Platelet-activating factor receptor initiates contact of Acinetobacter baumannii expressing phosphorylcholine with host cells

Abstract

Adhesion is an initial and important step in Acinetobacter baumannii causing infections. However, the exact molecular mechanism of such a step between A. baumannii and the host cells remains unclear. Here, we demonstrated that the phosphorylcholine (ChoP)-containing outer membrane protein of A. baumannii binds to A549 cells through platelet-activating factor receptor (PAFR), resulting in activation of G protein and intracellular calcium. Upon A. baumannii expressing ChoP binding to PAFR, clathrin and β-arrestins, proteins involved in the direction of the vacuolar movement, are activated during invasion of A. baumannii. PAFR antagonism restricts the dissemination of A. baumannii in the pneumonia model. These results define a role for PAFR in A. baumannii interaction with host cells and suggest a mechanism for the entry of A. baumannii into the cytoplasm of host cells.

FIGURE 1.

Expression of ChoP on A. baumannii. A and D, Western blot analysis of ChoP in A. baumannii 77wt, 77R, ATCC 17978wt, and PSAB03 strains. B, ChoP expression by A. baumannii was then quantified by FACS. Histograms illustrate the fluorescence intensities of control cells (A. baumannii without TEPC-15 antibody and with the FITC-tagged secondary antibody) when compared with those incubated with TEPC-15 and FITC-tagged secondary antibody. C, serum bactericidal assay on A. baumannii 77wt and 77R strains, grown to mid-log phase and treated for 60 min in 10% normal human serum. The percentage of survival is the number of cfus remaining when compared with controls in which complement activity was inactivated. Data are representative of three independent experiments.

FIGURE 2.

Role of ChoP in the A. baumannii adhesion to and internalization by lung epithelial cells. A549 cells were infected with 10⁸ cfu/ml A. baumannii ChoP⁺ or ChoP⁻ for 2 h. A, an assay of adherence and invasion was performed as described under “Experimental Procedures.” The adherence or invasion of A. baumannii ChoP⁻ is expressed as the percentage of total A. baumannii ChoP⁺ adhered to or internalized by A549 cells. B, immunostaining for A549 cells and A. baumannii OMPs and ChoP was performed and imaged by immunofluorescence microscopy. OMPs and ChoP (white lines) of A. baumannii strains were detected with mouse anti-A. baumannii OMPs and TEPC-15 antibodies and labeled with Alexa Fluor 488-tagged secondary antibodies (green). Blue staining shows the location of nuclei of A549 cells. C and D, adherence and invasion of ChoP⁺ or ChoP⁻, respectively, were determined in A549 cells pretreated with anti-ChoP TEPC-15 antibody. The effect of ChoP antibody on adherence and invasion of A. baumannii is expressed as the percentage of total nontreated A. baumannii ChoP⁺ adhered to or internalized by A549 cells. Data are representative of three independent experiments. *, between ChoP⁺ and ChoP⁻, ChoP⁺ + ChoP antibody, or ChoP⁻ + ChoP antibody. CTL, control; Ab, antibody.

FIGURE 3.

Involvement of PAFR in the adherence/internalization of A. baumannii and in the cell death of lung epithelial cell induced by A. baumannii. A549 cells were pretreated with PAFR antagonist (PAFR ant, 10, 50, 100 and 200 nm) or mouse anti-human PAFR antibody (Ab, 25 μg/ml) and infected with 10⁸ cfu/ml A. baumannii ChoP⁺ or ChoP⁻. A, assay of adherence and invasion of A. baumannii ChoP⁺ for 2 h was performed as described under “Experimental Procedures.” The effect of PAFR antagonist and antibody on adherence or invasion of A. baumannii is expressed as the percentage of total nontreated A. baumannii ChoP⁺ adhered to or internalized by A549 cells (CTL). B, immunostaining for A. baumannii OMPs in infected A549 cells for 2 h was performed and imaged by immunofluorescence microscopy. The A. baumannii ChoP⁺ strain was detected by mouse anti-OMP antibody and labeled with Alexa Fluor 488-tagged secondary antibody (green). The percentage of ChoP⁺ strain associated with A549 cells was calculated as ((number of A549 cells attached by ChoP⁺ strain colonies/number of total A549 cells) × 100). C and D, the A. baumannii ChoP⁺ or ChoP⁻ strain cytotoxicity was determined by monitoring the mitochondrial reduction activity using the MTT assay for 24 h. Representative results of three independent experiments are shown, and data are the means ± S.D. p < 0.05: *, between nontreated and treated groups, #, between ChoP⁺ treated groups.

