Bacterial membrane perturbation by native versus dry-heated lysozyme

Antimicrobial drug resistance causes public health problems worldwide. Scientific research for the discovery ofnovel antimicrobial molecules is thus highly stimulated [1]. Particular attention is given to antimicrobialmolecules that limit drug resistance development and have a large spectrum. Antimicrobial peptides and proteinsare thus good candidates. Some of them act on the bacterial cell wall because of their physico-chemicalproperties; when amphiphilic and positively charged, these molecules are able to interact with the negativelycharged bacterial surface[2]. These antimicrobial peptides and proteins can thus perturb the bacterial cellenvelope, enter the cell, and/or act on intracellular targets [2, 3]. Hen egg white lysozyme is a marketed protein,available at an affordable price (100€/kg). This small protein (14.4 kDa) is largely studied and widely known forits muramidase activity against the peptidoglycan of Gram-positive bacteria, the Gram negative bacteria beingprotected by their outer membrane [4]. However, lysozyme is also described as being active against some Gram-negative bacteria, especially becauseit permeabilizes the bacterial membranes [4, 5]. But few is known aboutthis activity, and the consequences on the bacterial membranes remains quite limited. Since dry-heated lysozyme, composed of a mixture of several isoforms, i.e.iso-aspartyle, native-like and succinimide lysozymes [6], is more positively charged, more hydrophobic and more tensio-active than native lysozyme [6], dry-heating could be away to increase lysozyme activity against the bacterial membranes, and especially against am-negative bacteria.To investigate the membrane activity of both native and dry-heated lysozyme, membrane permeability measurements using a mutant E. coli ML-35p and measurements of membrane potential perturbation were performed. These experiments revealed that native and dry-heated lysozyme induce different consequences on the bacterial membranes. Native lysozyme permeabilizes both outer and inner membranes. Pore formation could be observed for the outer membrane, but not for the inner membrane. Moreover, the membrane potential of the inner membrane is modified in the presence of native lysozyme. Dry-heated lysozyme induces an overall stronger disturbance than native lysozyme, by giving rise to the formation of more and/or larger pores in the outer membrane, by permeabilizing more rapidly the inne r membrane, and by leading to a stronger disturbance of membrane potential. We could also establish that each of the dry-heated lysozyme isoforms affords a specific disturbance of either the outer or the inner membrane. Succinimide lysozyme strongly increases the outer membrane permeability, but mildly modifies the permeability of the inner membrane. On the contrary, iso-aspartyle lysozyme modifies more strongly the inner than the outer membrane permeability. This was confirmed by membrane potential perturbation caused by iso-aspartylelysozyme. Both isoforms thus prove complementar