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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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Host cell entry and cytotoxicity of rAbOmpA fragments.(A) HeLa cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. The cells were fixed, permeabilized, and stained with anti-rabbit AbOmpA antibody, followed by Alexa Fluor® 568-conjugated rabbit IgG (red). DAPI was used to stain the nuclei (blue). The subcellular localization of AbOmpA was observed by confocal microscopy. Magnification: ×400. (B) The differentiated U937 cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. Cell viability was determined by a WST-1 assay. Untreated control cells (⧫), rAbOmpA22-170 (•), rAbOmpA1-356 (X), and rAbOmpA221-339 (▪).
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pone-0017027-g006: Host cell entry and cytotoxicity of rAbOmpA fragments.(A) HeLa cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. The cells were fixed, permeabilized, and stained with anti-rabbit AbOmpA antibody, followed by Alexa Fluor® 568-conjugated rabbit IgG (red). DAPI was used to stain the nuclei (blue). The subcellular localization of AbOmpA was observed by confocal microscopy. Magnification: ×400. (B) The differentiated U937 cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. Cell viability was determined by a WST-1 assay. Untreated control cells (⧫), rAbOmpA22-170 (•), rAbOmpA1-356 (X), and rAbOmpA221-339 (▪).

Mentions: In order to determine which AbOmpA domains were directly responsible for host cell death, three recombinant proteins, including rAbOmpA22-170, rAbOmpA221-339, and rAbOmpA1-356, were generated and then micelles harboring each rAbOmpA fragment were constructed to insert rAbOmpA proteins into the membrane or lumen according to their hydrophobicity. These micelles were treated to HeLa cells for 24 h, after which the cellular distribution of rAbOmpA was assessed. Three different rAbOmpA proteins were internalized by host cells (Fig. 6A). Next, the cytotoxicity of different rAbOmpA proteins was assessed in the macrophages. Micelles composed of rAbOmpA22-170 and rAbOmpA1-356 induced cell death at a concentration of ≥5 µg/ml, whereas rAbOmpA221-339 did not induce cell death at a concentration of ≤100 µg/ml (Fig. 6B). These results indicate that the N-terminal resides of AbOmpA are 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)

Host cell entry and cytotoxicity of rAbOmpA fragments.(A) HeLa cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. The cells were fixed, permeabilized, and stained with anti-rabbit AbOmpA antibody, followed by Alexa Fluor® 568-conjugated rabbit IgG (red). DAPI was used to stain the nuclei (blue). The subcellular localization of AbOmpA was observed by confocal microscopy. Magnification: ×400. (B) The differentiated U937 cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. Cell viability was determined by a WST-1 assay. Untreated control cells (⧫), rAbOmpA22-170 (•), rAbOmpA1-356 (X), and rAbOmpA221-339 (▪).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017027-g006: Host cell entry and cytotoxicity of rAbOmpA fragments.(A) HeLa cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. The cells were fixed, permeabilized, and stained with anti-rabbit AbOmpA antibody, followed by Alexa Fluor® 568-conjugated rabbit IgG (red). DAPI was used to stain the nuclei (blue). The subcellular localization of AbOmpA was observed by confocal microscopy. Magnification: ×400. (B) The differentiated U937 cells were treated with different forms of micelles containing rAbOmpA fragments for 24 h. Cell viability was determined by a WST-1 assay. Untreated control cells (⧫), rAbOmpA22-170 (•), rAbOmpA1-356 (X), and rAbOmpA221-339 (▪).
Mentions: In order to determine which AbOmpA domains were directly responsible for host cell death, three recombinant proteins, including rAbOmpA22-170, rAbOmpA221-339, and rAbOmpA1-356, were generated and then micelles harboring each rAbOmpA fragment were constructed to insert rAbOmpA proteins into the membrane or lumen according to their hydrophobicity. These micelles were treated to HeLa cells for 24 h, after which the cellular distribution of rAbOmpA was assessed. Three different rAbOmpA proteins were internalized by host cells (Fig. 6A). Next, the cytotoxicity of different rAbOmpA proteins was assessed in the macrophages. Micelles composed of rAbOmpA22-170 and rAbOmpA1-356 induced cell death at a concentration of ≥5 µg/ml, whereas rAbOmpA221-339 did not induce cell death at a concentration of ≤100 µg/ml (Fig. 6B). These results indicate that the N-terminal resides of AbOmpA are 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