-30-,,#VXV! *'6V 0L,# CW R CW R#!#.2-0 C T& C *!'3+ !ADPR 1"#,-1',#+W_ VD'.&-1.& 2# R' -1# !7!*'/3# C M C *M-"3*',# C +KII C T& ! *+-"3*',V"#.#,"#,2 .0-2#', )', 1# CAV CAVM-*',# CCP C* 2&0', C- 2#" P'21 CCRW RM!#.2#30 9 CVC !&'+'-)',# "# 27.# W CCV C* 2&0',VC- 2#" V#1'!3*#1 CDC C72-*71',#1 DM.#," ,2#1 "3 C&-*#12M0-* C"!VT C#** "'4'1'-, !-,20-* .0-2#', VT CDE C* 2&0', D#.#,"#,2 E,"-!72-1'1 C&AT C&-*',# !#27*20 ,1$#0 1# CHIPS CH#+-2 6'1 I,&' '2-07 P0-2#', -$ ST 30#31 CICR C *!'3+ I,"3!#" C *!'3+ 0#*# 1#" CIE C* 2&0', I,"#.#,"#,2 E,"-!72-1'1 C*$ C*3+.',% F !2-0 CMH 5-+.*#6#+9 (#30+"_<'12-!-+. 2' '*'2M+ CMH I -3 II C-+.*#6# M (#30 "_<'12-!-+. 2' '*'2M I -3 II C- C- %3* 1# CT 1-31V3,'2M B "# * 2-6',# !&-*M0'/3# CXCR 0M!#.2#30 9 CVXVC !&'+'-)',# DAG "' !7*%*7!M0-* DCMVSCV SQUV"'! 0 -67+M2&-67V .V2M20 V13*$-, 2# "# N V! *'6V 0L,# DMSO D'+M2&7*13*$-67"# DRG ,#30-,#1 1#,1-0'#*1 "3 % ,%*'-, 1.', * 6'4 DSF D'$$#0#,2' * S! ,,',% F*3-0#1!#,!# DTT D'2&'-2&0#'2-* E . E620 !#**3* 0 "�#,!# .0-2#', ECWR !-,!#,20 2'-, #$$'! !# +M"' ,# ECL E620 C#**3* 0 L--. ECM E620 C#**3* 0 M 20'6 EDIN E.'"#0+ * C#** D'$$#0#,2' 2'-, I,&' '2-01 #DNA ADN #620 !#**3* '0# EEAS E 0*7 E,"-1-+# A,2'%#, S EF E"#+ F !2-0 E$ E620 !#**3* 0 $' 0',-%#, ',"',% .0-2#', EGTA E2&7*L,# G*7!-*V '1+J V +',-+M2&7*+M2�+EQ+EQ+E_Q+E_+.M201!M2 2# EHDT E.1VSW H-+-*-%7 D-+ ',V!-,2 ',',% .0-2#', T ELISA E,87+#VL',)#" I++3,-S-0 #,2 A11 7 E+. ECM ',"',% .0-2#', ERAD E,"-.* 1+'! R#2'!3*3+VA11-!' 2#" D#%0 " 2'-, ERC E,"-!72'! R#!7!*',% C-+. 02+#,2 ERK E620 !#**3* 0 1'%, *VR#%3* 2#" K', 1#1 ESCRT E,"-1-+ * S-02',% C-+.*#6#1 R#/3'0#" $-0 T0 ,1.-02 ESIVMS E*#!20-1.0 7 I-,'8 2'-, M 11 S.#!20-+#207 ET E"#+ T-6', ET1 E6$-*' 2'4# T-6',1 FWM F*3-0#1!M',# WVM *M_+'"# U#6+-+VZZ+,+Q+U#++-+WTS+,+ F .A F' 0',-%#, ',"',% .0-2#', A F! F0 %+#,21 !-,12 ,21 "#1 ,2'!-0.1 K!IM! F! $0 %+#,2 -$ I%A 0#!#.2-0 I K!LM1 F! $0 %+#,2 -$ I%G 0#!#.2-01 FERM P0-2#', VTSQ E80',Q R "'6',Q A," M-#1', F*3-VU AM F*3-VU A!M2-67M M2&7*#12�+U#6+-++VZZ+,+Q+U#++-+WUR+,+ $MLP NV$-0+7*VM#2&'-,7*VL#3!7*VP&#,7* * ,',# F, . 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Tawk, 2† Ca 2+ channels or glutamate receptors. Drugs tar- Benoît-Joseph Laventie, 2†‡ Bernard Poulain 1 and geting Sarco-Endoplasmic Reticulum Ca 2+-ATPase Gilles Prévost 2 (SERCA) or H +-ATPase and antagonists of the 1INCI – UPR-CNRS 3212 ; Neurotransmission et store-operated Ca 2+ entry complex blunted, or sig- sécrétion neuroendocrine ; 5, rue Blaise Pascal; nificantly reduced, the leukotoxin-induced eleva- F- 67084 Strasbourg cedex, France. tion in intracellular Ca 2+. Moreover, activation of 2Université de Strasbourg , Physiopathologie et the ADP-ribosyl cyclase CD38 was also required Médecine Translationnelle EA-4438 ; Hôpitaux to initiate the release of Ca 2+ from acidic stores. Universitaires de Strasbourg , Institut de Bactériologie , These findings suggest that, prior to forming a 3, rue Koeberlé; F-67000 Strasbourg, France. pore at the plasma membrane, leukotoxin HlgC/ HlgB triggers a multistep process which initiates Summary the release of Ca 2+ from lysosomes, modifies the steady-state level of reticular Ca 2+ stores and Headache, muscle aches and chest pain of mild to finally activates the Store-Operated Calcium Entry medium intensity are among the most common complex. clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These Introduction nociceptive responses of the body raise the ques- tion of how bacterial infection impinges on the Staphylococcus aureus is a common host in human flora nervous system. Does S. aureus , or its released that asymptomatically colonizes one in three healthy indi- virulence factors, act directly on neurones? To viduals; children having higher persistent carriage rates address this issue, we evaluated the potential than adults (Wertheim et al ., 2005). However, this bacte- effects on neurones of certain bi-component leu- rium can be a serious pathogen, and is now the second kotoxins, which are virulent factors released by the cause of hospital and community-acquired infections bacterium. The activity of four different leukotox- worldwide (David and Daum, 2010; DeLeo et al ., 2010; ins was verified by measuring the release of gluta- David et al ., 2011). The severity and locations of mate from rat cerebellar granular neurones. The S. aureus infections display broad variation. Contamina- bi-component g-haemolysin HlgC/HlgB was the tion typically progresses from a local infection (furuncle, most potent leukotoxin, initiating transient rises cellulitis, wound infection) to systemic propagation in intracellular Ca 2+ concentration in cerebellar (bacteraemia) and metastatic infections (pneumonia, neurones and in primary sensory neurones from endocarditis, osteomyelitis, septic arthritis). The emer- dorsal root ganglia, as probed with the Fura-2 Ca 2+ gence and spread of community-acquired, drug-resistant indicator dye. Using pharmacological antagonists (CA-MRSA) and extremely virulent strains reinforces the of receptors and Ca 2+ channels, the variations morbidity of S. aureu s infections (Chambers and DeLeo, in intracellular Ca 2+ concentration were found 2009; David and Daum, 2010). Pathogenesis of the independent of the activation of voltage-operated bacterium relies on an arsenal of virulence-associated factors, some of which specifically produce toxin-induced diseases (e.g. bullous impetigo, toxic shock syndrome, staphylococcal scaled skin syndrome and food-borne Received 21 August, 2012; revised 18 October, 2012; accepted 6 gastroenteritis) (Archer, 1998; Lowy, 1998). November, 2012. *For correspondence. E-mail jover@inci-cnrs. One distinctive feature of CA-MRSA strains is the unistra.fr; Tel. ( +33) 3 88 45 66 47; Fax ( +33) 3 88 60 16 64. bearing of genes for the Panton-Valentine leukocidin †These authors equally contributed to the work. ‡Present address: Biozentrum; University of Basel; Klingelbergstrasse 50/70; CH - 4056 (PVL), a bi-component toxin encoded by lukS-PV and Basel, Switzerland. lukF-PV from an integrated bacteriophage, rarely carried © 2012 Blackwell Publishing Ltd cellular microbiology 2 E. Jover et al . by S. aureus strains prior to the 1990s (Woodin, 1960; Results Finck-Barbançon et al ., 1991; Rahman et al ., 1992; Staphylococcal leukotoxins trigger glutamate release Kaneko et al ., 1998). The activity of PVL plays a role in from cerebellar granular neurones pathogenesis under conditions involving host suscepti- bility factors and infected tissues (Gillet et al ., 2002; Cerebellar granular neurones have been shown to act in Crémieux et al ., 2009). Moreover, S. aureus has the response to the presence of certain pore-forming toxins capacity to produce several homologous two-component (Lonchamp et al ., 2010). Thus, to examine whether leukotoxins that contribute to the bacterium’s power of neurones sense and react to S. aureus leukotoxins, infection, including the ubiquitous g-haemolysins (Hlg, glutamate release by cerebellar granular neurones was encoded by hlgA , hlgB and hlgC ) (Prévost et al ., 1995), quantified after a 10 min exposure to the toxins. Dose– the leukotoxin LukE–LukD (Gravet et al ., 1998), PVL, response curves for a-toxin, leukotoxins HlgC/HlgB and LukM and LukF ′-PV (Choorit et al ., 1995). These toxins HlgA/HlgB and the PVL are shown in Fig. 1A. HlgC/HlgB assemble as pore-forming octamers on the surface of was the most potent of the four leukotoxins, reaching a susceptible target cells, such as neutrophils, monocytes half-maximal effect at a concentration of 3 nM and and macrophages, and can thereby alter host cell func- maximum glutamate release at 0.1 mM. This maximum tions or cause cytolysis (Prévost et al ., 2001; Jayasinghe was 2.4 times higher than the total release generated by and Bayley, 2005). Genes encoding several cytolytic a 5 s depolarization pulse of 60 mM KCl, but only 35% toxins, notably g-haemolysin subunits HlgA, HlgB and of the total-cell glutamate content, as assessed using HlgC, can be upregulated in human blood over time a 10 min osmotic shock in 10 mM Tris-HCl, pH: 7.4 (Malachowa et al ., 2011). (Fig. 1B). Alpha-toxin (0.1 mM), leukotoxin HlgA/HlgB Major advances have been achieved over the last (0.1 mM) and PVL (0.1 mM) were less effective than HlgC/ decade in the understanding of the structure and mecha- HlgB, releasing equivalent amounts of glutamate as that nisms of pore formation by two-component toxins from generated by the 5 s 60 mM KCl depolarization pulse S. aureus and from other origins (Menestrina et al ., 2003; (Fig. 1A). The release due to leukotoxin HlgC/HlgB was 2+ Gonzalez et al ., 2008). In contrast, much less is known dependent on an increase in free [Ca ]i, since pre- regarding the overall cell targets and responses to incubation of neurones with the intracellular calcium che- S. aureus leukotoxins. The increase in free intracellular lator BAPTA-AM (10 mM) (30 min loading followed by calcium or the loss of cellular potassium are examples of 20 min hydrolysis) strongly reduced the amount of gluta- early cellular reactions to the presence of pore-forming mate released into the medium (Fig. 1B). The HlgC or toxins, an ion imbalance that may lead to the activation of HlgB subunits separately failed to induce glutamate a variety of signalling cascades (Woodin and Wieneke, release from granular neurones (Fig. S1a). 1963; Staali et al ., 1998; Kloft et al ., 2009; Kao et al ., To determine whether HlgC/HlgB-treated neurones 2011). undergo lysis, the amount of glucose-6-phosphate dehy- In the present study, the question of the sensitivity of drogenase (G6PDH) enzymatic activity released into the neurones to bi-component leukotoxins of S. aureus is medium was quantified. An example of G6PDH activity addressed with the aim of deciphering the mechanisms measured after incubation at increasing concentrations of of cell response. Cell cultures from rat cerebellar cortex HlgC/HlgB leukotoxin is illustrated in Fig. 1C. Significant and primary sensory neurones from dorsal root ganglia enzymatic activity was detected only after 20 min incuba- were used to demonstrate the neurotoxic activity of PVL, tion with 0.1 mM leukotoxin, suggesting that high toxin a-toxin and g-haemolysins. The four virulence-associated concentration treatment may have been fatal for some factors maintained neuronal integrity while triggering a cells. However, assessment of the number of pyknotic 2+ [Ca ]i increase in cerebellar neurones, which was fol- nuclei in cultures labelled with Hoechst-33258 revealed lowed by the release of glutamate. The g-leukotoxin HlgC/ no significant difference between control (three inde- HlgB was found to be the most potent on both types of pendent cultures, > 400 nuclei, 6.5% pyknotic) and 6 nM cultured neurones. The leukotoxin-induced rises in free leukotoxin-treated cells after a 15 min incubation period 2+ 2+ intracellular ([Ca ]i) were initiated by a discharge of Ca (three independent cultures, > 400 nuclei, 5.8% pyknotic). from acidic stores and followed by a Ca 2+-induced Ca 2+ Membrane permeation of ethidium bromide has been pro- release from the reticulum. These effects were further posed for monitoring pore formation at the cell membrane amplified by the activation of the store-operated Ca 2+ due to leukotoxin (Staali et al ., 1998). Time-lapse video entry complex. The initial signal linking leukotoxin binding microscopy was used to record the infiltration of ethidium to the acidic stores was due to the activation of the ADP bromide in neurones incubated in the presence of 8 nM ribosyl cyclase CD38. This cellular response to the pres- leukotoxin HlgC/HlgB-Alexa-488. Visual inspection of ence of the leukotoxin in neuronal cells preceded the neurones incubated 30 min in the presence of toxin formation of toxin pores into the plasma membrane. revealed that ethidium bromide-related fluorescence © 2012 Blackwell Publishing Ltd, Cellular Microbiology S. aureus leukotoxins intracellular Ca 2+ signalling 3 Fig. 1. Staphylococcus aureus leukotoxins trigger glutamate release from cerebellar granular neurones. A. Cultured cells were challenged for 10 min with the indicated concentrations of leukotoxin HlgA/HlgB ( ), leukotoxin HlgC/HlgB ( ), a-toxin ( ) or PVL ( ). Leukotoxin HlgC/HlgB was the most active, showing a half-maximum concentration effect at nanomolar concentrations. The other three toxins triggered glutamate release at higher concentrations only; the glutamate measured in these instances was similar to the amount measured after a 10 s pulse of 60 mM KCl depolarization. B. Prior to addition of the toxin, cells were loaded with 10 mM of the membrane-permeable intracellular calcium chelator BAPTA-AM ( ), which led to a significant decrease in the amount of glutamate release. Control leukotoxin HlgC/HlgB ( ). The upper reference value (6.51 mM, blue dotted line) stands for the total-cell glutamate content. C. Glucose-6-phosphate dehydrogenase enzymatic activity measured in culture media after a 10 min incubation of neurones in the presence of leukotoxin HlgC/HlgB. Only the highest concentration (0.1 mM) demonstrated a significant amount of released enzymatic activity. Results in (B) (glutamate) and (C) (G6PDH) are from of the same experiment. The brown dotted line represents glutamate (A and B) or G6PDH activity (C) measured in the bathing media of control cells. The green dotted line in (A) and (B) corresponds to glutamate released by a 10 s depolarization of neurones in 60 mM KCl. The blue dotted line in (B) and (C) stands for total glutamate (B) or total G6PDH (C) content measured after a 10 min osmotic shock of neurones in 10 mM Tri-HCl buffer (pH 7.4). remained low, constant and consistently extracellular cells, Fig. 2C) and in 28% of DRG sensory neurones at (Fig. S2). These results together with the reduction in 4 nM (three cultures, 84 cells, Fig. 2D). glutamate release due to the Ca 2+ chelator BAPTA favour The typical reaction of a neurone to the presence of the notion that leukotoxin-induced glutamate release is leukotoxin HlgC/HlgB was a progressive elevation in free 2+ 2+ triggered by a rise in free [Ca ]i, independently of the [Ca ]i which led to a transient peak and returned to a new 2+ formation of a pore. [Ca ]i, resting value (Fig. 2A and B). In a same culture plate, the effect of the leukotoxin on different granular neurones occurred at various unrelated moments. The leukotoxin-induced rise in [Ca 2+] is i However, the response of DRG sensory neurones was concentration dependent both fast and synchronous for all responding cells of the 2+ We monitored [Ca ]i variations in cerebellar granular neu- same plate. For comparison purposes, the response of rones and in sensory neurones from dorsal root ganglia each neurone was characterized by the peak value of 2+ 2+ (DRG), using the Fura-2 Ca indicator dye, to analyse the [Ca ]i (amplitude) and the time needed by the cell to 2+ activity of leukotoxins HlgC/HlgB and HlgA/HlgB and PVL reach the [Ca ]i peak after the addition of the leukotoxin (Fig. 2). The subunits HlgC or HlgB separately failed to (latency). These values are shown in box plots with the 2+ induce [Ca ]i variations (Fig. S1b). Leukotoxin HlgC/HlgB mean, the median and percentiles. 2+ 2+ (2 nM) induced a rise in [Ca ]i in 86% of granular neu- In granular neurones, the amplitude of the [Ca ]i peak rones (12 cultures, 618 cells, Fig. 2A) and in 65% of DRG was dependent on leukotoxin concentration, as illustrated neurones at 4 nM (9 cultures, 191 cells, Fig. 2B). In con- in examples in Fig. 3A–C. Moreover, the consolidation of 2+ trast, leukotoxin HlgA/HlgB activated a rise in [Ca ]i in results from 44 independent experiments confirmed that 2+ 20% of granular neurones at higher concentration (20 nM; mean [Ca ]i peak amplitude values increased, while the three experiments, 130 cells). PVL (2 nM) was active in latency of the response decreased with increasing con- 24% of recorded granular neurones (13 cultures, 419 centrations of leukotoxin, as shown in Table 1 and Fig. 3D © 2012 Blackwell Publishing Ltd, Cellular Microbiology 4 E. Jover et al . 2+ Fig. 2. Leukotoxins induce [Ca ]i changes in cerebellar granular and in dorsal root ganglion neurones. A. Multiple examples of recordings of cerebellar neurones challenged with 2 nM leukotoxin HlgC/HlgB. The amplitude of the 2+ [Ca ]i peak induced by the leukotoxin was variable as was the latency of the response. Boxes along the axis show the median and percentiles for both peak amplitude and latency for 47 cells recorded simultaneously. The mean is indicated by the red trace. 2+ B. [Ca ]i variation caused by leukotoxin HlgC/HlgB in dorsal root ganglion neurones (4 nM toxin). The box presentation corresponds to 36 neurones from three different dishes recorded the same day. 2+ C. PVL (2 nM) induced [Ca ]i changes in 25% of recorded cerebellar neurones; in certain instances, the same cell presented a few 2+ [Ca ]i transient peaks. D. Responses of DRG neurones to different applications of 2 nM PVL; contrary to the g-leukotoxin HlgC/HlgB effects, PVL was able to induce different responses within the same cell during the recording period. Less than 25% of DRG cells from the same culture plate were sensitive to the leukocidin. The vertical stroke at approximately 200 s indicates the time of addition of the toxin. In (D), the three vertical strokes indicate three independent additions of PVL. In (A)–(D), all traces represent individual sample recordings. and E. However, the dispersion of both parameters was ionotropic glutamate receptors were antagonized with high among neurones within a same plate, which sug- 10 mM CNQX (AMPA receptors) or 10 mM D-AP5 (NMDA 2+ gests that a multistep process may be necessary to receptors) while recording [Ca ]i variations induced by 2+ 2+ disrupt [Ca ]i homeostasis and that its activation may leukotoxin HlgC/HlgB. Amplitude and latency of [Ca ]i depend on a particular cell’s status. variations are presented in Fig. 4A and B and in Table 2 (mean values). The presence of these drugs either alone or in various combinations, prior to addition of the leuko- The increase in free [Ca 2+] is independent of i toxin, did not considerably alter the amplitude of the [Ca 2+] voltage-operated Ca 2+ channels or glutamate i peak (Fig. 4A). However, blockade of all VOCC signifi- receptor activity cantly shortened the latency of the leukotoxin effect by Activation of any of the various types of voltage-operated 45% ( P < 0.05), effect observed also when Na + channels Ca 2+ channels (VOCC) or glutamate receptors may lead and glutamate receptors were additionally blocked 2+ 2+ to rise in [Ca ]i in granular neurones (Randall and Tsien, (Fig. 4B, Table 2). When the P/Q-type Ca channel 1995; Brickley et al ., 2001). To investigate whether leuko- antagonist w-AgaTk was removed from the cocktail, the toxin HlgC/HlgB activates a Ca 2+-permeable channel or latency of the response was four times longer (853 Ϯ 14 s receptor, VOCC were antagonized with either 5 mM nifed- versus 210 Ϯ 20 s, P < 0.05). The selective blockade of ipine (L-Type channels), 0.1 mM w-GVI-A (N-Type chan- voltage-gated Na + channels (0.3 mM TTX) doubled the nels) or 0.1 mM w-AgaTK (P/Q-Type channels) whereas latency of the response (436 Ϯ 8 s versus 210 Ϯ 20 s, © 2012 Blackwell Publishing Ltd, Cellular Microbiology S. aureus leukotoxins intracellular Ca 2+ signalling 5 2+ Fig. 3. The mean of the [Ca ]i peak amplitude and the response latency to leukotoxin HlgC/HlgB are concentration dependent. 2+ A–C. Representative recordings of [Ca ]i changes in cerebellar neurones challenged with three different concentrations of leukotoxin HlgC/HlgB (A: 2 nM, B: 4 nM, C: 8 nM). The dot around 200 seconds in (A), (B) and (C) indicates the addition point of the toxin. These examples suggest a toxin concentration-dependent effect that was further established by pooling the values from 44 independent experiments. Boxes are the median and percentiles and include the mean value (red line). All other traces represent individual sample recordings. 2+ D and E. (D) [Ca ]i peak amplitude, (E) latency. The mean values are significantly different as determined by a Kruskal–Wallis analysis of variance and Dunn’s Pairwise Multiple Comparison Procedures (*** P < 0.005). 