Platelet-activating Factor Receptor Initiates Contact of Acinetobacter baumannii Expressing Phosphorylcholine with Host Cells

Background: The mechanism of interaction between Acinetobacter baumannii and host cells remains unclear. Results: The presence of ChoP in A. baumannii increased bacterial adherence/invasion in A549 cells. Inhibitions of PAFR, G protein, and vacuole formation decreased this bacterial adherence/invasion. PAFR inhibition decreased lung bacterial loads in vivo. Conclusion: We define a role for PAFR and ChoP in A. baumannii interaction with host cells. Significance: This will aid in studying how A. baumannii invades host cells. 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.

that interacts with host cells and induces biofilm formation on abiotic surfaces (3,4) is responsible for the ability of A. baumannii to adhere and invade epithelial cells (5). Several other A. baumannii surface proteins and intracellular proteins have been identified and characterized. For many of them, isogenic mutants are less virulent in vitro and in vivo. Mutants deficient in OmpA, lipopolysaccharide (LPS), K1 capsular polysaccharide, penicillin-binding protein, and phospholipase D are less virulent in vitro and in vivo (4,(5)(6)(7)(8)(9).
In other Gram-negative bacteria, such as Pseudomonas aeruginosa and Haemophilus influenzae, mutants lacking phosphorylcholine (ChoP) expression in outer membrane show reduced interaction with host cells and virulence (10,11). The location of ChoP is different depending on bacterial species. Thus, it has been observed with LPS, fimbriae, capsules, and teichoic and lipoteichoic acids (12)(13)(14)(15) and can contribute to bacterial pathogenicity via a direct contact with host cells. Recently, it was demonstrated that ChoP plays an important role in H. influenzae biofilm maturation (16). In addition, binding of bacteria positive for ChoP to platelet-activating factor receptor (PAFR) mediates its adhesion to and invasion into human cells (10,11,17,18). This binding has been studied extensively in vitro. Typical of G protein-coupled receptors, PAFR activation by ChoP results in an initiation of a two-host cell signal cascade. Firstly, after the coupling of bacteria with PAFR, the G protein and phospholipase C (PLC) recruitment to the PAFR site is activated, and intracellular Ca 2ϩ is released after induction of inositol triphosphate (19). The intracellular Ca 2ϩ increase was shown to be involved in cell death induced by various pathogens (20,21). Secondly, the trafficking of vacuole has been shown to recycle the bacterial coupling of PAFR to the apical surface with further transmigration to the basolateral surface of the cell, with delivery of viable bacteria across endothelia and epithelia (22). In this second pathway, adapter proteins such as clathrin and ␤-arrestin-1 and ␤-arrestin-2 play a role in the direction of the vacuolar movement because they tether the bacteria-occupied PAFR to the vesicular trafficking system (23). Once ␤-arrestins target PAFR to endocytosis and/or transcytosis, they recruit and activate extracellular signal-regulated kinase 1 (ERK-1)/ERK-2 and mitogen-activated protein (MAP) kinase (23). In the case of A. baumannii, the adhesion to and invasion of this bacterium into eukaryotic cells is mediated by pili and fimbrial-like structure (24,25). A zipperlike mechanism (receptor-mediated entry) was suggested to be induced during the A. baumannii adherence to epithelial cells (5). Lee et al. (26) have shown that the A. baumannii surface protein activated the MAPKs including ERK, c-Jun N-terminal kinases, and p38 MAPK. Thus, circumstantial evidence suggests that A. baumannii uptake has characteristics of a PAFRmediated event. In this work, we sought to study whether A. baumannii induced the PAFR-mediated uptake pathway in vitro and in a pneumonia model. Moreover, we aimed to determine the involvement of clathrins and ␤-arrestins in this pathway.

EXPERIMENTAL PROCEDURES
Expanded details of all methods are given in the supplemental Materials and Methods.
Bacterial Strains and Growth Conditions-A. baumannii clinical isolate 77wt, collected in Spain in 2000 (27), its derivative ciprofloxacin-resistant strain 77R, and six other A. baumannii clinical strains were used (28,29). ATCC 17978wt and its derivative porinD mutant were also used (supplemental Table S1) (30). All strains were grown in a Mueller Hinton Broth or Luria Bertoni (LB).
