Dry-Heating of Lysozyme Increases Its Activity against Escherichia coli Membranes

Abstract

For food as well as for medical applications, there is a growing interest in novel and natural antimicrobial molecules. Lysozyme is a promising candidate for the development of such molecules. This protein is largely studied and known for its muramidase activity against Gram-positive bacteria, but it also shows antimicrobial activity against Gram-negative bacteria, especially when previously modified. In this study, the activity of dry-heated lysozyme (DH-L) against Escherichia coli has been investigated and compared to that of native lysozyme (N-L). Whereas N-L only delays bacterial growth, DH-L causes an early-stage population decrease. The accompanying membrane permeabilization suggests that DH-L induces either larger pores or more pores in the outer membrane as compared to N-L, as well as more ion channels in the inner membrane. The strong morphological modifications observed by optical microscopy and atomic force microscopy when E. coli cells are treated with DH-L are consistent with the suggested disturbances of membrane integrity. The higher hydrophobicity, surface activity, and positive charge induced by dry-heating could be responsible for the increased activity of DH-L on the E. coli membranes.

Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: Lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane

“…Membrane permeabilization has been suggested as one of the mechanisms responsible for this activity [8,28]. This assumption was recently confirmed by our group who demonstrated that N-L causes the formation of pores and ion channels in the outer and cytoplasmic membranes, respectively [9,11]. Pore formation due to N-L implies that interactions occur between the protein and the E. coli outer membrane.…”

“…Native lysozyme (N-L) has been shown active against Gram-negative bacteria such as E. coli [11,27]. Membrane permeabilization has been suggested as one of the mechanisms responsible for this activity [8,28].…”

“…Moreover, dry-heated lysozyme (DH-L) has a higher antimicrobial activity and higher membrane disruption potential than N-L [11]. This improved activity is supposed to be related to the modified physicochemical properties of DH-L. DH-L is more hydrophobic, flexible and surface active than N-L, but its secondary and tertiary structures remain intact [29,30].…”

“…The disruption of both the outer and cytoplasmic membranes of Escherichia coli by native lysozyme has been recently demonstrated in our laboratory [9]. Especially, pore formation in the outer membrane of E. coli has been described [11]. Moreover, pore size and/or quantity were higher with dry-heated lysozyme as compared to the native protein [11].…”

“…Especially, pore formation in the outer membrane of E. coli has been described [11]. Moreover, pore size and/or quantity were higher with dry-heated lysozyme as compared to the native protein [11]. The physicochemical modifications resulting from dry-heating are an increased positive charge at physiological pH as well as increased flexibility and hydrophobicity while preserving the secondary and tertiary structure; these modifications could be responsible for the enhanced antibacterial activity of dryheated lysozyme, similarly to what has been described for lysozyme modification by enzyme hydrolysis, heat denaturation, or fusion with several chemical moieties [7,[12][13][14][15][16][17].…”