2+ P < 0.05). Altogether, these results rule out both VOCC kinetics of the leukotoxin effect, a role for resting [Ca ]i or and glutamate receptors as direct passageways for extra- membrane potential cannot be excluded. cellular Ca 2+ influx induced by leukotoxin. However, given To ascertain the contribution of extracellular Ca 2+, neu- the impact of the blockade of these channels on the rones were equilibrated in buffers of various extracellular Table 1. Peak amplitude and latency values are dependent on toxin concentration . Mean [Ca 2+] Latency, Secondary [Ca 2+] HlgC/HlgB peak Ϯ SEM seconds Ϯ SEM rise, % of cells 2 nM leukotoxin HlgC/HlgB (14 plates, 396 cells) 178 nM Ϯ 4 687 s Ϯ 21 4 nM leukotoxin HlgC/HlgB (24 plates, 862 cells) 355 nM Ϯ 8 551 s Ϯ 15 54% cells 8 nM leukotoxin HlgC/HlgB (6 plates, 178 cells) 416 nM Ϯ 17 221 s Ϯ 18 98% cells Mean values of the peak amplitudes and latencies of the experiment shown in Fig. 3D and E. Dunn’s Pairwise Multiple Comparison shows that the differences among all mean values are statistically significant. © 2012 Blackwell Publishing Ltd, Cellular Microbiology 6 E. Jover et al . 2+ Fig. 4. The rise in free [Ca ]i evoked by leukotoxins in cerebellar neurones is independent of voltage-operated Ca 2+ channels or glutamate receptor opening. (A) Peak amplitude and (B) latency of recordings carried out in a: control (56 cells) or in the presence of HlgC/HlgB (4 nM) and the following blockers/antagonists: b: (5 mM nifedipine + 0.1 mM w-GVI-A + 0.1 mM w-Aga-TK; 88 cells), c: (0.3 mM TTX; 24 cells), d: (5 mM nifedipine + 0.1 mM w-GVI-A + 0.3 mM TTX + 10 mM CNQX + 10 mM APV; 26 cells), e: (5 mM nifedipine + 0.1 mM w-GVI-A + 0.1 mM w-Aga-TK + 0.3 mM TTX + 10 mM CNQX + 10 mM APV; 21 cells). None of the differences in mean values for peak amplitude were statistically significant when examined by the Kruskal–Wallis analysis of variance. All the differences in mean latency values were statistically significant ( P < 0.001) with respect to the control value (box a); pairwise multiple comparison showed a non-significant difference among values for b and e. 2+ 2+ Ca concentration ([Ca ]e) prior to incubation with the ments) responded to the presence of leukotoxin HlgC/ leukotoxin HlgC/HlgB. As shown in Fig. 5A, the mean HlgB (4 nM) by displaying a transient peak of low 2+ 2+ [Ca ]i peak was reduced by 80% in 30 mM [Ca ]e relative amplitude (Fig. 5B: 160 Ϯ 8 nM versus 362 Ϯ 37 nM; to controls (43 Ϯ 2 nM versus 227 Ϯ 7 nM; n = 50, n > 100, P < 0.001) whereas the latency of the response P < 0.001), with no significant change observed for the was prolonged (1447 Ϯ 44 s versus 413 Ϯ 6 s; n > 100, 2+ latency of the effect. Leukotoxin-induced [Ca ]i changes P < 0.001). Inhibition of vesicular H-ATPase by 0.2 mM could still be observed in neurones incubated in 3 mM bafilomycin A abolished nearly all leukotoxin-induced 2+ 2+ 2+ [Ca ]e but not in cells bathing in Ca -free buffer (0 mM [Ca ]i movements; approximately half of the recorded 2+ 2+ Ca , 5 mM EGTA), where resting [Ca ]i remained cells showed delayed low-amplitude transient increases 2+ constant. in [Ca ]i that barely reached twice the amplitude of resting 2+ 2+ 2+ The contribution of Ca stored in internal compart- [Ca ]i. To confirm that Ca release from acidic organelles ments was assessed by incubating the neurones in the indeed contributed to the leukotoxin effect, osmotic presence of 1 mM thapsigargin (endoplasmic reticular lysosomal degradation was induced with Glycyl-1- stores) or 0.2 mM bafilomycin A (acidic stores) (Fig. 5B) phenylalanine 2-naphthylamide (GPN) (Churchill et al ., prior to toxin application. In the presence of thapsigargin, 2002). Application of GPN (200 mM, 10 min) essentially 56% of the cells (mean of seven independent experi- blunted the leukotoxin effect; only 11% of neurones Table 2. Mean values and statistical significance of recordings with blunted voltage-operated Ca 2+ channels (VOCC) and voltage-gated Na + channels (VGNC) . Pairwise comparison Mean [Ca 2+] Latency of mean latencies 8 nM HlgC/HlgB peak Ϯ SEM s Ϯ SEM (Holm-Sidak method) a: control (65 cells) 270 Ϯ 15 nM 210 Ϯ 20 s a versus b: P < 0.001; a versus c: P < 0.001; a versus d: P < 0.001; a versus e: P < 0.001 b: blockade of L-, N- and P/Q-Type VOCC by 5 mM nifedipine + 0.1 mM 265 Ϯ 12 nM 116 Ϯ 4 s b versus c: P < 0.001; w-GVI-A + 0.1 mM w-Aga-TK (88 cells) b versus d: P < 0.001; b versus e: P = 0.839 c: blockade of VGNC by 0.3 mM TTX (24 cells) 263 Ϯ 22 nM 436 Ϯ 8 s c versus d: P < 0.001; c versus e: P < 0.001 d: blockade of L- and N-Type VOCC, VGNC and Glu-R by 5 mM nifedipine + 0.1 mM 196 Ϯ 10 nM 853 Ϯ 14 s d versus e: P < 0.001 w-GVI-A + 0.3 mM TTX + 10 mM CNQX + 10 mM D-AP5 (26 cells) e: blockade of L-, N- and P/Q-Type, VGNC and Glu-R: 5 mM nifedipine + 0.1 mM 232 Ϯ 14 nM 111 Ϯ 6 s w-GVI-A + 0.1 mM w-Aga-TK + 0.3 mM TTX + 10 mM CNQX + 10 mM D-AP5; 21 cells Mean values and statistical significance of peak amplitudes and latencies of the experiment shown in Fig. 4. The pairwise comparison between the means of the peak amplitude did not reveal significant statistical differences, whereas mean latencies were significantly different, but for experiments b and e only. © 2012 Blackwell Publishing Ltd, Cellular Microbiology S. aureus leukotoxins intracellular Ca 2+ signalling 7 2+ 2+ Fig. 5. Both extracellular and intracellular Ca stores contribute to leukotoxin-induced [Ca ]i changes in cerebellar neurones. 2+ 2+ 2+ A. Averaged traces of four recordings obtained at different extracellular Ca concentrations ([Ca ]e) showing that, in a Ca -free medium, 2+ 2+ neurones (35 cells) do not react to the presence of the leukotoxin. In low [Ca ]e (3 mM), an increase in free [Ca ]i can be observed (mean of 41 recorded neurones). Control recordings with 1.25 mM Ca 2+ (34 cells) are shown as well as recordings with 30 mM Ca 2+ (47 cells). The boxes show the distribution values for control and 30 mM Ca 2+ recordings. B. Average traces of cells recorded upon interruption of reticular Ca 2+ refilling by blockade of the SERCA pump (1 mM thapsigargin; two experiments, 66 cells) or upon interruption of acidic compartment Ca 2+ refilling through the blockade of H-ATPase (0.2 mM bafilomycin; two experiments, 80 cells). The boxes show the distribution values for control and thapsigargin recordings. 2+ C. Lysosomal destruction by 0.2 mM Glycyl-1-phenylalanine 2-naphthylamide (GPN) prevented leukotoxin-induced [Ca ]i changes. Two different experiments are shown where the mean of control traces are compared with the mean traces of neurones recorded after addition of GPN. The boxes correspond to the values of control recordings (87 cells). In all panels, the addition point of the toxin is indicated by a vertical stroke. 2+ 2+ produced low-amplitude transient variations in [Ca ]i but totally abolished the second Ca rise present in 96% 2+ or displayed a late and moderate increase in [Ca ]i of control cells challenged with 4 nM leukotoxin (not after addition of the leukotoxin (three cultures, 87 cells, shown). The SOCE antagonist econazole (5 mM), totally 2+ 2+ Fig. 5C). These observations suggest that Ca released prevented the toxin-induced rise in [Ca ]i (Fig. 6A). from acidic compartments may couple with reticular stores In addition, incubation of cells for 15 min in 100 mM through a Ca 2+-induced Ca 2+ release (CICR) mechanism. 2-aminoethoxydiphenylborane (2-APB), a cell-permeable Moreover, the contribution of Ca 2+ from both the external modulator of inositol (1,4,5)-P3-induced Ca 2+ release tar- medium and intracellular stores points towards the Store- geting SOCE, decreased the peak amplitude by 40% Operated Ca 2+ Entry (SOCE) complex as a final outcome (203 Ϯ 18 nM versus 335 Ϯ 17 nM; n > 20, P < 0.001) of Ca 2+ mobilization initiated by the leukotoxin. and increased the latency of the response by more than 10-fold (1149 Ϯ 47 s versus 100 Ϯ 1 s; n > 20, P < 0.001) (Fig. 6B). Cells maintained for 15 min in the presence Leukotoxin HlgC/HlgB alters the store-operated Ca 2+ of 90 mM dentrolene, which also blocks ryanodine and entry complex Ins(1,4,5)-P3 receptors, showed a 57% reduction in 2+ Neuronal [Ca ]i variations were monitored in the pres- amplitude (144 Ϯ 4 nM versus 335 Ϯ 17 nM; n > 40, ence of drugs or ions known to interfere with SOCE (Bird P < 0.001) and a sixfold delay in latency (608 Ϯ 25 s et al ., 2008; Varnai et al ., 2009). A highly effective neu- versus 100 Ϯ 1 s; n > 40, P < 0.001) (Fig. 6B). The pyra- tralization was observed when 100 mM Gd 3+ was added a zole derivative YM-58483 has been reported as an inhibi- few minutes prior to the leukotoxin (Fig. 6A). At lower tor of the Ca 2+ release-activated Ca 2+ (CRAC) channels 3+ 2+ concentration, Gd (50 mM) reduced the [Ca ]i amplitude (Ishikawa et al ., 2003). Pre-incubation of neurones in by 44% (291 Ϯ 20 nM versus 519 Ϯ 20 nM; n > 50, 10 mM YM-58483 significantly modified the leukotoxin P < 0.001) and slightly affected the time of the response, effect by reducing the amplitude of the Ca 2+ peak by 70% © 2012 Blackwell Publishing Ltd, Cellular Microbiology 8 E. Jover et al . Fig. 6. Disruption of the Store-Operated Ca 2+ entry complex strongly inhibits the effect of leukotoxin HlgC/HlgB in cerebellar neurones. A. Mean traces of cells recorded in the presence of 5 mM econazole (24 cells) and 100 mM gadolinium (38 cells). The control experiment corresponds to the mean of 35 cells. B. Mean traces of cells recorded in the presence of drugs that target SOCE by also interfering with Ins(1,4,5)-P3 Ca 2+ mobilization (2-APB, 100 mM) or ryanodine receptors (dentrolene, 90 mM). Pre-treatment with these agents significantly reduced the effect of g-leukotoxin HlgC/HlgB. Control experiment, 38 cells; 2-APB, 13 cells; dentrolene, 49 cells. This experiment was reproduced three times. C. incubation of neurones in the presence of LY-294002 (50 mM), which inactivates phosphatidylinositol 3 and 4 kinase and modifies membrane PIP2 content, thus strongly affecting the cellular response to leukotoxin action. Control boxes present the median of 110 recorded cells and LY-294002 (red lines) recordings are the mean of 52 and 63 treated cells. The boxes in the three panels correspond to the values of control recordings. Addition of the toxins is indicated by a vertical stroke in all panels. (142 Ϯ 10 nM versus 458 Ϯ 31 nM; n > 26, P < 0.001), et al ., 2009). The consequences of leukotoxin HlgC/HlgB while the latency of the effect remained unaltered. CRAC activity on the redistribution of interacting partners of the channels are formed in the plasma membrane by the SOCE complex were investigated through immunolabel- association of Orai1 monomers, mediated by the reticular ling of neurones with antibodies raised against STIM1 protein STIM1 that senses the ER Ca 2+ filling state and Orai1. Figure 7 illustrates the differences in labelling (Collins and Meyer, 2011 and references therein). The observed for STIM1 in control cells relative to cells incu- interaction between STIM1 and Orai1 is regulated by bated for 10 min with 4 nM leukotoxin. In treated cells, phosphatidylinositol 4,5-bisphosphate (PIP2) (Korzenio- labelling became partially punctuated suggesting that a wski et al ., 2009; Walsh et al ., 2009). In order to verify redistribution of STIM1 is also an effect of leukotoxin whether the disruption of the STIM1–Orai1 interaction HlgC/HlgB. Taken together, the above results from phar- alters the leukotoxin effect, experiments were conducted macological manipulation as well as immunolabelling 2+ in which phosphatidylinositol kinases (PI3K and PI4K) allow to decompose [Ca ]i movements induced by the were inhibited by LY-294002 (50 mM, 15 min), thereby leukotoxin as a succession of events beginning by the potentially modifying the PIP2 content, while recording release of acidic Ca 2+ stores and resulting in the stimula- 2+ leukotoxin-induced [Ca ]i changes. Under these condi- tion of the SOCE complex. tions, only 6% of the 176 recorded cells (three independ- ent experiments) responded to the leukotoxin through a 2+ 2+ The HlgC/HlgB-induced rise in [Ca ]i is initially low amplitude rise in [Ca ]I whereas in controls, 90% of 2+ dependent on ADP-ribosyl cyclase CD38 signalling the 155 recorded cells mobilized [Ca ]i (Fig. 6C). Following the Ca 2+ depletion, the translocation of STIM1 We next focused on the early signalling which could be molecules to endoplasmic reticulum–plasma membrane activated by the leukotoxin in the plasma membrane and junctions activates the Orai1-formed CRAC channels; induce the release of Ca 2+ from acidic stores. Lysosomes simultaneously the formation of STIM1 puncta can be release Ca 2+ through two-pore channels that are observed using fluorescence labelling methods (Walsh opened by nicotinic acid adenine dinucleotide phosphate © 2012 Blackwell Publishing Ltd, Cellular Microbiology S. aureus leukotoxins intracellular Ca 2+ signalling 9 Fig. 7. Distribution of STIM1 and Orai1 molecules of the SOCE complex before and after incubation of neuronal cells with 4 nM HlgC/HlgB-Alexa 488. Confocal acquisition images of cerebellar neurones labelled with the anti-Orai1goat polyclonal antibody D15 (A1, B1) and the anti-Stim1 mouse monoclonal antibody A-8 (A2, B2). Panels A3 and B3 correspond to merged images with Hoechst 33258-labelled nuclei. Cells in row A were fixed prior to incubation for 10 min in the presence of 4 nM leukotoxin HlgC/HlgB; cells in row B were fixed after incubation with the toxin. (NAADP), an intracellular messenger produced by the CD38 antibody or Alexa 594-conjugated Cholera toxin-B ADP-ribosyl cyclase CD38 (Zhu et al ., 2009; Cosker subunit (Fig. 8C and D). The anti-CD38 labelling was et al ., 2010). Although efficient antagonists of CD38 activ- observed at the edge of the cells, showing a dotted aspect ity are not currently available, it is known that b-NAD that partially appeared superimposed with leukotoxin rapidly induces the internalization of the enzyme and labelling. However, the fluorescence of Alexa 488-HlgC/ reduces its activity (Zocchi et al ., 1999). Hence, we HlgB leukotoxin appears located preferentially in the assessed the potential effect of preventing granular neu- cytosol. Labelling with the Alexa 594-Cholera toxin, which rones from being activated by the leukotoxin by incubating binds membrane gangliosides, surrounded the cells and the latter for 10 min in the presence of 5 mM b-NAD prior was clearly segregated from the Alexa 488-leukotoxin to the addition of the toxin. In four independent experi- fluorescence. These results suggest that the leukotoxin ments, only 26% of recorded cells showed a transient rise may be partially trapped in intracellular organelles 2+ in [Ca ]i, along with a significant reduction in mean peak together with CD38. amplitude (191 Ϯ 14 nM versus 444 Ϯ 18 nM; n = 23, Taken together, the results presented here can be P < 0.001) and a delayed latency (1147 Ϯ 31 s versus better understood if confronted to the mechanism pro- 537 Ϯ 35 s; n = 23, P < 0.001) (Fig. 8A). Similar effects posed for the endolysosomal Ca 2+ signalling in a variety of were observed using 2.5 mM NADP or 1 mM NAADP cells (Morgan et al ., 2011). It is suggested that NAADP added 10 min prior to the toxin (Fig. 8A). NAADP likely initiates a multistep process by releasing Ca 2+ from acidic induces a desensitization of the lysosomal two-pore stores, which triggers the further release of reticular Ca 2+ channel (Zhu et al ., 2009) that may prevent leukotoxin and finally causes the activation of CRAC channels. The signalling. Challenge of the granular neurones by 5 mM HlgC/HlgB leukotoxin could initiate the same signalling 2+ b-NAD or 2.5 mM NADP did not produce [Ca ]i peaks, but pathway, as we suggest through the schematic represen- 2+ induced a slight increase in [Ca ]i level. tation of Fig. 9. The enzymatic properties of CD38 can be altered by antibodies directed against its C-terminal portion (Ferrero et al ., 2004; Hara-Yokoyama et al ., 2008). When cells Discussion where incubated for 20 min in the presence of an anti- S. aureus leukotoxins are a critical threat to the CD38 C-terminal goat polyclonal antibody (M-19 antibody, nervous system Santa Cruz Biotechnology), the response to the leuko- toxin was significantly delayed (842 Ϯ 25 s versus Staphylococcus aureus has been shown to efficiently 378 Ϯ 27 s; n = 94, P < 0.001) along with a reduction in invade human brain microvascular endothelial cells, 2+ the peak of [Ca ]i (259 Ϯ 16 nM versus 430 Ϯ 18 nM; which results in tissue damage, brain abscesses and n = 94, P < 0.001) (Fig. 8B). meningeal inflammation (Pedersen et al ., 2006; Sheen Granular neurones incubated in Alexa 488-tagged et al ., 2010). We carried out this work in cerebellar granu- HlgC/HlgB leukotoxin (4 nM) were observed by confocal lar neurones to assess whether leukotoxins may cause microscopy after fixation and paired labelling the anti- damage to nervous tissue. Rat granular neurones have © 2012 Blackwell Publishing Ltd, Cellular Microbiology 10 E. Jover et al . 2+ Fig. 8. Leukotoxin-induced [Ca ]i movements in cerebellar neurones require ADP-ribosyl cyclase CD38 activity. A. CD38 internalization by addition of 5 mM b-NAD prior to adding leukotoxin HlgC/HlgB significantly reduced its effect. The results combine data from three independent experiments. Control line and boxes (101 cells); recordings of b-NAD pre-treated cells are the mean of the 25% responding cells. Recordings of cells pre-treated with 2.5 mM or 1 mM NAADP are also shown. The boxes represent control and 5 mM b-NAD recordings. B. Effect of incubation of granular neurones in the presence of the anti-CD38 goat polyclonal antibody prior to addition of 4 nM leukotoxin. The decrease in mean peak amplitude and the reduction in response latency were statistically significant ( P < 0.001). Control, 190 cells; CD38 antibody, 94 cells. C. Granular neurones labelled with HlgC/HlgB-Alexa 488 and Cholera toxin-B-subunit-Alexa 594 conjugated. D. Neurones labelled with HlgC/HlgB-Alexa 488 and anti-CD38 goat polyclonal antibody M-19 revealed by a donkey anti-goat Alexa 594-conjugated secondary antibody. been used for cellular studies on bacterial toxins while intracellular Ca 2+ rise and pore formation as it has been glutamate release assays have aided in investigating their observed in neutrophils through the introduction of muta- neurotoxicity (Foran et al ., 2003; Lonchamp et al ., 2010). tions in PVL (Baba Moussa et al ., 1999). Moreover, PVL The use of these cells allowed us to establish that differ- can confer an enhanced bactericidal capacity to human ent S. aureus leukotoxins can represent a serious threat neutrophils at very low concentrations, independently of to neurones by inducing a rise in free intracellular Ca 2+ the formation of a pore (Graves et al ., 2012). The activity and, as a consequence, the release of glutamate. The of the HlgC/HlgB leukotoxin is concentration dependent, 2+ increase in free [Ca ]i was also measured in DRG neu- which may suggest that minute amounts of toxin can be rones, highlighting the fact that the activity of leukotoxins sensed by neurones and induce intracellular signalling. A is not restricted to a single class of neurones. The neuronal early response to leukotoxin could have the g-haemolysin HlgC–HlgB was found to be the most positive outcome of initiating cellular protection (Aroian potent, both in cerebellar and in DRG neurones, inducing and van der Goot, 2007), conceivably in association with important cellular effects while preserving the integrity of noxious stimuli. the plasma membrane. The pharmacological manipula- Neurones preferentially use VOCC and ionotropic neu- 2+ 2+ tions, which can prevent free [Ca ]i movements through rotransmitter receptors as Ca influx pathways, while for the disruption of internal stores as well as acting on the neutrophils the SOCE complex is the main Ca 2+ signalling plasma membrane, together with the lack of permeability system. This difference could obscure the fact that both 2+ to ethidium bromide, parallel to the [Ca ]i increase, cells types are sensitive to the leukotoxin. Interestingly, strongly supports the notion that the formation of a pore is the Ca 2+ activated movement shown herein demonstrates not the first cellular effect induced by leukotoxins in a reasonable link between internal Ca 2+ stores and neurones. Cellular differences between neurones and membrane signalling proteins, through pathways which neutrophils may explain an expanded delay between are otherwise observed separately in neurones. The © 2012 Blackwell Publishing Ltd, Cellular Microbiology S. aureus leukotoxins intracellular Ca 2+ signalling 11 Fig. 9. Schematic representation of the multistep signalling mechanism triggered by g-leukotoxin HlgC/HlgB on neuronal cells. Upon binding (1) to a receptor complex, likely associated with lipid rafts, the bi-component leukotoxin triggers the formation of NAADP by ADP-ribosyl 2+ 2+ cyclase CD38. NAADP, acting on the two-pore Ca channels of lysosomes (2) elicits the release of Ca2 + into the cytosol. Free [Ca ]i can further be increased (3) through a Ca 2+-induced Ca 2+ release mechanism from reticular stores. Finally, reticular Ca 2+ depletion activates the Store-Operated Ca 2+ Entry complex formed by STIM1 and Orai1 (4). ADP-ribosyl cyclase CD38 has been detected in rat brain by leukotoxins in neurones. It is of interest to note that in (Yamada et al ., 1997; Ceni et al ., 2003), where the sig- mild S. aureus pathologies, such as chronic rhinosinusitis, nalling molecule NAADP has been proposed as a stimu- associated headaches and facial pain/pressure nega- lator of neurite outgrowth (Brailoiu et al ., 2005). The tively affect quality of life and impair daily activities release of Ca 2+ from either lysosomes or Ryanodine- (Singhal et al ., 2011). sensitive pre-synaptic stores has been shown to enhance the efficiency of neurotransmitter release (Collin et al ., 2005; McGuinness et al ., 2007). In cortical and hypocam- A multistep Ca 2+ signalling mechanism that could be pal neurones, differential regulatory roles of intracellular shared by other toxins Ca 2+ levels have been observed for STIM1 and STIM2 (Gruszczynska-Biegala et al ., 2011), and the depletion of An increase in free intracellular Ca 2+ has already been Orai1 impairs the rhythmic firing flight motoneurones in described as an early response for the activity of different drosophila (Venkiteswaran and Hasan, 2009). A further pore forming toxins. Intracellular Ca 2+ movements are question will be to ascertain whether endogenous activa- regarded as a prerequisite for the pre-pore to pore tran- tor(s) would be able to stimulate this ‘leukotoxin signalling sition of membrane bound bi-component S. aureus leuko- multistep process’ in the nervous system. The future iden- toxins (Woodin and Wieneke, 1963; Staali et al ., 1998). tification of the molecular complex that binds the leuko- The a-haemolysin from Escherichia coli has been shown 2+ toxins will help to find the answer, as it will contribute to to induce [Ca ]i oscillations in renal epithelial cells that assess the consequences of the Ca 2+ imbalance caused are sensitive to L-type Ca 2+ channel blockers (Uhlen et al ., © 2012 Blackwell Publishing Ltd, Cellular Microbiology 12 E. Jover et al . 