Rescue of Escherichia coli cells from UV-induced death and filamentation by caspase-3 inhibitor.

Abstract

Escherichia coli cells have been observed earlier to display caspase-3-like protease activity (CLP) and undergo programmed cell death (PCD) when exposed to gamma rays. The presence of an irreversible caspase-3 inhibitor (Ac-DEVD-CMK) during irradiation was observed to increase cell survival. Since radiation is known to induce SOS response, the effect of a caspase-3 inhibitor on SOS response was studied in E. coli. UV, a well-known SOS inducer, was used in the current study. Cell filamentation in E. coli upon UV exposure was found to be inhibited by ninefold in the presence of a caspase-3 inhibitor. CLP activity was found to increase twofold in UV-exposed cells than in control (non-treated) cells. Further, bright fluorescing filaments were observed in UV-exposed E. coli cells treated with FITC-DEVD-FMK, a fluorescent dye tagged with an irreversible caspase-3 inhibitor (DEVD-FMK), indicating the presence of active CLP in these cells. Unlike caspase-3 inhibitor, a serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), was not found to improve cell survival after UV treatment. Additionally, a SOS reporter system known as SIVET (selectable in vivo expression technology) assay was performed to reconfirm the inhibition of SOS induction in the presence of caspase-3 inhibitor. SIVET assay is used to quantify cells in which the SOS response has been induced leading to a scorable permanent selectable change in the cell. The SIVET induction frequency (calculated as the ratio of SIVET-induced cells to total viable cells) increased around tenfold in UV-exposed cultures. The induction frequency was found to decrease significantly to 51 from 80% in the cells pre-incubated with caspase-3 inhibitor. On the contrary, caspase-3 inhibitor failed to improve cell survival of E. coli ΔrecA and E. coli DM49 (SOS non-inducible) cells post UV treatment. Summing together, the results indicated a possible linkage of SOS response and the PCD process in E. coli. The findings also indicated that functional SOS pathway is required for CLP-like activity; however, the exact mechanism remains to be elucidated.