INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Pseudomonas aeruginosa is an important nosocomial pathogen with an impressive arsenal of secreted and cell-associated virulence factors (42). Although considered a primarily extracellular pathogen, some clinical and laboratory isolates are internalized by epithelial cells (11, 12, 14). Invasive strains cause disease (11), but the role of invasion in the pathogenic process is unclear. Bacterial internalization may actually benefit the host by serving as a defense mechanism. Shedding of epithelial cells harboring intracellular P. aeruginosa may help clear the bacteria from the site of infection (40), and phagocytosis by macrophages may help eliminate the pathogen and stimulate adaptive immune responses.

Initial analyses suggested that P. aeruginosa strains were either exclusively cytotoxic or exclusively invasive (14), but further investigations have revealed that at least one strain, lung isolate PA103, has the capacity to be either cytotoxic or invasive (24). Wild-type PA103 is cytotoxic and noninvasive, but isogenic mutants defective in type III secretion are no longer cytotoxic and are efficiently internalized by multiple eukaryotic cell types (8, 24). PA103 expresses the type III secreted effector proteins ExoT and ExoU but not ExoS and ExoY, which have been identified in other P. aeruginosa strains (52). An isogenic PA103 mutant lacking the type III secreted effector ExoU is not cytotoxic and is not internalized (24). These results prompted the hypothesis that ExoT or an additional unknown type III secreted effector protein negatively regulates invasion of eukaryotic cells (8, 24).

P. aeruginosa joins a growing list of gram-negative bacteria that regulate their internalization by delivering effector proteins to the host cell via a type III secretion system. Examples of other bacteria with these properties include enteropathogenic Escherichia coli (EPEC), Yersinia species, and Salmonella enterica serovar Typhimurium. EPEC inhibits its own uptake by macrophages by a type III secretion-dependent mechanism that coincides with phosphotyrosine dephosphorylation of a subset of host proteins. The dephosphorylation activity was inhibited by pervanadate (an inhibitor of tyrosine phosphatases), but no tyrosine phosphatase activity could be detected (18). In Yersinia species, the invasin protein interacts with high affinity with the 1 integrin receptors on the host cell, resulting in bacterial uptake (26, 27). Two Yersinia type III secreted effectors, YopE and YopH, function as anti-internalization factors (38, 41). YopH is a potent tyrosine phosphatase whose targets include the focal adhesion protein p130^cas and focal adhesion kinase (4, 19, 20, 38). The invasion of epithelial cells by S. enterica serovar Typhimurium is exquisitely regulated by the type III secreted effectors SopE and SptP. SopE catalyzes the conversion of the inactive GDP-bound forms of CDC42 and Rac to the active GTP-bound forms by virtue of its guanine nucleotide exchange activity (21). The increases in CDC42 and Rac activities lead to a disruption of the actin cytoskeleton (ruffling), which promotes bacterial internalization (5). The SopE-induced ruffling is terminated and thus tightly regulated by SptP. The N-terminal portion of SptP shares homology with YopE of Yersinia and with the type III secreted effector proteins ExoS and ExoT of P. aeruginosa; all four effectors have a conserved arginine finger domain characteristic of GTPase-activating proteins (GAPs) (16).

While this work was in progress, Cowell and coworkers demonstrated that ExoT inhibits internalization of P. aeruginosa by corneal epithelial cells (6). The closely related protein ExoS has been shown to function as an anti-internalization factor (6, 15). Bacteria secreting either of these proteins also induce rounding and/or detachment of some epithelial cell types in culture (48). We build upon these results and demonstrate that ExoT can inhibit the internalization of P. aeruginosa by polarized epithelial cells and by macrophage-like cells in culture. We hypothesize that the anti-internalization activity of ExoT results from its ability to act as a GAP. In support of this hypothesis, we demonstrate that the arginine finger domain present in ExoT and characteristic of GAPs contributes to its anti-internalization activity as well as its ability to alter the actin cytoskeleton. Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger does not disrupt translocation. Finally, we demonstrate that the virulence of PA103UT is abrogated compared to that of PA103U, revealing that the ability of P. aeruginosa to regulate its internalization is important for its pathogenic potential.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and media. Bacterial strains were routinely cultured in Luria-Bertani (LB) broth or Vogel-Bonner minimal medium (VBM) with antibiotics as needed for cloning purposes. The following antibiotics and concentrations were used: ampicillin, 100 µg/ml for E. coli; carbenicillin, 200 µg/ml for P. aeruginosa; and gentamicin, 15 µg/ml for E. coli and 100 µg/ml for P. aeruginosa. All antibiotics and chemicals were purchased from Sigma (St. Louis, Mo.) except where noted. A list of strains and constructs used in this study is shown in Table 1. All cloning steps were performed with XL2-Blue ultracompetent E. coli (Stratagene). Primer bases shown in lowercase letters are not homologous to the chromosomal sequence.