2000), although the contribution of the L-type channel of Ca 2+ from internal stores are now starting to be identi- was subsequently challenged (Koschinski et al ., 2006). In fied (Cosker et al ., 2010). The more potent of these mol- the present study, the pharmacological neutralization of ecules is NAADP, which is synthesized by the ADP-ribosyl voltage-operated Ca 2+ channels, including L-type chan- cyclase CD38 (Malavasi et al ., 2008) and activates the nels, as well as glutamate receptors revealed that these two-pore Ca 2+ channels from lysosomes (Zhu et al ., channels are not a primary pathway for the S. aureus 2009). Our various approaches in shutting down CD38 2+ leukotoxin-induced rise in [Ca ]i. However, the latency to activity by mimicking published protocols (Zocchi et al ., 2+ reach the [Ca ]i peak was affected by combinations of 1999; Ferrero et al ., 2004; Hara-Yokoyama et al ., 2008), drugs known to be active on voltage-operated channels, successfully reduced or suppressed the leukotoxin effect, which may suggest an influence of either the resting strengthening the probable relationship between leuko- 2+ 2+ [Ca ]i, or the membrane potential, on the kinetics of leu- toxin HlgC/HlgB binding and Ca release from acidic kotoxin activity. The vacuolating cytotoxin A (VacA) from stores. Summarizing the pharmacological manipulations Helicobacter pylori induces the oscillation of free cytosolic that alter the granular neurones response to HlgC/HlgB Ca 2+ through the mobilization of internal stores (De leukotoxin, Fig. 9A illustrates a multistep process evoked Bernard et al ., 2005). The leukotoxin HlgC–HlgB shares by the endolysosomal Ca 2+ signalling (Morgan et al ., the feature of mobilizing internal stores. Its activity was 2011). The cell membrane molecule (or molecules) that significantly inhibited when reticular Ca 2+ stores were bind the toxin needs to be identified; here is represented depleted. Even though the total reticular Ca 2+ load by a ‘schematic receptor’. The step ‘binding’ (1) must be 2+ remains low, and free cytosolic [Ca ]i slightly changes connected to the activation of the CD38 ADP-ribosyl upon blocking the SERCA pump with thapsigargin (Pinilla cyclase, since the toxin activity is significantly inhibited by et al ., 2005), the reticulum is an important link for the b-NAD acting on CD38 or the action of an anti-CD38- response process activated by the toxin. A drastic inhibi- specific antibody. Step 2 comprises the NAADP mediated tion was observed when H-ATPase, which contributes to activation of Ca 2+ release from the endolysosomal com- the refilling of acidic Ca 2+ stores, was blocked by Bafilo- partment, as suggested by the total inhibition caused by mycin A, or upon disruption of lysosomes by GPN. There- Bafilomycin A or GPN. The local release of Ca 2+ triggers fore, we propose that an initial release of Ca 2+ from the further Ca 2+ release from the thapsigargin-sensitive reticu- acidic stores may be followed by a Ca 2+-induced Ca 2+ lar store (step 3). The final step (4) involves the activation release from the endoplasmic reticulum. The Ca 2+ deple- of CRAC channels induced by the Ca 2+ reticular stores tion of the endoplasmic reticulum, sensed by the stromal depletion. interacting molecule STIM1, subsequently leads to the It would be of interest to determine whether other bac- assembly of Orai1 molecules to form the store-operated terial toxins mobilizing intracellular Ca 2+ initiate a related CRAC channel (Varnai et al ., 2009). Noteworthy, listeri- mechanism, regardless of other effects they may induce. olysin O, produced by Listeria monocytogenes , mobilizes Similarly to S. aureus bi-component leukotoxins that Ca 2+ from the extracellular milieu as well as from various evoke intracellular Ca 2+ rise in neurones, even though types of intracellular stores (Gekara et al ., 2007) whereas they are known to discriminate between target cells Streptolysin O, from Streptococcus pyogenes , induces (Menestrina et al ., 2003). Irrespective of their similarities, long-lasting intracellular Ca 2+ oscillations through the acti- the g-haemolysins HlgA–HlgB and HlgC–HlgB share their vation of the SOCE complex, which are modulated by the F-component, the S-components HlgC and LukS-PV can level of expression of STIM1 and Orai1 (Usmani et al ., compete for a same binding site (Prévost et al ., 1995; 2012). Another leukotoxin, secreted by Actinobacillus Meyer et al ., 2009), S. aureus leukotoxins share an initial 2+ 2+ actinomycetemcomitans, induces free [Ca ]i movements intracellular Ca mobilization which may induce a vari- if the molecules needed for its activity are previously able final outcome depending on the cell type or on the clustered into lipid rafts (Fong et al ., 2006). In the present specific toxin. A question to focus on will be how study, the inhibition of S. aureus leukotoxin activity bi-component bacterial toxins initiate cell signalling. It through LY294002 inactivation of PI3K and PI4K may is tempting to speculate about some multi-molecular suggest a similar prerequisite for leukotoxin HlgB/HlgC. system, which should integrate the characterized cellular However, the relationship between membrane PiP2 responses that are independent of the pore forming content and the regulation of SOCE (Korzeniowski et al ., ability, such as cytotoxicity (Genestier et al ., 2005; Zivko- 2009; Walsh et al ., 2009), could also be an alternative vic et al ., 2011), enhanced bactericidal capacity (Graves explanation for such inhibition. et al ., 2012) and Ca 2+ mobilization from acidic stores. The contribution of intracellular acidic stores as an early A comparative study of the responses of cultured step of Ca 2+ mobilization induced by a bacterial toxin is a neurones, alveolar macrophage and neutrophils to the novel finding. It is worth mentioning that signalling mol- S. aureus bi-component leukotoxins would be of valuable ecules linking plasma membrane receptors to the release interest. © 2012 Blackwell Publishing Ltd, Cellular Microbiology S. aureus leukotoxins intracellular Ca 2+ signalling 13 Experimental procedures pipettes of decreasing tip diameter. After removal of tissue debris, cells were centrifuged and the pellet resuspended in culture Ethic statement medium. Cells were seeded on either poly- L-lysine-coated (10 mg ml -1) 24-well plates (Corning) at a density of 3.5 ¥ 10 5 cells All experiments have been conducted using protocols designed per well for glutamate release studies, in 35 mm glass bottom according to the European and French guidelines on animal Petri dishes for microfluorimetry, or on 11-mm-diameter glass experimentation and approved by the direction of the Bas-Rhin coverslips for immunocytochemical studies. Cells were then veterinary office, Strasbourg, France; authorization number incubated at 37°C in a 5% CO -air humidified atmosphere. The 67-312 to E.J. 2 culture medium consisted of B27-supplemented Neurobasal medium (Gibco/Life Technologies) and 25 mM KCl, 2 mM Animals L-glutamine and penicillin/streptavidin antibiotics. Growth medium was further complemented with 1 mM insulin, 20 nM Sprague-Dawley rats were obtained from the Institut des Neuro- progesterone, 0.1 mM putrescine, 80 mg ml -1 transferrin and sciences Cellulaires et Intégratives (INCI) animal facilities 0.5 mM sodium selenite. Cultures were also supplemented with (French government agreement No. B-67-482-25). All proce- 5 mM cytosine b-D-arabinofuranoside 24 h after plating in order to dures regarding housing, feeding and sacrifice of the animals prevent glial proliferation; culture media were renewed every 3 were in accordance with approved European guidelines (No. days. Experiments were performed after 8-day differentiation in 86/609/CEE) on animal care and experimentation. vitro to allow neuronal stabilization of intracellular Ca 2+ stores (Mhyre et al ., 2000). Toxins Dorsal root ganglion neurones The S. aureus a-toxin, g-haemolysins HlgC/HlgB and HlgA/HlgB and the Panton – Valentine leukocidin LukS-PV/LukF-PV where Dorsal root ganglia from as many spinal levels as possible were purified as described previously (Werner et al ., 2002). The fluo- collected from euthanized P5 rats. The cells were dissociated rescent derivatives for HlgB were prepared as previously from the ganglia as described previously (Jover et al ., 2005). The described (Finck-Barbançon et al ., 1991). cells were seeded on poly- L-lysine-coated bottom-glass Petri dishes at a density of 1 ¥ 10 5 cells per dish. Culture media con- sisted of complete Neurobasal medium supplemented with Drugs and chemicals 100 ng ml -1 Nerve Growth Factor (Alomone Labs, Israel). The All standard chemicals, the glutamate receptor antagonists culture medium was renewed 4 days after plating. Experiments CNQX and APV, the calcium blockers and modulators nifedipine, were performed using neurones cultured for 4–7 days. dantrolene, 2-Aminoethoxydiphenyl borate D-AP5, Bafilomycin A1, Glycyl-1-phenylalanine 2-naphthylamide and the PI3Kinase antagonist LY-294002 were purchased from Sigma-Aldrich Glutamate release and quantification (Saint-Quentin Fallavier, France). N-[4-[3,5- Bis (trifluoromethyl)- Prior to toxin incubation or KCl stimulation, the medium was 1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxa- removed and the cell monolayer washed three times in Hanks mide (YM-59483) was from Tocris Bioscience (Bristol, UK). balanced salt solution (HBSS) pre-warmed at 37°C. The appro- The voltage-gated channel blockers Tetrodotoxin, w-Conotoxin priate toxin concentration was added for an incubation of 10 min. GVI-A, w-Agatoxin TK as well as ryanodine and thapsigargin For KCl stimulation, cells were maintained in the depolarizing were from Alomone Labs (Israel). Fura2-AM was from TEFLabs buffer (same as incubation buffer but with 60 mM KCl and 73 mM (Austin, TX), while Pluronic-127 and BAPTA-AM were from NaCl) for less than 10 s, before collecting the media for glutamate Molecular Probes/Life Technologies (Saint Aubin, France). Stock determination. solutions of the drugs were prepared in their appropriate solvent Glutamate cycling assay was based on the Amplex Red (ethanol, dimethyl sulfoxide or water) at 1000 ¥ the final working Glutamic Acid method (Chapman and Zhou, 1999). Typically, concentration. The Ca 2+-free bath solution used in selected reactions were performed in 96-well plates (Falcon). To 50 ml of experiments was prepared by omitting CaCl from the standard 2 samples was added an equal volume of reagent mix containing solution and adding 10 mM EGTA. 0.25 U ml -1 HRP, 0.08 U ml -1 glutamate oxydase, 0.5 U ml -1 glutamate pyruvate synthetase, 0.2 mM L-alanine and 100 mM Cerebellar granular neurones 10-acetyl-3,7-dihydroxyphenoxazine (AnaSpec, Fremont, CA) in 0.1 mM Tris-HCl buffer pH 7.4. The cycling reaction was allowed Cell culture media and reagents, enzymatic solutions and antibi- to proceed at 37°C for 30 min and the increase in resorufin otics were from Gibco/Life Technologies (Saint Aubin, France). fluorescence was measured using a Mithras LB 940 fluorescence Insulin, progesterone, putrescine and human apo-transferrin plate reader (Berthold Technologies, Thoiry, France) at 530 nm were from Sigma (Saint Quentin Fallavier, France). Cerebellar excitation and 590 nm emission. cortices from postnatal day 5 (P5) rats were incubated for 5 min Results are expressed as means Ϯ SEM from at least four at 37°C in trypsin 0.05%–EDTA 0.02% solution, followed by independent well measurements for each concentration of toxin. 20 min at 37°C in papain 10 U ml -1 and DNase (250 U ml -1). Glutamate determination was performed in triplicate. Statistical Enzymes were inactivated by adding fetal calf serum (10%) and significance was determined by one-way analysis of variance removed by three successive washes in culture media. The followed by a Student’s t-test using SigmaPlot for Windows tissue was mechanically dissociated through polished Pasteur Version 11.0. Differences were considered significant at P < 0.05. © 2012 Blackwell Publishing Ltd, Cellular Microbiology 14 E. Jover et al . Glucose-6-phosphate dehydrogenase pixel basis for each image pair. The calcium concentration was then estimated by the formula (Grynkiewicz et al ., 1985): Quantification of cell death was performed using the fluorescence-based micro-plate assay (Batchelor and Zhou, 2+ [Ca ] i =Kd ××β ( RR − min ) ( R max − R ) 2004) which measures glucose-6-phosphate dehydrogenase 2+ (G6PD) activity released into the media. The same supernatant where Kd is the dissociation constant of Fura-2 for Ca (924 nM, calculated in our cultures), b = (I380 max )/( I380 min ) collected for glutamate determination was also used for G6PDH Ϯ activity. Reactions were conducted in a 100 ml final volume (96- (2.08 0.03, n = 39). Values of Rmin and Rmax were well plates). Experimental samples were mixed 1:1 (v/v) with determined by periodical calibrations (Grynkiewicz et al ., reagent mix containing 15 mM resazurin, 2 mM Glucose-6- 1985). Phosphate, 0.5 mM NADP and 0.5 U ml -1 diaphorase in 100 mM Tris-HCl buffer, pH 7.5. The reaction was carried out at 37°C for Statistics 30 min and the increase in fluorescence was measured as described for the glutamate assay. Data are presented as box-plots with median, 10th, 25th, 75th and 90th percentiles and the outlying points; the mean line is also shown. Statistical significance was determined by Kruskal–Wallis one-way analysis of variance (SigmaPlot for Immunocytochemistry Windows version 11.0). Differences were considered significant The subcellular localization of leukotoxin in granular neurones at P < 0.05. was detecting using the HlgB subunit tagged with Alexa Fluor 488 C5 maleimide (Life Technologies). Cells were incubated at room Acknowledgements temperature for 1–10 min in the presence of 1 nM native or 5 nM tagged HlgC/HlgB. Neurones were then fixed in 4% (v/v) para- The authors are particularly grateful to Daniel Keller for the formaldehyde and 4% (w/v) sucrose in phosphate-buffered saline skilful preparation of leukotoxins. Thanks to Dr Sophie Reibel (PBS) for 15 min at room temperature and subsequently rinsed and her team at the animal facilities of the Institut Fédératif with PBS. Neurones were permeabilized and non-specific des Neurosciences IFR37/Strasbourg and to the Imaging epitopes were blocked in PBS buffer containing 0.1% (v/v) Triton facility of the IFR37. Special thanks to Dr Nancy Grant X-100, 1% (w/v) bovine serum albumin and 5% (v/v) normal goat (CNRS UPR3212, Strasbourg) for her valuable comments and serum (Chemicon-Millipore, Molsheim France) for 30 min prior to discussions. 2 h incubation at room temperature with antibodies diluted in the The experimental work was supported by grants from EA-4438 same buffer. The following primary antibodies were used: the and Scientific Council from the Université de Strasbourg, and anti-STIM1 (N-19) goat polyclonal antibody and A8 mouse mono- from UMR 3212. The funding agencies had no role in any of the clonal antibody; the anti-mouse CD38 (2Q1628) rat monoclonal study design, data collection and analysis, decision to publish antibody and M-19 goat polyclonal antibody (Santa Cruz Biotech- or preparation of the manuscript. None of the results obtained nology). The plasma membrane was labelled by the cholera toxin are part of a patent or commercial product. M.Y.T. and B.-J.L. B subunit conjugated to Alexa Fluor 594 (Life Technologies).After were supported by a doctoral fellowship from the Ministère de three washes in PBS, secondary antibodies tagged with Alexa l’Enseignement Supérieur et de la Recherche and from the Fon- 488 or Alexa 546 (Molecular Probes/Life Technologies, 1:1000) dation Fouassier respectively. E.J. and B.P. are permanent staff were incubated in PBS for 2 h at room temperature. 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(2009) Role of phosph- oinositides in STIM1 dynamics and store-operated calcium Additional Supporting Information may be found in the online entry. Biochem J 425: 159–168. version of this article: Werner, S., Colin, D.A., Coraiola, M., Menestrina, G., Monteil, H., and Prevost, G. (2002) Retrieving biological activity Fig. S1. The HlgC or HlgB subunits separately fail to induce from LukF-PV mutants combined with different S compo- glutamate release or free intracellular [Ca 2+] changes in granular nents implies compatibility between the stem domains of neurones. these staphylococcal bicomponent leucotoxins. Infect a. Dose–response curves of glutamate released by granular neu- Immun 70: 1310–1318. rones challenged for 10 min with the indicated concentrations of Wertheim, H.F.L., Melles, D.C., Vos, M.C., van Leeuwen, W., leukotoxin HlgC/HlgB ( ), HlgC subunit alone ( ) or HlgB subunit van Belkum, A., Verbrugh, H.A., and Nouwen, J.L. (2005) alone ( ). The green line shows the glutamate measured after a The role of nasal carriage in Staphylococcus aureus infec- 10 s pulse of 60 mM KCl depolarization. tions. Lancet Infect Dis 5: 751–762. b. averaged traces of records obtained in the presence of 2 nM Woodin, A.M. (1960) Purification of the two components of HlgC/HlgB leukotoxin (51 cells, blue line), 2 nM HlgC subunit leucocidin from Staphylococcus aureus . Biochem J 75: alone (60 cells, green line) and 2 nM HlgB subunit alone (31 cells, 158–165. dark red line). The boxes show the distribution values of [Ca 2+] Woodin, A.M., and Wieneke, A.A. (1963) The accumulation of peak amplitude (vertical box) and peak latency (horizontal box) calcium by the polymorphonuclear leucocyte treated with from the control records. staphylococcal leucocidin and its significance in extrusion Fig. S2. Confocal microscopy records of living granular neu- of protein. Biochem J 87: 487–495. rones incubated in 12 mM Ethidium bromide and Leukotoxin Yamada, M., Mizuguchi, M., Otsuka, N., Ikeda, K., and Taka- HlgC/HlgB. Optical sections of 1.68 mm of cells bathing in HBSS hashi, H. (1997) Ultrastructural localization of CD38 immu- containing Ethidium bromide (12 mM) were observed on a Leica noreactivity in rat brain. Brain Res 756: 52–60. SP5-II confocal microscope (fields of magnification ¥ 63, initial Zhu, M.X., Ma, J., Parrington, J., Calcraft, P.J., Galione, A., pixel size 79.4 nm). Images of 1024 ¥ 1024 pixels were acquired and Evans, A.M. (2009) Calcium signaling via two-pore using 500–545 nm (green channel), 589–750 nm (red channel) channels: local or global, that is the question. Am J Physiol and Nomarsky-DIC. Line 1: sample images after 3 min incubation Cell Physiol 298: C430–C441. in 12 mM Ethidium Bromide; 1A shows the living cells autofluo- Zivkovic, A., Sharif, O., Stich, K., Doninger, B., Biaggio, M., rescent signal in the green channel, 1B is the Ethidium Bromide Colinge, J., et al . (2011) TLR 2 and CD14 mediate innate red fluorescence signal and 1C shows the recorded cells. Line 2 immunity and lung inflammation to staphylococcal Panton- shows the same cells 15 min after the addition in the bath of Valentine leukocidin in vivo . J Immunol 186: 1608–1617. HlgC/HlgB-Alexa-488 tagged leukotoxin (4 nM final); 2A green Zocchi, E., Usai, C., Guida, L., Franco, L., Bruzzone, S., channel, 2B red channel and 2C Nomarsky-DIC image. Line 3 Passalacqua, M., and De Flora, A. (1999) Ligand-induced shows the same two cells after 20 more min incubation in 8 nM internalization of CD38 results in intracellular Ca 2+ mobili- final concentration of HlgC/HlgB-Alexa-488 tagged; 3A green zation: role of NAD + transport across cell membranes. fluorescence, 2B Ethidium Bromide red fluorescence and 2C FASEB J 13: 273–283. 