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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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Mitochondrial membrane potential of HEp-2 cells and TMRE fluorescence intensity at a single cell level after 24-h incubation with: a Eagle minimum essential medium, bA. calcoaceticus MPU M12, cA. calcoaceticus MPU M7. ΔΨm was assessed after TMRE staining. The fluorescence was visualized by laser confocal microscopy
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Fig3: Mitochondrial membrane potential of HEp-2 cells and TMRE fluorescence intensity at a single cell level after 24-h incubation with: a Eagle minimum essential medium, bA. calcoaceticus MPU M12, cA. calcoaceticus MPU M7. ΔΨm was assessed after TMRE staining. The fluorescence was visualized by laser confocal microscopy

Mentions: While TEM observations manifested that the mitochondria found in epithelial cells are morphologically disrupted during Acb-infection, the functional nature of these organelles within the infected epithelium remains uncertain. Therefore, we determined the effect of the strains on mitochondrial transmembrane potential (ΔΨm). We observed that infected HEp-2 cells, in the range from 56.3 ± 3.1 to 89.6 ± 4.1 % exhibited a reduced level of TMRE fluorescence, compared to only 6.8 ± 2.1 % of the control ones incubated with a non-pathogenic E. coli strain. The number of the cells with reduced level of fluorescence increased to the range from 67.1 ± 2.8 to 97.3 ± 2.1 % at 48 h after infection. We determined the fluorescence intensity at a single cell level (Fig. 3). The average pixel intensity for each cell was assessed. We determined the membrane potential in viable, apoptotic, and oncotic cells. The fluorescence was the highest in control viable cells (Fig. 3a) in comparison with apoptotic (Fig. 3b) and oncotic ones (Fig. 3c). The control cells had mean fluorescence above 250 FU. Acinetobacter spp.-infected apoptotic cells revealed decline in TMRE fluorescence in comparison with the controls to the minimum level of 65 ± 27 FU at 24 h. Interestingly, the lowest values of fluorescence intensity in the range from 51 ± 22 to 24 ± 11 FU were noticed in oncotic cells. The reduction of intensity of ΔΨm-sensitive dye TMRE in Acinetobacter spp.-infected cells indicated mitochondrial depolarization. The Pearson test revealed positive correlation between the ApI and loss of ΔΨm (r = −0.58, P < 0.01).Fig. 3


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)

Mitochondrial membrane potential of HEp-2 cells and TMRE fluorescence intensity at a single cell level after 24-h incubation with: a Eagle minimum essential medium, bA. calcoaceticus MPU M12, cA. calcoaceticus MPU M7. ΔΨm was assessed after TMRE staining. The fluorescence was visualized by laser confocal microscopy
© Copyright Policy
Related In: Results  -  Collection

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

Fig3: Mitochondrial membrane potential of HEp-2 cells and TMRE fluorescence intensity at a single cell level after 24-h incubation with: a Eagle minimum essential medium, bA. calcoaceticus MPU M12, cA. calcoaceticus MPU M7. ΔΨm was assessed after TMRE staining. The fluorescence was visualized by laser confocal microscopy
Mentions: While TEM observations manifested that the mitochondria found in epithelial cells are morphologically disrupted during Acb-infection, the functional nature of these organelles within the infected epithelium remains uncertain. Therefore, we determined the effect of the strains on mitochondrial transmembrane potential (ΔΨm). We observed that infected HEp-2 cells, in the range from 56.3 ± 3.1 to 89.6 ± 4.1 % exhibited a reduced level of TMRE fluorescence, compared to only 6.8 ± 2.1 % of the control ones incubated with a non-pathogenic E. coli strain. The number of the cells with reduced level of fluorescence increased to the range from 67.1 ± 2.8 to 97.3 ± 2.1 % at 48 h after infection. We determined the fluorescence intensity at a single cell level (Fig. 3). The average pixel intensity for each cell was assessed. We determined the membrane potential in viable, apoptotic, and oncotic cells. The fluorescence was the highest in control viable cells (Fig. 3a) in comparison with apoptotic (Fig. 3b) and oncotic ones (Fig. 3c). The control cells had mean fluorescence above 250 FU. Acinetobacter spp.-infected apoptotic cells revealed decline in TMRE fluorescence in comparison with the controls to the minimum level of 65 ± 27 FU at 24 h. Interestingly, the lowest values of fluorescence intensity in the range from 51 ± 22 to 24 ± 11 FU were noticed in oncotic cells. The reduction of intensity of ΔΨm-sensitive dye TMRE in Acinetobacter spp.-infected cells indicated mitochondrial depolarization. The Pearson test revealed positive correlation between the ApI and loss of ΔΨm (r = −0.58, P < 0.01).Fig. 3

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