Immunoblot analysis. Five milliliters of cultured bacteria was centrifuged at 6,000 × g at 4°C for 20 min. Proteins were recovered from the supernatants by ammonium sulfate precipitation (final concentration of 55%). After incubation on ice for 18 h, precipitated proteins were concentrated by centrifugation at 13,000 × g at 4°C for 20 min. The pellet was boiled in a solution containing 50 µl of 10 mM NaCl and 50 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 5 min, and 20 µl of each sample was electrophoresed on an SDS-8% polyacrylamide gel (33). Proteins were electrotransferred to nitrocellulose for immunoblot analysis, and ExoT was detected using a rabbit polyclonal ExoT antiserum (24). Goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugate (Gibco BRL, Gaithersburg, Md.) diluted 1:5,000 was used as a secondary antibody. Detection was performed using the enhanced chemiluminescence (ECL) system (Amersham Corp., Arlington Heights, Ill.).

HeLa cell rounding assay. HeLa cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle medium with 4.5 g of glucose per liter (DME-H21) obtained from the University of California, San Francisco, Cell Culture Facility (UCSF-CCF) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco BRL). Cells were plated at 2 × 10⁵ cells/well in 24-well plates and grown overnight at 37°C in the presence of 5% CO₂. Cells were washed twice with minimal essential medium-Eagle's medium containing Hanks' buffered saline solution (HBSS) (MEM; Sigma Chemical Co.), 20 mM HEPES buffer (pH 8.0), 3.5% sodium bicarbonate, and 0.6% bovine serum albumin (MEM-etc.). HeLa cell wells were inoculated with 5 × 10⁶ bacteria and incubated for 6 h at 37°C in room air. Rounding was assessed by visual inspection of the wells at ×100 and ×200 magnifications on a Nikon Eclipse TE200 microscope. Images were captured using a charge-coupled device (CCD) camera and SPOT imaging software (Diagnostic Instruments).

Fluorescence microscopy of infected HeLa cells. HeLa cells were infected by the same procedure used for the rounding assay described above, except that the wells contained 12-mm-diameter acid-etched glass coverslips. After 6 h of infection, wells were washed twice with ice-cold phosphate-buffered saline (PBS). Cells were fixed by rocking them in 0.4% paraformaldehyde in PBS at room temperature for 30 min. Wells were rinsed once with PBS, and Quench (PBS with 75 mM NH₄Cl and 20 mM glycine) was applied for 10 min, with rocking at room temperature. Fixed cells were stored in PBS for 18 h at 4°C. Cells were permeabilized in PBS with 0.7% (wt/vol) fish scale gelatin, 0.005% (wt/vol) saponin, and 100 µg of RNase A per ml (PBS-FSG-SAP) for 30 min with rocking at 37°C. Coverslips were placed cells down on a 50-µl drop of PBS-FSG-SAP with unpurified anti-ExoU polyclonal antiserum (diluted 1:500) in a sealed humid container at 37°C for 2 h. Although the strains used in these experiments do not produce ExoU, the antiserum contains additional antibodies, likely to lipopolysaccharide, which result in sufficient labeling of the bacteria. Coverslips were returned to their original wells and washed with PBS-FSG-SAP for 10 min four times, with rocking at 37°C. Coverslips were placed face down on 50-µl drops of PBS-FSG-SAP with a 1:500 dilution of goat anti-rabbit-fluorescein isothiocyanate antiserum (Jackson Laboratories, Bar Harbor, Maine) and a 1:200 dilution of Texas Red X-phalloidin (Molecular Probes, Eugene, Oreg.) in a sealed humid container at 37°C for 45 min. Coverslips were returned to their original wells and washed with PBS-FSG-SAP twice for 10 min with rocking at 37°C. The coverslips were then washed with PBS containing 0.1% (wt/vol) Triton X-100 twice for 5 min with rocking at 37°C. Coverslips were rinsed once with PBS and dipped in distilled H₂O before being mounted on glass slides with a ProLong Antifade Kit (Molecular Probes). Cells were observed, and images were captured at ×1,000 magnification under oil immersion using a Nikon Eclipse E800 fluorescence microscope. Images were captured using a CCD camera and SPOT imaging software.