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Mira Y. Tawk 1, Gaëlle Zimmermann-Meisse 1, Jean-Louis Bossu 2, Cristina Potrich 3, Tristan Bourcier 1, Mauro Dalla Serra 3, Bernard Poulain 2, Gilles Prévost 1* , Emmanuel Jover 1* 1 Université de Strasbourg - Hôpitaux Universitaires de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg, EA7290 Virulence Bactérienne précoce. Institut de Bactériologie, 3 rue Koeberlé ; F-67000 Strasbourg 2 INCI M UPR-CNRS 3212 ; Physiologie des réseaux de neurones, 5 rue Blaise Pascal, F- 67084 Strasbourg cedex 3 National Research Council of Italy - Institute of Biophysics & Bruno Kessler Foundation, via Sommarive 18, 38123 Trento, Italy * Corresponding authors (Emails: [email protected] ; [email protected] ) Running title: Leukotoxins internalization and Ca 2+ mobilization ABSTRACT . A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the pore-forming family. These findings raise the new question of whether the receptors are normally activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureus Panton-Valentine Leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement-derived peptide, their activity was explored on C5aR-expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected cells and initiated mobilization of intracellular Ca 2+ . HlgC/HlgB required the presence of robust intracellular acidic Ca 2+ stores in order 2+ 2+ to evoke a rise in free [Ca ]i, while the LukS-PV/LukF-PV directly altered reticular Ca stores. Intracellular target specificity was conferred by the F-subunit formign the complex. Furthermore, internalization of C5aR with the two leukotoxin components (S- and F-subunits) was required for the 2+ initiation of [Ca ]i mobilization. Electrophysiological recordings on living cells demonstrated that LukS- PV/LukF-PV did not alter the membrane resistance of C5aR-expressing cells, which was confirmed in planar lipid bi-layers and in liposomes. The present observations suggest that part of the pore forming process occurs in distinct intracellular compartments rather than at the plasma membrane. Keywords : Staphylococcus aureus , Panton and Valentine Leukocidin, HlgC/HlgB, human neutrophils, Fura-2, liposomes, electrophysiological recordings, immunocytochemistry, confocal microscopy. Author Summary Staphylococcus aureus virulence is achieved by a large variety of cell-threatening activities carried out by a wide range of released factors. Among these, bi-component pore-forming leukotoxins are exhibiting greater sophisticated effects than previously reported. Two Staphylococcus aureus leukotoxins bind the C5a complement-derived peptide receptor, divert its internalization process and release intracellular Ca 2+ from specific internal stores. The Panton and Valentine leukotoxin induces the release of Ca 2+ from reticular stores whereas HlgC/HlgB, which acts more swiftly, triggers Ca 2+ release from acidic stores. While generally recognized to complete cell lysis through the formation of a pore in the plasma membrane, LukS-PV/LukF-PV is unable to alter the membrane resistance of C5aR-expressing cells or of planar lipid bilayers. Conversely, HlgC/HlgB can form pores in bilayers as well as alter cell membrane resistance, although its pore-forming activity competes with internalization process in the cells. The HlgB subunit determines the aptitude of the two associated components to insert and disrupt membranes. These newly-revealed properties thus provide a novel understanding of physiological effects observed for sublytic concentrations of leukotoxins. INTRODUCTION Staphylococcus aureus has the ability to cause a large variety of human infections, from mild boils and subcutaneous abscesses to severe toxic shock syndrome, necrotizing pneumonia and sepsis, among others [1]. The bacterium colonizes mucosal and skin surfaces and escapes host immunity through the production of adhesion proteins [2,3], superantigens [4,5], cytolysins and pore-forming toxins [6,7]. Of the latter, -toxin and the -lysin assemble as single-component polymers to constitute a pore [8,9,10]; the -toxin is a neutral sphingomyelinase that lyses erythrocytes [11], while the other toxins are members of the -barrel pore-forming leukotoxin family and act through the combination of two subunits commonly known as the fast- (F) and slow-eluted (S) components [12]. To date the latter group includes six S. aureus class-S and five class-F proteins naturally assembling as: "-Hemolysins HlgA/HlgB or HlgC/HlgB, the Panton MValentine Leukocidin LukS-PV/LukF-PV, $%&'($%&)R -PV, LukE/LukD and LukA/LukB [13,14], which significantly differ in their specific cell targets [7,15,16]. The subunits of the -barrel leukotoxins are secreted as separate molecules, which will act synergistically to alter a target cell [17]. It is currently acknowledged that the class-S component binds to +,O%./0%1, receptor P and that the subsequent binding of the F-subunit initiates the assembly of a pre-pore structure [18]. The class-S components LukS-PV and HlgC associate with human neutrophils by binding a membrane molecule expressed during differentiation to the metamyelocyte stage [19,20]. In addition, a link has been suggested between the toxin binding to the receptor and the activation of a Ca 2+ conductance, which allows the oligomerization of S- and F-subunits and the formation of hetero- octameric pores [21,22,23,24]. However, the characterization of intracellular Ca 2+ movements induced by HlgC/HlgB [25] and the demonstration of human neutrophils activation by sublytic concentrations of LukS-PV/LukF-PV [26], both challenge the view of the formation of a membrane pore after activation of a Ca 2+ conductance. In the past few years, various active membrane proteins have emerged as receptors for S. aureus virulence factors. The ADAM-10 metalloproteinase is the predominant high affinity receptor for -toxin. ADAM-10 allows the initialization of the sequence of events by which the toxin is transformed into a cytolytic pore [27,28]. The CCR5 G protein coupled receptor is deemed to be necessary and sufficient for the killing of T cells, macrophages and dendritic cells by leukotoxin LukE/LukD, [29], although when targeting neutrophils, the same leukotoxin binds the chemokine receptors CXCR1 and CXCR2 [30]. Notwithstanding the above, the identification of steps leading to cell lysis and the process of pore formation after binding of the receptor remain elusive events. LukS-PV/LukF-PV-induced lung inflammation has been suggested to be mediated through its binding to the CD14/TLR2 complex [31], although this observation has recently been challenged by data showing that the human complement receptors C5aR and C5L2 act as host targets for LukS-PV, mediating both toxin binding and cytotoxicity [32]. Herein, we confirm that the HlgC/HlgB and the Panton MValentine Leukocidin both bind to human neutrophils and to the stably-transfected U937-C5aR cell line. Conversely, their binding can be antagonized by the C5a peptide and their activity modified by the synthetic C5aR antagonist W-54011 [33]. Moreover, after binding to C5aR, the toxins trigger a rise in intracellular Ca 2+ , which differs from that of C5a-induced Ca 2+ mobilization. Notably, particular intracellular Ca 2+ -releasing compartments are related to each toxin, as suggested by the initial depletion of distinct Ca 2+ stores preventing the 345/.R6,177183 . This compartment-associated Ca 2+ specificity is supported by the F-subunit, since the LukS-PV/HlgB complex initiates Ca 2+ release from acidic stores as is the case of HlgC/HlgB while the 2+ HlgC/LukF-PV complex initiates a rise in free [Ca ]i from the same compartment as LukS-PV/LukF- PV. Considering that both LukS-PV/LukF-PV and HlgC/HlgB are endocytosed shortly after binding of the F component, our results suggest that part of the pore forming process possibly occurs in distinct intracellular compartments rather than at the plasma membrane. RESULTS Luk S-PV/L uk F-PV and HlgC/HlgB bind to the C5a Receptor. Previous work from our group showed that the leukotoxin HlgC S-subunit competes for the same binding site as the LukS-PV S-subunit of LukS-PV/LukF-PV [34]. The human complement receptor C5aR has been proposed to act as the host target of LukS-PV/LukF-PV, mediating both toxin binding and cytotoxicity [32]. In light of the above, binding properties of both leukotoxins S-subunits were verified on transfected U937-C5aR cells and compared with the affinity association constants in human neutrophils. The results, summarized in table 1, show that the affinity of the two leukotoxins for C5aR was highly similar in natural neutrophils and in transfected cells. Moreover, the presence of the natural ligand C5a or the antagonist W-54011, did not affect the binding properties of the leukotoxins. Non transfected cells U937 were unable to bind either of the toxins. Although the affinity of the toxins for the receptor was not affected by the presence of well-defined C5aR ligands, maximum binding was nevertheless reduced as summarized in table 2. The same concentrations of C5a (1.2 nM) or W-54011 (20 nM) reduced the binding of LukS-PV/LukF-PV by 55 % in neutrophils and by 30 % in transfected cells. Interestingly, the binding of HlgC/HlgB was slightly increased in the presence of W-54011. The high affinity antagonist of the C3a receptor, SB- 290157 [35], had no significant effect on leukotoxins binding. Both L uk S-PV/L uk F-PV and HlgC/HlgB mobilize intracellular Ca 2+ upon binding to C5aR. The association of C5aR to its natural agonist C5a leads to the activation of the trimeric G !i2 " complex, which in turn triggers phosphoinositide 3-kinase (PI3K) activity, amplifies the canonical phospholipase C 2-PI3K pathway and, finally, impinges on the release of reticular Ca 2+ [36,37]. 2+ Herein, free [Ca ]i movements were recorded in C5aR-expressing cells (human neutrophils and transfected U937 cells) in order to compare the activity of the C5a peptide with that of the two 2+ leukotoxins. We first verified the failure of isolated S-components to induce [Ca ]i elevation (figure 1A: 2.5 nM LukS-PV, orange trace; 5 nM HlgC, clear blue trace). When cells were challenged with concentrations slightly above the dissociation constant (K d), all three active molecules induced a 2+ 2+ variation in free [Ca ]i, although differing in shape, latency and buffering to resting [Ca ]i values 2+ (figure 1, A and B). The mobilization of [Ca ]i due to 1.2 nM C5a was fast, transient and returned to 2+ the initial resting value within a few minutes (figure 1A and 1B, green trace). The free [Ca ]i increase triggered by 0.5 nM HlgC/HlgB began after a short latency, reached a maximum at a slow rate and decreased to a new steady state value above the initial resting concentration (figure 1A and 1B, dark 2+ blue trace). Finally, the rise in free [Ca ]i induced by 0.25 nM LukS-PV/LukF-PV was slow to start and gradually reached a long lasting plateau (figure 1A and 1B, red trace). These observations suggest that C5aR occupancy by each active molecule triggers activation of a distinct Ca 2+ release mechanism. As a result, and to clarify how C5aR contributes to the leukotoxin 2+ effect, [Ca ]i changes were recorded in the presence of the small molecules W-54011 (20 nM) or SB- 290157 (1µM), well-known antagonists of C5aR and C3aR respectively [38,39]. The effect of 1.2 nM C5a in neutrophils was completely abolished by the presence of 20 nM W-54011 (figure 1C, red trace) while reduced to 46.5 % ± 13.1 (n=3) in U937-C5aR cells (figure 1D, red trace). The presence of 1 µM SB-290157 slightly sharpened the C5a response in neutrophils, reducing the area under the curve by 19.1 % ± 16 (n=3) (figure 1C, green trace). In the transfected cells, SB-290157 increased the amplitude of the C5a response (area under curve: +18.47 % ± 7.3, n=3) (figure 1D, green trace). When cells were pre-incubated with 1 µM thapsigargin (a SERCA inhibitor) or 1 µM ionomycin (a reticular Ca 2+ ionophore), C5a signaling was completely abolished, both in neutrophils and in transfected cells, thus confirming the reticular origin of the Ca 2+ increase (figures 1C and 1D). As expected, non-transfected U937 cells were entirely unaffected by the presence of 12 nM C5a, 0.1 µM LukS-PV/LukF-PV or 0.1 µM HlgC/HlgB (figure 1D insert). The C5aR antagonist W-54011 (20 nM) reduced the LukS-PV/LukF-PV effect by 71.7 % ± 7 in human neutrophils (n=3) (figure 1E, red trace) and by 34.9 % ± 15 in U937-C5aR cells (n=3) (figure 1F, red trace). A reduced latency of the LukS- PV/LukF-PV effect was observed in the presence of 1 µM SB-290157 with a non significant alteration on the area under the curve, both in neutrophils (figure 1E, green trace) and in U937-C5aR cells (figure 1F, green trace). Surprisingly, 20 nM W-54011 amplified the activity of HlgC/HlgB up to 149.3 % ± 23 of baseline level (n=3) in neutrophils and up to 181.3 % ± 23 (n=3) in U937-C5aR cells (figure 1G and 1H, red trace). A non significant reduction of the effect was always observed however in at least 3 independent experiments in the presence of 1 µM SB-290157 (figures 1G and 1H, green trace). These results strengthen the notion of C5aR fostering the cellular activity of the two leukotoxins, although the outcome of their effects is significantly different in addition to being divergent from C5a activity. Hence, given that the activity of leukotoxins is reduced but not abolished in the absence of extracellular Ca 2+ (supplementary figure 1), we hypothesized that the increase in free 2+ 2+ [Ca ]i may stem from the release of Ca from an unidentified intracellular compartment. This concept is also strengthened by the confirmation that a commonly-used test to assess membrane pore formation (i.e. ethidium bromide diffusion into the cell) was only achieved when cells were bathed in a Ca 2+ -free buffer [20] (supplementary figure 2). The leukotoxins HlgC-HlgB and LukS-PV/LukF-PV induce a rise in intracellular Ca 2+ from diverse intracellular compartments In a previous study, we had described the effect of HlgC/HlgB in rat granular neurons, where the resulting complex triggers the release of Ca 2+ from intracellular acidic stores [25]. Herein, a similar pharmacological analysis was conducted to identify putative Ca 2+ stores targeted by staphylococcal leukotoxins in human neutrophils. In order to determine their participation, established Ca 2+ compartments [40] were pharmacologically altered, namely: i) the reticular store, which releases Ca 2+ through IP3 and ryanodine receptors, was emptied through the use of the SERCA blocker thapsigargin [41], ii) the action of ionomycin enabled to reveal a complementary neutral compartment [42] and iii) the contribution of acidic organelles was tested through the action of bafilomycin A1 (a V- type H +-ATPase inhibitor) or glycyl-phenylalanine 2-naphthylamide (GPN), a cathepsin C substrate, which drives the blockade of lysosomes by altering their osmolarity [43]. As indicated above, the effect of C5a can be prevented by the presence of 1µM thapsigargin or ionomycin in the bath (figure 1C and D), which confirms the expected release of Ca 2+ from reticular stores [44,45]. In neurons, HlgC/HlgB activity impinges on the acidic Ca 2+ stores, which release Ca 2+ through a nicotinic acid adenine dinucleotide phosphate (NAADP)-activated mechanism [25]. Herein, 2+ in the presence of the NAADP competitor Ned-19 [46], the free [Ca ]i change due to HlgC/HlgB was increased by 159.3 % ± 18 (figure 2A1) whereas the LukS-PV/LukF-PV effect was unaltered in the presence of the drug (figure 2A2), hence supporting a link between HlgC/HlgB and a specific acidic + 2+ store. Moreover, H -ATPase blockade by bafilomycin A reduced the rise in [Ca ]i due to HlgC/HlgB by 66.6 % ± 21 (figure 2B1) while slightly accelerated the LukS-PV/LukF-PV response with no modification of the area under the curve (figure 2B2). Similarly, the alteration of lysosomal activity by GPN reduced the effect of HlgC/HlgB to 61.5 % ± 10 (n=3) (figure 2C1) but did not alter the LukS- PV/LukF-PV effect (figure 2C2). The presence of 1 µM thapsigargin (figures 2D1 and 2D2) or 1 µM ionomycin (figures 2E1 and 2E2) significantly reduced the effect of both leukotoxins. The drop in HlgC/HlgB effect due to the blockage of SERCA by thapsigargin reached 59.9 % ± 2 (n=3) of the maximum and 55.7 % ± 8 (n=3) for LukS-PV/LukF-PV. Ionomycin completely abolished the effect of both toxins on internal stores. Finally, the pyrazole derivative YM-58483, a specific store-operated Ca 2+ entry (SOCE) blocker [47], inhibited HlgC/HlgB activity to 71.9 % ± 17 (n=3) (figure 2F1) whereas it did not alter the effect of LukS-PV/LukF-PV, although the latency time was shortened (figure 2F2). 2+ All of these effects were similarly expressed when comparing maximum [Ca ]i ratios between control and drug-treated cells as shown in table 3. These results confirm that, acting early through acidic intracellular compartments, HlgC/HlgB activity is similar in human neutrophils and in rat cerebellar neurons [25]. LukS-PV/LukF-PV, however, appears to target neutral stores leaving the acidic stores unaltered. Nevertheless, in order to understand how two leukotoxins which bind the same receptor differ with regard to the release of intracellular Ca 2+ emanating from different compartments, we next focused our attention to the F-Subunits and their ability to target these various compartments. The F subunit of leukotoxins determines the intracellular compartment releasing Ca 2+ . Given that the two S-subunits, which directly associate with C5aR, can form active compounds with either LukF-PV or HlgB [48], the cellular activity of atypical subunit associations was verified in order to discriminate the specific contribution of the F-subunit to Ca 2+ release. When 1.5 nM HlgC 2+ complemented with 15 nM Luk-F-PV was added to neutrophils, a delay in the increase in free [Ca ]i was observed when compared with HlgC/HlgB (0.5 nM each), with an overall response profile similar to the usual reaction to LukS-PV/LukF-PV (figure 3A, compared to figure 1A blue and red traces). 