Deletion of the SREC-I cytoplasmic domain does not prevent gonococcal uptake.

<p>CHO cells were transfected with pEGFPN1 (control) and constructs overexpressing wildtype SREC-I-GFP (SREC-I), SREC-IY818A-GFP (SREC-IY818A), and SREC-I-GFP lacking the cytoplasmic domain (SREC-IΔCD). (<b>A</b>) 24 h post transfection these cells were infected with N927 (PorB_IA, P⁻) at a MOI of 50. Invasive bacteria were enumerated by confocal microscopy from 50 randomly chosen cells using differential immunostaining. (<b>B</b>) Whole-cell lysates were analyzed by western blotting using an anti-GFP and anti-Actin antibody. (<b>C</b>) CHO cells transfected with plasmid encoding SREC-I (white bars) or SREC-IΔCD (black bars) were either left untreated (-) or treated with 25 µg/ml Nystatin for 1 h and infected with N927 (PorB_IA, P⁻) at a MOI of 50. Invasive bacteria were counted from 50 randomly chosen cells using differential immunostaining. The graphs show the mean ± SD of two independent experiments. p<0.05: *; p<0.01: **.</p

PI3 kinase is required for N927 invasion and is recruited to caveolae.

<p>(<b>A</b>) Activation of PI3K shown by phosphorylation of Akt. Whole cell lysates of Chang cells infected with either N927 (PorB_IA, P⁻) or N138 (PorB_IB, P⁺) at an MOI 50 for 30 min were subjected to SDS PAGE and Western blot using anti-phospho-Akt, anti-Akt and anti-Actin antibodies. (<b>B</b>) Chang cells were pretreated for 1 h with PI3K inhibitors LY294002 (LY, 10 µM) or Wortmannin (WM, 1 µM) and infected with N927 (MOI 10, 30 min). Adherence (white bars) and invasion (black bars) were quantified by gentamicin protection assay. The number of adherent and invasive bacteria of untreated control cells was set as 100%. The graph shows mean values ± SD of three independent experiments performed in duplicates. p<0.01: ** (<b>C</b>) Distribution of signaling molecules in membrane rafts of infected cells. Chang cells were subjected to subcellular fractionation after infection either with N927 (PorB_IA, P⁻) or N138 (PorB_IB, P⁺) (MOI 20) for 1 h (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003373#ppat.1003373.s011" target="_blank">Text S1</a>). Flotillin was detected as marker for the membrane raft fraction (fraction 4–5) separated from most cellular proteins (fraction 8–12). Caveolin, SREC-I (multiple bands represent differentially glycosylated forms), Flotillin and PI3K, were detected by Western blot analysis. (<b>D</b>) CHO cells stably transfected with either SREC-I WT (CHO-SREC-I) or empty vector control (CHO-pEGFP-N1) were infected with N927 (PorB_IA, P⁻) at an MOI of 50 or treated with 2 µg/ml acLDL (Invitrogen) for 5 min. Whole cell lysates were subjected to SDS-PAGE and Western blot analysis using anti-phospho-Akt and anti-Tubulin antibodies. (<b>E</b>) Graphical representation of SREC-I gonococci interaction in Caveolae. Upon infection with N927 SREC-I localizes in cholesterol, sphingolipid and caveolin-1 rich membrane rafts. PLCy1 and PI3K are recruited to phosphorylated Cav1 (Tyrosin 14) and initiate the signaling cascade leading to endocytic uptake of the gonococci. Adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003373#ppat.1003373-Parton1" target="_blank">[25]</a>.</p

Caveolin is required for N927 invasion.

<p>(<b>a</b>) AGS cells (AGS) and a transgenic AGS cell line expressing caveolin-1 (AGS Cav1wt) were infected with N927 (PorB_IA, P⁻) at MOI 50. Adherent (adh.) and intracellular (inv.) bacteria were quantified by gentamicin protection assays. The number of adherent and invasive bacteria of AGS cells was taken as 100%. The graph shows mean values ± SD of three independent experiments done in duplicates. White bars: adherent bacteria; black bars: intracellular bacteria p<0.01: ** (<b>b</b>) AGS cell expressing an HA-tagged wt (AGS Cav1wt) or mutant (AGS Cav1Y14F) caveolin were analyzed by Western blot using an HA antibody. (<b>c</b>) Intracellular N927 (PorB_IA, P⁻) of the experiment shown in figure (D) were quantified by differential immunofluorescence. p<0.01: **. (<b>D</b>) AGS cells were transiently transfected with HA-tagged Cav1 (AGS-Cav1) or Cav1Y14F (AGS-Cav1Y14F) and infected with N927 at MOI 25. Adherent (pink) and intracellular (red; white arrows) bacteria were detected by differential immunofluorescence. Caveolin expression was visualized with an HA antiserum and a Cy2-conjugated secondary antibody (green). Scale bar: 10 µm (<b>E</b>) SREC-I is recruited to N927 (PorB_IA, P⁻)(white arrows), but not N138 (PorB_IB, P⁺). Chang cells were infected with SNARF-labeled bacteria at an MOI 25. SREC-I was detected with a polyclonal serum against SREC-I and a Cy2-conjugated secondary antibody. Co-localisation of SREC-I and gonococci was analyzed by confocal fluorescence microscopy. Scale bar: 10 µm.</p

PKD1 is required for invasion of N927.

