ExoY activity does not noticeably affect microtubule disassembly.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M (upper panels) or wild type ExoY (middle panels). Following infection, cells were either fixed before microtubule disassembly was induced (left) or at 1 (middle) or 2 minutes (right) after being placed at 0°C. As shown, disassembly was complete in both control (Ctr), ExoY^K81M and ExoY⁺ expressing cells by 2 minutes after transfer to 0°C. Bar = 10 µm.</p

ExoY activity affects microtubule assembly in PMVECs.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺. Untreated control cells (Ctr) and infected cells were then placed on ice to induce microtubule disassembly. Microtubule re-growth was initiated by transferring the cells to 37°C. [<b>A.</b>] Individual coverslips were fixed either at the time of transfer (T = 0) or at varying times after transfer to 37°C. The coverslips then were labeled with antitubulin antibodies. Cells at 4 and 8 minutes post-transfer are shown; microtubule growth is apparent by 4 minutes and peripheral microtubules are resolved by 8 minutes in untreated and K81M infected cells. Microtubule re-growth lagged significantly in cells intoxicated with wt ExoY. Bar = 10 µm. [<b>B.</b>] Polymerized (P) and soluble unpolymerized (S) tubulin levels were quantified by immunoblot analysis using antitubulin antibodies. To obtain soluble and polymer fractions, control cells and cells infected with bacteria expressing either ExoY^K81M or ExoY⁺ were extracted at 8 minutes post-transfer to 37°C. The ratio of polymerized tubulin to soluble tubulin was significantly less in cells containing wild type ExoY [0.10±0.06 <i>vs.</i> 0.36±0.10 (K81M) and 0.41±0.09 (Ctr); n = 4; P<0.05 compared to both K81M and untreated cells]. [<b>C.</b>] Tau levels are unchanged following cold treatment to disassemble microtubules. Cells were treated with cold and then whole extracts were collected from control cells (Ctr) and from cells that were intoxicated with either ExoY⁺ or ExoY^K81M. The extracts were then probed for tau levels using polyclonal anti-tau antibody.</p

ExoY activity causes a decrease in microtubules in PMVECs.

<p>[<b>A</b>] PMVECs infected with <i>P.aeruginosa</i> expressing either non-functional K81M mutant ExoY (ExoY^K81M; center) or wild type ExoY (right) were observed following processing for anti-tubulin immunofluorescence microscopy. Uninfected cells are also shown (left). Bar = 10 µm. [<b>B</b>] Levels of polymerized tubulin (P) and unpolymerized soluble tubulin (S) were quantified by immunoblot analysis using antibody against α-tubulin. Extracts obtained from untreated cells (Ctr) and from PMVECs infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺ are shown. The ratio of polymerized tubulin to soluble tubulin was significantly less in cells containing wild type ExoY⁺ [0.29±0.06 <i>vs.</i> 0.44±0.05 (ExoY^K81M) and 0.49±0.08 (Ctr); n = 5; P<0.05 compared to both ExoY^K81M and untreated control].</p

ExoY activity does not noticeably affect microtubule assembly from PMVEC centrosomes.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M (middle panel) or ExoY⁺ (left panel). Untreated cells (Ctr) and infected cells were extracted to remove soluble proteins and then purified rat brain tubulin was added and microtubule nucleation was initiated by incubating at 37°C for 15 min. The preparations then were fixed and labeled with antitubulin antibodies. Centrosome nucleation of microtubules is shown by arrows. Bar = 10 µm.</p

Phosphorylation causes Tau to dissociate from microtubules.

<p>[<b>A.</b>] Untreated cells (Ctr) as well as cells that were infected with P. aeruginosa expressing either ExoY^K81M or ExoY⁺ were incubated, and then extracts were prepared and soluble fractions and cell ghosts containing intact microtubules were collected. Both microtubule-associated (Mt) and soluble free Tau (S) were assayed by immunoblot using a pan-Tau antibody. Significantly less tau was present in cells ghosts prepared from cells intoxicated with ExoY⁺ compared to those prepared from either untreated or K81M-intoxicated cells [0.22±0.09 <i>vs.</i> 0.44±0.08 (Ctr) and 0.46±0.04 (K81M); n = 4; P<0.05 compared to both K81M and untreated cells]. [<b>B.</b>] <b>Tau co-pellets with taxol-stabilized microtubules.</b> Extracts were prepared from untreated control PMVECs (Ctr) and from cells infected with bacteria expressing either ExoY⁺ (middle) or ExoY^K81M (right), microtubules were assembled by addition of taxol, and then the assembled microtubules were pelleted through a sucrose cushion. The pelleted microtubules were then probed by immunoblot using polyclonal anti-tau antibody (top). The blot was then stripped and re-probed using antitubulin antibody (bottom).</p

Phosphorylation of tau is essential for disrupted microtubule assembly.

<p>Over-expression of a non-phosphorylatable form of Tau (S214A mutant) rescues PMVECs from effects of ExoY⁺ on microtubule assembly. PMVECs were stably transfected with a cDNA encoding a form of Tau mutated at the PKA phosphorylation site and then infected with <i>P. aeruginosa</i> encoding either ExoY^K81M or ExoY⁺. Microtubules were disassembled by incubation on ice, and then microtubule re-assembly was initiated by transferring cells to 37°C. [<b>A.</b>] S214A Tau-expressing control cells (Ctr) and cells infected with either ExoY^K81M or ExoY⁺ were fixed and labeled with antitubulin antibodies either at the time of transfer to 37°C (top) or at 8 minutes post-transfer (bottom). Bar = 10 µm. [<b>B.</b>] Polymerized (P) and soluble unpolymerized (S) tubulin levels were quantified in S214A-expressing cells at 8 minutes post-transfer to 37°C. Extracts were prepared from control cells (Ctr) and from cells infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺. There was no significant difference in the ratio of microtubule polymer to soluble tubulin when cells infected with bacteria expressing ExoY⁺ were compared to those expressing ExoY^K81M or uninfected control cells [0.35±0.8 <i>vs.</i> 0.38±0.06 (K81M) and 0.44±0.11 (Ctr); n = 5].</p

ExoY intoxication disrupts Tau localization.

<p>Tau localization in control PMVECs (Ctr) and cells intoxicated with bacteria producing either ExoY^K81M or wild-type ExoY⁺. The cells then were fixed and processed for immunofluorescence using both tubulin (green) and tau (red) antibodies. Images were collected at the same focal plane, the images were merged and co-localization of tubulin and tau can be seen (arrows) in the control and ExoY^K81M infected cells. No co-localization was observed in cells intoxicated with ExoY⁺. Bar = 10 µm.</p

Flowchart of patient screening.

The number of patients screened, excluded, and recruited to the study are listed for both the culture-positive and culture-negative arms.</p

Tau, boiled tau, Aβ₄₂, and cytotoxicity levels in culture-positive and culture-negative patient lavage fluid.

(A) Tau, (B) boiled tau, (C) Aβ42, and (D) cytotoxicity were assayed as described in the Methods for culture-positive and culture-negative patients. Data are expressed as mean ± SEM, n are listed below groups; * represents p < 0.05.</p

Tau, boiled tau, Aβ₄₂, and cytotoxicity levels are similar in different bacterial phenotypes.

(A) Tau, (B) boiled tau, (C) Aβ42, and (D) cytotoxicity were assayed as described in the Methods in different phenotypes of bacteria: Gram-positive, Gram-negative, or both. Data are expressed as mean ± SEM, n are listed below groups. (TIF)</p