A Helix-Loop-Helix Peptide at the Upper Lip of the Active Site Cleft of Lysozyme Confers Potent Antimicrobial Activity with Membrane Permeabilization Action

Abstract

Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98 -112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loophelix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.

Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein

“…Both Asp and Asn residues, and not Glu and Gln, have been implicated in the formation of reverse turns, possibly because these amino acids easily form hydrogen bonds with the peptide backbone (44,45). Together, these data indicate that the hydrophilic region between HR1 and HR2 indeed facilitates a reverse turn, and they suggest that 2B adopts a helix-loop-helix structure, a commonly found structural feature in membrane-active proteins (29,46).…”

“…20). The 2B protein contains two hydrophobic regions, designated HR1 (amino acids [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] and HR2 (amino acids 63-80), of which the first is predicted to form a cationic amphipathic ␣-helix (21,22). The amphipathic ␣-helix displays characteristics typical for the group of lytic polypeptides, which may build membrane-integral pores upon the formation of multimeric transmembrane complexes (23)(24)(25).…”

Transfer of egg white proteins with reference to lysozyme during the development ofMeleagris gallopavo(Galliformes: Phasianidae) embryos

“…Alternatively, lysozyme at later stages of development may undergo enzymatic hydrolysis by certain proteases that produce fragments of the enzyme with enhanced activity. It has been shown that the hydrolysis of lysozyme by different proteolytic enzymes, such as clostripain, pepsin and trypsin, caused the exposure of the antibacterial peptides of the enzyme, and as a result increased its antibacterial activity (Pellegrini et al 1997;Ibrahim et al 2001Ibrahim et al , 2002Ibrahim et al , 2005Mine et al 2004).…”

Salmonella in Shell Eggs: Mechanisms, Prevention and Detection

“…Ovortransferrin is considered a major antimicrobial ingredient of the egg whites because it chelates iron, which is a critical growth factor for microorganisms such as Salmonella [61][62][63]. Ovotransferrin and lysozyme are known to generate pores on the surface of gram-negative bacteria resulting in membrane permeabilization [64,65]. However, there are various studies showing that Salmonella cells survive effectively inside of egg whites using their defense system.…”

“…For example, Lu et al [66] reported that damaged DNA by egg whites can be restored by yafD, xthA and rfbH genes of Salmonella. Other researchers have shown that Salmonella is more adept at thriving in egg albumen compared with other microorganisms because of the distinctive genes related to their cell wall formation and metabolism [64,67,68]. Moreover, using the siderophore, Salmonella can effectively uptake iron to survive in egg white [69,70].…”