ANTIMICROBIAL PEPTIDES (RICH IN DISULFIDE BRIDGES)

Antimicrobial peptides (AMPs), key molecules in the innate immunity of all living organisms, represent one of the most promising alternatives to fight antibiotic resistance, a major public health problem.

We are working on a fine understanding of the 3D structures (structural diversity, structure-activity relationships, evolution of structures via phylogeny) and the fine understanding of the mechanisms of action of some promising original AMPs, to help designing new antimicrobial compounds.

The four examples below (double defensins from birds, defensins from the king penguin, defensins from oysters, defensins from butterflies) illustrate the complementary nature of the techniques we combine with NMR—phylogeny, imaging, micro-scale thermophoresis (MST) and calorimetry (ITC) – to dissect these mechanisms of action at the molecular and atomic scale.

Avian-double-beta-defensins

We determined the 3D structure of the first avian-double-β-defensin, Gga-AvBD1, from the chicken Gallus gallus, defining a novel 3D fold. We studied several biological activities, including antibacterial, antiparasitic and antiviral, and assessed the contribution of each domain to these activities (left, www.pnas.org/cgi/doi/10.1073/pnas.1912941117). Finally, we showed that the gene coding for this double-defensin, present in all the bird genomes currently available (over 300), does not originate from a duplication-fusion (https://doi.org/10.3390/biology11050690 ).

Ancestral oyster defensins

We have solved the 3D structure of two big-defensins from the Crassostrea gigas oyster, which are active in a saline environment (400 mM NaCl) against a broad spectrum of bacteria (example Cg-BigDef1, left, https://doi.org/10.1128/mBio.01821-19). These two-domain PAMs comprise a β-defensin-like domain (in green) rich in disulfide bridges and a highly conserved hydrophobic N-terminal domain structured in helix-tower-helix (in brown). The latter is essential for antimicrobial activity in a saline environment, and governs the self-association of the entire molecule by forming “nanonets” to trap bacteria (right). We have also demonstrated synergy between the domains and remarkable complementarity in their antimicrobial spectrum (https://doi.org/10.1128/mBio.01821-19, https://doi.org/10.3390/md20120745).

King penguin defensins

An essential first step towards the rational engineering of highly effective antibacterial peptides for clinical use is to understand in detail how nature has optimized AMPs. Data on non-lytic AMPs (i.e. those that do not lyse the bacterial membrane to destroy the bacteria), such as β-defensins, are scarce and therefore remain poorly understood. We study the mechanism of action of defensins, on the example of the king penguin defensin AvBD103b, by performing fluorescence microscopy experiments on living E. coli bacteria that allow us to monitor in real time the slightest permeabilization of their inner and outer membranes (https://doi.org/10.3390/ijms23042057).

Defensins from butterflies

Defensin ETD151 was optimized from butterfly defensins. We demonstrated that the first step in its multifaceted mechanism required the presence of glucosylceramides (GlcCers), fungal membrane lipids that are crucial for fungal growth and virulence, and therefore considered promising targets. We have shown that there is a direct molecular interaction between ETD151 and fungal GlcCer (https://doi.org/10.1073/pnas.2415524122), and we are studying it using a multidisciplinary approach that combines micro-scale thermophoresis (MST), isothermal titration calorimetry (ITC), solution NMR, and solid-state NMR. We have shown that methyl, which is specific to fungal GlcCers, plays a major role in this interaction (https://doi.org/10.1016/j.jbc.2025.110587 ).