A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria.

Lysozymes are ancient and important components of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall polymer. Bacteria engaging in commensal or pathogenic interactions with an animal host have evolved various strategies to evade this bactericidal enzyme, one recently proposed strategy being the production of lysozyme inhibitors. We here report the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in gram-negative bacteria. First, a lysozyme inhibitor was isolated by affinity chromatography from a periplasmic extract of Salmonella Enteritidis, identified by mass spectrometry and correspondingly designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme). A pliC knock-out mutant no longer produced lysozyme inhibitory activity and showed increased lysozyme sensitivity in the presence of the outer membrane permeabilizing protein lactoferrin. PliC lacks similarity with the previously described Escherichia coli lysozyme inhibitor Ivy, but is related to a group of proteins with a common conserved COG3895 domain, some of them predicted to be lipoproteins. No function has yet been assigned to these proteins, although they are widely spread among the Proteobacteria. We demonstrate that at least two representatives of this group, MliC (membrane bound lysozyme inhibitor of c-type lysozyme) of E. coli and Pseudomonas aeruginosa, also possess lysozyme inhibitory activity and confer increased lysozyme tolerance upon expression in E. coli. Interestingly, mliC of Salmonella Typhi was picked up earlier in a screen for genes induced during residence in macrophages, and knockout of mliC was shown to reduce macrophage survival of S. Typhi. Based on these observations, we suggest that the COG3895 domain is a common feature of a novel and widespread family of bacterial lysozyme inhibitors in gram-negative bacteria that may function as colonization or virulence factors in bacteria interacting with an animal host.

Role of the lysozyme inhibitor Ivy in growth or survival of Escherichia coli and Pseudomonas aeruginosa bacteria in hen egg white and in human saliva and breast milk.

Ivy is a lysozyme inhibitor that protects Escherichia coli against lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth of E. coli MG1655 and Pseudomonas aeruginosa PAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-type E. coli was able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 10(4) CFU ml(-1) of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of the ivy mutant to grow like the parental strain. P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.

Detection of a lysozyme inhibitor in Proteus mirabilis by a new reverse zymogram method.

A reverse zymogram method for the detection of bacterial lysozyme inhibitors was developed. This method was validated by using a periplasmic protein extract of Escherichia coli containing a known inhibitor and subsequently led to the detection of a new proteinaceous hen egg white lysozyme inhibitor in Proteus mirabilis.

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

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.

Sensitization of outer-membrane mutants of Salmonella typhimurium and Pseudomonas aeruginosa to antimicrobial peptides under high pressure.

High pressure can sensitize gram-negative bacteria to antimicrobial peptides or proteins through the permeabilization of their outer membranes; however, the range of compounds to which sensitivity is induced is species and strain dependent. We studied the role of outer-membrane properties in this sensitization by making use of a series of rough and deep rough mutants of Salmonella enterica serovar Typhimurium that show an increased degree of lipopolysaccharide (LPS) truncation, along with Pseudomonas aeruginosa PhoP and PhoQ mutants with altered outer-membrane properties. The outer-membrane properties of P. aernginosa were also modulated through the use of different Mg2- concentrations in the growth medium. Each of these strains was challenged under high pressure (15 min at 270 MPa for Salmonella Typhimurium and 15 min at 100 MPa for P. aerttginosa) in phosphate buffer with lysozyme (100 microg/ml), nisin (100 IU/ml), lactoferricin (20 microg/ml), and HEL96-116 (100 microg/ml), a synthetic lysozyme-derived peptide, and sensitization levels were compared. The results obtained indicated that outer-membrane properties affected high-pressure sensitization differently for different compounds. LPS truncation in Salmonella Typhimurium was correlated with increased sensitization to lysozyme (up to 1.5 log10 units) and nisin (up to 1.2 log10 units) but with decreased sensitization to lactoferricin under pressure. For P. aeruginosa, the pattern of sensitization to lactoferricin and nisin resembled that of polymyxin B at atmospheric pressure, suggesting that pressure induces the self-promoted uptake of both peptides. Sensitization to HEL96-116 was not affected by outer-membrane properties for either organism. Hence, outer-membrane permeabilization by high pressure cannot be explained by a single unifying mechanism and is dependent on the organism, the outer-membrane properties, and the nature of the antimicrobial compound. On the basis of these findings, the use of antimicrobial cocktails targeting different bacteria and fractions of bacterial populations may enhance the efficacy of high pressure as a preservation treatment.

Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure.

A different behavior was observed in three gram-positive bacteria exposed to hen egg white lysozyme by plate counts and phase-contrast microscopy. The inactivation of Lactobacillus johnsonii was accompanied by spheroplast formation, which is an indication of peptidoglycan hydrolysis. Staphylococcus aureus was resistant to lysozyme and showed no signs of peptidoglycan hydrolysis, and Listeria innocua was inactivated and showed indications of cell leakage but not of peptidoglycan hydrolysis. Under high hydrostatic pressure, S. aureus also became sensitive to lysozyme but did not form spheroplasts and was not lysed. These results suggested the existence of a nonlytic mechanism of bactericidal action of lysozyme on the latter two bacteria, and this mechanism was further studied in L. innocua. Elimination of the enzymic activity of lysozyme by heat denaturation or reduction with beta-mercaptoethanol eliminated this bactericidal mechanism. By means of a LIVE/DEAD viability stain based on a membrane-impermeant fluorescent dye, the nonlytic mechanism was shown to involve membrane perturbation. In the absence of lysozyme, high-pressure treatment was shown to induce autolytic activity in S. aureus and L. innocua.