DNA Procedures-Plasmid DNA was isolated and amplified using specific primers of porinD gene (supplemental Table S2). Isolated DNAs were separated by agarose gel electrophoresis and visualized by SYBR Safe DNA gel stain (Invitrogen).
Generation of porinD Knock-out from A. baumannii Strain ATCC 17978wt-To construct a porinD knock-out from A. baumannii ATCC 17978wt strain, an internal porinD 559-bp fragment obtained by PCR amplification with primers porinD IntUp and porinD IntLw (supplemental Table S2) was cloned into pGEM-T (31) to give plasmid porinD-pGEM-T. The resulting construct incorporated in Escherichia coli DH5␣ strain was purified and electroporated into A. baumannii ATCC 17978wt strain to knock out its porinD gene by allelic replacement. Transformants were selected on LB agar plates containing 80 g/ml ticarcillin. porinD gene disruption within the resulting strain, designated PSAB03, was verified by PCR using a combination of primers matching the upstream region of porinD gene and the pGEM-T Easy vector.
Human Cell Culture and Infection-Type II pneumocyte cell line A549 derived from human lung carcinoma was grown in supplemented DMEM medium. The cells were seeded 24 h in 96-well plates for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma) prior to infection with A. baumannii 77wt and 77R strains at a multiplicity of infection of 100.
Immunofluorescence-A549 cells plated on coverslips, transfected or not with siRNA of ␤-arrestin-1, ␤-arrestin-2, and control, and infected with A. baumannii 77wt and 77R at 37°C for 2 h were removed and washed five times with cold PBS.
ChoP Immunoblotting-Outer membrane fractions from ATCC 17978 wt, PSAB03, 77wt, 77R, and six other A. baumannii clinical strains were isolated as described previously (29). Western blot was performed using 10% SDS-PAGE with anti-ChoP TEPC-15 mAb as primary antibody and horseradish peroxidase-conjugated goat anti-mouse IgG antibody as secondary antibody. For peptide mass fingerprinting, a SimplyBlue TM Saf-eStain-stained band representing ChoP-containing OMP was excised from SDS-PAGE gel and analyzed by MALDI-TOF-TOF (MS-MS/MS).
␤-Arrestin Immunoblotting-Proteins removed from transfected A549 cells were collected. Western blot was performed using 10% SDS-PAGE with rabbit anti-human ␤-arrestin-1/2 as primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG as secondary antibody.
Flow Cytometry-FACS analysis was used to determine ChoP expression in 77wt and 77R strains. A single color containing only FITC-labeled ChoP of bacteria was analyzed, and the percentage of FITC-labeled ChoP of bacteria was quantified. A minimum of 10,000 events were analyzed in each experiment.
Serum Bactericidal Assay-Complement-mediated serum bactericidal activity was determined in normal human serum as described previously (34).
Pneumonia Infection Model-A pneumonia model was used as described previously by our group (35). Two groups of nine mice were anesthetized intraperitoneally with 5% (w/v) sodium thiopental and were inoculated with 50 l of 77wt (7.67 log cfu/ml), in the presence or absence of PAFR antagonist (1 g/mouse).
Histopathological Study-Lung histopathology studies were performed as described previously by our group (35).
Statistical Analysis-The group data are presented as means Ϯ S.D. A Student's t test was used to determine the differences between means. The difference was considered significant at p Ͻ 0.05. The SPSS (version 15.0) statistical package was used (SPSS Inc., Chicago, IL).

