Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

Abstract

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.

Endolysins as Antimicrobials

“…coli O157:H7, Shigella flexneri, Yersinia enterocolitica, and Salmonella typhimurium (Nakimbugwe et al, 2006). The λ lysozyme outperformed the PG hydrolase in a bacterial inactivation assay by almost 2 and 5 logs in skim milk and banana juice, respectively.…”

“…While this may not have direct human applications, it does have potential applications for decontamination and food processing. HHP has several advantages: it can be bactericidal alone (Briers et al, 2008;Hauben et al, 1996;Masschalck et al, 2000;Masschalck et al, 2001;Nakimbugwe et al, 2006), it does not use heat so it will not compromise the quality of foodstuffs, and most importantly, it is not considered to be a food additive. However, generating the required high pressures (200 to 500 MPa) can pose a cost hurdle.…”

“…However, generating the required high pressures (200 to 500 MPa) can pose a cost hurdle. HHP has been used with a variety of antibacterials including nisin, lactoferrin, and several PG (Briers et al, 2008;Hauben et al, 1996;Masschalck et al, 2000;Masschalck et al, 2001;Nakimbugwe et al, 2006). …”

“…Nakimbugwe and colleagues tested HHP in conjunction with six individual PG hydrolases, including phage endolysins from λ and T4, on ten different bacterial strains (five each of Gramnegative and -positive) (Nakimbugwe et al, 2006). Both phage endolysins were active on four out of five of the Gram-negative bacteria and Bacillus subtilis, though the λ-derived endolysin showed greater activity on most of the strains.…”

Structure-Based Modification of a Clostridium difficile-Targeting Endolysin Affects Activity and Host Range

“…Species specificity was confirmed with turbidity reduction assays on a variety of species and C. difficile strains obtained from culture collections as listed previously (26). E. coli K-12 cells were treated with choloroform to remove the outer membrane by the method of Nakimbugwe et al (27), and their sensitivity was tested in lysis assays with 30 g crude protein extracts prepared in 20 mM sodium phosphate buffer, pH 6 (26), using chicken egg white lysozyme (Sigma) as a positive control. Lysis assays were performed in duplicate in 300-l volumes.…”