Proteomics study of anthrax lethal toxin‐treated murine macrophages

Abstract

The anthrax lethal toxin (LeTx) is composed of two proteins, protective antigen and lethal factor, which bind and enter the cell through a host receptor termed the anthrax toxin receptor (ATR). In the cell, LeTx targets p38, part of the MAP kinase signaling pathway. The toxin appears to initiate an apoptotic pathway in infected cells, indicating additional downstream targets of the toxin. We have applied a proteomics approach to investigate these downstream targets and the affected processes. In this study we have used an improved strategy for fractionation based on protein pI, off-gel electrophoresis, employed in conjunction with relative quantitation using the mass labeling approach. In our survey, 67 proteins were observed and quantified from the cytosol of RAW 264.7 cells with respect to control versus toxin-treated cells. Many of these proteins are involved in the oxidative stress response, as well as apoptosis, and thus likely to be relevant to the effects of anthrax in infected cells. Our results indicate that the tumor necrosis factor-alpha-mediated pathway is compromised in intoxicated cells. The knowledge of such changes and the pathways leading to the changes should be of great value in understanding and combating this disease.

Early response in macrophages by exposure to a low concentration of anthrax lethal toxin

“…TNF receptor-associated factor 1 (Traf1) suppresses caspase-8 activity and TNF-induced apoptosis 31 . Ctsb was found to be downregulated two-fold at 180 min in LeTx-treated murine macrophages in a study by Kuhn et al 30 . However, in spite of the down-regulation of the Ctsb gene, the cell survives from TNF-induced apoptosis by the induction of Traf1 (Table 2).…”

“…Other transcription factors, including Klf2 and Nfil3, were also upregulated in our study. Cathepsin B (Ctsb) plays a role as a positive regulator of cell death 30 and has been shown to be released from lysosomes following the activation of caspase-8 by treatment with TNF-α. Macrophages treated with a low concentration of LeTx (0.1 μg/mL of PA and 10 -6 μg/mL of LF) secrete TNF-α 7 .…”

“…RAW 264.7 cells (6×10 4 ) were plated in a 96-well plate. After 24 hours, each well was treated with 0.1 μg/mL of PA (fixed concentration) and 0.004, 0.008, 0.016, 0.032, or 0.1 μg/mL of LF for various time periods (20,30,60,90,120,180, and 240 min) and finally, 25 μL of MTT (5 μg/mL in PBS) were added to all of the wells. Data were expressed as the mean ±standard error.…”

“…Western blot analysis carried out against Akt, phospho-Akt, GSK-3β, and phospho-GSK-3β (Ser9) on either PA or LeTx treated murine macrophage cell lysates at different time points(30, 60, and 180 min). Only PA treated macrophages were used as a reference.…”

Anthrax Toxins

“…Many studies of this type assess events at time points where macrophages are well beyond the point of no return, mostly dead, even by each study ' s own death response curves, and thus are describing the sequelae of severe stress and cell death rather than LT -specifi c events (Comer et al, 2005b ;Kuhn et al, 2006 ;Sapra et al, 2006 ). Even when attempts are made to investigate events at time points where a smaller percentage of cells are dead, the fact that any portion of cells have succumbed is clearly indicative that most of what is being studied is the macrophage stress responses to the lytic event (Chandra et al, 2005 ).…”