MDCK cell invasion assay. MDCK cells were cultured in minimal essential medium-Eagle's medium with Earle's buffered saline solution (UCSF-CCF) with 5% FBS. Polarized monolayers were prepared by adding 10⁶ MDCK cells to 12-mm-diameter, 0.4-µm-pore-size Transwell filter supports (Corning Costar Corp., Cambridge, Mass.) and incubating them for 3 days at 37°C with 5% CO₂. MDCK cell monolayers were washed once with MEM-etc. and 10⁷ bacteria were added to the upper chamber of the transwell. The bacteria were incubated with the MDCK cells for 2 h at 37°C with 5% CO₂ to allow for invasion. The MDCK cell monolayers were then washed once with MEM-etc. and incubated for 2 h at 37°C with 5% CO₂ in the presence of 400 µg of amikacin per ml to kill extracellular bacteria. Monolayers were then washed with MEM-etc., and the filters were excised and placed in Falcon 2059 tubes, where the cells were lysed by vortexing them with glass beads in Ca²⁺- and Mg²⁺-free HBSS (UCSF-CCF) with 0.25% (wt/vol) Triton X-100 (Sigma). Lysates were plated on LB agar and incubated for 18 h at 37°C to quantify the CFU of internalized bacteria. Invasion counts for the various mutants were normalized to that for PA103pscJ::Tn5 for strains without plasmids and to that for PA103UT+pUCP20 for strains with plasmids.

J774.1 invasion assays. J774.1 macrophage-like cells were cultured in DME-H21 with 10% FBS. Cells were plated in 24-well culture plates at 1.25 × 10⁵ cells/well and incubated for 18 h at 37°C with 5% CO₂. The J774.1 cells were washed once with DME-H21 with 10% FBS, 10⁷ bacteria were used to inoculate each well, and the plate was incubated at 37°C with 5% CO₂ for 2 h. Amikacin was added to the infected wells to a final concentration of 400 µg/ml, and the plates were incubated an additional 2 h at 37°C with 5% CO₂. We have noted marked detachment of the J774.1 cells when infecting with ExoS- or ExoT-expressing bacteria; thus, removal of the antibiotic-containing medium from the wells prior to release of intracellular bacteria was performed as follows. Infected J774.1 cells were scraped off the bottom of the wells using a cell scraper, and the suspension was placed in an Eppendorf tube. The cells were microcentrifuged at 14,000 rpm in an Eppendorf 5417C centrifuge for 2 min, the supernatant was removed, and the pellet was resuspended in Ca²⁺- and Mg²⁺-free HBSS with 0.25% Triton X-100. Suspensions were returned to their original wells and incubated for 30 min at room temperature. Serial dilutions were plated for counts of internalized bacteria on LB agar. An aliquot of the lysed cell suspension from each well was assayed for total lactate dehydrogenase (LDH) activity by measuring pyruvate reduction as instructed by the manufacturer (Sigma, procedure 500). The total LDH activity per well was used as a measure of total J774.1 cells left in the well at the end of the assay. The number of internalized bacteria counted for each well was divided by the total LDH units measured for that well. These ratios were normalized to that obtained with PA103pscJ::Tn5 for non-plasmid-containing strains or normalized to that obtained with PA103UT+pUCP20 for plasmid-containing strains. The average percentages of invasion and standard errors of the means are shown.

