Assessing the impact of xenobiotics on live cells: novel approaches

With the ever increasing number and variety of anthropogenic xenobiotics, there is a growing need to characterize their impact on non-target organisms at the molecular level.

We have assessed the impact of exposing bacteria to the commonly used herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), at sublethal levels using advanced microscopy and metabolomics. Escherichia coli is a common gut bacteria also found in the environment, and Rhizobium leguminosarum viciae 3841 (Rlv) fixes nitrogen as a beneficial soil organism. Low level 2,4-D exposure gave rise to envelope remodelling, altered phenotypes, generation of reactive oxygen species (ROS) and changes to central metabolism consistent with oxidative stress in Rlv and E. coli. While E. coli adapted by reducing central metabolic activity in response to 2,4-D, Rlv increased the activity of many integral pathways of cellular metabolism and showed the potential to assimilate 2,4-D. We identified biomarkers of stress in E. coli and the auxin-like response in Rlv that we used to develop high-content correlative microscopy assays.

Correlative real-time live cell imaging by atomic force microscopy (AFM) in quantitative imaging (QI) mode integrated with laser scanning confocal microscopy (LSCM) shows exposure to 1 mM 2,4-D directly inhibits E. coli divisome assembly within seconds. We observed immediate (within seconds) and significant changes in the localization cell division proteins (FtsZ, FtsA), associated with the immediate arrest of cell division, DNA damage and initiation of the SOS response (SulA intensity), with concomitant changes to cell surface roughness, elasticity and adhesion, in a time-dependent manner. Similar results were observed for compounds that are known to alter cell membrane potential. We propose that 2,4-D blocks cell division in E. coli within seconds by affecting the higher order assembly of the divisome through FtsZ/FtsA-mediated inhibition of Z- ring formation as a direct result of altered membrane potential.

We have recently demonstrated our high-content correlative AFM-QI-LSCM assays to be broadly applicable for assessing cytotoxic impact on a variety of cell types, with proof-of-principle in bacteria, yeast and human cells 

Speaker: Dr. Tanya Dahms, Department of Chemistry and Biochemistry, University of Regina