<p>(<b>A</b>) Chang cells were transfected with siRNAs against PKD1 (siPKD1) and luciferase (siLuc) as control. The cells were infected with N927 (PorB_IA, P⁻; MOI 10) for 30 min 72 h after transfection of siRNAs. Intracellular (inv., black bars) and adherent (adh., white bars) bacteria were quantified by gentamicin protection assay and the number of adherent or invasive bacteria of control cells (siLuc) was set to 100%. Shown are the means ± SD of three independent experiments performed in duplicates. p<0.01: ** (<b>B</b>) Knock down of PKD1 in Chang cells was verified by Western blotting. β-tubulin was used as loading control. (<b>C</b>) Endogenous levels of phosphorylated PKD1 (pPKD1) were assayed after infection with N927 (MOI 100) or treatment with the known activator 12-O-Tetradecanoylphorbol 13-acetate (TPA, 0.2 µM) for 30 min. Whole cell lysates were analyzed by immunoblotting using phospho-specific PKD1 antibody (detects pSer744 and pSer748). (<b>D</b>) PKD1 was overexpressed by transfection of PKD-HA expression construct (Addgene; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003373#ppat.1003373-Storz1" target="_blank">[41]</a>) in Chang cells and phosphorylation was detected as described in (C).</p

Abl1 and PLCγ1 are essential for N927 invasion.

<p>(<b>A</b>) Chang cells were pretreated for 1 h with the Abl1 inhibitor Imatinib (10 µM) and subsequently infected with N927 (PorB_IA, P⁻) at an MOI 10 for 30 min. Adherence (white bars) and invasion (black bars) were quantified by gentamicin protection assays. The number of adherent (adh.) and invasive (inv.) bacteria of untreated control cells was set to 100%. The bar chart shows mean percentages ± SD of three independent experiments each performed in duplicate. (<b>B</b>) shRNA-mediated downregulation of PLCγ1 in Hela cells results in decreased internalization of N927 (PorB_IA, P⁻). Control cells (shLuci) as well as shPLCγ1 cells (shPLCy1-1, shPLCy1-2) were infected with strain N927 (MOI 10, 30 min) and adherence (white bars) as well as invasion (black bars) were analyzed by gentamicin protection assays. The numbers of adherent and invasive bacteria of control cells (shLuci) were set to 100%. Shown are mean percentages ± SD of three independent experiments performed in duplicate. (<b>C</b>) Chang cells were either left untreated (-) or pretreated for 30 min with PLCγ1 inhibitor U73122 (10 µM) and infected with N927 (MOI 10, 30 min). Adherence (white bars) and invasion (black bars) were quantified by gentamicin protection assays. The numbers of adherent (adh.) and invasive (inv.) bacteria of untreated control cells were set to 100%. The graph shows mean values ± SD of three independent experiments performed in duplicates. (<b>D</b>) PLCγ1 co-precipitates with Cav1 in N927-infected cells and untreated cells. Chang cells were infected with either N927 (PorB_IA,P⁻) or N138 (PorB_IB,P⁺) MOI 20 for 1 h. Endogenous Cav1 was precipitated from infected and not infected control (no inf) cells and co-precipitated PLCγ1 was detected by Western blot. p<0.01: **.</p

Phospho-Tyr14-Cav1 binding partners identified by MALDI-TOF/TOF after streptavidin pulldown of biotin-labeled Tyr14-Cav1 peptides.

<p>Phospho-Tyr14-Cav1 binding partners identified by MALDI-TOF/TOF after streptavidin pulldown of biotin-labeled Tyr14-Cav1 peptides.</p

Neisseria MS11 derivatives.

a<p>Pn, non-revertible loss of piliation; P+, piliated; P−, non-piliated; Opa−, no detectable Opa expression;</p>b<p>Shown are the genes and their orientation introduced into the genome of strain MS11; The plasmid pTH6 (with or without opa genes) are integrated into the Neisseria p<i>tetM</i>25.2 plasmid. Arrowheads indicate 5′ end (> or <) and 5′ to 3′ orientation (>) of genes.</p

Identification of new Cav1 pTyr14 interaction partners.