Fractionation of infected HeLa cells. HeLa cells were plated at approximately 10⁶ cells per 10-cm-diameter dish in DME-H21 with 10% FBS and incubated at 37°C in the presence of 5% CO₂ for 36 h. On the day of the assay, an extra plate was trypsinized and a count of 4 × 10⁶ cells per plate was obtained using a hemacytometer. Cells were washed once with MEM-etc. without albumin, and each plate was infected with 2 × 10⁷ bacteria, a multiplicity of infection (MOI) of approximately 5. The infected cells were incubated for 1.5 h at 37°C in the presence of 5% CO₂; this time point was chosen to minimize HeLa cell detachment due to the cell rounding effects of ExoT.

Animal experiments. Bacteria were grown for 24 h in MINS medium (25 mM KH₂O₄, 95 mM NH₄Cl, 50 mM monosodium glutamate, 110 mM disodium succinate, 10 mM trisodium nitrilotriacetic acid, 2.5% glycerol, 5 mM MgSO₄, 18 µM FeSO₄) or LB broth at 37°C with shaking and then washed and resuspended in PBS; the growth conditions of the bacteria do not affect the outcome of the virulence experiments (J. Engel, unpublished results). Equal numbers of bacteria of PA103 and PA103T (5.0 × 10⁶ CFU of each strain), PA103U and PA103UT (5.0 × 10⁷ CFU of each strain), or PA103exoU::Tn5 and PA103U/T(R149K) (5.0 × 10⁷ CFU of each strain) were resuspended in a total of 50 µl of PBS and instilled into the nares of anesthetized 6- to 8-week-old female BALB/c mice. Twenty-four hours later, the animals were sacrificed, at which time the animals were near death. The right lobes of the lung and of the liver were removed under sterile conditions, placed in 1 ml of LB broth, and homogenized. Serial dilutions were plated onto LB agar plates containing no antibiotic or 50 µg of gentamicin per ml [PA103 and PA103U carry no antibiotic resistance markers, whereas PA103T and PA103UT carry a Gm^r gene that replaces part of the exoT gene; PA103U/T(R149K) does not carry a resistance marker, whereas PA103exoU::Tn5 has a transposon carrying a Gm^r gene inserted into the exoU gene]. A competitive index was calculated by obtaining the ratio of the number of cells of the ExoT mutant strain to that of the wild type (with respect to ExoT expression) recovered from the lung or liver and comparing it to the same ratio obtained with the infecting inoculum (roughly 1.0 but calculated precisely for each experiment). A competitive index greater than 1.0 indicates that the ExoT mutant strain colonized better than the wild type, and a competitive index less than 1.0 indicates that the ExoT mutant strain was less efficient than the wild type in colonization. All animal experiments were carried out in accord with the policies of the Committee on Animal Research at UCSF.

Statistical analysis. Statistical analysis was performed using InStat 1.12 software.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

ExoT is required for inhibition of internalization of PA103 by multiple cell types. Because bacterial invasion is difficult to assess in a cytotoxic strain, the anti-invasive properties of ExoT were studied in an exoU mutant background. A strain containing an in-frame deletion of the exoU gene, PA103U, was constructed as described in Materials and Methods. Reverse transcriptase-PCR analysis demonstrated that the mutation did not affect transcription of the downstream spcU gene (data not shown and reference 9). This mutant was then used to construct an exoU exoT double mutant (PA103UT) (see Materials and Methods). Figure 1 demonstrates that ExoT secretion was not detected by immunoblot analysis of PA103UT (Fig. 1, lane 3) but was restored by complementation with a vector carrying the exoT gene cloned from PA103 (strain PA103UT+pExoT) (Fig. 1, lane 8).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Immunoblot analysis of secreted ExoT. Bacterial strains were cultured under the same conditions used for phenotypic assays (LB broth with 200 µg of carbenicillin per ml for plasmid-containing strains, standing for 18 h at 37°C). Proteins secreted into the culture medium were precipitated with ammonium sulfate, electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose, and hybridized with rabbit anti-ExoT polyclonal antiserum. Goat anti-rabbit IgG horseradish peroxidase conjugate was used as a secondary antibody, and detection was performed using the ECL system. Lane 1, PA103; lane 2, PA103U; lane 3, PA103UT; lane 4, PA103U/T(R149G); lane 5, PA103U/T(R149K); lane 6, PA103pscJ::Tn5; lane 7, PA103UT+pUCP20; lane 8, PA103UT+pExoT; lane 9, PA103UT+pExoT(R149G); lane 10, PA103UT+pExoT(R149K).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   ExoT inhibits internalization of P. aeruginosa by MDCK cells. Polarized monolayers of MDCK cells were infected apically at an MOI of 10 to 20 with the isogenic mutants of P. aeruginosa strain PA103 shown. (A) For strains containing no episomal vectors, the number of internalized bacteria for each well was normalized to the average number of internalized PA103pscJ::Tn5 bacteria. The normalized values were averaged to calculate the percentages of invasion, and the standard errors of the means for 17 to 18 experimental points are shown. The actual average numbers of internalized bacteria per well for PA103pscJ::Tn5 and standard errors of the means for the three experimental rounds shown were 691.67 ± 64.16, 955.00 ± 134.46, and 591.83 ± 119.14. (B) For strains containing episomal vectors, the number of internalized bacteria for each well was normalized to the average number of internalized PA103UT+pUCP20 bacteria. The normalized values were averaged to calculate the percentages of invasion, and the standard errors of the means for 10 to 12 experimental points are shown. The actual average numbers of internalized bacteria per well for PA103UT+pUCP20 and standard errors of the means for the two experimental rounds shown were 1,911.67 ± 484.49 and 3,873.33 ± 292.64.

