Solid-State Nmr Of Intact Bacteria Reveals The Effect Of Stress And Antimicrobial Agents

The worldwide problem of bacterial resistance requires the development of novel antibiotics against bacterial pathogens. It is thus essential to determine the action mechanism of these drugs, and in particular, to understand their interaction with the bacterial cell wall - the first barrier crossed or, in some cases, targeted. 2H solid-state (SS) NMR is a valuable tool to probe, at a molecular level, the effect of antimicrobial agents on the organization and dynamics of membrane phospholipids. These studies are usually performed on model membranes, but “in-cell” SS-NMR is advantageous as it takes into account the complexity of the cell wall in the interaction. We developed for the first time a protocol to deuterate the membrane phospholipids of Vibrio splendidus - a Gram(-) marine bacterium. To do so, we used deuterated palmitic acid with oleic acid to better preserve the natural membrane saturated/unsaturated lipid ratio. We characterized the membrane fluidity in vivoduring different growth stages by 2H SS-NMR using magic-angle spinning (MAS) and spectral moment analysis. Our results show greater membrane fluidity during the stationary growth phase compared to the exponential phase under labelling conditions. The difference in membrane fluidity is related to a change in fatty acid chain composition, indicating that bacteria adapt to cope with the “stress” induced by the labelling conditions. Then in vivo 2H SS-NMR was used to study the interaction of a promising antimicrobial pigment called “marennine” on V. splendidus membranes. Our results suggest that marennine would act through a stiffening mechanism that could involve interaction with lipopolysaccharides in the outer membrane. Altogether, our work shows that 2H SS-NMR is a useful tool to reveal the effect of stress and interactions on the membrane of intact microorganisms.