Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling

Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection

Länderstudie Deutschland

Faulstich P, Graeßner G, Bade-Becker U, Gorys B. Länderstudie Deutschland. In: Hanft A, Knust M, eds. Weiterbildung und lebenslanges Lernen in Hochschulen. Eine internationale Vergleichsstudie zu Strukturen, Organisation und Angebotsformen. Münster: Waxmann; 2007: 87-164

Structure of BamA, an essential factor in outer membrane protein biogenesis

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1–β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer

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

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

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