ExoT inhibits bacterial internalization by macrophages. In addition to contacting epithelial cells, P. aeruginosa is likely to interact with macrophages in vivo (31, 43). The ability of the bacterium to prevent its internalization by professional phagocytes, thus avoiding destruction and antigen presentation, likely provides a survival benefit and enhances pathogenesis. Bacterial uptake into macrophages may occur by pathways alternate or additional to those employed by epithelial cells. We tested the ability of ExoT to inhibit internalization of P. aeruginosa by the J774.1 macrophage-like cell line. Preliminary experiments demonstrated that ExoS and ExoT cause rounding and detachment of J774.1 cells. J774.1 cells are also susceptible to type III secretion-dependent apoptotis-like damage (22), potentially confounding quantitation of bacterial internalization. To correct for this, following cocultivation of the bacteria with the adherent cells, the medium was spun to collect any detached cells. The cell pellet was combined with the remaining adherent cells prior to lysis of the cells to release internalized bacteria. In addition, the amount of internalized bacteria calculated for each well was normalized to the total number of J774.1 cells in that well as measured by total LDH activity released during the lysis step.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   ExoT inhibits internalization of P. aeruginosa by J774.1 macrophage-like cells. J774.1 cells were infected with isogenic mutants of P. aeruginosa strain PA103, shown at an MOI of 40 to 60. For each infected well, the number of internalized bacteria was divided by the value for total LDH activity released upon lysis of the cells in that well to normalize for J774.1 cell detachment from the culture plate during infection (internalized CFU/LDH). Averages ± the standard errors of the means for five to six experimental points are shown. (A) For strains containing no episomal vectors, the internalized CFU/LDH value for each well was normalized to the average internalized CFU/LDH value for PA103pscJ::Tn5 in experiments performed with J774.1 cells of the same passage. The normalized CFU/LDH values were averaged to calculate the percentages of invasion, and the standard errors of the means are shown. The actual average number of internalized CFU per well ± the standard error of the mean for PA103pscJ::Tn5 in the representative experimental results shown was 3.87 × 106 ± 4.44 × 104, and the average LDH units per well was 9.05 × 104 ± 3.91 × 103, yielding an average internalized CFU/LDH of 43.1 ± 1.35. (B) For strains containing episomal vectors, the internalized CFU/LDH for each well was normalized to the average internalized CFU/LDH for PA103UT+pUCP20. The actual average number of internalized CFU per well ± the standard error of the mean for PA103UT+pUCP20 in the representative experimental results shown was 1.26 × 106 ± 6.91 × 104, and the average value for LDH units per well was 4.96 × 104 ± 2.33 × 103, yielding an average internalized CFU/LDH and standard error of the mean of 25.54 ± 1.65.

