HepG2 cells infected with Klebsiella pneumoniae show DNA laddering at apoptotic and necrotic stages.
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.
Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen Hua 1 Road, Kwei San, Tao Yuan, 333, Taiwan.
Pei-Yin
PY
Yang
Wen-Xin
WX
Chen
Fong-Yin
FY
Chang
Hui-Wen
HW
Chen
Chun-Hung
CH
Lin
Yu-Chun
YC
Hsu
June Hsieh
JH
Wu
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D007711:Klebsiella pneumoniae; D008100:Liver Abscess / Q000235:genetics / Q000382:microbiology / Q000473:pathology*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009336:Necrosis; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D013049:Spectrin
D000255:Adenosine Triphosphate; D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; D002352:Carrier Proteins; D020169:Caspases; D003851:Deoxyribonucleases; D008840:Microfilament Proteins; D011065:Poly(ADP-ribose) Polymerases; D000075223:Poly-ADP-Ribose Binding Proteins; D013049:Spectrin; C494829:AIFM1 protein, human; C506592:APAF1 protein, human; C109664:caspase-activated DNase inhibitor; C113696:DFFB protein, human