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Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles.

Jin JS, Kwon SO, Moon DC, Gurung M, Lee JH, Kim SI, Lee JC - PLoS ONE (2011)

Bottom Line: Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs.The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death.In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea.

ABSTRACT
Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606(T) secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA(22-170) was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

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Flow cytometric analysis of cell death induced by the OMVs from A. baumannii ATCC 19606T and the ΔompA mutant.The differentiated U937 cells were treated with various concentrations (0, 10, 20, 50, and 100 µg/ml) of OMVs and stained with Annexin V and PI. Upper panel, control cells without OMVs for 24 h. Middle panel, the cells were treated with OMVs from A. baumannii ATCC 19606T for 24 h. Lower panel, the cells were treated with OMVs from the ΔompA mutant for 24 h. Representative data from three independent experiments are shown. In the graph, cells in right upper and lower parts are apoptotic cells and cells in left upper part are necrotic cells.
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pone-0017027-g005: Flow cytometric analysis of cell death induced by the OMVs from A. baumannii ATCC 19606T and the ΔompA mutant.The differentiated U937 cells were treated with various concentrations (0, 10, 20, 50, and 100 µg/ml) of OMVs and stained with Annexin V and PI. Upper panel, control cells without OMVs for 24 h. Middle panel, the cells were treated with OMVs from A. baumannii ATCC 19606T for 24 h. Lower panel, the cells were treated with OMVs from the ΔompA mutant for 24 h. Representative data from three independent experiments are shown. In the graph, cells in right upper and lower parts are apoptotic cells and cells in left upper part are necrotic cells.

Mentions: In order to determine whether A. baumannii OMVs induced host cell damage, macrophages were treated with various concentrations of OMVs for 24 h and then stained with Annexin V and propidium iodide (PI). The U937 cells were used in this study because macrophages had low threshold concentrations of AbOmpA for cell death as compared with epithelial cells (HEp-2 cells) and fibroblast cells (Cos-7 cells) [13]. Flow cytometric analysis demonstrated no cell death at ≤20 µg/ml (protein concentrations) of OMVs, but 50 and 100 µg/ml of OMVs did induce host cell death (Fig. 5, middle panel). Based on previous studies demonstrating that AbOmpA directly induced apoptotic cell death, OMVs were prepared from the ΔompA mutant and their ability to induce cytotoxicity was compared to that of the OMVs from wild-type A. baumannii. The OMVs from the ΔompA mutant did not induce cell death evenly at a concentration of 100 µg/ml (Fig. 5, lower panel). These results suggest that AbOmpA associated with A. baumannii OMVs is directly responsible for host cell death.


Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles.

Jin JS, Kwon SO, Moon DC, Gurung M, Lee JH, Kim SI, Lee JC - PLoS ONE (2011)

Flow cytometric analysis of cell death induced by the OMVs from A. baumannii ATCC 19606T and the ΔompA mutant.The differentiated U937 cells were treated with various concentrations (0, 10, 20, 50, and 100 µg/ml) of OMVs and stained with Annexin V and PI. Upper panel, control cells without OMVs for 24 h. Middle panel, the cells were treated with OMVs from A. baumannii ATCC 19606T for 24 h. Lower panel, the cells were treated with OMVs from the ΔompA mutant for 24 h. Representative data from three independent experiments are shown. In the graph, cells in right upper and lower parts are apoptotic cells and cells in left upper part are necrotic cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046175&req=5

pone-0017027-g005: Flow cytometric analysis of cell death induced by the OMVs from A. baumannii ATCC 19606T and the ΔompA mutant.The differentiated U937 cells were treated with various concentrations (0, 10, 20, 50, and 100 µg/ml) of OMVs and stained with Annexin V and PI. Upper panel, control cells without OMVs for 24 h. Middle panel, the cells were treated with OMVs from A. baumannii ATCC 19606T for 24 h. Lower panel, the cells were treated with OMVs from the ΔompA mutant for 24 h. Representative data from three independent experiments are shown. In the graph, cells in right upper and lower parts are apoptotic cells and cells in left upper part are necrotic cells.
Mentions: In order to determine whether A. baumannii OMVs induced host cell damage, macrophages were treated with various concentrations of OMVs for 24 h and then stained with Annexin V and propidium iodide (PI). The U937 cells were used in this study because macrophages had low threshold concentrations of AbOmpA for cell death as compared with epithelial cells (HEp-2 cells) and fibroblast cells (Cos-7 cells) [13]. Flow cytometric analysis demonstrated no cell death at ≤20 µg/ml (protein concentrations) of OMVs, but 50 and 100 µg/ml of OMVs did induce host cell death (Fig. 5, middle panel). Based on previous studies demonstrating that AbOmpA directly induced apoptotic cell death, OMVs were prepared from the ΔompA mutant and their ability to induce cytotoxicity was compared to that of the OMVs from wild-type A. baumannii. The OMVs from the ΔompA mutant did not induce cell death evenly at a concentration of 100 µg/ml (Fig. 5, lower panel). These results suggest that AbOmpA associated with A. baumannii OMVs is directly responsible for host cell death.

Bottom Line: Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs.The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death.In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea.

ABSTRACT
Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606(T) secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606(T) induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA(22-170) was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

Show MeSH
Related in: MedlinePlus