The arginine finger domain of ExoT is important for anti-internalization activity. The N-terminal region of ExoT is homologous to those of ExoS, YopE, and SptP (16). The region of homology lies in a motif, GXXRXSG, characteristic of and required for GTPase activity in GAPs. In particular, the invariant arginine residue is critical to GAP activity (54). ExoS, YopE, and SptP have demonstrable GAP activities which require the invariant arginine residue in biochemical assays utilizing purified substrates in vitro (3, 16, 50). Mutation of the conserved arginine in SptP abolishes the ability of S. enterica serovar Typhimurium to restore the normal appearance of the actin cytoskeletons of epithelial cells following infection (16). We hypothesized that ExoT might also exert effects on the host cell actin cytoskeleton through GAP activity. In accordance with our hypothesis, it has been recently reported that a truncated version of ExoT (amino acids 78 to 237) possesses GAP activity toward RhoA, Rac1, and CDC42 in vitro (32).

The arginine finger domain of ExoT is required for cell rounding and cytoskeleton disruption. Previous reports have implicated both ExoS and ExoT in cell rounding and in the disruption of the actin cytoskeleton in CHO cells; these biological effects might be predicted in a protein with GAP activity towards the small GTPases Rho, Rac, and/or Cdc42. PA103UT expressing either ExoT or ExoS caused cell rounding when it was incubated with CHO cells (48). Similarly, transfection of ExoS into CHO cells altered the cytoskeletal architecture (37).

View larger version (122K): [in this window] [in a new window]   FIG. 4.   The conserved residue R149 contributes to the cell rounding activity of ExoT. HeLa cells were infected with isogenic strains of P. aeruginosa at an MOI of 25 for 6 h. (A) No bacteria; (B) PA103pscJ::Tn5; (C) PA103U+pUCP20; (D) PA103UT+pUCP20; (E) PA103UT+pExoS; (F) PA103UT+pExoT; (G) PA103UT+pExoT(R149G); (H) PA103UT+pExoT(R149K). Infected cells were observed by phase-contrast microscopy. The larger images were captured at a ×100 magnification, and the inset images were captured at a ×200 magnification using a CCD camera and SPOT imaging software.

View larger version (80K): [in this window] [in a new window]   FIG. 5.   The conserved residue R149 contributes to the ability of ExoT to disrupt the actin cytoskeletons of infected cells. HeLa cells were infected with isogenic strains of P. aeruginosa at an MOI of 25 for 6 h. (A and B) No bacteria; (C and D) PA103pscJ::Tn5; (E and F) PA103U+pUCP20; (G and H) PA103UT+pUCP20; (I and J) PA103UT+pExoS; (K and L) PA103UT+pExoT; (M and N) PA103UT+pExoT(R149G); (O and P) PA103UT+pExoT(R149K). Infected cells were fixed, permeabilized, and stained with Texas Red-labeled phalloidin to visualize actin (red). In addition, the fixed and permeabilized cells were labeled with rabbit anti-ExoU antiserum and a secondary goat anti-rabbit-fluorescein isothiocyanate antibody to visualize P. aeruginosa (green). Note that although the bacterial strains shown do not express ExoU, the anti-ExoU antiserum contains additional antibodies, likely to lipopolysaccharide, which result in sufficient labeling of the bacteria. Labeled cells were observed by fluorescence microscopy, and the images shown were captured at a ×1,000 magnification with a CCD camera and SPOT imaging software. (A, C, E, G, I, K, M, and O) Actin staining only; (B, D, F, H, J, L, N, and P) merged image of both actin (red) and bacterial (green) staining.

ExoT is translocated into the host cell cytoplasm. ExoT protein release into the culture medium is known to be type III secretion dependent (48, 52, 53); however, translocation of the protein into the cytoplasm of host cells has not yet been demonstrated. One explanation for the partial loss of the anti-internalization and cell rounding ability of the ExoT R149 mutants is that the mutation causes a defect in translocation to the host cell cytoplasm. In order to test this possibility and to demonstrate translocation of wild-type ExoT, cellular fractionation of infected HeLa cells followed by Western blot analysis was performed.

