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HepG2 cells infected with Klebsiella pneumoniae show DNA laddering at apoptotic and necrotic stages

Apoptosis volume 17, pages 154–163 (2012)Cite this article

Abstract

Virulent Klebasiella pneumoniae (KP) inflicts severe liver abscesses in infected patients. This study investigated how the bacterial infection affected cell survival at the molecular level, in a cultured cell model. A strain of KP highly virulent in mice was isolated from a patient with liver abscess, and was used to infect HepG2 cells. The infected cells were examined for their viability, DNA fragmentation, and proteins involved in apoptosis or necrosis. We found that the infection decreased the viability of HepG2 cells at 4 hours (h) to 12 h post infection (pi). DNA ladders appeared 6–16 h pi and flow cytometry analysis showed apoptosis at 3–5 h pi, secondary necrosis at 6–9 h pi and primary necrosis at 8–9 h pi. Cleavages of Caspase 7, Caspase 9, α-Fodrin, and PARP were evident at 2–4 h pi. At 7 h pi, we observed the following: increased nuclear AIF, the release from mitochondria of cytosolic Apaf-1 and Cyt c, increased DFF40 expression, decreased DFF45, decreased BcL-xL and the release of Endo G from mitochondria to nucleus. Cellular ATP concentration decreased at 4–8 h pi, accompanied by increased Calpain-2 expression. In summary, infected HepG2 cells underwent apoptosis early after infection and progressed to secondary necrosis and primary necrosis. Nuclear fragmentation corresponded to Caspase 7 activation and the appearance of Endo G and DFF40 in the nucleus, with a concomitant decrease in DFF45. Mitochondrial release of Cyt c together with activation of Caspase 9 and Apaf-1 in cytosol was also observed. Early-hour cleavage of poly(ATP-ribose) polymerase (PARP) followed by the later activation of PARP corresponded to the appearance of DNA laddering, and the depletion of cellular ATP was associated with the appearance of necrosis.

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Abbreviations

AIF:

Apoptosis inducing factor

Apaf-1:

Apoptotic protease-activating factor 1

Cyt c :

Cytochrome c

DFF:

DNA fragmentation factor

Endo G:

Endonuclease G

h:

Hour

KP:

Klebsiella pneumoniae

min:

Minute

Nec-1:

Necrostatin-1

PARP:

Poly(ADP) polymerase

pi:

Post infection

QVD-OPH:

Quindyl valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone

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Acknowledgments

The work was supported by Grants, NSC93-2320-B-182-024, NSC96-3112-B-182-005 and NSC97-3112-B-182-001 from National Science Council of Taiwan and a Grant CMRPD180461 from Chang Gung University of Taiwan.

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Affiliations

  1. Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen Hua 1 Road, Kwei San, Tao Yuan, 333, Taiwan

    Pei-Yin Yang, Wen-Xin Chen, Fong-Yin Chang, Hui-Wen Chen & June Hsieh Wu

  2. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

    Chun-Hung Lin

  3. Department of Industrial Design, Chang Gung University, Kwei San, Tao Yuan, Taiwan

    Yu-Chun Hsu

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  1. Pei-Yin Yang
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Correspondence to June Hsieh Wu.

Electronic supplementary material

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Supplementary Fig. 1. PARP activity assay in HepG2 cell infected with KpL1. (JPG 740 kb)

10495_2011_666_MOESM2_ESM.jpg

Supplementary Fig. 2. Flow cytometry analysis after QVD-OPH or necrostatin-1 treatment in HepG2 cells infected with KpL1. (JPG 815 kb)

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Yang, PY., Chen, WX., Chang, FY. et al. HepG2 cells infected with Klebsiella pneumoniae show DNA laddering at apoptotic and necrotic stages. Apoptosis 17, 154–163 (2012). https://doi.org/10.1007/s10495-011-0666-1

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Keywords

  • Annexin V/PI staining
  • Flow cytometry
  • Cell viability
  • Western blot