ChoP-containing OMP Expression on A. baumannii-The
Western blot analysis of OMPs isolated from seven A. baumannii clinical strains, with ChoP-specific mAb TEPEC-15, demonstrated the presence of a 43-kDa band that showed ChoPcontaining OMP expression by these strains (supplemental Fig.  S1). In the derivative ciprofloxacin-resistant strain (77R) of one of these strains (77wt), no detectable ChoP-containing OMP expression was apparent (Fig. 1A). The corresponding band of ChoP-containing OMP of 77wt was excised from a parallel SDS-PAGE and subjected to MS-MS/MS analysis and was found to correspond to porinD of A. baumannii (data not shown). To confirm that porinD contains ChoP, we used the allelic exchange strategy to delete the porinD gene from A. baumannii ATCC 17978wt strain. The presence or absence of porinD gene in A. baumannii ATCC 17978wt and PSAB03 strains was confirmed by PCR, showing that the PSAB03 strain is deficient in porinD gene (supplemental Fig. S2). The Western blot analysis of OMPs isolated from ATCC 17978wt and PSAB03 strains demonstrated the presence and absence of ChoP in ATCC 17978wt and PS03 strains, respectively (Fig.  1D). Flow cytometry analysis of both intact A. baumannii strains incubated with anti-ChoP mAb TEPC-15 antibody showed that the 77wt strain was positive for ChoP in 55.27 Ϯ 17.7% of all the cells analyzed. In contrast, 77R was only positive for ChoP in 6.83 Ϯ 2.11% of all the cells analyzed (Fig. 1B). These Western blot and flow cytometry results were further confirmed by serum bactericidal activity experiments. Previously, it was demonstrated that ChoP contributes to H. influenzae susceptibility to serum bactericidal activity (34). A. baumannii 77wt and 77R strains differing in expression of ChoP were compared for their ability to survive human serum bactericidal effect. 77wt strain survival in human serum for 60 min was 47.52 Ϯ 8.73%, whereas 77R strain survival was 94.22 Ϯ 12.55% (Fig. 1C). For the rest of the results of this study, we will name the 77wt strain positive for ChoP as ChoP ϩ and name the 77R strain negative for ChoP as ChoP Ϫ .
ChoP Involvement in A. baumannii Adhesion to and Internalization by Lung Epithelial Cells-To evaluate the involvement of ChoP in A. baumannii adherence and invasion of A549 cells, we studied the adherence and invasion of ChoP ϩ and ChoP Ϫ of A549 cells for 2 h. We showed that ChoP ϩ adhered to and invaded A549 cells 95% more than ChoP Ϫ (Fig. 2A). Infected A549 cells immunostaining with ChoP ϩ or ChoP Ϫ using the anti-A. baumannii OMPs antibody or the anti-ChoP mAb TEPC-15 antibody showed ChoP ϩ attachment to A549 cells. In contrast, infected A549 cells with ChoP Ϫ showed an absence of ChoP Ϫ attachment to A549 cells (Fig. 2B). We then determined the relationship between ChoP expression and A. baumannii adherence to and internalization by A549 cells. Anti-ChoP mAb TEPC-15 antibody incubation with ChoP ϩ for 1 h significantly reduced ChoP ϩ adhesion to and internalization by A549 cells to 25.29 Ϯ 7.68 and 23.06 Ϯ 11.97%, respectively (Fig. 2C). In contrast, pretreatment of A549 cells with unspecific antibody to ChoP, a mouse IgG, did not significantly reduce the adherence of ChoP ϩ to A549 cells (data not shown). As negative control, ChoP Ϫ incubation with anti-ChoP mAb TEPC-15 antibody did not significantly decrease ChoP Ϫ adhesion to and invasion of A549 cells (Fig. 2D). Altogether, we found that ChoP expression in A. baumannii was involved in A. baumannii adhesion to and invasion of lung epithelial cells.
PAFR Involvement in the Adherence/Internalization of A. baumannii and in the Cell Death of Lung Epithelial Cell Induced by A. baumannii-To evaluate the involvement of PAFR in A. baumannii ChoP ϩ adherence to and invasion of A549 cells, we studied the PAFR antagonist and anti-PAFR antibody effect on ChoP ϩ adherence to and invasion of A549   (Fig. 3A). Similarly, pretreatment of A549 cells with 25 g/ml anti-PAFR antibody significantly reduced ChoP ϩ adherence to and invasion of A549 cells to 52.32 Ϯ 14.8 and 56.26 Ϯ 26.56%, respectively (Fig. 3A). Immunostaining of infected A549 cells with ChoP ϩ and pretreating with 200 nM PAFR antagonist and 25 g/ml anti-PAFR antibody showed reduction in ChoP ϩ attachment to A549 cells from 53.92 Ϯ 1.74% to 23.52 Ϯ 8.08 and 27.08 Ϯ 7.73%, respectively (Fig. 3B). In addition, we evaluated the involvement of PAFR in the cell death induced by A. baumannii. For this, we studied the PAFR antagonist and anti-PAFR antibody effect on cell death of A549 cells induced by ChoP ϩ and ChoP Ϫ for 24 h by MTT assay analysis. We showed that pretreatment of A549 cells with 100 and 200 nM PAFR antagonist and 25 g/ml anti-PAFR antibody significantly protects A549 cells from cell death induced by ChoP ϩ from 68.11 Ϯ 1.08% to 82.18 Ϯ 2.69% and from 87.29 Ϯ 7.97 to 87 Ϯ 10.85%, respectively (Fig. 3C). We further confirmed these results by MTT assay analysis of ChoP Ϫ -infected A549 cells and pretreated with PAFR antagonist and antibody. As shown in Fig. 3D, pretreatment of A549 cells with PAFR antagonist and antibody did not significantly increase cell viability in comparison with non-pretreated and ChoP Ϫ -infected A549 cells. From these data, we found that PAFR play an important role in the adherence, internalization, and cell death induced by A. baumannii expressing ChoP.