2+ Moreover, when 0.5 nM LukS-PV was complemented with 1 nM HlgB, the free [Ca ]i increase occurred much faster than the effect observed with LukS-PV/LukF-PV (0.25 nM for each subunit) (figure 3B, compared to figure 1A blue and red traces). Furthermore, the activity of HlgC/LukF-PV was unaltered in cells incubated in the presence of GPN (figure 3C) as previously observed for the usual LukS-PV/LukF-PV response (figure 2C2). Finally, the association of LukS-PV with HlgB presented a sensitivity to GPN similar to that of HlgC/HlgB association (figure 3D, Table 3). These results strengthen the notion of a Ca 2+ -activating specificity displayed by leukotoxins and defined by the F- subunit, in addition to suggesting the need of a particular environment for each F-subunit to act accordingly. Only HlgB confers pore forming capabilities to the leukotoxin complex. The pharmacological sensitivity that differentiates cell responses to each leukotoxin suggests that they also differ in the bearing of pore formation. We therefore investigated pore formation in planar lipid bilayers [49], in large unilamellar vesicles [48] and through determination of membrane resistance in living cells [50]. The S- and F-subunits of the leukotoxins were added in various associations to the cis-side of stable bilayers comprised of diphytanoyl-phosphatidylcholine (DPhPC) after which the current between the two separate chambers was measured. The HlgC/HlgB leukotoxin formed pores with a unitary conductance of 175.6 ± 15.8 pS (n=280), which is consistent with previously published values [49,51]; the LukS-PV/HlgB association also formed membrane pores of 184.8 ± 19.5 pS (n=16) unitary conductance (figure 4A and 4B). Conversely, the LukS-PV/LukF-PV and the association of HlgC with LukF-PV never assembled as transmembrane pores in this membrane lipid composition. The threshold concentration required to establish a pore was obtained in large unilamellar vesicles (100 nm in diameter), prepared with a 1:1 mixture of dioleoylphosphatidylcholine/cholesterol (DOPC/Ch), and containing 80 mM calcein (pH 7.0). The calcein-related fluorescence released from vesicles enabled to reveal the pores formed by the various associations of leukotoxin subunits. HlgC/HlgB triggered calcein release with a half-concentration effect of 0.5 nM while the combination of LukS-PV and HlgB also generated calcein release with a half concentration effect above 1.5 nM. Conversely, both the LukS-PV/LukF-PV and the HlgC/LukF-PV associations at high concentrations (100 nM) failed to induce calcein release from the vesicles (figure 4C). Lipid bilayers and liposomes do not include proteins; therefore, the above results apparently challenge the need of a specific protein receptor for the leukotoxins. Therefore, the trans-membrane resistance (voltage-clamp, whole cell configuration) was recorded in C5aR-transfected cells and in the original U937 cell line in order to detect changes due to leukotoxin treatment, while maintaining the membrane potential at M60 mV. Membrane resistance (Rm = V/I, according to Ohms law) was monitored by analyzing the trans-membrane current traces (I) corresponding to changes in trans-membrane potential (V). After a control determination of Rm, the studied leukotoxin was applied to the bath and Rm determined every minute for a minimum of ten minutes. No significant changes were observed in Rm of U937-C5aR transfected cells bathed in a 2 nM solution of LukS-PV/LukF-PV. Figure 5A1 shows a typical example while figure 5A2 summarizes eight independent experiments before and after LukS- PV/LukF-PV addition. In contrast, HlgC/HlgB induced an abrupt decrease in membrane resistance after a 5-min incubation of the cells in the presence of 2 nM of the toxin. Figure 5B1 illustrates an example of a cell treated with HlgC/HlgB while figure 5B2 depicts the resting membrane resistance values before and after addition of the toxin for eight independent cells. Furthermore, certain cells incubated in the presence of the combination of subunits LukS-PV and HlgB (4 nM each) also generated changes in membrane resistance of C5aR-expressing cells. Figure 5C1 illustrates a responding cell while figure 5C2 summarizes the results of resting membrane resistance recordings. Concentrations up to 10 nM HlgC/HlgB failed to induce any modification in membrane resistance in U937 cells (figure 5B1). These results highlight the need of a different environment for each F-subunit in order to efficiently induce the surge in free intracellular Ca 2+ into the cytosol of a cell expressing the appropriate receptor for the S-subunit. Both HlgC/HlgB and LukS-PV/LukF-PV are internalized by neutrophils. Altogether, the above results suggest that the association of either LukS-PV or HlgC to C5aR fails to achieve intracellular Ca 2+ movements while the association of the F-subunit to the complex initiates the release of Ca 2+ from diverse internal compartments. To achieve this, the internalization of C5aR in a complex with leukotoxins could provide the latter the possibility of triggering Ca 2+ release from internal stores. To verify this hypothesis, specific antibodies raised against each S-subunit were used to observe leukotoxin location in human neutrophils following various treatments. Cells incubated in the presence of leukotoxins were fixed and fluorescently stained with the appropriate antibodies, together with the B-subunit of Cholera toxin (Alexa-488 labeled) to label the plasma membrane. A Leica SP5-II confocal microscope was used to acquire single optical slices of random areas from different preparations. The proportion of cells showing an intense labeling of the membrane was determined for S-subunits alone (figure 6A1: 2 nM LukS-PV and figure 6B1: 2 nM HlgC); cells presenting degradation (condensed nucleus) were discarded from the analysis. The labeling after addition of the corresponding F subunit (figure 6A2: 2 nM LukS-PV/LukF-PV and figure 6B2: 2 nM HlgC/HlgB) showed a translocation of membrane labeling towards the cytosol. A reduction in the proportion of cells with intense membrane labeling was also observed; from 82 % for LukS-PV alone to 52 % after 5 min in the presence of LukS-PV/LukF-PV and 31 % after 10 min. The labeling of cells treated with the HlgC/HlgB subunits followed a similar distribution pattern. However, cell degradation occurred more rapidly than during LukS-PV/LukF-PV incubation with plasma membrane degradation appearing first, as opposed to condensation of the nuclei. Clearly-damaged cells were not included and cells were never incubated for more than 5 min in the presence of the two components in Ca 2+ - containing buffer. Nevertheless, a significant reduction in membrane-labeled cells was already present after a 5 min incubation with the toxin (figure 6C2). Conversely, cells incubated with the toxins in a Ca 2+ -free buffer displayed an intense labeling of the plasma membrane suggesting an absence of internalization (figure 6A3: 2 nM LukS-PV/LukF-PV and figure 6B3: 2 nM HlgC/HlgB). Discussion It is widely accepted that staphylococcal leukotoxins form pores on particular lipid bi-layers or in unilamellar vesicles [52,53]. However, they specifically target certain cells of the myeloid lineage, while remaining harmless in other cells [19,20]. Moreover, of the two leukotoxins HlgC/HlgB and LukS- PV/LukF-PV, which have been described as recognizing the same membrane receptor on human neutrophils [16], only HlgC/HlgB was able to form pores in lipid bi-layers [48]. This divergence is partially clarified by the finding of human complement receptors C5aR and C5L2 as mediators of LukS-PV/LukF-PV action in neutrophils [32]. C5aR also acts as a receptor for HlgC/HlgB and, by comparing its activity with that of LukS-PV/LukF-PV, we show herein that both leukotoxins induce a rise in intracellular free Ca 2+ by diverting the C5aR signaling process. The two leukotoxin S-subunits recognize and compete with each other for C5aR expressed in human neutrophils and in stably transfected U937-C5aR cells. The presence of 1.2 nM C5a reduces their binding without altering their affinity for the receptor, which is in agreement with published data highlighting an interaction of the S- subunit with the C5aR N-terminus [32]. There is also a second low-affinity site of interaction between the C5a C-terminus and the core region of the receptor which is critical for receptor activation and which can be challenged by the non peptide antagonist W-54011 [54]. The presence of 20 nM of the antagonist reduced the binding of LukS-PV while the binding of HlgC with the receptor remained unaltered, hence suggesting differences in the interaction of each leukotoxin with the receptor. Other known protein ligands for C5aR include the human ribosomal protein homodimer S19 (hRP S19) [55], a 17 kDa molecular chaperone of Gram-negative bacteria Skp [56] and the Chemotaxis Inhibitory Protein of S. aureus (CHIPS) [57]. C5a and hRP S19 act as chemo-attractants and secretagogues [55] while Skp behaves as a pure chemo-attractant factor [58] and CHIPS works as a potent antagonist [59]. Thus, the two leukotoxins expand the number of natural proteins that bind C5aR, while acting differentially on the receptor. It could be expected from binding data that isolated S- subunits act as antagonists on C5aR, which is never the case for complete leukotoxins known to stimulate neutrophils and to cause the secretion of chemotactic factors [17,60]. Isolated S-subunits 2+ clearly bound C5aR and the addition of a F-subunit was required to observe changes in [Ca ]i, which differed to changes due to C5a (figure 1A and 1B). However, combination of diverse concentrations of LukS-PV and LukF-PV led to the observation that 1 nM LukS-PV primed human neutrophils for the intracellular production of H 2O2 activated by fMLP or PMA, an effect reversed by LukF-PV in a concentration dependent manner (see figure 8 in [61]). Besides, the immunolabeling results showed that isolated S-subunits remain associated with the extracellular side of the membrane, while binding of both S- and F-subunits to C5aR brings about internalization of the toxin-receptor complex. All this, which is in keeping with the activation of an unexpected signaling pathway, calls for a careful attention to be given to a potential signaling triggered by the binding of a single leukotoxin S-subunit to C5aR. Herein we focused on intracellular Ca 2+ concentration changes occurring when C5aR is simultaneously engaged by both S- and F- leukotoxin subunits which, as shown from the pharmacological analysis, differed from receptor activation by C5a. In instances where the C5aR antagonist W-54011 was in direct competition with the leukotoxins, the outcome on LukS-PV/LukF-PV was one of inhibition, while outcome on HlgC/HlgB was rather that of potentiation. Aside from the 2+ dissimilar kinetics between the rise in free [Ca ]i induced by each toxin, the manipulation of distinct intracellular Ca 2+ stores also differentiates these leukotoxins with regard to their outcome. The alteration of acidic store integrity resulted in a decreased activity of HlgC/HlgB, while leaving LukS- PV/LukF-PV effect unaltered. Conversely, the modification of reticular stores influenced the effect of both leukotoxins. The block of SOCE markedly reduced the extent of HlgC/HlgB-induced Ca 2+ 2+ mobilization, while LukS-PV/LukF-PV-induced [Ca ]i movements were accelerated. Interestingly, the targeting of either one of the internal Ca 2+ stores relied on the F-subunit being present in the complex. As for HlgC/HlgB, the association of HlgB to LukS-PV required a robust acidic store to generate Ca 2+ changes, while the HlgC/LukF-PV combination necessitated a well Ca 2+ -loaded reticular compartment as in the case of LukS-PV/LukF-PV. As expected, homologous associations of leukotoxins resulting from individual genetic loci were the most potent, while heterogeneous associations worked at higher concentrations. The present results advocate for a reconsideration of the sequence of events leading to neutrophil lysis. It is generally acknowledged that after the first S-subunit binds to the receptor, the association of four pairs of S- and F-subunits form a pre-pore that will transform in a fully open pore if 2+ the latter is accompanied by an increase in free [Ca ]i [19,24]. However, the unveiled relationship between a particular F-subunit and a distinct intracellular compartment, aimed to release Ca 2+ , impairs such frame of thinking. The presence of HlgB, associated to a generic S-subunit, was sufficient to induce membrane resistance reduction in artificial lipid membranes and in C5aR expressing cells. Interestingly, the staphylococcal -toxin and HlgB are closely related in their structure, both containing a rim domain with a preponderance of exposed aromatic residues and a binding site for phospholipid head groups located in a cleft, which enables the fusion of these leukotoxins to lipids [62]. Likewise to -toxin, which needs the presence of ADAM10 to initiate self assembly and to engage cell membrane damage [27,28], HlgB likely begins altering membrane integrity through its high affinity association with the C5aR-S-subunit complex. Nevertheless, a subsequent internalization may buffer the process and ultimately transfer the membrane damaging ability of HlgC/HlgB to the endosome. A comparable mechanism it has been suggested for the -toxin, which could be internalize to prevent pore-complexes to be formed [63]. A similar intracellular progression is likely followed by the LukS-PV/HlgB association. However, inherent properties of LukS-PV/LukF-PV, due to LukF-PV association to a particular receptor [16], may preserve membrane integrity prior to accessing of the complex to a particular environment. This hypothesis, which calls for future studies to be confirmed, is consistent with i) the absence of pore formation in liposomes and cells carrying the receptor, ii) the internalization of LukS-PV/LukF-PV-C5aR complex and iii) the observed delay before the onset of free 2+ [Ca ]i modifications and pharmacological block. The need for leukotoxins of a receptor that transport them into the cell sheds further light on the fact that sublytic concentrations activate human neutrophils, but fail to lyse the latter [26]. This is of critical importance since low concentrations of staphylococcal leukotoxins are likely those who support certain forms of virulence in S. aureus infections. The challenge remains to understand what becomes of a cell having internalized a few leukotoxin molecules after their respective binding to C5aR. This particular threat not only addresses blood cells, but all tissues expressing the C5a receptor, particularly neurons [25,64,65]. Furthermore, a pathogenic intracellular mechanism may start clarifying the controversy about LukS-PV/LukF-PV as a major virulence factor and stresses the need for finding new antivirulence therapies [66,67]. MATERIALS AND METHODS Ethics statement. Buffy coats donators were adult volunteers that provided informed consent. Written !"#$%#&'()$'!"**%!&%+',-'&.%'O'0&),*1$$%2%#&'34)# )1$'+6'7)#8'97&4)$,"648:'34)#!%;'P:'(.1!.'=%>&'&.%' information confident. Drugs and chemicals. Thapsigargin, Ionomycin, Bafilomycin A1, Glycyl-1-phenylalanine 2- naphthylamide and Triton X-100 were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). (YM-59483) and (trans-Ned-19) were from Tocris Bioscience (Bristol, United Kingdom). (W- 54011) and (SB-290157) were from Calbiochem (EMD Millipore, MA, United States) while Fura-2 acetoxymethyl ester (Fura-2/AM) was from Molecular Probes-Invitrogen (Fisher Scientific, Illkirch, France). Human polymorphonuclear cell (hPMN) preparation. PMNs were prepared from buffy coats obtained from healthy donors of either sex within 24 hours after blood donation (Etablissement Français du Sang, Strasbourg, France) as previously described [68]. Undifferentiated U937 and U937-C5aR cell cultures. Undifferentiated U937 and U937-C5aR cells, which respectively do not express or stably express the C5aR receptor [69], were a generous gift from Pr. J.A. van Strijp (Utrecht University, The Netherlands). Cells were cultured as 50 ml cultures at 37°C under a 5% CO 2 atmosphere in 250 ml flasks in RPMI-1640 medium supplemented with 10% (v/v) decomplemented fetal calf serum (Life Technologies, Carlsbad, USA) and 0.1% (w/v) of both penicillin and streptomycin (InVitrogen, Paisley, UK). Leukotoxin purification. The Staphylococcus aureus HlgC/HlgB and the Panton and Valentine leukocidin LukS-PV/LukF-PV were purified as previously described [70]. Leukotoxin mutations and purification were performed as previously described [34]. Leukotoxin labeling. The mutated proteins HlgB S27C, LukS-PV G10C and LukF-PV-Cys were fluorescein-labeled to obtain HlgB*, LukS-PV* and LukF-PV*: 500 µL of 50 µM HlgB S27C, LukS-PV G10C or LukF-PV-Cys were mixed with a 20-fold excess of fluorescein 5-maleimide (Molecular Probes, Eugene, OR) and were incubated for 1h 30 min in darkness at room temperature in a total volume of 500 µl in 0.1 M HEPES, 0.2 M NaCl, 1 mM EDTA (pH 7.8). The mixtures were then desalted and the coupling yield for HlgB*, LukS-PV* or LukF-PV* determined through the ratio of fluorescein 9@AB nm = 80,000 cm -1 mol -1 ) concentration to protein concentration obtained from Bradford titration (Bio-Rad, Ivry sur Seine, France). Only leukotoxins with a coupling yield higher than 0.95 but less than 1 were used in this study. Spectrofluorimetry. Variations in intracellular free Ca 2+ levels were determined by recording the Fura-2 fluorescence contained in hPMNs as previously described [68]. Briefly, cells were loaded with 4 µM Fura-2 AM (FluoProbes ®, Interchim S.A., Montluçon) in EGTA buffer for 45 min in darkness, at room temperature. Thereafter, hPMNs were washed twice and then suspended (3.5 x 10 6 cells/ml) in EGTA buffer. Two ml of hPMNs were incubated for 5 min with 1.1 mM CaCl 2 in a 4-ml Polystyrene cuvette (1-cm light path). Variations in fluorescence intensity were recorded at 37°C with a dual- excitation spectrofluorometer (Deltascan; PTI, United States) operated in a ratio mode for Fura-2 at excitation wavelengths of 340 and 380 nm (slit widths, 4 nm) and an emission wavelength of 510 nm (slit widths, 4 nm). To compare the effects of different toxins in various conditions, the fluorescent signal was standardized by giving the background, or zero value, before the addition of the toxin and the 100 % value to the fluorescence measured after the addition of Triton X-100 (0.05 %, final concentration) which equilibrates the internal Ca 2+ concentration with that of buffer content. Results are expressed as percentage of the maximum, after subtraction of the background value. Planar lipid bilayer experiments . Solvent-free planar lipid bilayers were prepared by the apposition on both sides of a 0.1 mm )>%4&64%'2)+%'1#')'BC')2'&.1!='E%F*"#'$%>&62' (pretreated with pentane:n- hexadecane 20:1) of two monolayers of diphytanoylphosphatidylcholine (DPhPC, >99% pure from Avanti Polar Lipids) spread from a 5 mg/ml lipid solution in pentane, as described previously [71]. Bicomponent couples were added on one side only (called cis ) to preformed bilayers; the voltage potential was applied on the cis side while the trans side was grounded. Macroscopic currents were recorded by a patch clamp amplifier (Axopatch 200; Axon Instruments). A PC equipped with a DigiData 1200 A/D converter (Axon Instruments) was used for data acquisition. Current traces were filtered at 100 Hz and acquired at 500 Hz using the Axoscope 8 software (Axon Instruments). Measurements were performed at room temperature as described previously [72]. Permeabilization of lipid vesicles. Large unilamellar vesicles (LUV) comprised of a 1:1 molar ratio of dioleoyl phosphatidylcholine/cholesterol (DOPC/Ch) were prepared as previously described [48] by the extrusion method. A suspension of multilamellar liposomes prepared in the presence of 80 mM calcein (Sigma), pH 7.0, was extruded 31 times through polycarbonate membranes with 100 nm pores. Vesicle permeabilization was assayed in a fluorescence microplate reader (Fluostar Galaxy, BMG Germany) with excitation and emission filters at 480 and 540 nm, respectively. The two components of the complex, always at the same concentration, were serially 2-fold diluted in 10 mM Tris/HCl, 20 mM NaCl and 0.1 mM EDTA, pH 7.0 and dispensed into wells of a 96-well microplate, after which 100 )l of calcein-loaded LUVs were added at a final lipid concentration of 5 )M. The time course of calcein release was recorded for 45 min at room temperature and expressed as the increase in fluorescence owing to the dequenching of the released dye when diluted in the external medium. Toxin-induced permeabilizing activity was calculated as: P(%) = 100(Ft-Fi)/(Fm-Fi) [73] where Fi is the initial fluorescence prior to adding the toxins, Ft the value at time t (i.e. 45 min), and Fm the maximal value after addition of 1 mM Triton X-100. Spontaneous release of calcein was negligible. Electrophysiological recording of passive plasma membrane resistance in U937-C5aR cells. For single cell electrophysiological recordings, a suspension of 10 4 cells (30 µl) was transferred to poly-Lysine coated culture dishes; after 5 min, 2 ml of HBSS were added to the cells and the dish moved to the fixed stage of a Nikon upright microscope. Five-5 M ! micro-electrodes were pulled from borosilicate glass capillaries (Clark Electromedical Instruments, Pangbourne, England), and filled with a solution containing: 135 mM CsMeSO 4, 1 mM MgCl , 5 mM NaCl, 10 mM Hepes/CsOH, 4 mM Mg- 2 ATP and 0.4 mM Na 2-GTP, pH = 7.3 adjusted with CsOH. For the electrode approach, cells were observed through a 40x objective, DIC mode. Whole cell membrane currents were recorded at RT (22-24°C) using the patch-clamp technique (Axopatch 200A amplifier; Axon Instruments, Foster City, CA). Cell transmembrane potential was held at -60 mV, while passive transmembrane resistance (R m) was determined from membrane current changes ( I) induced by a series of hyperpolarizing and depolarizing square pulses ( V, 200 ms duration, with stepwise 5 mV increments), under voltage- clamp. This protocol was repeated once every minute throughout the duration of the experiment. The recorded signals were digitized at 20 KHz using a Digidata 1320 (Axon Instruments) and stored on a PC hard drive. Off-line analyses were performed using pClamp-8 (Axon Instruments) software, with previously filtered at 1.5 KHz. For each set of data (i.e. one protocol), R m was determined by averaging the V/ I values. Flow cytometry determinations. Flow cytometry data were obtained using a