How to MOdulate the SAlt-stability of antimicRobial DEFensins – MOSAR-DEF

The project relied on a wide combination of approaches, combining synthetic chemistry, structural biology, next-generation sequencing, cell biology, microbiology, mutagenesis, and super-resolution microscopy.

- Synthetic chemistry enabled the production of sufficient quantities of each big defensin (several tens of milligrams) and the generation of modular big defensins that can be labeled with fluorophores using click chemistry.

- Structural biology allowed determination of the three-dimensional structures of the different big defensins and identification of amino acids potentially involved in bacterial recognition.

- Next-generation sequencing (16S barcoding) was used to demonstrate in vivo the role of big defensins in shaping the microbiota of their mollusk hosts. Single-cell sequencing, combined with cell biology, identified the immune cells producing big defensins in these hosts.

- Microbiology was used to identify bacteria sensitive to big defensins, characterize their mechanisms of action, and test the ability of bacteria to develop resistance.

- Mutagenesis was applied to generate bacterial mutants in order to test the molecular targets of big defensins on their bacterial species.

- Super-resolution microscopy was used to identify, at fine scale, the cellular targets of big defensins within bacteria.

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- Structural diversity of big defensins: an advantage for better host protection

Big defensins are active against a wide range of bacteria belonging to the microbiota of their natural hosts, oysters. Their great structural diversity broadens their antimicrobial spectrum and creates synergistic effects, suggesting that such diversification may have evolved in these filter-feeding organisms (which feed and breathe by filtering seawater) constantly exposed to a vast diversity of marine bacteria. Sequencing of the oyster microbiota showed that big defensins finely regulate bacterial populations within oyster tissues. At the cellular level, the immune cells producing big defensins 1 and 2 are mainly hyalinocytes, a subtype of cells devoid of granules.

- Mechanism of action: targeting and aggregating bacteria

Detailed studies of the interactions between big defensin 1 and Staphylococcus aureus revealed that the bacterial cell wall is its main target. Upon recognition, big defensin 1 self-assembles, trapping bacteria in thick defensive networks called “nanonets.” The bacteria are then killed by a mechanism that remains only partially understood. Recognition of the bacterial wall involves the N-terminal domain of the peptide, and teichoic acids play a key role among its molecular targets. The absence of these major wall components renders bacteria resistant. The N- and C-terminal domains of big defensin 1 act synergistically to kill bacteria: the C-terminal domain, structurally similar to vertebrate β-defensins, is carried like a cargo by the N-terminal domain to approach the bacterial surface. This N-terminal domain is also essential for nanonet formation and is believed to have been maintained in oysters due to adaptation to high-salinity environments.

- Therapeutic potential: activity against resistant bacteria but limited stability in biological fluids

Big defensins are active against multidrug-resistant bacteria, including MRSA (S. aureus) strains isolated from cystic fibrosis patients. Their antimicrobial activity remains stable at high salt concentrations (up to 400 mM NaCl), supporting their potential use in treatments combining antimicrobial and saline aerosol administration. Moreover, these molecules showed no pro-inflammatory activity. However, their stability in biological fluids proved limited (<2 h), especially in pulmonary secretions. Finally, experimental evolution assays showed that S. aureus can adapt, becoming less sensitive to big defensin after about ten generations under single-agent in vitro treatment.

Although our work does not yet support the use of big defensins as a plausible antibiotic treatment for S. aureus infections, it opens several promising research directions inspired by the study of these remarkable molecules:

- Targeting the cell wall and trapping bacteria: The N-terminal domain of big defensins has been lost in nearly all species that do not live in saline environments. Human defensins, for instance, play various immune roles but no longer function as antimicrobials at the salt concentrations found in human serum (150 mM). Our work has shown that this ancestral domain possesses remarkable properties: it enables targeting of the bacterial cell wall and the formation of molecular networks that trap bacteria independently of salt concentration. We believe that exploring its potential role in transporting antibiotics or antimicrobial peptides to the bacterial wall could help enhance or even restore their activity. This will require further investigation of both direct and indirect interactions of this domain with teichoic acids, essential components of Gram-positive bacterial cell walls that we have identified as potential targets.

- Antimicrobial cocktails: Given the shortage of new antibiotics to treat infections, it would be unwise to propose new treatments that could generate resistance as rapidly as current antibiotics. Big defensin 1 has shown that it can induce bacterial resistance when used alone. It will therefore be important to compare the rate of resistance emergence to that observed with existing antibiotics. Furthermore, nature offers inspiration: in oysters, big defensins act as cocktails of diverse molecules. It will thus be essential to test, first in vitro, whether administering big defensin cocktails slows down the emergence of resistance. Additional in vitro assays could test big defensins in combination with other antimicrobials, such as antibiotics. Finally, in vivo testing could be carried out, where big defensins would act alongside mouse antimicrobial peptides, potentially reducing resistance development in a more natural context.

The preclinical development of cationic antimicrobial peptides as an alternative to antibiotics is precluded, among other processes, by their inactivation by salts. Taking inspiration from marine biodiversity, our goal is to turn salt-sensitive human defensins into salt-stable defensins. This ambitious goal is made possible thanks to (i) recent discoveries from our consortium on the structure/activity of big-defensins, the marine ancestors of vertebrate ß-defensins (ii) new methodologies in chemical synthesis and engineering of such molecules, and (iii) the multidisciplinary team gathered in the MOSAR-Def consortium that brings together peptide chemists, biochemists, structural biologists, microbiologists (marine and human) and cell physiolgists. The project will contribute to significant knowledge on the mechanisms of defensins active both at normal and high salt concentrations, and their therapeutic applications. This represents a step further to unravel alternatives to conventional antibiotics treatments.