Interactions in complex biological systems by nuclear magnetic resonance

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The cell wall is the first barrier encountered by drugs and contaminants and, as such, is targeted or traversed by bioactive molecules that can alter its architecture with critical consequences on the cell. Likewise organelles are delimitated by membranes where important toxicity mechanisms take place. Therefore understanding, for example, how antibiotics interact with bacteria, or pollutants enter microalgae, will help address bacterial drug resistance or environmental issues. It is thus crucial to have in situ nanometer-level information on interactions occurring with the cell constituents. The general objective of our research program is thus to develop new solid-state nuclear magnetic resonance (SS-NMR) approaches to characterize intact cells and study their molecular interactions in vivo. In-cell SS-NMR is challenging because of the tremendous number of signals that are produced by a huge number of cell constituents. Therefore the short-term objectives of our research program are to establish strategies to achieve spectral selectivity and improve signal assignment in complex cell systems, and to develop methodologies to monitor molecular interactions with cells. To do so, we will focus on human and marine bacteria as well as microalgae. Two approaches will be used: (1) exclusive isotopic labelling of specific cell constituents; and (2) unspecific cell labelling combined to NMR signal filtering. To facilitate peak assignment, cell constituents (such as cell wall, chloroplasts or mitochondria) will be isolated. This reconstructive approach will facilitate data collection and analysis in whole cells. The long term objective of this program is to expand our SS-NMR strategy to the study of molecular interactions with higher organisms. “Whole-cell” NMR is the stepping stone towards “whole-organism” NMR. Our research program is innovative because it will extend the emerging field of in-cell NMR to the study of cell interactions to which very few research groups in the world have contributed. Efforts in establishing new methodologies have the potential to expand applications to several complex biological systems. This is worthwhile since NMR can inform on structure and interactions in a non-destructive manner. Our long-term program opens the possibility to cover several human and animal pathogens, terrestrial and aquatic microorganisms, and even animal cell lines. It will help provide a novel toolbox for the design of new antibiotics, such as antimicrobial peptides that we will study. Because of global warming, aquatic pathogens subject to multi-drug resistance are increasingly threatening humans and the water biota. By studying aquatic bacteria, our program will help support aquaculture an important sector for the economy of Canadian coastal regions. It will also enable close characterization of microalgal cells, thus helping their biotechnological exploitation.