Identification of survival factors in LPS-stimulated anthrax lethal toxin tolerant RAW 264.7 cells through proteomic approach

Abstract

Anthrax lethal toxin (LeTx; a combination of protective antigen and lethal factor) is secreted by the vegetative cells of Bacillus anthracis and is cytotoxic for certain macrophage cell lines. First-time exposure of murine macrophage cells (RAW 264.7) to lethal toxin (LeTx) (0.1+0.1 mg/mL) caused extensive cell death with a survival rate of approximately 40%, but upon secondary exposure to LeTx and lipopolysaccharide (LPS) (1 mu g/mL), after a few passages, these cells had a survival rate of approximately 100%. The present study assessed protein expression changes after LPS exposure to LeTx-intoxication-resistant RAW 264.7 cells. To analyze the protein expression profile of LPS-treated LeTx-intoxication-resistant RAW 264.7 cells, we employed matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDITOF MS), and later, Ingenuity Pathways Analysis (IPA) was applied to establish the molecular networks. Among the differentially expressed proteins were voltage dependent anion channel 1, jip3 protein, heat shock protein 4, tubulin beta, 26S protease regulatory subunit 4, and DNA polymerase delta subunit 4 (DNA pol delta) were significant. The molecular network signatures and functional proteomics obtained in this study may facilitate the evaluation of potential pathways such as PI3K signaling, NF-kappa B signaling, and LPSinduced MAPK signaling for the recovery of LPSinduced LeTx-intoxication-resistant RAW 264.7 cells.