FIGURE 4.

Activation of G protein coupled to PLC during invasion and cell death induced by A. baumannii. A549 cells were pretreated with U73122 (5 μm) or U73343 (5 μm) and infected with 10⁸ cfu/ml A. baumannii ChoP⁺ or ChoP⁻. A, assay of adherence and invasion of A. baumannii ChoP⁺ for 2 h was performed as described under “Experimental Procedures.” The effect of U73122 or U73343 on adherence or invasion of A. baumannii is expressed as the percentage of total nontreated A. baumannii ChoP⁺ adhered to or internalized by A549 cells. CTL, control. B, intracellular Ca²⁺ mobilization was monitored for 10 min in nontreated or pretreated A549 cells with U73122 and infected with ChoP⁺ strain for 5 min. C and D, A. baumannii ChoP⁺ or ChoP⁻ strain cytotoxicity was determined by monitoring the mitochondrial reduction activity using the MTT assay for 24 h. Representative results of three independent experiments are shown, and data are the means ± S.D. p < 0.05: *, between nontreated and treated groups, #, between ChoP⁺ treated groups.

FIGURE 5.

Role of clathrin and β-arrestins in A. baumannii internalization by lung epithelial cells. A and C, A549 cells were pretreated with monodansylcadaverine (MDC, 30 μm) or chlorpromazine (CPZ, 5 μm) or transfected with control, β-arrestin-1, and β-arrestin-2 siRNA and infected with 10⁸ cfu/ml A. baumannii ChoP⁺ for 2 h. An assay of adherence and invasion of A. baumannii ChoP⁺ assay was performed as described under “Experimental Procedures.” The effect of monodansylcadaverine-, CPZ-, or siRNA-mediated β-arrestin depletion on adherence or invasion of A. baumannii is expressed as the percentage of total nontreated A. baumannii ChoP⁺ adhered to or internalized by A549 cells. B, immunoblot analysis of β-arrestin expression in control, β-arrestin-1 (β-arr-1), and β-arrestin-2 (β-arr-2) siRNA-transfected A549 cells. Values shown are expressed as the percentage of level of each siRNA of β-arrestin in control-transfected A549 cells. D, immunostaining for A549 cells and A. baumannii OMPs was performed in mock and control or β-arrestin-1 or β-arrestin-2 siRNA-transfected A549 cells and imaged by immunofluorescence microscopy. β-Arrestins of A549 cells and OMPs of A. baumannii strains were detected by rabbit anti-human β-arrestins and mouse anti-A. baumannii OMPs antibodies and labeled with Alexa Fluor 594- and Alexa Fluor 488-tagged the secondary antibodies (red and green), respectively. Blue staining shows the location of the nuclei of A549 cells. Representative results of three independent experiments are shown, and data are the means ± S.D. p < 0.05: *, between nontreated and treated groups. Sc: scramble.

FIGURE 6.

Role of PAFR in the dissemination of A. baumannii in the pneumonia model and in the histological changes. A, mice were intratracheally inoculated with the ChoP⁺ strain in the presence or absence of PAFR antagonist (PAFR ant, 1 μg). CTL, control. B and C, bacterial loads were determined at 6 h of infection. B and C, representative lung slides of mice infected with ChoP⁺ (B) or infected with ChoP⁺ + PAFR antagonist (C) after 6 h of infection. D and E, histopathological score evaluated alveolar and vascular inflammation (D) and percentages of total alterations (E). See “supplemental Materials and Methods” for full description; sections are representative of at least three mice per group. Pictures were taken under 100× magnifications. Data are the means ± S.D. p < 0.05: *, between nontreated and treated groups.

FIGURE 7.

A proposed scheme for the mechanism of A. baumannii adherence and invasion in human lung epithelial cells. We propose that ChoP and PAFR enhance the adherence and invasion of A. baumannii in A549 cells. A. baumannii expressing ChoP adhere to A549 cells via PAFR, which thereafter activates a cascade of pathways composed of G protein-coupled PLC, clathrin, and β-arrestins, required for A. baumannii invasion. IP₃: inositol triphosphate.