FACSort cytometer (Becton Dickinson, Le Pont de Claix, France) equipped with a 15-mW argon laser tuned to 488 nm. hPMNs were classically distinguished by forward and side light scatter. Binding of toxins on hPMNs was determined by measuring fluorescein fluorescence of labeled toxins. Fluorescence intensities were recorded in the FL1 channel (emission wavelength, 530 nm). The FACSort cytometer was adjusted in such a way that calibrated fluorescent microbeads (Immuno-Brite; Coulter Corporation, Hialeah, FL) displayed the same fluorescence intensity for each experiment. Resulting mean fluorescein fluorescence intensity was expressed in standardized fluorescence units. Immunocytochemistry. The subcellular localization of leukotoxins in hPMNs was detected using rabbit polyclonal antibodies independently raised against the HlgC, HlgB, LukS-PV and LukF-PV subunits [16,74]. Cells, attached to poly-lysine treated coverslips, were incubated at room temperature for 5 min in the presence of S-subunits alone or the complete leukotoxin and, after washing, were fixed in 4 % (v/v) paraformaldehyde and 4% (w/v) sucrose in phosphate-buffered saline (PBS) for 5 min at room temperature. Cells were then incubated for 30 min in 150 mM glycine in PBS, (for reducing tissue auto-fluorescence), permeabilized for 5 min in chloroform at M 20 °C and washed in ethanol before rehydration in PBS. Fixed cells were incubated overnight in blocking buffer (10 % FCS, 5mg/ml BSA in PBS), then transferred for 60 min in a 1 µg/ml PBS solution of specific primary antibodies, containing 5 % FCS and 1 mg/ml BSA,. After repeated washings, cells were incubated with Alexa-labeled secondary antibodies (Life Technologies) for 60 min, then washed and incubated 30 min in the presence of a 0.3 µg/ml solution of Alexa-488-labelled Cholera toxin B-subunit (Life Technologies) and 10 µg/ml Hoechst 32258 (Sigma) for labeling of the plasma membrane and cell nuclei, respectively. Coverslips mounted in Mowiol were stored at 4°C until observation with a Leica SP5-II confocal microscope. Statistical analysis Results are expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. Microsoft Excel 2007 was used to calculate the mean of each experimental condition and GraphPad Prism (version 5 for Windows) for preparation of graphs and statistical analysis. Statistical significance of the difference between differently immunolabelled groups was evaluated by one-way Anova followed by Tukey multiple-group comparisons. Differences were considered significant at p < 0.05. Acknowledgements: The authors are particularly grateful to Daniel Keller for constant support, recollection of unpublished results in frequent scientific discussions and the skilful preparation of leukotoxins. The authors thank the Institut Fédératif des Neurosciences IFR37/Strasbourg and the Imaging facility of the IFR37. Thanks to the team of C. Moog (Mécanisme de neutralisation du VIH par les anticorps, UMR-1110) for sharing human neutrophils from buffy coats. The authors also thank Pr. J.A. van Strijp (Utrecht University, The Netherlands) for generously providing U937-C5aR-transfected cells. The authors are extremely grateful to Dr P. Pothier (Sherbrooke, Québec Canada) for his excellent work straightening our defective English. References 8 LQ17 FD ^ _ S :].7CQHQHH%s :%`V%s IJ`VH 1QJs8 NV1 EJ$C J MVR 7 R 8 8 FQs V` TJ5 GVQ$.V$:J JA5 G:JVs. K5 HQQ@ M ^_ AR.Vs1QJ5 1J0:s1QJ :JR V0:s1QJ7 .V I:J7 `%JH 1QJs Q` .V s%``:HV ]`Q V1Js Q` S :].7CQHQHH%s :%`V%s 8 N: %`V RV01V1s M1H`QG1QCQ$7 7 R 8 8 PQ]Q0 L5 KQ0:Cs@1 J5 G`:JR1 G5 B:$JQC1 F5 AI1V0: MR ^_ T.`VVRD1IVJs1QJ:C H%I:J S@1J MQRVCs Q JRV`s :JR S :].7CQHQHH%s :%`V%s S@1J CQCQJ1<: 1QJ :JR IJ`VH 1QJ8 F`QJ 1V`s 1J III%JQCQ$7 8 8 L:]]1J E5 FV`$%sQJ AJ ^ _ G`:IR]Qs1 10V Q61H s.QH@ s7JR`QIVs8 T.V L:JHV IJ`VH 1Q%s D1sV:sVs 7 R 8 8 MHCQ`I1H@ JK5 :`1QQR JM5 SH.C1V0V` PM ^_ TQ61H S.QH@ S7JR`QIV :JR B:H V`1:C S%]V`:J 1$VJs7 AJ ]R: V8 AJJ%:C RV01V1 Q` M1H`QG1QCQ$7 7 R8 8 A`H.V` GL ^ _ S :].7CQHQHH%s :%`V%s 7 A VCCRA`IVR P: .Q$VJ8 CC1J IJ`VH D1s 7 R 8 8 :JRVJVsH. 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All traces of the left panels (panels A-C-E-G) are from recordings in human neutrophils and traces in the right panels (panels B-D-F-H) were obtained in U937-C5aR transfected cells. The effects of 1.2 nM C5a (green), 0.25 nM LukS-PV/LukF-PV (red) and 0.5 nM HlgC/HlgB (blue) are compared in panels A (human neutrophils) and B (U937-C5aR cells). Control recordings indicating the lack of activity of the isolated S-subunits are also shown in A (5 nM HlgC, light blue trace, 2.5 nM LukS-PV orange trace). The black trace in panels A and B depicts the recorded baseline in human neutrophils and U937-C5aR cells in the absence of toxins. Panels C to H show the response of human neutrophils (C, E and G) and U937-C5aR cells ( D, F and H) to 1.2 nM C5a, 0.25 nM LukS-PV/LukF-PV or 0.5 nM HlgC/HlgB (dark blue), in the presence of 20 nM W-54011 (red) or 1 µM SB-290157 (green). The light blue trace in panels C and D represent the response to 1 µM thapsigargin and the grey trace in panel D to 1 µM ionomycin. The inset in panel D shows the lack of response of the non-transfected U937 cell line to the presence of 12 nM C5a (orange), 0.1 µM LukS-PV/LukF-PV (green) or 0.1 µM HlgC/HlgB (red) and the control recording (blue). Results are normalized to the maximum Fura-2 2+ fluorescent signal obtained after bathing the cells in 0.05 % Triton X-100 to equilibrate [Ca ]i with that of the buffer concentration, after which data are expressed as percentage of this value. Figure 2: The origin of free intracellular Ca 2+ stems from separate compartments and depends on the identity of the leukotoxin. As indicated, the panels on the left show recordings of 0.5 nM HlgC/HlgB leukotoxin and panels on the right show recordings of 0.25 nM LukS-PV/LukF-PV in human neutrophils (blue traces in all cases). The red traces depict recordings in cells incubated with various drugs prior to addition of the corresponding leukotoxin: panels A 1-2 are for the NAADP antagonist Ned19 (100 µM), panels B 1-2 the H +-ATPase blocker bafilomycin (100 nM), panels C 1-2 the lysosomal disrupter glycyl- phenylalanine 2-naphthylamide GPN (50 µM), panels D 1-2 1 µM thapsigargin, panels E 1-2 1 µM ionomycin and panels F 1-2 the SOCE blocker pyrazole derivative YM-58483 (10 µM). Results are normalized to the maximum Fura-2 fluorescent signal obtained after bathing the cells in 0.05 % Triton 2+ X-100 to equilibrate [Ca ]i with that of buffer concentration, then expressed as a percentage of this value. Figure 3: The F-subunit of the HlgC/HlgB and LukS-PV/LukF-PV bicomponent leukotoxins carries the specificity for releasing Ca 2+ from acidic stores (HlgB) or neutral stores (LukF-PV). A: HlgC/HlgB (both 0.5 nM, in blue) and HlgC/LukF-PV (1.5/15 nM, in red). B: LukS-PV/LukF-PV (both 0.25 nM, in blue) and LukS-PV/HlgB (0.5/1 nM, in red). C: control recording of 1.5/15 nM HlgC/LukF-PV (in blue) and GPN-treated cells prior to the addition of HlgC/LukF-PV (in red). D: control recording of 0.5/1nM LukS-PV/HlgB subunits (in blue) and GPN-treated cells prior to the addition of LukS-PV/HlgB (in red). Results are normalized to the percent maximum Fura-2 fluorescent 2+ signal obtained after bathing the cells in 0.05 % Triton X-100 to equilibrate [Ca ]i with that of buffer concentration. The use of these concentrations highlights the similarity between the responses of HlgC/LukF-PV and the LukS-PV/LukF-PV and those between HlgC/HlgB and LukS-PV/HlgB. Figure 4: The presence of HlgB is necessary for pore formation in lipid bilayers. A: Time course of pore opening in diphytanoyl-phosphatidylcholine lipid bilayers challenged by the presence of 4 nM HlgC/HlgB (black trace) or LukS-PV/HlgB (gray trace) in the cis -side. Both leukotoxins were consistently able to form pores, although with a different time scale: during a longer recording period, the current measured for LukS-PV/HlgC was 10 to 20 times lower that for the HlgC/HlgB combination. Buffer composition was 100 mM KCl, 20 mM Hepes, pH 7.0. Final concentration of the toxin was 4 nM for each component; applied voltage was +40mV. B: Histograms of a single pore conductance (G) for HlgC/HlgB (black bars) and LukS-PV/HlgB (grey bars). The mean conductance was 175.6±15.8 pS (n=280 pores) and 184.8±19.5 pS (n=16 pores) for HlgC/HlgB and LukS-PV/HlgB respectively. C: calcein was released from dioleoylphosphatidylcholine/cholesterol (1:1 molar ratio) vesicles by increasing concentrations of HlgC/HlgB (squares), LukS-PV/HlgB (circles) and HlgC/LukF-PV (triangles). P% (y axis) is the permeabilizing activity, as defined in material and methods. Figure 5: The HlgB F-subunit confers to the two-component leukotoxins the capability of dropping the membrane resistance of U937-C5aR transfected cells. A: the membrane resistance of C5aR-transfected cells recorded in the presence of 2 nM LukS- PV/LukF-PV remained constant over a 10-min incubation period. A1 shows the Rm recording of a representative cell. The histogram in A2 assembles the Rm values of eight independently recorded cells prior to and 10 min after the addition of 2 nM LukS-PV/LukF-PV. B: the Rm of C5aR-transfected cells dropped dramatically in the presence of 2 nM HlgC/HlgB, whereas it remained elevated in non- transfected U937 cells. B1 shows the membrane resistance recording of a C5aR cell incubated in the presence of 2 nM HlgC/HlgB (black) compared to a representative recording of a non-transfected U937cell (in red) recorded in the presence of 10 nM HlgC/HlgB. B2 summarizes membrane resistance values for ten independent experiments prior to and after the addition of 2nM HlgC/HlgB (10 min). C: C5aR-transfected cells were challenged by a 4 nM solution of LukS-PV/HlgB combination. Membrane resistance dropped in 50 % of the cells after an 8-min incubation with the latter combiantion while in the remaining half, membrane resistance remained unchanged for more than 10 min. C1 shows the Rm recording a C5aR cell incubated in the presence of the LukS-PV/HlgB combination and C2 the Rm values prior to addition of LukS-PV/HlgB and after a 10-min incubation in seven independent experiments. Figure 6: The association of the F-subunit to the S-subunit attached to the C5a receptor facilitates the internalization of the resulting complex. After a 10-min incubation of human neutrophils in the presence of 2 nM of isolated S-subunits, labeling with toxin-specific antibodies against Luk-S PV (panel A1) and HlgC (panel B1 ) was associated with the plasma membrane. The addition of the normally-associated F-subunit for 2 to 5 min induced an attenuation of membrane labeling likely due to the internalization of the complex. A2: representative example of cells labeled after a 5-min incubation in a 2 nM solution of LukS-PV/LukF-PV; A3: cells labeled after a 10-min incubation in a 2 nM LukS-PV/LukF-PV solution in Ca 2+ free buffer. B2: representative example of cells labeled after a 5-min incubation in 2 nM HlgC/HlgB; B3: cells labeled after a 10-min incubation in a 2 nM HlgC/HlgB solution in Ca 2+ free buffer. The labeled neutrophils presented in panels A1 and B1 are representative of the aspect of counted cells included in the histograms presented in C1 and C2 , which summarize the percentage of human neutrophils exhibiting fluorescent staining with the specific antibodies associated with the plasma membrane under various incubation conditions indicated under each column. Antibodies are specific for the S-subunits. Supplementary figure 1: Leukotoxins that bind C5aR do not require extracellular Ca 2+ to induce 2+ 2+ [Ca ]i increase. Recordings of LukS-PV/LukF-PV ( A) or HlgC/HlgB-induced ( B) rise in [Ca ]i in human neutrophils in 1 mM Ca 2+ -containing buffer (blue traces) or in Ca 2+ -free-EGTA buffer (red traces). Supplementary figure 2: Extracellular Ca 2+ is required for the permeated leukotoxin membrane to allow Ethidium Bromide leaking into human neutrophils. Mean fluorescence intensity of human neutrophils staining by Ethidium bromide diffusion due to LukS-PV/LukF-PV ( A) or HlgC/HlgB ( B) incubation. Blue traces illustrate recordings in Ca 2+ -free-EDTA buffer, while red traces were obtained in 1 mM Ca 2+ containing buffer. Data were obtained using a FACSort cytometer equipped with a 15- mW argon laser tuned to 488 nm. Ethidium bromide fluorescence was recorded at a 650 nm wavelength. F1$%`V F1$%`V F1$%`V F1$%`V F1$%`V F1$%`V Supplementary figure 1 Supplementary figure 2 Tables Human Neutrophils: Control 1.2 nM C5a 20 nM W-54011 LukS-PV-Fluorescein labeled 0.21 ± 0.04 nM 0.35 ± 0.05 nM 0.49 ± 0.08 nM LukS-PV-Fluorescein/ Luk-F-PV 0.17 ± 0.02 nM 0.30 ± 0.02 nM 0.48 ± 0.02 nM LukS-PV / LukF-PV-Fluorescein labeled 15.35 ± 1.78 nM 15.17 ± 2.44 nM 18.3 ± 2.44 nM Ki HlgC competition / LukS-PV-Fluorescein labeled 0.16 ± 0.05 nM irrelevant irrelevant HlgC / HlgB Fluorescein labeled 0.87 ± 0.12 nM 1.16 ± 0.15 nM 0.68 ± 0.10 nM U937-C5aR : Control 1.2 nM C5a 20 nM W-54011 LukS-PV-Fluorescein labeled 0.32 ± 0.13 nM 0.43 ± 0.17 nM 0.37 ± 0.16 nM LukS-PV-Fluorescein/ LukF-PV 0.29 ± 0.05 nM 0.38 ± 0.08 nM 0.58 ± 0.12 nM LukS-PV / LukF-PV-Fluorescein labeled 35.34 ± 9.13 nM 35.16 ± 0.42 nM 29.22 ± 8.05 nM Ki HlgC competition / LukS-PV-Fluorescein labeled 0.18 ± 0.14 nM irrelevant irrelevant HlgC / HlgB Fluorescein labeled 2.16 ± 0.42 nM 2.21 ± 0.45 nM 1.33 ± 0.24 nM Table 1. - Dissociation constants of the S-subunits of LukS-PV/LukF-PV and HlgC/HlgB in human neutrophils and in the U937-C5aR cell line alone or in the presence of the C5a peptide and the W- 54011 antagonist. Human Neutrophils C5a 1.2 nM W-54011 20 nM SB-290157 1µM 0.25 nM LukS-PV-Fluorescein labeled 45 ± 9 % (4) 42 ± 8 % (5) 103 ± 6 % (2) 0.25 nM LukS-PV-Fluorescein labeled + 0.25 nM LukF-PV 69 ± 3 % (3) 67 ± 9 % (5) 100 ± 6 % (2) 0.5 nM HlgC + 0.5 nM HlgB Fluorescein labeled 75 ± 6 % (4) 111 ± 3 (5) % 100 ±0.8 % (2) U937-C5aR 1 nM LukS-PV-Fluorescein labelled 71 ± 4 % (4) 72 ± 4 % (4) 99 % (1) 1 nM LukS-PV-Fluorescein labeled + 1 nM LukF-PV 83 ± 5 % (5) 73 ± 5 % (5) 98 ± 0.8 % (2) 0.5 nM HlgC + 0.5 nM HlgB-Fluorescein labeled 92 ± 3 % (3) 109 ± 5 % (7) 103 ± 3 % (2) Table 2. - Effect of the C5a peptide and the antagonist W-54011 on leukotoxin binding to C5aR in human neutrophils and in the U937-C5aR cell line. The high affinity antagonist of the C3a receptor, SB-290157 [35], had no significant effect on leukotoxin binding. LukS- LukS- HlgC/LukF- HlgC/HlgB PV/LukF-PV PV/HlgB PV Drug Target Compound 52,9 +/- 5,8 92,7 +/- 2,6 Bafilomycin Acidic Endo- (n=2) (n=2) lysosome 61,9 +/- 2,0 95,2 +/- 4,8 82,8 +/- 11,4 110,3 +/- 8 GPN (n=4) (n=3) (n=3) (n=3) Two-pore channel 155,3 +/- 12,0 102,5 +/- 7,5 Ned-19 Endosome (n=3) (n=3) 50,4 +/- 3,5 55,1 +/- 4,8 Thapsigargin (n=3) (n=4) Neutral reticulum 18,3 +/- 5,5 29,8 +/- 8,2 Ionomycin (n=5) (n=4) 64,9 +/- 13,1 87,6 +/- 5,8 SOCE YM-58483 (n=3) (n=3) Table 3. - Effect of internal Ca 2+ stores alteration by various drugs on the leukotoxins-induced rise in free intracellular [Ca 2+ ]. The results represent the mean values +/- SEM of the percentage of the 2+ maximum [Ca ]i in cells incubated with the respective drugs versus untreated cells in the same experiment. SOCE: store-operated Ca 2+ entry. 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VS0 #, 1#,!# #2 #, .0M1#,!# "# !#1 "#36 *#3!-2-6',#1!,_#12!. 1!1'%,'$'! 2'4#T SXZ IDENTIFICATION DES RESIDUS ESSENTIELS A LA FIXATION DE LUKSVPV A CWAR P-30 * *' '1-, "# CW 9 CW RQ 3, +-"L*# "# *' '1-, 9 "#36 1'2#1 M2M .0-.-1MQ " ,1 *#/3#* *#1 0M1'"31 1'/3#1 " ,1 *# ,-7 3 "# CW ',2#0 %'11#,2 " -0" 4#! *#1 0M1'"31 !'"#1 " ,1 *#620M+'2M NV2#0+', *# "# CW RQ 13'4'# "# *',2#0 !2'-, "# *#620M+'2M CV2#0+', *# "# CW 4#! 3,# .-!&# "# *' '1-, $-0+M# . 0 "#1 0M1'"31 &7"0-.&- #1 "#1 "-+ ',#1 20 ,1+#+ 0 , '0#1 #2 "#1 0M1'"31 !& 0%M1 9 * 1# "#1 ECL1 "3 0M!#.2#30T C#22# "#36'L+# ',2#0 !2'-, #12 #11#,2'#**# .-30 * !2'4 2'-, "3 0M!#.2#30 M-,) #2 *TQ TRRYR P-12+ #2 *TQ TRRWR S'!'*' ,- #2 *TQ S[[VR Z& ,% #2 *TQ S[[YT S. ,, #2 *T -,2 +-,20M /3# L3)SVPV ',2#0 %'2 9 * $-'1 4#! * 0M%'-, NV2#0+', *# "# CW R #2 * . 02'#!!#,20 *#!#2!/3#!!#22#!"#0,'L0#!#12!,M!#11 '0#!9!*_ !2'4'2M!"#!$-0+ 2'-,!"#!.-0#! S. , #2 *TQ TRSUT C-,!#0, ,2 L3)SVPVQ 3,#!120 2M%'#!"_!_* ,',#!Q! ,,',%! !. 0!+32 %M,L1#!"'0'%M#! M2M 32'*'1M#Q $', "# "M*'+'2#0 3, "-+ ',# 1203!230 * 0#1.-,1 *# "# * $'6 2'-, "# L3)SVPV 9 CW RT C#22# 2#!&,'/3# 4'1# 9 "M2#0+',#0 * !!-,20' 32'-,!"_3,!0M1'"3!1.M!'$'/3#!#,!*#!+32 ,2T!?_ !'"#! +',M! "#! 13 12'232'-,! #12! %M,M0 *#+#,2! *__* ,',#Q! "-,2! * ! !& ',#! * 2M0 *#! #12! 3,! +M2&7*#! 0#* 2'4#+#,2 .#3 0M !2'$ #2 .#3 #,!-+ 0 ,2T C#22# -.M0 2'-, #12 0M.M2M#Q $', "# !0' *#0 "#1 "-+ ',#1 #,2'#01 "# * .0-2M',#T L#1 -3!*#1 "3 "-+ ',# R'+ "# L3)SVPV -,2 M2M !' *M#1 .3'1/3# !_#12!*#!"-+ ',#!/3'!#,20#!#,!!-,2 !2! 4#!!*#1!+#+ 0 ,#1!"#1!!#**3*#1T L#1 !'"#1 +',M1 9 +32#0 -,2 M2M !&-'1'1 13'2# 9 * !-+. 0 '1-, "# 1M/3#,!# "# L3)SVPV 4#! *#1 320#1 .0-2M',#1 "# !* 11# SQ #2 ,-2 ++#,2 H*%C /3' #,20# #, !-+.M2'2'-, 3 +N+# 1'2# +#+ 0 , '0#T ARTICLE N5U S RESIDUES ESSENTIAL FOR PANTONVVALENTINE LEUKOCIDIN S COMPONENT BINDING TO ITS CELL RECEPTOR SUGGEST BOTH PLASTICITY AND ADAPTABILITY IN ITS INTERACTION SURFACE SX[ Residues Essential for Panton-Valentine Leukocidin S Component Binding to Its Cell Receptor Suggest Both Plasticity and Adaptability in Its Interaction Surface Benoit-Joseph Laventie 1¤ , Fre ´de ´ric Gue ´rin 2,3 , Lionel Mourey 2,3 , Mira Y. Tawk 1, Emmanuel Jover 1, Laurent Maveyraud 2,3 *, Gilles Pre ´vost 1* 1 Universite´ de Strasbourg-CHRU, Fe´de´ration de Me´decine Translationnelle de Strasbourg, EA 7290 Virulence bacte´rienne pre´coce, Institut de Bacte´riologie, Strasbourg, France, 2 Institut de Pharmacologie et Biologie Structurale (IPBS), Centre National de la Recherche Scientifique (CNRS), Toulouse, France, 3 Universite´ de Toulouse, Universite´ Paul Sabatier, IPBS, Toulouse, France Abstract Panton-Valentine leukocidin (PVL), a bicomponent staphylococcal leukotoxin, is involved in the poor prognosis of necrotizing pneumonia. The present study aimed to elucidate the binding mechanism of PVL and in particular its cell- binding domain. The class S component of PVL, LukS-PV, is known to ensure cell targeting and exhibits the highest affinity 210 29 for the neutrophil membrane ( Kd,10 M) compared to the class F component of PVL, LukF-PV ( Kd,10 M). Alanine scanning mutagenesis was used to identify the residues involved in LukS-PV binding to the neutrophil surface. Nineteen single alanine mutations were performed in the rim domain previously described as implicated in cell membrane interactions. Positions were chosen in order to replace polar or exposed charged residues and according to conservation between leukotoxin class S components. Characterization studies enabled to identify a cluster of residues essential for LukS- PV binding, localized on two loops of the rim domain. The mutations R73A, Y184A, T244A, H245A and Y250A led to dramatically reduced binding affinities for both human leukocytes and undifferentiated U937 cells expressing the C5a receptor. The three-dimensional structure of five of the mutants was determined using X-ray crystallography. Structure analysis identified residues Y184 and Y250 as crucial in providing structural flexibility in the receptor-binding domain of LukS-PV. Citation: Laventie B-J, Gue´rin F, Mourey L, Tawk MY, Jover E, et al. (2014) Residues Essential for Panton-Valentine Leukocidin S Component Binding to Its Cell Receptor Suggest Both Plasticity and Adaptability in Its Interaction Surface. PLoS ONE 9(3): e92094. doi:10.1371/journal.pone.0092094 Editor: Binh An Diep, University of California, San Francisco, United States of America Received December 12, 2013; Accepted February 18, 2014; Published March 18, 2014 Copyright: ß 2014 Laventie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from EA4438 to GP (Direction de la Recherche et des Etudes Doctorales, and BJL was awarded by the French Ministery of Superior Education and Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (LM); [email protected] (GP) ¤ Current address: Division of Molecular Microbiology Biozentrum, University of Basel, Basel, Switzerland Introduction Four of these leukotoxins are involved in human pathogenicity. Panton-Valentine leukocidin (PVL) is associated with necrotizing Staphylococcus aureus largely relies on the secretion of toxins and skin infections, such as boils [17,18], and plays an important role other virulence factors such as superantigens and proteases for its in the poor prognosis of necrotizing pneumonia [19,20,21]. While virulence, targeting various actors of innate immunity [1,2]. the two c-haemolysins HlgA/HlgB and HlgC/HlgB are not Staphylococcal leukotoxins, a subfamily of pore-forming toxins, associated with a specific disease, they