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Acinetobacter calcoaceticus-baumannii complex strains induce caspase-dependent and caspase-independent death of human epithelial cells.

Krzymińska S, Frąckowiak H, Kaznowski A - Curr. Microbiol. (2012)

Bottom Line: We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death.Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)).Bacterial infection caused generation of nitric oxide and reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Adam Mickiewicz University, ul. Umultowska 89, 61-614 Poznan, Poland. sylkrzym@amu.edu.pl

ABSTRACT
We investigated interactions of human isolates of Acinetobacter calcoaceticus-baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A. calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A. calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.

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Apoptosis and necrosis of infected HEp-2 cells. The cells were stained with propidium iodide and AO (100 μg/ml) and observed in the fluorescence microscope; The cells were incubated with A Eagle culture medium; BA. calcoaceticus MPU M12; the arrows pointed: a live, b apoptotic, c necrotic cells. Magnifications: A ×250, B ×200
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Fig1: Apoptosis and necrosis of infected HEp-2 cells. The cells were stained with propidium iodide and AO (100 μg/ml) and observed in the fluorescence microscope; The cells were incubated with A Eagle culture medium; BA. calcoaceticus MPU M12; the arrows pointed: a live, b apoptotic, c necrotic cells. Magnifications: A ×250, B ×200

Mentions: Live uninfected and infected cells showed green fluorescence (Fig. 1A). In contrast, red nuclei appeared in HEp-2 cells after 24 h of infection with Acb strains, indicating that EtBr and AO entered the cells (Fig. 1B). The results suggested that the infection is involved in pore formation in the cell membrane, leading to the uptake of EtBr and osmotic lysis. The highest ApI, ranging from 56.1 to 65.1 % at 24 h after infection, was observed in cells incubated with four (40 %) A. calcoaceticus and four (57 %) A. baumannii strains. The lowest ApI, between 18.6 and 32.1 %, was expressed by one (14 %) A. strain and three (30 %) A. calcoaceticus strains. The percentage of apoptotic cells increased at 48 h post infection (Table 2). The highest ApI ranging from 67.1 to 78.8 % was observed in HEp-2 cells infected with one (14 %) A. baumanii and three (30 %) A. calcoaceticus strains. The lowest index, ranging from 21.4 to 36.1 %, was revealed by cells infected with one (14 %) A. baumannii and four (40 %) A. calcoaceticus strains. The mean ApI of the negative control was 9.6 ± 1.8 %, whereas for the UV-irradiated positive control it reached 94.6 ± 5.1 %. The Pearson linear correlation test revealed positive correlation between the ApI and AdI of cells infected with Acb complex strains (r = 0.51, P < 0.01). The results were confirmed by treatment of HEp-2 cells with cytochalasin D, which inhibits actin polymerization and therefore, bacterial invasion of the cells. The incubation did not inhibit cell killing by apoptosis, which suggested that bacterial invasion of epithelial cells was not responsible for the cell death. Moreover, the Pearson coefficient increased for correlations between the apoptotic index and cell-contact cytotoxicity (r = 0.73, P < 0.01).Fig. 1


Acinetobacter calcoaceticus-baumannii complex strains induce caspase-dependent and caspase-independent death of human epithelial cells.

Krzymińska S, Frąckowiak H, Kaznowski A - Curr. Microbiol. (2012)

Apoptosis and necrosis of infected HEp-2 cells. The cells were stained with propidium iodide and AO (100 μg/ml) and observed in the fluorescence microscope; The cells were incubated with A Eagle culture medium; BA. calcoaceticus MPU M12; the arrows pointed: a live, b apoptotic, c necrotic cells. Magnifications: A ×250, B ×200
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401494&req=5

Fig1: Apoptosis and necrosis of infected HEp-2 cells. The cells were stained with propidium iodide and AO (100 μg/ml) and observed in the fluorescence microscope; The cells were incubated with A Eagle culture medium; BA. calcoaceticus MPU M12; the arrows pointed: a live, b apoptotic, c necrotic cells. Magnifications: A ×250, B ×200
Mentions: Live uninfected and infected cells showed green fluorescence (Fig. 1A). In contrast, red nuclei appeared in HEp-2 cells after 24 h of infection with Acb strains, indicating that EtBr and AO entered the cells (Fig. 1B). The results suggested that the infection is involved in pore formation in the cell membrane, leading to the uptake of EtBr and osmotic lysis. The highest ApI, ranging from 56.1 to 65.1 % at 24 h after infection, was observed in cells incubated with four (40 %) A. calcoaceticus and four (57 %) A. baumannii strains. The lowest ApI, between 18.6 and 32.1 %, was expressed by one (14 %) A. strain and three (30 %) A. calcoaceticus strains. The percentage of apoptotic cells increased at 48 h post infection (Table 2). The highest ApI ranging from 67.1 to 78.8 % was observed in HEp-2 cells infected with one (14 %) A. baumanii and three (30 %) A. calcoaceticus strains. The lowest index, ranging from 21.4 to 36.1 %, was revealed by cells infected with one (14 %) A. baumannii and four (40 %) A. calcoaceticus strains. The mean ApI of the negative control was 9.6 ± 1.8 %, whereas for the UV-irradiated positive control it reached 94.6 ± 5.1 %. The Pearson linear correlation test revealed positive correlation between the ApI and AdI of cells infected with Acb complex strains (r = 0.51, P < 0.01). The results were confirmed by treatment of HEp-2 cells with cytochalasin D, which inhibits actin polymerization and therefore, bacterial invasion of the cells. The incubation did not inhibit cell killing by apoptosis, which suggested that bacterial invasion of epithelial cells was not responsible for the cell death. Moreover, the Pearson coefficient increased for correlations between the apoptotic index and cell-contact cytotoxicity (r = 0.73, P < 0.01).Fig. 1

Bottom Line: We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death.Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)).Bacterial infection caused generation of nitric oxide and reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Adam Mickiewicz University, ul. Umultowska 89, 61-614 Poznan, Poland. sylkrzym@amu.edu.pl

ABSTRACT
We investigated interactions of human isolates of Acinetobacter calcoaceticus-baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A. calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A. calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.

Show MeSH
Related in: MedlinePlus