Activation of G Protein Coupled to PLC during Invasion and
Cell Death Induced by A. baumannii-To evaluate the involvement of G protein coupled to PLC in A. baumannii ChoP ϩ adherence to and invasion of A549 cells, we studied the effect of G protein coupled to PLC inhibitor U73122 on ChoP ϩ adherence to and invasion of A549 cells for 2 h. U73122 was found to be effective in blocking the ChoP ϩ invasion of A549 cells to 16.06 Ϯ 8.49%. However, the total of the cell-adhered bacteria did not differ between non-pretreated and U73122-pretreated A549 cells, indicating that the inhibition was not due to inefficient binding ChoP ϩ to A549 cells. In contrast a control inactive analog compound, U73343, which did not show significant ability to block ChoP ϩ invasion in A549 cells, also failed to show inhibition of bacteria adhered to A549 cells (Fig. 4A). To further confirm that inhibition of ChoP ϩ invasion into U73122-pretreated A549 cells was the result of G protein coupled to PLC inhibition, intracellular Ca 2ϩ mobilization in baumannii ChoP ϩ or ChoP Ϫ . A, assay of adherence and invasion of A. baumannii ChoP ϩ 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 ϩ 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 ϩ strain was detected by mouse anti-OMP antibody and labeled with Alexa Fluor 488-tagged secondary antibody (green). The percentage of ChoP ϩ strain associated with A549 cells was calculated as ((number of A549 cells attached by ChoP ϩ strain colonies/number of total A549 cells) ϫ 100). C and D, the A. baumannii ChoP ϩ or ChoP Ϫ 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 Ϯ S.D. p Ͻ 0.05: *, between nontreated and treated groups, #, between ChoP ϩ treated groups.
ChoP ϩ -infected A549 cells pretreated with U73122 was monitored. We demonstrate that U73122 abolished almost completely the intracellular Ca 2ϩ increase induced by ChoP ϩ (Fig.  4B).
As mentioned in the Introduction, intracellular Ca 2ϩ increase has been shown to be involved in cell death. Therefore, we studied the effect of U73122 and U73343 on cell death of A549 cells induced by ChoP ϩ and ChoP Ϫ for 24 h. As shown in Fig. 4C, pretreatment of A549 cells with U73122 exhibited significant protection of A549 from cell death induced by ChoP ϩ from 66.18 Ϯ 5.87% to 90.38 Ϯ 6.31%. In contrast, U73343pretreated ChoP ϩ -infected A549 cells showed similar cell death to that observed with non-pretreated and ChoP ϩ -infected A549 cells. We further confirmed these results by MTT assay analysis of ChoP Ϫ -infected A549 cells and A549 cells pretreated with U73122 and U73343. As shown in Fig. 4D, pretreatment of A549 cells with U73122 and U73343 did not significantly increase the cell viability in comparison with nonpretreated ChoP Ϫ -infected A549 cells. From these data, we found that G proteins coupled to PLC play a role in the adher-ence, internalization, and cell death induced by A. baumannii expressing ChoP.

