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Science 325 (5936): 83-87

Copyright © 2009 by the American Association for the Advancement of Science

Meningococcal Type IV Pili Recruit the Polarity Complex to Cross the Brain Endothelium

Mathieu Coureuil1,*, Guillain Mikaty1, Florence Miller2,3, Hervé Lécuyer1,4, Christine Bernard1, Sandrine Bourdoulous2,3, Guillaume Duménil1,{dagger}, René-Marc Mège5, Babette B. Weksler6, Ignacio A. Romero7, Pierre-Olivier Couraud2,3, and Xavier Nassif1,4

1 Université Paris Descartes, Faculté de Médecine, INSERM (U-570), 75015 Paris, France.
2 Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 75015 Paris, France.
3 INSERM, U567, 75014 Paris, France.
4 AP-HP, Hôpital Necker-Enfants Malades, Paris F-75015, France.
5 INSERM UMR-S 839, Université Pierre et Marie Curie-Paris6, Institut du Fer à Moulin, 75005 Paris, France.
6 Weill Cornell Medical College, New York, NY 10021, USA.
7 Department of Life Sciences, The Open University, Walton Hall, Milton Keynes MK7 6AA, UK.

Figure 1
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Fig. 1. N. meningitidis recruits ectopic junctionlike domains beneath colonies. (A) VE-cadherin (green), the main component of the endothelial AJ, colocalized with actin (red) beneath N. meningitidis (Nm) colonies (top row). Two other AJ components, p120-catenin and β-catenin, and three components of the TJ, ZO-1, ZO-2, and claudin-5, are recruited under N. meningitidis colonies (bottom row). Arrow indicates a bacterial colony. Scale bars indicate 10 µm. (B) Yellow fluorescent protein (YFP)–tagged Par6 (par6-YFP) or myc-tagged Par3 (par3-myc), both green, are recruited underneath N. meningitidis colonies where they colocalize with actin (red). Areas outlined in white indicate the presence of a N. meningitidis colony. Scale bars, 10 µm. The formation of these ectopic early junctionlike domains is not found underneath all N. meningitidis colonies. Signaling underneath bacterial microcolonies required a minimal number of 20 bacteria per colony to be detected by immunofluorescence, with around 40 to 50% of microcolonies containing 40 to 50 bacteria. The average number of colonies signaling after 2 hours of infection is 40%.


Figure 2
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Fig. 2. The Cdc42-Par3/Par6/PKC{zeta} pathway controls the formation of ectopic early junctionlike domains. Data are expressed as mean ± SEM. (A) Knockdown of Cdc42 was performed by using specific small interfering RNA duplexes (Cdc42 siRNA). Cells were cotransfected with par6-YFP or par3-myc. Knockdown of Cdc42 by RNAi reduced the recruitment of par6-YFP and par3-myc by fourfold. *t test (P < 0.005). (B) Knockdown of Cdc42, Par6 and Par3 were performed as described (27) (Cdc42 siRNA, Par6 siRNA, and Par3 siRNA). Scramble siRNA and siCONTROL were used as control for Cdc42/Par6 and Par3 knockdown, respectively. Knockdown of Cdc42 by RNAi reduced the recruitment of VE-cadherin, p120-catenin, and actin by 2.2-fold, 2.3-fold, and 2.5-fold, respectively. See also fig. S3. Knockdown of Par6 by RNAi reduced the recruitment of VE-cadherin, p120-catenin, and actin by 2.7-fold, 2.4-fold, and 2.4-fold, respectively. Knockdown of Par3 by RNAi reduced the recruitment of VE-cadherin by twofold. *t test (P < 0.01), **t test (P < 0.002). (C and D) HCMEC/D3 cells were either incubated with 3 µM or 6 µM of PKC{zeta}-PS or PKC{eta}-PS (control) or left untreated. (C). PKC{zeta}-PS (6 µM) reduced VE-cadherin, p120-catenin, and actin recruitment by 8.5-fold, 5-fold, and 4.9-fold, respectively. *t test (P < 0.001). (D) HCMEC/D3 cells were transfected with either par6-YFP or par3-myc. Par3-myc recruitment was reduced 9-fold by 6 µM PKC{zeta}-PS, but par6-YFP recruitment was not affected [*t test (P < 0.001), **t test (P < 0.01)].


Figure 3
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Fig. 3. P120-catenin is key to the recruitment of both actin and AJ proteins. (A and B) VE-cadherin silencing was performed by stable expression of a VE-cadherin shRNA (VEC shRNA). (A) Recruitment of β-catenin, p120-catenin, and actin was determined by immunofluorescence. Knockdown of VE-cadherin had no effect on the recruitment of p120-catenin and actin but reduced β-catenin recruitment by 20-fold. *t test (P < 0.001). Data are expressed as mean ± SEM. (B) In VEC-shRNA–expressing cells, p120-catenin was still recruited beneath N. meningitidis colonies, where it colocalized with actin (top), whereas β-catenin was no longer recruited (bottom). Areas outlined in white indicated the location of a N. meningitidis colony. Scale bars, 10 µm. (C) Silencing of p120-catenin was performed by using a specific siRNA duplex (p120 siRNA). Recruitment of VE-cadherin and actin was determined by immunofluorescence. Knockdown of p120-catenin reduced VE-cadherin and actin recruitment by 10-fold and 4-fold, respectively. *t test (P < 0.001). Data are expressed as mean ± SEM.


Figure 4
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Fig. 4. N. meningitidis–induced PKC{zeta} activity facilitates cell-cell junction opening. (A) The permeability coefficient of Lucifer Yellow was measured 4 hours postinfection by N. meningitidis (Nm) or its nonpiliated isogenic strain (Nm {Delta}pilE), or after treatment by PKC{zeta}-PS or PKC{eta}-PS (6 µM). N. meningitidis induced a 1.55-fold increase compare with control. D-mannitol, which disrupts all cell-cell junctions, induced a 3.1-fold increase. *t test (P < 0.001). (B) HCMEC/D3 cells were incubated with 6 µM of PKC{zeta}-PS or of PKC{eta}-PS (control). (a) VE-cadherin localization was analyzed on the basolateral cross section of N. meningitidis–infected cells. Yellow arrowheads and areas outlined in yellow indicate gaps between cells. Areas outlined in red indicate the presence of N. meningitidis colonies. Blue bars marked 1 to 4 refer to Z-axis reconstruction images 1 to 4 in (b). Scale bars, 20 µm. (b) Z-axis reconstructions from a stack of 0.12-µm interval images show that VE-cadherin is apically relocalized underneath N. meningitidis colonies (white arrows) only in cells treated with PKC{eta}-PS (control). (C) HCMEC/D3 cells grown on 3.0µm pore size inserts were treated or not with PKC{zeta}-PS and incubated with N. meningitidis or its nonpiliated isogenic strain (N.meningitidis {Delta}pilE). Size and quantity of gaps observed 4 hours after infection are calculated as described (27). (D) Diffusion of N. meningitidis through an hCMEC/D3 monolayer, 4 hours postinfection, is 3.2-fold higher than diffusion of N. meningitidis in the presence of 6 µM PKC{zeta}-PS and 16.5-fold higher than diffusion of its nonpiliated derivative (Nm {Delta}pilE). The rate of N. meningitidis internalization, determined by gentamicin protection assay, is very low (1 colony-forming unit in 3.5 x 105), identical to that of a control without cells, thus excluding a possible transcytosis of bacteria. Data are expressed as fold increase of N. meningitidis diffusion and calculated as described (27). Data from (B) to (D) are one representative experiment of three independent duplicates. Data are expressed as mean ± SEM.


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