View larger version (40K): [in this window] [in a new window]   FIG. 6.   ExoT is translocated into the host cell cytoplasm. HeLa cells were infected at an MOI of 5 with PA103UT carrying the plasmids shown. After infection for 1.5 h, the medium was removed and separated into supernatant (soluble proteins) and pellet (nonadherent bacteria) fractions. The adherent HeLa cells were lysed and separated into lysate (cytoplasm) and pellet (cell membranes and adherent or internalized bacteria) fractions. Proteins from all fractions were resuspended in SDS-PAGE sample buffer, and 40 µl of each sample (representing 16% of each fraction) was electrophoresed on SDS-8% and SDS-13% polyacrylamide gels. Proteins were electrotransferred to nitrocellulose and hybridized with rabbit polyclonal anti-ExoT antiserum (for 8% gels) or rabbit polyclonal anti-sigma E antiserum (for 13% gels). Goat anti-rabbit IgG-horseradish peroxidase conjugate was used as a secondary antibody, and detection was performed using the ECL system.

ExoT is required for full virulence in an animal model of acute pneumonia. The role of P. aeruginosa internalization in the pathogenesis of disease is unclear. P. aeruginosa strains that are invasive, but not cytotoxic, can cause disease (11). In some cell lines, there is evidence that the cystic fibrosis transmembrane receptor mediates internalization (39). It has been suggested that the loss of internalization by airway epithelial cells contributes to the chronic colonization and recurrent acute pneumonias seen in cystic fibrosis patients and that internalization followed by epithelial cell sloughing is a host defense mechanism (39). The generation of isogenic ExoT mutant strains of wild-type PA103 and PA103U afforded the opportunity to test the role of ExoT in virulence in a mouse model of acute pneumonia.

View larger version (25K): [in this window] [in a new window]   FIG. 7.   ExoT is required for full virulence in an animal model of acute pneumonia. Equal amounts of the indicated pairs of strains of bacteria were inoculated into the nares of mice. The lungs and livers of the mice were harvested 24 h later and homogenized, and serial dilutions were plated on selective medium to obtain counts of each bacterial strain. A competitive index was calculated by obtaining the ratio of the ExoT mutant strain counts to wild-type (with respect to ExoT expression) bacterial counts recovered from the lung or liver and comparing it to the same ratio obtained with the infecting inoculum (roughly 1.0 but calculated precisely for each experiment). A competitive index greater than 1.0 indicates that the ExoT mutant strain colonized better than the wild type, and a competitive index less than 1.0 indicates that the ExoT mutant strain was less efficient than the wild type in colonization. The number of animals used for each experiment is shown. Compared to PA103U, PA103UT was significantly impaired in its ability to colonize the liver (P = 0.06, Student's two-tailed t test).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Like many other gram-negative pathogens, P. aeruginosa can regulate its uptake into host cells. The work presented here, in conjunction with the results of Cowell et al. (6), conclusively identifies ExoT as the anti-internalization factor that can inhibit the internalization of P. aeruginosa by multiple cell types, including polarized and nonpolarized epithelial cells and tissue culture macrophages. Interestingly, of the pathogens examined so far, each one utilizes a unique approach to modulating its internalization. The S. enterica serovar Typhimurium SptP protein is a GAP for Cdc42 and Rac (16), while a type III secreted protein of EPEC may result in the dephosphorylation of one or more host tyrosine-phosphorylated proteins (18). As discussed below, ExoT may function as a Rho GAP to inhibit bacterial internalization.

An additional and intimately related activity of ExoT revealed by these experiments and others (48) is its ability to induce rounding and detachment of some but not all eukaryotic cell types in vitro. Phalloidin staining of HeLa cells incubated with isogenic ExoT mutants reveals that ExoT induces cell rounding, loss of stress fibers, the appearance of peripheral areas of condensed actin, and residual needle-like extensions that may reflect prior sites of host cell-extracellular matrix contact. The actin condensation is polar but does not appear in close proximity to the adherent bacteria. The pathway of actin condensation, the identification of other proteins in the structure, and the cause for the polar localization of the collapsed actin are important biological questions to pursue. In addition, the reason why some cell types are resistant to the cell rounding effects but sensitive to the anti-internalization activity of ExoT, such as polarized MDCK cells, remains to be explored. It is intriguing to speculate that this resistance reflects known differences between cell types in the regulation of cortical actin that contacts tight junctions in polarized epithelium (7).