<p>(<b>a</b>) Pull-down of Cav1-pY14 interaction partners. Biotin-labeled Cav1 peptides either with (Cav1 pY14 peptide) or without (Cav1 peptide) phosphorylated Tyr14 were used as baits to precipitate interacting proteins from AGS cell lysates using a streptavidin-agarose pulldown assay. Precipitates were separated by SDS-PAGE and proteins visualized by silver staining. The proteins enriched in the Cav1 pY14 pulldown (indicated by numbers; see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003373#ppat-1003373-t001" target="_blank">table 1</a>) were identified by Maldi-MS/MS. (<b>b</b>) Western blot analysis of proteins precipitated from ME-180 cells using either biotin-labeled phosphorylated or non-phosphorylated Cav1 peptides as bait. PLCy1 as well as the regulatory p85 and catalytic p110 subunit of PI3K, are only detected in precipitates of the phosphorylated peptide with the respective primary antibodies. (<b>c</b>) PI3K-p85 interacts with Cav1 in N927 infected cells. Chang cells were infected with either N927 (PorB_IA, P⁻) or N138 (PorB_IB, P⁺) at an MOI 20 for 1 h or not infected (no inf). Endogenous Cav1 was precipitated and co-precipitated proteins were detected by Western blot with antibodies specific for Cav1 or PI3K-p85. (<b>D</b>) Relative amount of PI3 kinase quantified from (C). The bars represent the mean values ± SD of three independent experiments. (<b>E</b>) PI3 kinase recruitment to caveolin depends on activity of PLCy1. The experiment was performed as described in (C). 30 min prior to infection the PLCy1 inhibitor U73122 (10 µM) was added to the cells. (<b>F</b>) Relative amount of p85 quantified from three independent experiments. Shown are the mean values ± SD. (<b>G</b>) Interaction of Cav1 with Vav2 tested under conditions as described in (C). (<b>H</b>) Relative amount of Vav2 quantified from the experiment shown in (G). The bar graph shows the mean value of three independent experiments ± SD. p<0.05: *; p<0.01: **.</p

Pili interfere with PorB_IA-dependent invasion.

<p>(<b>A</b>) Chang cells were infected with N2009 (PorB_IA, P⁺) or N2010 (PorB_IA, P⁻) at MOI 10 for 1 h and then analyzed by gentamicin protection assays. The number of adherent (white bars) and invasive (black bars) bacteria of the strain N2010 was taken as 100%. The graph shows mean values ± SD of two independent experiments performed in duplicates. (<b>B</b>) Intracellular N2009 are highly enriched for non-piliated variants. Piliated (white bars, Pili+) and non-piliated (black bars, Pili−) gentamicin resistant N2009 gonococci recovered from the experiment shown in (A) were quantified for their pilus phenotype with a steromicroscope. (<b>C</b>) Chang cells were infected at an MOI of 10 for 30 min with N927 (PorB_IA, P⁻) or with N2013 (<i>recA</i>ⁱ, PorB_IA, P⁺), a <i>rec</i>A mutant which cannot undergo <i>pil</i>E recombination. The bar chart shows the results of a gentamicin protection assay as the mean values ± SD of three independent experiments done in duplicates. The number of adherent (white bars) and invasive (black bars) bacteria of N927 was set to 100%. p<0.01: ** (<b>D</b>) Model of the molecular events underlying the switch from local to invasive gonococcal infection.</p

<i>Ctr</i>-induces EphA2 activation and receptor internalization which associates with pPI3K during early infection.

<p>(A) HeLa cells uninfected (UN) or infected with <i>Ctr</i> (MOI-50-75) for 30 min or 3 h were analyzed via FACS for surface EphA2 under non permeabilised or total EphA2 under permeabilised condition. Controls were indicated on the left curve chart and corresponding samples on the right curve chart of the same experiment. (UN: uninfected, Iso: isotype and perm: permeabilised). (B) Result of experiment shown in A demonstrated as bar chart. Shown is the mean ± SD of three independent experiments normalized to UN perm. *P<0.05, ns: non-significant. Error bars show mean ± SD. (C) HeLa cells were UN or infected with <i>Ctr</i> (MOI-50) for the indicated time points. The cells were immunobloted against pEphA2 and Actin. The blot was stripped and reprobed for total EphA2. (D) HeLa cells were treated with control or rhEphrin-A1 for 3 h and were harvested for WB analysis. (E) HeLa cells were UN or infected with EB for the indicated time points and immunoprecipitated (IP) with α-EphA2 or α-p85-PI3K antibodies. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%) or (F) immunostained using α-EphA2 and α-pPI3K for 2 h at RT followed by secondary staining with anti-mouse Alexa fluor 488 and anti-rabbit Alexa fluor 647 for the microscopic analysis, respectively. Magnification is indicated in size bar.</p