Exploring the mechanisms of membrane insertion of native and dry-heated lysozyme: use of [i]E. coli[/i] lipopolysaccharide monolayers

National audience; Antibiotic resistance causes public health problems and stimulates research for novelantimicrobials. Particular attention is given to molecules that limit drug resistancedevelopment.1Hen egg white lysozyme acting on the bacterial cell envelope through itsphysico-chemical properties is thus a good candidate.2,3However, its antimicrobial effectcaused by membrane permeabilization on Gram-negative bacteria remains limited. But somephysico-chemical modifications of the lysozyme can modify its membrane activity, increasinglysozyme antimicrobial properties against E. coli ; dry-heating is able to induce suchmodifications. 4 Especially, we previously highlighted that native (N-L) and dry-heatedlysozyme (DH-L) disrupt the outer membrane of E. coli , but in different ways.3,4 The mode of insertion into the bacterial outer membrane and molecular interactions remains unknown.This was thus investigated using an E. coli lipopolysaccharide monolayer (LPSM) membranemodel, mimicking the outer leaflet of the bacterial outer membrane. The interactions betweenlysozyme and LPSM were studied by tensiometry, ellipsometry, atomic force microscopy(AFM) and Brewster angle microscopy (BAM). Both N-L and DH-L are able to insert into aLPSM. As expected, electrostatic interactions between the negatively charged LPSM and bothpositively charged forms of lysozyme were observed. Furthermore, we could establish that N-L and DH-L insertion into the LPSM depends on the presence of the polysaccharide moieties.These polysaccharide chains might increase the space between the lipid headgroups, enablinglysozyme insertion. Moreover, dry-heating increases the lysozyme affinity for the LPSM.Microscopic observations (BAM and AFM) show that the LPSM reorganizes and reorients inthe presence of DH-L, in contrast to N-L. Dry-heating thus improves the lysozyme insertion,which might explain the increased activity on the outer membrane of E. coli , resulting in ahigher antimicrobial effec