Our work further suggests a mechanism for the capacity of ExoT to both inhibit bacterial internalization and modify the host cell cytoskeleton. As noted previously, the N-terminal portion of ExoT has an arginine finger domain suggestive of GAP activity. Furthermore, it has recently been shown that the N-terminal portion of ExoT can act as a GAP for Rho, Rac1, and CDC42 in vitro (32). We now demonstrate that the arginine finger domain, and thus GAP activity, is critical to the function(s) of ExoT. Mutation of the conserved arginine to a glycine or lysine partially diminished the anti-internalization activity of ExoT-producing bacteria. Consistent with this partial loss of function, less cell rounding and far less disruption of the actin cytoskeleton were observed in infections with strains expressing the arginine mutant ExoT proteins than in infections with the strain expressing wild-type ExoT.

There are several possible explanations for the residual anti-internalization activity, induction of cell rounding, and actin cytoskeleton rearrangements observed for the ExoT(R149G) or ExoT(R149K) mutant protein. The mutations may simply interfere with the secretion and/or translocation of ExoT into eukaryotic cells. However, immunoblot analysis demonstrates that plasmid-expressed R149 mutant proteins and wild-type ExoT are secreted when the bacteria are grown under inducing conditions in the absence of eukaryotic cells (Fig. 1). Fractionation of infected HeLa cells demonstrated that the R149 mutant ExoT proteins were translocated into the host cell cytoplasm as efficiently as wild-type ExoT (Fig. 6). Moreover, we have found that when the R149 mutant ExoT proteins are introduced directly into HeLa cells by transient transfection, only minimal cell rounding and actin cytoskeleton disruption are observed and GAP activity is lost (B. Kazmierczak, unpublished data). Similar mutations introduced at the conserved arginine residue of the closely related proteins ExoS and Yersinia YopE or the Salmonella homolog SptP result in loss of GAP activity in vitro (3, 16, 17, 50). Together, these findings suggest that the arginine finger motif is critical to ExoT function but that other domains of ExoT or other type III secreted proteins also contribute to inhibition of internalization and modulation of the actin cytoskeleton. If other type III secreted proteins contribute to this process, they cannot be ExoS or ExoY, as PA103 does not synthesize these proteins. Experiments to test these hypotheses are in progress.

Our demonstration that the putative GAP domain of ExoT is important to its function is consistent with recent investigations into the pathway by which PA103 enters epithelial cells. We have found that P. aeruginosa internalization is likely regulated by small GTPases, as it is inhibited by Clostridium difficile toxin B (B. I. Kazmierczak, K. Mostov, and J. Engel, unpublished data). This bacterial toxin inhibits the Rho, Rac, and CDC42 families of small GTPases that have been shown to effect actin cytoskeleton rearrangements (28). Our work further suggests that Rho is sufficient to stimulate P. aeruginosa internalization but that additional toxin B-sensitive GTPases may also be activated upon P. aeruginosa entry (30). We hypothesize that the target of ExoT GAP activity is one or more toxin B-sensitive GTPases whose activities are required for P. aeruginosa internalization. Support for this notion comes from the observation by others that ExoT exhibits GAP activity towards RhoA, Rac, and CDC42 in vitro (32). Identification of the exact intracellular target(s) of the ExoT anti-internalization factor may provide additional information about the process of internalization itself.

As previously noted by others (6), an interesting paradox is observed in naturally occurring strains that secrete both ExoS and ExoT. While most or all clinical isolates appear to encode the ExoT gene, only a subset of clinical isolates harbor the ExoS gene (13). Such strains thus potentially produce two anti-internalization factors, and yet they appear much more invasive than strains that produce only ExoT, even when the gene encoding the cytotoxin ExoU is inactivated in the ExoT-secreting strains. For example, both the ExoS and ExoT genes encoded by strain 388 confer anti-internalization activity and cause epithelial cell rounding when they are expressed in PA103UT (48). Yet strain 388 is highly invasive despite having the ability to secrete both proteins in vitro (52) and to at least translocate ExoS into eukaryotic cells (49) (evidence for translocation of ExoT by strain 388 has not been reported). One possible explanation for these observations is that ExoS can antagonize the activity of ExoT. In fact, this possibility is suggested by our finding that expression of ExoS derived from 388 can antagonize the anti-internalization act