Role of Clathrin and ␤-Arrestin in A. baumannii Internalization by Lung Epithelial Cells-After bacterial invasion by bind-
ing to PAFR, clathrin and ␤-arrestins would be expected to participate in bacterial uptake. We examined the effect of clathrin inhibitors on ChoP ϩ adherence to and invasion of A549 cells for 2 h. Monodansylcadaverine (MDC) and chlorpromazine (CPZ), both specific inhibitors of assembly and recycling of clathrin-coated vesicles, were found to be effective in blocking ChoP ϩ invasion to 57.46 Ϯ 17.56 and 45.54 Ϯ 16.49%, respectively. However, total cell-adhered bacteria did not differ between nontreated and monodansylcadaverine-or CPZtreated A549 cells, indicating that the inhibition was not due to inefficient binding of ChoP ϩ to A549 cells (Fig. 5A). Moreover, to determine the role of ␤-arrestins in A. baumannii internalization by A549 cells, we evaluated the effect of ␤-arrestin small interfering RNA (siRNA)-transfected A549 cells on ChoP ϩ adherence to and invasion of A549 cells for 2 h. We first tested the ability of ␤-arrestin-1 or ␤-arrestin-2 siRNA to deplete the ␤-arrestin levels in A549 cells. ␤-Arrestin-1 or ␤-arrestin-2 siRNA transfections reduce the expression of targeted ␤-arrestin by 35 and 31%, respectively, when compared with either nonsilencing or control siRNA-transfected A549 cells (Fig. 5B). Both ␤-arrestin-1 and ␤-arrestin-2 siRNA-transfected A549 cells were found to be effective in blocking the ChoP ϩ invasion to 50.79 Ϯ 13.21 and 53.68 Ϯ 12.19%, respectively. However, total cell-adhered bacteria did not differ between control and ␤-arrestin-1 or ␤-arrestin-2 siRNA-transfected A549 cells, indicating that inhibition was not due to inefficient binding of ChoP ϩ to A549 cells (Fig. 5C). In contrast, control siRNA-transfected A549 cells did not show significant blocking of the ChoP ϩ invasion in A549 cells (Fig. 5C). Immunostaining studies showed that in A549 cells infected with ChoP ϩ , ␤-arrestin was colocalized with ChoP ϩ in A549 cells (Fig. 5D). Similar results are observed with control siRNA-transfected A549 cells. Both ␤-arrestin-1 and ␤-arrestin-2 siRNA-transfected A549 cells prevent the colocalization between ␤-arrestin and ChoP ϩ and, consequently, ChoP ϩ invasion of A549 cells (Fig. 5D). Taken together, we demonstrated that clathrin and ␤-arrestins were involved in the invasion of lung epithelial cells by A. baumannii expressing ChoP.

Role of PAFR in the Dissemination of A. baumannii in Pneumonia
Model-To study the role of PAFR on A. baumannii dissemination in vivo, we used the pneumonia model to infect mice with ChoP ϩ and treated them with PAFR antagonist. Lung colonization and infection by ChoP ϩ were attenuated to nearly 1.5 log cfu/ml by PAFR antagonist. Indeed, PAFR antagonist significantly reduced bacterial loads in infected lungs from 7.61 Ϯ 0.28 to 6.34 Ϯ 0.17 log cfu/ml (Fig. 6A). Furthermore, ChoP ϩ induced a high level of alveolar inflammation and vascular congestion scores 3.33 Ϯ 1.15 and 3.67 Ϯ 0.85, respectively, whereas treatment with PAFR antagonist in ChoP ϩ -infected mice decreased these scores to 1.67 Ϯ 0.58 and 1.67 Ϯ 0.58 for both alveolar inflammation and vascular congestion (Fig. 6, B-D). Total lung alterations were reduced in ChoP ϩinfected mice and in mice treated with PAFR antagonist in comparison with ChoP ϩ -infected mice (72.5 Ϯ 17.68% versus 40 Ϯ 13.23%) (Fig. 6E). From these data, we suggest that PAFR plays an important role in A. baumannii dissemination and pulmonary injury induction.

DISCUSSION
The present study provides new data highlighting the nature of the mechanism involved in the adherence to and invasion of host cells by A. baumannii. Here, we present the first evidences of the essential role played by PAFR in A. baumannii expressing ChoP adherence and invasion in human lung epithelial cells and dissemination of A. baumannii in the pneumonia model.