are nonetheless expressed appear to simultaneously confer to S. aureus high virulence and by over 99% of S. aureus strains [22,23] and are thought to increase protection against the host’s immune system. With the exception the severity of the infection [24,25]. LukE/LukD has been of a-hemolysin, which is homo-heptameric [3], leukotoxins are reported as a dermonecrotic toxin and involved in bullous bipartite toxins, formed by the non-covalent association of two impetigo [26]. The toxic action of leukotoxins results from a distinct proteins, a class S and a class F component of complex mechanism which has been described in the case of approximately 31 and 34 kDa, respectively, into a likely octameric HlgA/HlgB [7,27,28,29] and is characterized by: (i) binding of the species [4,5,6,7]. To date, 7 bipartite leukotoxins have been S class component on the target cell membrane, which requires identified in S. aureus : Panton-Valentine leukocidin [8], LukM/ the presence of a specific receptor, (ii) recruitment of the F class LukF’-PV [9], two c-hemolysins [10,11], LukE/LukD [12] and a component, (iii) dimerization possibly accompanied by conforma- variant thereof [13], and more recently, LukH/LukG [14] also tional rearrangement, (iv) formation of an octameric prepore, and named LukAB [15]. S. intermedius has also been shown to express a (v) pore formation across the membrane. During this process, both LukS-I/LukF-I leukotoxin, [16], while certain related genes can be class S and F proteins are faced with a dual environment: a found in other Staphylococcus species. hydrophilic milieu, when secreted by the bacteria upon infection, and a hydrophobic milieu, when forming the pore in the PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues membrane. Independently of pore formation, leukotoxins are able S204 and M234–R263, with the least conserved regions being to rapidly activate cellular signalization [30,31], including an Y60–R73, F163–E197 and T240–V261, corresponding to loops in increase in intracellular calcium concentration and chemokine the LukS-PV structure (Fig. 1B, C). Positions corresponding to secretion [32]. polar or charged residues, with their side-chain oriented towards At the molecular level, sequence identity varies from 55 to 79% the exterior of the protein (as depicted on the structure of wild-type within a given class, when excluding LukH (LukA) or LukG LukS-PV (PDBID 1T5R), Fig. 1C), were thus selected for (LukB). When these proteins are included in the comparison, mutation as these residues are more likely to be involved in sequence identities drop to about 30–34% [14]. Similarity across interactions either with the target cell membrane or with a classes remains below 30%. The three-dimensional structure of the receptor at the cell surface. soluble forms of several leukotoxin components are known [33,34,35,36] and display a similar fold, organized around a Production, purification and characterization of LukS-PV central domain formed by two six-stranded antiparallel b-sheets mutants (Fig. 1). This so-called cap or core b-sandwich domain is the most With the exception of the D195A and R241A mutants, all conserved region, and is the location where most protein-protein proteins were produced and purified to homogeneity, as assessed interactions found in the pre-pore and pore occur. Two additional by Coomassie-blue stained SDS-PAGE. For the D195A and structural domains are also found: the rim domain anchors the R241A mutants, production yields were too low to allow for protein to the membrane surface [7,37,38,39], while the stem purification, likely due to mutation-induced aggregation of the domain, closely apposed to the core b-sandwich in the soluble form, fusion protein. contributes two b-strands to the pore b-barrel. Interestingly, the rim domain is the least conserved domain, possibly resulting in Binding capacities of LukS-PV mutants variable cell specificities, depending on the leukotoxin involved. Binding capacities of wild-type or mutant LukS-PV to hPMNs PVL displays a narrow cellular spectrum, restricted to human were evaluated by competition experiments with a functional and rabbit polymorphonuclear neutrophil leukocytes, PMNs, fluorescein-labeled LukS-PV G10C mutant (LukS-PV*). The K of monocytes, and macrophages [31]. The binding of LukS-PV has d LukS-PV* for hPMNs was measured at 0.066 60.005 nM using been shown to be a prerequisite for LukF-PV binding and the flow cytometry (Fig. 2A), which is in good agreement with subsequent activation of PMNs. Native or recombinant LukS-PV previously published data [31]. The concentration of wild-type or displays a K as low as 0.07 nM on neutrophils and 0.02 nM on d LukS-PV mutants required for 50% inhibition of labeled monocytes [31] which is the highest known affinity for leukotoxins. competitor binding (EC ) was calculated, allowing to derive K Since the binding of LukS-PV to the membrane is a saturable 50 i process, the necessary presence of a LukS-PV receptor on the cell values (Fig. 2B and Table 1). surface was thus proposed [31,40]. This was recently confirmed by Among the 17 tested mutations, twelve did not significantly alter Spaan et al . who showed that the C5a receptor is required for the LukS-binding to hPMNs, with the corresponding proteins binding of LukS-PV to human neutrophils [41]. This receptor is displaying a binding capacity only affected by a factor smaller also likely involved in the binding of HlgC, since both LukS-PV than 25-fold. The mutants exhibiting significantly decreased and HlgC were shown to compete for a common receptor [31]. In binding abilities with respect to wild-type LukS-PV were: R73A, the case of LukE, receptors CCR5, CXCR1 and CXCR2 have Y184A, T244A, H245A, and Y250A, with Ki values ranging from been identified as binding partners on the cell surface [42,43], 1.8 to 6.2 nM, i.e. a 69- to 238-fold increase compared to the Ki whereas LukH/LukG (LukA/LukB) binds to the CD11b subunit value of wild-type LukS-PV. No single mutation was found to of the integrin Mac-1 [44]. completely prevent binding of LukS-PV (Table 1). In order to identify the LukS-PV residues involved in binding to Undifferentiated U937 cells were mostly insensitive to the the C5a receptor at the cell surface, an alanine scanning site- binding of LukS-PV, for concentrations of LukS-PV* up to . directed mutagenesis strategy was adopted. Indeed, mutation into 500 nM, yielding a calculated Kd 20 mM. By contrast, U937 cells alanine is considered to allow good conservation of molecular expressing C5aR, the LukS-PV receptor, bound LukS-PV* with a structures, whereas the removal of polar or charged residues often Kd of 0.32 nM, thus 5-fold higher than for hPMNs. Furthermore, alters the interacting capacity of proteins. Charged or polar the five LukS-PV mutants R73A, Y184A, T244A, H245A and residues from the rim domain or residues that are conserved in Y250A were strongly affected in their binding to U937-C5aR with LukS-PV and HlgC, but not in S component of other leukotoxins, calculated Ki ranging from 4 to 12 nM, compared to 1.12 nM for were more specifically targeted. Nineteen residues were selected wild-type LukS-PV, thus confirming comparable influences of and investigated for their ability to bind to neutrophils as well as these mutations to those tested with hPMNs. activate the latter and form a functional pore. Five of the most impaired mutants were crystallized and their three-dimensional Ability of LukS-PV mutants to activate neutrophils structure determined. Activation of Fluo3-loaded hPMNs was evaluated by the ability of LukS-PV mutants to induce variations in intracellular calcium Results concentration [40,46]. A limiting concentration of wild-type LukS- PV or mutants was used (0.02 nM) with an excess of LukF-PV Selection of mutated positions (10 nM). This concentration of LukF-PV was chosen as it is closed LukS-PV and HlgC were previously shown to display similar to the reported Kd value for the binding of LukF-PV to LukS-PV cellular spectra and to compete for a common membrane site [47] Wild-type LukS-PV and mutants led to an almost immediate when binding to cells [31]. Moreover, the rim domain was shown increase in intracellular calcium concentration. Only four mutants, to likely interact with the cell membrane within the pore [7,45]. R73A, Y184A, T244A and Y250A, displayed an increased lag- Therefore, the sequences of the rim domains of class S components time prior to calcium entry and a significantly reduced calcium were compared, in order to identify conserved positions in LukS- entry slope, i.e. less than 55% of the LukS-PV control ( p,0.001, PV and HlgC, but not in other class S proteins (Fig. 1A). The rim one-way ANOVA) (Fig. 3 and Table 1). LukS-PV mutants Y181A, domain encompasses three stretches of residues: S56–F76, A160– K182A, Y246A and N248A also had a significantly decreased PLOS ONE | www.plosone.org 2 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues Figure 1. Positions selected for mutations in LukS-PV. A. Sequence alignment of the three stretches of residues constituting the rim domain of class S components of leukotoxins. Numbering corresponds to the mature LukS-PV protein. Red asterisks indicate positions selected for mutation. Strictly conserved residues are indicated on a red background, while similar residues in group 1 (LukS-PV and HlgC) or in group 2 (all others) are PLOS ONE | www.plosone.org 3 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues indicated with red letters. The secondary structure of LukS-PV is indicated above the alignment, colored according to the corresponding structural domain: b-sandwich (cyan) and rim (purple). GenBank accession numbers are LukS-PV: CAA51251.1, HlgC: AAA26638.1, HlgA: AAA26637.1, LukE: CAA73667.1, LukE-V: BAB47174.1, LukS-I: CAA55782.1, LukM: BAA97866.1. B. Schematic representation of the three dimensional structure of LukS-PV [35], PDB entry 1T5R, highlighting the three structural domains: b-sandwich (cyan), stem (orange) and rim (purple). C. Stereo view of the C a trace of wild-type LukS-PV. Residues selected for mutations are displayed as sticks and labeled. doi:10.1371/journal.pone.0092094.g001 calcium entry slope, from 65% to 75% of the control ( p,0.05, increased hPMN activation, also displayed a slightly increased one-way ANOVA). LukS-PV Y191A was the only mutant to pore-forming capacity (111%) compared to LukS-PV. display a significantly increased ability to activate neutrophils, with a calcium entry slope of 135% of the control ( p,0.01, one-way Structure determination of LukS-PV mutants ANOVA). Six of the seventeen studied mutants were subjected to structural analysis: Y184A, T244A, H245A, Y246A, N248A, Pore-forming capacity of LukS-PV mutants and Y250A. Crystals were obtained for all of the above but those The pore-forming ability of the LukS-PV mutants was obtained for the H245A LukS-PV mutant diffracted very poorly investigated by the measurement of ethidium entry through the and were systematically split; therefore, the structure of this pore using flow cytometry. All tested mutants formed pores, most mutant could not be solved. with a slightly decreased ability (Fig. 4 and Table 1). Four mutants, The T244A, Y246A and N248A mutated proteins crystallized R73A, Y184A, T244A and Y250A, had a significantly affected in identical conditions (50% PEG 200, 0.1 M MES-NaOH, pore-forming activity, with at least a 50% reduction in ethidium pH 7.50). The corresponding crystals belonged to the P43212 bromide entry at 30 min. All other mutants had a pore forming space group, with similar cell parameters (a = b ,94 A ˚ and activity close to that observed with wild-type LukS-PV. Interest- c,308 A ˚ ) and 4 molecules per asymmetric unit. These crystals ingly, the LukS-PV Y191A mutant, which already exhibited diffracted X-rays at medium resolution (2.50 to 2.80 A ˚ ). Crystals Figure 2. Binding properties of LukS-PV* and LukS-PV mutants to hPMNs and U937-C5aR cells. A. Flow cytometry measurement of LukS- PV* and fluorescein-labeled LukS-PV G10C binding to human PMNs and U937-C5aR cells ( n = 3). B. Graphic representation of the Ki values obtained for wild-type or mutant LukS-PV. The dotted line corresponds to the value of wild-type LukS-PV. Error bars represent the 95% confidence interval. Statistical analysis: **: p,0.01, ***: p,0.001 ( n = 3). doi:10.1371/journal.pone.0092094.g002 PLOS ONE | www.plosone.org 4 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues Table 1. Binding properties as well as cellular and pore-forming activities of wild-type and LukS-PV mutants 1. + Relative affinity Ca 2 entry slope Ethidium entry (30 min) Mutation Ki695% (pM) ± confidence interval to LukS-PV (% of max/s) ± SEM (% of control) ± SEM LukS-PV 26 61.1 - 1.4 60.05 100 (120 623) - T244A 2 6,190 6560 238 0.50 60.093 25 65.9 (11,000 62,000) (92) R73A 2 5,080 61,000 195 0.66 60.065 41 611 (12,000 65,200) (100) Y250A 2 2,970 6370 114 0.77 60.17 46 614 (5,700 6680) (48) Y184A 2 1,800 6110 69 0.31 60.056 11 64.3 (4,100 6470) (34) H245A 2 1,800 6260 69 1.1 60.059 80 66.7 (4,200 6900) (35) R242A 619 692 24 1.2 60.076 83 64.3 F260A 427 630 16 1.2 60.11 88 62.2 Y246A 365 647 14 1.0 60.098 80 66.7 Y181A 155 612 6 0.95 6 0.045 54 65.2 N248A 74 68.2 2.8 0.93 60.078 77 67.6 K182A 68 65.2 2.6 1.0 60.062 79 63.4 H257A 56 64.5 2.2 1.3 60.12 96 65.5 H173A 45 61.3 1.7 1.2 60.073 73 65.8 D190A 44 62.6 1.7 1.3 60.04 90 63 Q186A 43 60.67 1.7 1.1 60.072 80 64.4 Y191A 41 62.9 1.6 1.9 60.16 111 62.4 R263A 38 61.3 1.5 1.4 60.068 90 62.8 1 Ki values, calcium entry slope and ethidium entry at 30 min were obtained for wild-type LukS-PV and all mutants with hPMNs. Ki values with U937-C5aR cells were obtained for wild-type LukS-PV and for the most affected mutants (values given in parenthesis). 2Mutations causing a significant decrease in LukS-PV affinity for hPMNs ( p,0.001, one-way ANOVA with Dunnett’s post test). doi:10.1371/journal.pone.0092094.t001 Figure 3. Biological activity of LukS-PV and corresponding mutants: rise of cytoplasmic calcium concentration due to human neutrophil activation. Values represent the calcium entry slope expressed in percent of maximum calcium fluorescence (after neutrophil lysis with Triton X-100 0.05% v/v) per second. Statistical analysis: ns, non-significant; *, p,0.05; **, p,0.01; ***, p,0.001 (one-way ANOVA with Dunnett’s post test, n = 6). doi:10.1371/journal.pone.0092094.g003 PLOS ONE | www.plosone.org 5 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues Figure 4. Pore-forming activity of LukS-PV and corresponding mutants. Flow cytometry analysis of ethidium entry in neutrophils 30 min after addition of LukS-PV (wild type or mutant, 0.02 nM) in combination with an excess of LukF-PV (10 nM). Neutrophils were incubated with 4 mM ethidium bromide prior to toxin addition. Values of ethidium entry are expressed as percent of the control (wild type LukS-PV) activity. Statistical analysis: ns, non-significant; *, p,0.05; **, p,0.01; ***, p,0.001 (one-way ANOVA with Dunnett’s post test, n = 3). doi:10.1371/journal.pone.0092094.g004 of the Y184A mutant were obtained at a lower pH 6.50, but the however only when using micromolar concentrations of LukS-PV conditions were otherwise identical. The latter belonged to the [48]. All of the experiments described in the present study were P41212 space group with cell parameters a = 104.86 A ˚ and performed at nanomolar concentrations of wild-type or mutant c = 106.87 A ˚ , 1 molecule per asymmetric unit, and diffracted up LukS-PV, ensuring that only specific binding to the surface of to 2.33 A ˚ . The Y250A mutant crystallized in 5% PEG 6000, neutrophils, i.e. to the C5a receptor, could occur. This was 0.1 M sodium citrate, pH 4.0. Crystals were orthorhombic, space confirmed through the use of undifferentiated U937 cells that do ˚ ˚ group P21212, with cell parameters a = 85.30 A , b = 84.29 A and not naturally express C5aR and do not significantly bind LukS- c = 38.09 A ˚ and 1 molecule per asymmetric unit. Crystals PV* even at protein concentrations up to 500 nM, whereas this ˚ diffracted up to 1.55 A . Since no crystal was isomorphous to the binding was observed with a Kd of 320 pM with U937 cells crystals of wild-type LukS-PV [35], molecular replacement was expressing human C5aR. performed in all cases. This allowed the identification of a clear Among the seventeen LukS-PV mutants investigated, eight and unique solution. Refinement statistics are provided in Table 2. mutants (H173A, Q186A, D190A, Y191A, R242A, H257A, The overall three-dimensional structures of the mutants were F260A and R263A) displayed only marginally altered biological very similar to the wild-type structure. Indeed, all secondary properties. Five mutants (Y181A, K182A, H245A, Y246A and structure elements were preserved and local differences were only N248A) were partially affected. Four LukS-PV mutants (R73A, observed in certain loops (Fig. 5A). The b-sandwich domain was the Y184A, T244A, and Y250) displayed significant alterations for all most conserved, with r.m.s. deviations after superposition of measured parameters, i.e. at least 50-fold decrease in affinity for ˚ + equivalent C a atoms below 0.63 A . The stem domain appeared hPMN membranes, 2-fold decrease in the slope of Ca 2 influx and ˚ more flexible with r.m.s. deviations ranging from 0.21 A to 2- to 10-fold reduction in pore-forming capacity. The most ˚ 2.21 A . dramatically reduced affinity of the LukS-PV mutants for the In the case of the rim domain, wild-type LukS-PV and mutants hPMN membrane led to a reduced amount of bound leukotoxin, T244A, Y246A and N248A displayed only small differences, with which resulted in a decreased number of both activated hPMNs ˚ ˚ r.m.s. deviations comprised between 0.20 A and 0.30 A , whereas and functional pores. However, none of the studied mutants was the structure of the rim domain of the Y184A and Y250A mutants completely inactive. Indeed, the K of wild-type LukS-PV was ˚ i were markedly different, with r.m.s. deviations of 1.10 A and 0.03 nM and even mutants displaying a 200-fold degradation in 1.28 A ˚ , respectively, when compared to the wild-type LukS-PV binding still displayed a Ki of about 10 nM, resulting in a structure (Fig. 5). These differences were mostly concentrated in dramatically reduced but still recordable biological activity. the V169–Q186 and T244–N248 loops, with mutants Y184A and Comparable variations of K were observed when LukS-PV Y250A being the most divergent. Of particular note, the rim region i mutants were tested for binding to recombinant undifferentiated was generally associated with weak electron density, and could not U937-C5aR (Figure 2). The absence of measurable binding to be completely built in most structures, except in the case of the undifferentiated U937 cells indicates that the presence of the C5a Y250A mutant. receptor is required for the initial binding of LukS-PV and for subsequent events of toxin formation. Discussion The rim domain is known to be responsible for the interaction of Specific binding of LukS-PV to the surface of human leukotoxins with target cell membranes while the structure of the neutrophils has been shown to require the presence of a functional c-hemolysin pore [7] indicates that the loops extending at the C5a receptor [41], and occurs with an apparent affinity varying bottom of the rim domain are likely involved in this interaction. In between 0.06 to 6 nM depending on the protein purification tag LukS-PV, this area would correspond to residues 181–186 and and the methodology used [31,40,41]. Non-specific binding to 241–251. Three of the four most sensitive residues (Y184, T244 cellular or artificial membranes has also been shown to occur, and Y250) are found in this region, at close distance from each PLOS ONE | www.plosone.org 6 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues Table 2. Crystallographic data and refinement statistics of the LukS-PV mutants 1. Y184A T244A Y246A N248A Y250A 40% PEG 200 NaMES 40% PEG 200 NaMES 40% PEG 200 NaMES 40% PEG 200 NaMES 5% PEG 6000 NaCitrate Crystallization condition 0.1 M pH 6.50 0.1 M pH 7.50 0.1 M pH 7.50 0.1 M pH 7.50 0.1 M pH 4.00 Data collection and processing Beamline ID29 ID23-EH2 ID14-EH1 ID23-EH2 ID14-EH4 Spacegroup P4 1212 P43212 P43212 P43212 P21212 Cell parameters (A˚) a = 104.87 c = 106.89 a = 94.47 c = 310.31 a = 94.19 c = 306.44 a = 93.99 c = 309.39 a = 85.30 b = 89.29 c = 38.09 Resolution limits (A˚) 47.60–2.20 (2.30–2.20) 46.70–2.75 (2.80–2.75) 47.05–2.50 (2.64–2.50) 46.99–2.80 (2.90–2.80) 30.84–1.55 (1.60–1.55) Nb. of observations 222,233 (27,688) 231,080 (4,712) 213,925 (28,588) 291,382 (8,514) 299,073 (22,054) Nb. of unique reflection 30,803 (3,759) 36,657 (1,606) 46,924 (6,622) 34,936 (3,263) 43,049 (3,855) Multiplicity 7.2 (7.4) 6.3 (2.9) 4.6 (4.3) 8.3 (2.6) 6.9 (3.6) Completeness 99.8 (99.7) 97.3 (83.0) 96.9 (95.7) 99.1 (94.6) 99.7 (99.9) Rsym 0.070 (0.915) 0.110 (0.834) 0.149 (0.827) 0.122 (0.649) 0.067 (0.753) I/ s 16.2 (2.7) 15.1 (2.0) 10.0 (1.8) 16.0 (1.9) 18.8 (3.2) Refinement Resolution limits (A˚) 47.60–2.20 46.70–2.75 47.05–2.50 46.99–2.80 30.84–1.55 Nb. of reflections 30,803 36,656 46,908 34,936 43,037 Rfactor 0.210 0.198 0.185 0.224 0.167 Rfree 0.218 0.246 0.228 0.245 0.189 Nb.ofmolecules/A.U. 1 4 4 4 1 Nb. of atoms Protein 2,171 8,804 8,761 8,719 2,242 Water 106 319 473 96 297 Others 12 none none none 30 R.m.s. deviations Bond lengths (A˚) 0.010 0.010 0.010 0.008 0.009 Bond angles ( u) 1.12 1.21 1.17 1.09 1.06 Peptide v angles ( u) 4.14 3.34 3.69 2.79 4.37 PDBID 4IYT 4IYC 4J0O 4IZL 4IYA 1Numbers in parentheses report statistics for the highest resolution shell. doi:10.1371/journal.pone.0092094.t002 other (Fig. 1C). Residue R73, the fourth most affected residue, is 240 (Thr in LukS-PV and Ile in HlgC), 241 (Arg in LukS-PV and located close to the b-sandwich domain (Fig. 1C). In close Lys in HlgC) and 243 (Thr in LukS-PV and Ser in HlgC). This proximity to residues Y184, T244 and Y250, residues R242, H245 T240-T243 stretch is strictly conserved among 40 known variants and Y246 also induced altered biological activities when mutated of LukS-PV. Such differences between LukS-PV, HlgC and other to alanine, albeit to a somewhat lesser extent. Binding affinities of class S components may contribute to differences in their target LukS-PV Y184, T244 and Y250 mutants underwent a 14- to 61- cell spectrum as well as the binding capacities of PVL and HlgC/ fold decrease with respect to the value obtained for the wild-type HlgB. protein. These results hence identify the bottom loop 240–250 as The three dimensional structures of five of the seventeen well as residue Y184 as being crucial for LukS-PV binding to the investigated LukS-PV mutants were obtained: Y184A, T244A, target cell membrane. Residues Y184, T244, Y250, R242, H245, Y246A, N248A, and Y250A. This provided a unique opportunity and Y246 are located on poorly conserved regions among to correlate their altered biological behavior to structural leukotoxins, except between LukS-PV and HlgC. Residues R73, properties. In order to sample all possible conformations of H245, Y246 and N248 are specific to the two proteins, whereas LukS-PV, all structures, including the multiple structures resulting Y184 of LukS-PV is replaced by a histidine residue in HlgC from non-crystallographic symmetry, were superimposed onto the (Figure 1A). In these two proteins, a four-residue insertion is found A-chain of the wild-type LukS-PV structure (PDB entry 1T5R). in the loop T240–Y250 compared to other class S components. With the 13 structures provided by this study, and since there are 8 This loop has furthermore been shown to be essential for c- molecules in the asymmetric unit of the wild-type crystals, 21 hemolysin activity [49] which has a different cell-target spectrum, structures could be compared (Fig. 5A). The comparison clearly although its direct role in LukS-PV and HlgC activity has never enabled to identify regions with the highest structural flexibility: been demonstrated until now. Indeed, our study provides clear the stem region and the three loops of the rim domain. In the evidence that the corresponding segment in LukS-PV is crucial for structure of the soluble form of both S and F leukotoxin its binding to the C5a receptor. In this region, the only differences components, the stem region is comprised of a 3-stranded in sequence between Luks-PV and HlgC are located at positions antiparallel b-sheet and a connecting loop (residues P121–F129 PLOS ONE | www.plosone.org 7 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues PLOS ONE | www.plosone.org 8 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues Figure 5. Structural variations of LukS-PV upon mutations in the rim domain. A. Superposition of the C a trace of all available structures of LukS-PV proteins, wild-type or mutants. The Y184A mutant structure is represented in green and the Y250A mutant structure in blue. For the other structures, the three structural domains are highlighted: b-sandwich (cyan), stem (orange) and rim (purple). B. Close-up stereo view of the rim domain of the LukS-PV wild-type structure, shown as stick (thin for main-chain and thick for side-chains). Residues labeled in red correspond to the most sensitive position as identified in this work. C. Stereo view of portion of the rim domain of wild-type LukS-PV (purple) and of the Y184A mutant structure (green). D. Stereo view of portion of the rim domain of wild-type LukS-PV (purple) and of the Y250A mutant structure (blue). Panels B to D, hydrogen bonds are represented as grey dots, the orientation of the image is the same as in A. Panels C and D, mutated positions are labeled in italic. doi:10.1371/journal.pone.0092094.g005 in LukS-PV), closely apposed against the b-sandwich domain. Our residue, Y184, also interacts with this loop. To date, no natural comparison of all available structures of the LukS-PV proteins mutation of LukS-PV has been reported that would affect its indicates that this connecting loop is highly flexible. In all specific binding to the membrane receptor. Recent results structures, except for the Y250A mutant, electron density was very obtained with LukE have identified regions Q180–A193 and weak in this region and the loop could not be built completely. L234–R268 as essential for LukE/LukD cytotoxicity, but only This structural flexibility is not surprising since the stem region has region Q180–A193 as being required for binding to CXCR1 and to undergo major structural rearrangement when it deploys into CXCR2 [43], whereas in the case of LukH (LukA), residue 323 the membrane and contributes two b-strands to the b-barrel pore. was found to be crucial for binding to CD11b [50] This suggests The rim domain is built from three stretches of residues: S56– that the mechanism of receptor recognition may not be unique P75, A160–H203, and N233–H257 [35]. Both S56–P75 and among all leukotoxins. Moreover, binding of the S component of N233–H257 segments are comprised of b-strands that extend leukotoxins to its cognate receptor is not sufficient in itself to result from/into the b-sandwich domain. They form a 4-stranded b-sheet in pore formation, which requires the recruitment of the F onto which residues A160–H203 are apposed. This latter stretch component and the formation of the adequate hetero-oligomer in of residues includes a small 2-stranded b-sheet (N161–T165 and order to form the prepore and, eventually, the pore. All of these G168–G172) and a long loop (H173–H203) that includes three steps, which potentially require specific and major interactions of short helical segments (P188–Y191, D195–E197, and P200– LukS-PV with the extracellular region of C5aR, remain to be H203). The conformation of this loop is stabilized by several intra- characterized at the molecular level. Such data along with a better loop hydrogen bonds, whereas only a few interactions with the understanding of these critical steps involved in LukS-PV binding other regions of the rim domain were found (Fig. 5B). In most are of crucial importance for pharmacological purposes. structures of LukS-PV, the rim domain conformation is well conserved, with the exception of the R241–A250 loop where Experimental Procedures certain local variations were observed (Fig. 5A). Other significant variations in the conformation of the rim domain were only found Bacterial strains and vectors in the structure of the Y184A mutant, where residues V179–Q186 Escherichia coli XL1 Blue cells [ recA1 endA1 gyrA96 thi1 hsdR17 were affected (Fig. 5C), as well as in the conformation of the supE44 relA1 lac (F 9 proAB lacIqZ DM15 Tn10 (tet r))] (Stratagene, Y250A mutant, where two loops (V179–Q186 and R241–A250) Agilent Technologies, Massy, France) were used as recipient cells showed structural variations (Fig. 5D). For both Y184A and for transformation with recombinant pGEX-6P-1 ,Panton-Val- Y250A mutants, crystallization was achieved in different condi- entine leukocidin genes (Genbank:X72700) (GE Healthcare Life 2 2 2 tions than those for wild-type LukS-PV and the other mutants, Science, France, [5]). E. coli BL21 [F , ompT, hsdS (rB , mB ), gal ] resulting in a different crystal packing. This alone suggests that the was used for over-expression of the glutathione-S-transferase structures of mutants Y184A and Y250A differ from the others, (GST) ,leukotoxin fusion genes, according to the manufacturer’s preventing crystallization in similar conditions. As the result of the instructions (GE-Healthcare). Y184A mutation, the hydrogen bond found in the wild-type LukS- PV structure between Y184OH and S249OG was lost, and the Alanine scanning site-directed mutagenesis ˚ loop was reorganized with a displacement of 6.0 A for the CA LukS-PV mutants were constructed by means of the Quick- atom at position 184 (Fig. 5C). In the Y250A mutant structure, the Change mutagenesis protocol (Stratagene) using Phusion Hot removal of the tyrosine side chain allowed the displacement of the Start DNA polymerase (Finnzyme, Espoo, Finland) and dedicated N248 side chain, which in turn drove the reorganization of oligonucleotides as previously described [5]. All mutated genes residues R241–Y250. Space was therefore provided for the were verified by DNA sequencing. displacement of the V179–Q186 loop, which adopted a confor- mation close to that observed in the case of the Y184A mutant Expression and purification of leukotoxins (Fig. 5D). Wild-type and mutant leukotoxins were expressed and purified The structural variations observed in this study suggest that the as previously described [30]. Briefly, recombinant BL21 E. coli cells rim domain of LukS-PV may have the ability to easily adapt its were grown in 2 6TY medium (bacto-tryptone 17 g/l, bacto-yeast conformation in order to bind to the C5a receptor present in the extract 10 g/l, NaCl 5 g/l), and protein expression was induced target cell membrane. Our results show that the rim domain of with 0.2 mM IPTG. GST-fusion proteins were purified by affinity LukS-PV likely displays the required plasticity for these events, chromatography on glutathione-Sepharose 4B (GE Healthcare), and that tyrosine residues 184 and 250 may be of paramount followed by a SP-sepharose cation-exchange run on a Fast Protein importance in this process, since the corresponding mutants are Liquid Chromatography AKTAPurifyer, after removal of the among the most affected. Moreover, the functional studies glutathione S-transferase tag with PreScission protease (GE performed herein indicate that loop 240–250 is crucial for the Healthcare). The identity and purity of proteins were confirmed binding of LukS-PV to C5aR. Indeed, three of the five most by radial gel immunoprecipitation against native antigens (0.6% sensitive residues identified (T244, H245 and Y250) are located in (w/v) agarose in phosphate buffered saline) and 10–15% (w/v) this loop and form a surface that could represent the principal SDS-PAGE. Proteins were stored at 280 uC. binding site for the receptor (Figure 6). The fourth most affected PLOS ONE | www.plosone.org 9 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues Figure 6. Molecular surface of the rim domain of LukS-PV. Residues identified in this study as important for the binding of LukS-PV on the C5a receptor ( Ki increased more than 50 fold upon mutation to Ala) are depicted in red. Mutated residues affecting binding to a lesser extent (i.e. increase in Ki by a factor between 5 and 50) are depicted in pink whereas residues for which no effect on binding was found upon mutation (increase in Ki less than 3 fold) are depicted in blue (Table 1). Two orthogonal views around a vertical axis are presented, with the orientation on the left being the same as in figure 5. doi:10.1371/journal.pone.0092094.g006 hPMN purification and activation of 3 610 3 hPMNs was measured using a FacSort flow cytometer Blood samples of anonymous healthy volunteer donors were (Becton-Dickinson, Le Pont de Claix, France) equipped with a 15- purchased at the ‘‘Etablissement Franc¸ais du Sang, Strasbourg, mW, 488-nm, argon-ion laser. Data were acquired using France’’ in accordance with convention n uSG-CLI-003. Human CellQuest Pro software (Becton-Dickinson). Ethidium fluorescence polymorphonuclear neutrophil leukocytes (neutrophils) were puri- of hPMNs after a 30-min incubation with PVL was calculated and fied from buffy coats as previously described [47], and suspended normalized with respect to the wild-type LukS-PV control. The at 5 610 5 cells/ml (unless specified otherwise) in 10 mM HEPES, results for 3 different donors were averaged and expressed as 140 mM NaCl, 5 mM KCl, 10 mM glucose, 0.1 mM EGTA percentages of fluorescence values recorded with dead cells. pH 7.3. Human PMN activation was monitored by following the Baseline values were obtained for each series of data from a + variation in intracellular free Ca 2 . Calcium changes were control without addition of toxin, and were systematically determined by recording the variations in emitted fluorescence subtracted from the results of other assays. of Fluo3-loaded neutrophils as previously described [31]. Briefly, H neutrophils were incubated with 2 mM Fluo3-AM (Molecular Leukotoxin binding assays Probes, Eugene, USA) for 45 min at room temperature, then Kd determination. The dissociation constants, Kd, were washed and resuspended twice in HEPES buffer. Five minutes determined using a binding saturation experiment with increasing prior to toxin addition, 1.1 mM CaCl 2 was added to hPMN concentrations of fluorescein-labeled LukS-PV* (0.01 nM to 6 suspensions (6 610 cells/ml). Variations in fluorescence intensity 10 nM for hPMNs; 0.1 nM to 500 nM for U937 cells), as of Fluo3 were recorded with a spectrofluorometer (Deltascan, PTI, previously described [31]. The amount of labeled protein bound to USA) at lEx = 488 nm and lEm = 530 nm. the cell surface was measured by flow cytometry as cell fluorescence at lEm = 530 nm and expressed as the percentage Undifferentiated U937 and U937-C5aR cell cultures of maximum fluorescence obtained at the highest concentration of Undifferentiated U937 and U937-C5aR cells, which respec- LukS-PV*. Experimental data were fitted using GraphPad Prism tively do not express or stably express the C5aR receptor [51], version 5.04 for Windows (GraphPad Software, San Diego, USA). were a generous gift from Pr. J.A. van Strijp (Utrecht University, Kd values were calculated by a non-linear regression using the The Netherlands). Cells were cultured as 50 ml suspension at ‘‘One site - Specific binding’’ equation. u 37 C under a 5% CO 2 atmosphere in 250 ml flasks in RPMI- Ki determination. Fluorescein-labeled LukS-PV* (1 nM) 1640 medium supplemented with 10% (v/v) of decomplemented was displaced by increasing concentrations of various non-labeled fetal calf serum (Life Technologies, Carlsbad, USA) and 0.1% (w/ LukS-PV or mutants (0.03 nM to 500 nM). The amount of LukS- v) of both penicillin and streptomycin (InVitrogen, Paisley, UK). PV* bound to the cell surface was measured by flow cytometry as the amount of cell fluorescence at lEm = 530 nm. Fifty percent Pore formation measurements effective concentrations (EC 50 ) were calculated by GraphPad Pore formation was revealed by the penetration of ethidium into Prism using the non-linear regression ‘‘one site binding’’ equation. cells [52]. Neutrophils (5 610 5 cells/ml) were pre-incubated for The equation of Cheng and Prusoff [53] was used to calculate the 10 min with 4 mM ethidium bromide. Measurements were inhibition constant, Ki, from the EC 50 value (parameters: Kd LukS- initiated directly after the simultaneous addition of 0.1 nM of PV* = 0.066 nM; [LukS-PV*] = 1 nM). the S component and 10 nM of LukF-PV. Ethidium fluorescence PLOS ONE | www.plosone.org 10 March 2014 | Volume 9 | Issue 3 | e92094 Leukocidin S Component Essential Binding Residues Structures were solved using the molecular replacement EC 50 method, using Phaser 2.3 [58], with the structure of the wild- Ki~ ½LukS {PV à type LukS-PV as starting model [35]. Refinement was performed 1z with Buster (GlobalPhasing, UK) and Coot softwares [59] Kd (Table 2). Acknowledgments Protein crystallization and structure determination The authors thank Raymonde Girardot and Daniel Keller for their skillful Proteins were conditioned in 50 mM MES-NaOH buffer, technical assistance, Christiane Moog (INSERM U1110) for human 50 mM NaCl, pH 6.5 at approximately 10 mg/ml. Crystallization PMNs, as well as Pr J.A van Strijp from Utrecht University (The trials were implemented using the sitting drop method and a Netherlands) for the generous gift of recombinant U937 cells. Authors also NanoDrop ExtY automated crystallization platform (Innovadyne) acknowledge Didier Colin for his help with data analysis. The equipment at 285 K. Drops were generated by mixing 200 nL of protein used for the crystallization experiments and X-ray crystallography is part of solution to the same volume of crystallization solution (Table 2). the Integrated Screening Platform of Toulouse (PICT, IBiSA). We thank Crystals were cryo-protected by brief immersion in the the staff of synchrotron beam lines ID14-1, ID14-4, ID23-2, and ID29 at the European Synchrotron Radiation Facility (Grenoble, France). crystallization solution supplemented with 20% (v/v) ethylene glycol before being transferred into a gaseous nitrogen flux at 100 K. All data collections were performed at the European Author Contributions Radiation Synchrotron Facility (ESRF, Grenoble, France). Data Conceived and designed the experiments: BJL EJ L. Mourey L. processing was initially performed using autoPROC [54], and Maveyraud GP. Performed the experiments: BJL FG MYT L. Mourey optimized with XDS [55] and SCALA [56] (Table 2). All L. Maveyraud. Analyzed the data: BJL FG MYT L. Mourey EJ L. subsequent operations were performed using the CCP4 program Maveyraud GP. Wrote the paper: BJL L. Mourey L. Maveyraud GP. suite [57]. References 1. Cheung GYC, Otto M (2012) The potential use of toxin antibodies as a strategy 16. 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PLOS ONE | www.plosone.org 12 March 2014 | Volume 9 | Issue 3 | e92094 DISCUSSION CONCLUSION L#1 *#3!-2-6',#1 -,2! 0#I3! !#! ,-+! . 0!#! /3#! *_-,! ! *-,%2#+.1! !-,1'"M0M! /3#! *#301! !' *#1! .0'+ '0#1 1-,2 *#1 *#3!-!72#1Q .-302 ,2 !#1 +-*M!3*#1 .#34#,2 311' %'0 130 .*31'#301 320#1 !' *#1 !#**3* '0#1T L#1 ,#30-,#1 #, %0 ', "3 !#04#*#2 0#.0M1#,2#,2 3,# "#1 !#1 !' *#1 -& * LPVQ *_I V2-6',#)#2)* )L V&M+-*71',# H*%A H*%B . 0 '11#,2 .*31 $ ' *#+#,2 !2'4#1 /3# H*%C H*%BQ /3' .0-4-/3# 3,# $-02# *' M0 2'-, "# G*32 + 2#T C#1 "'$$M0#,!#1 0#," '#,2 ',2M0#11 ,2 "# !-+. 0#0 *#1 #$$#21 "# H*%C H*%B 9 !#36 "# * LPVQ !-,1'"M0M# * .*31 !2'4# 130 *# 27.# !#**3* '0# *# .*31 1#,1' *#T N-20# M23"# +-,20M /3# + *%0M "#1)0#11#+ * ,!#1Q)!#1)"#36)*#3!-2-6',#1Q),_-,2). 1)#6 !2#+#,2)*#)+N+#)+-"#)"_ !2'-,T) 7*) 4 '2)M2M)+-,20M)/3#)*_ !2'-,)"#1)*#3!-2-6',#1)!-++#,!#). 0)* )$'6 2'-,)"#)* )1-31 V3,'2M "# !* 11# S 9 * +#+ 0 ,# .* 1+'/3# "#1 !#**3*#1 !' *#1 C-*', #2 *TQ S[[VT RM!#++#,2Q 3,# ..0-!&#) 1M#)130)* ),#320 *'1 2'-,). 0)"#1) ,2'!-0.1)1.M!'$'/3#1) ).#0+'1)*_'"#,2'$'! 2'-,)"#) CW RQ !-++# 0M!#.2#30 "# L3)SVPV S. , #2 *TQ TRSUT D# .*31Q H*%C #,20# #, !-+.M2'2'-, 4#! 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�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former des NETs (Neutrophil Extracellular Traps) non !"#$%&'()*R&'" %,)- /0,#'-&)1&)1 2&,'&)1&) R#--%,#" )#, née assuré par les neutrophiles (Cheng and Palaniyar, 2013), 3)1&) R567),%/ 0#8&)/3%9&8"):08)1&')-3 /% &')0,"#-#/83;#&,,&')&'")&<:% ' )10,') le milieu extracellulaire via 1&')9 '#/% &'):3%8)#--3;# #'&8) R0=&,")#,2&/"#&%<( En effet en présence de S. aureus , le noyau polylobé des PMNs devient rapidement arrondi et condensé, les membranes nucléaires internes et externes se séparent en même temps que la formation de vésicules, et se remplissent d'ADN nucléaire. Puis les vésicules sont extrudées intactes dans l'espace extracellulaire où elles libèrent la chromatine. Ce processus indépendant du mécanisme oxydatif, est très rapide (5-60 min), et ne nécessite pas la lyse des neutrophiles ou même la rupture de la membrane plasmique (Pilsczek et al., 2010). La LPV, comme LukG/LukH (Malachowa et al., 2013), semble induire ce mécanisme, ce qui pourrait être approfondi et recherché pour les autres leucotoxines (Pilsczek et al., 2010). E,$',Q * .-3013'2# "# * 0#!�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�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