We showed that A. baumannii expressing ChoP adhere to human lung epithelial cells via PAFR, which thereafter activate a cascade of pathways composed of G protein-coupled PLC, clathrin, and ␤-arrestins, which are required for invasion of A. baumannii (Fig. 7).
This study showed that ChoP-containing OMP is among the factors involved in the adherence to and invasion of epithelial cells by A. baumannii, consistent with other factors described in other pathogens (36,37). Many results concerning the ability of pathogens expressing ChoP to invade human cells were related to different species and even different compartments from the same bacteria (10,11,38). Here, we showed that ChoP was present in OMPs of A. baumannii. Several studies have suggested that loss of ChoP might result in bacterial invasion dysfunction (10,11,19), which is not enough to avoid the human defense systems. We demonstrated that A. baumannii lacking ChoP, even if they expressed OMPA (data not shown), adhere to and invade epithelial cells much less strongly than A. baumannii expressing ChoP.
ChoP binding to PAFR is known to mediate the internalization of bacteria by human cells (10,17). In our study, we focused on determining the role of PAFR in A. baumannii adherence and invasion of epithelial cells. Previously, we have shown that A. baumannii was able to adhere to and invade lung epithelial cells (39). Here, we showed that A. baumannii expressing ChoP acts through PAFR, which are functionally linked to the G protein signal pathway, and that the inhibition of G protein-coupled PLC prevented A. baumannii invasion of epithelial cells. Previous studies have determined that G protein coupled to PLC inhibition blocks the bacterial invasion of human endothelial and epithelial cells (40 -42). Binding of bacteria to PAFR is known to increase inositol triphosphate and mobilization of intracellular Ca 2ϩ (19), which is known to involve Ca 2ϩ influx through ionic channels leading to cell death (20,21). There is now considerable evidence that increase of intracellular Ca 2ϩ  plays an important role in the cell death mediated by calpain activation, a substrate for Ca 2ϩ (39,43,44). In this study, we demonstrated that A. baumannii increased intracellular Ca 2ϩ , and we showed that inhibition of G protein coupled to PLC decreases Ca 2ϩ influx and cell death induced by A. baumannii. Along the same lines, it has been shown that interaction of E. coli and Listeria monocytogenes with human endothelial and epithelial cells triggers PLC activation followed by increase of inositol triphosphate and intracellular Ca 2ϩ (40,45).
On the other hand, several studies have identified clathrin and ␤-arrestins as key signaling players in endocytosis and transcytosis (46,47). However, only a few studies have reported that clathrin and ␤-arrestin-1 mediate bacterial invasion of epithelial cells (23,48). To our knowledge, the present study provides the first evidence for clathrin and ␤-arrestin-1 and especially ␤-arrestin-2 involvement in A. baumannii invasion of epithelial cells. Firstly, we demonstrated that inhibition of clathrin prevented A. baumannii invasion of A549 cells. Secondly, we showed that ␤-arrestin-1 and ␤-arrestin-2 siRNA inhibited A. baumannii invasion of A549 cells.
Importantly, in the in vivo pneumonia model caused by A. baumannii, treatment with PAFR antagonist reduces bacterial loads in lungs, which indicates that PAFR participates in A. baumannii dissemination in vivo. Previous studies have determined that in PAFR knock-out and PAFR antagonisttreated mice, bacterial loads in blood, cerebrospinal fluid, and lung in Streptococcus pneumoniae and P. aeruginosa pneumonia and meningitis models were reduced (10,23,49,50); meanwhile our data are the first to demonstrate functionally that PAFR is essential for A. baumannii dissemination in vivo. Moreover, we showed that PAFR antagonist pretreatment reduced but did not abolish A. baumannii dissemination in vitro and in vivo, indicating that other receptors and/or components of eukaryotic cells might mediate this residual A. baumannii invasion and dissemination. Alternatively, the residual A. baumannii invasion could be mediated by cytoskeletal rearrangement (5), fibronectin binding (51), and/or by laminin receptor (52); in addition, the last is known to initiate bacterial contact with the blood-brain barrier in experimental meningitis models. However, further investigations are needed to highlight these other specific mechanisms involved in A. baumannii dissemination.
In summary, we have demonstrated that PAFR play an important role in A. baumannii expressing ChoP adherence and in invasion of epithelial cells that requires G protein-coupled PLC, clathrin, and ␤-arrestin activation.