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.

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.

Inactivation of gram-negative bacteria in milk and banana juice by hen egg white and lambda lysozyme under high hydrostatic pressure.

The effect of hen egg white lysozyme (HEWL) and bacteriophage lambda lysozyme (LaL) in combination with high pressure (HP) treatment on the inactivation of four gram-negative bacteria (Escherichia coli O157:H7, Shigella flexneri, Yersinia enterocolitica and Salmonella typhimurium), was studied in skim milk (pH 6.8; a(w) 0.997) and in banana juice (pH 3.8; a(w) 0.971). In the absence of lysozymes, S. flexneri was more sensitive to HP in milk than in banana juice, while the opposite was observed for the other three bacteria. In combination with HP treatment, LaL was more effective than HEWL on all bacteria in both milk and banana juice. Depending on the bacteria, inactivation levels in banana juice were increased from 0.4-2.7 log units by HP treatment alone to 3.6-6.5 log units in the presence of 224 U/ml LaL. Bacterial inactivation in milk was also enhanced by LaL but only by 0.5-2.1 log units. Under the experimental conditions used, LaL was more effective in banana juice than in milk, while the effectiveness of HEWL under the same conditions was not significantly affected by the food matrix. This effect could be ascribed to the low pH of the banana juice since LaL was also more effective on E. coli in buffer at pH 3.8 than at pH 6.8. Since neither LaL nor HEWL are enzymatically active at pH 3.8, we analysed bacterial lysis after HP treatment in the presence of these enzymes, and found that inactivation proceeds through a non-lytic mechanism at pH 3.8 and a lytic mechanism at pH 6.8. Based on these results, LaL may offer interesting perspectives for use as an extra hurdle in high pressure food preservation.

Comparison of bactericidal activity of six lysozymes at atmospheric pressure and under high hydrostatic pressure.

The antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (Enterococcus faecalis, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Micrococcus lysodeikticus) and five gram-negative bacteria (Yersinia enterocolitica, Shigella flexneri, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella typhimurium). 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). T4L, LaL and GEWL were highly pure as evaluated by silver staining of SDS-PAGE gels and zymogram analysis while CFL was only partially pure. At ambient pressure each gram-positive test organism displayed a specific pattern of sensitivity to the six lysozymes, but none of the gram-negative bacteria was sensitive to any of the lysozymes. High pressure treatment (130-300 MPa, 25 degrees C, 15 min) sensitised several gram-positive and gram-negative bacteria for one or more lysozymes. M. lysodeikticus and P. aeruginosa became sensitive to all lysozymes under high pressure, S. typhimurium remained completely insensitive to all lysozymes, and the other bacteria showed sensitisation to some of the lysozymes. The possible applications of the different lysozymes as biopreservatives, and the possible reasons for the observed differences in bactericidal specificity are discussed.

Inactivation of Escherichia coli by high-pressure homogenisation is influenced by fluid viscosity but not by water activity and product composition.

The inactivation of Escherichia coli MG1655 by high-pressure homogenisation (HPH) at pressures ranging from 100 to 300 MPa was studied in buffered suspensions adjusted to different relative viscosities (1.0, 1.3, 1.7, 2.7 and 4.9) with polyethylene glycol 6000 (PEG 6000). The water activity of these suspensions was not significantly affected by this high molecular weight solute. Bacterial inactivation was found to decrease with increasing viscosity of the suspensions, an effect that was more pronounced at higher pressures. To study the effect of water activity, series of E. coli suspensions having a different water activity (0.953-1.000) but the same relative viscosity (1.3, 1.7, 2.7 and 4.9) were made using PEG of different molecular weights (400, 600, 1000 and 6000), and subjected to HPH treatment. The results indicated that water activity does not influence inactivation. Finally, inactivation of E. coli MG1655 by HPH in skim milk, soy milk and strawberry-raspberry milk drink was found to be the same as in PEG containing buffer of the corresponding viscosity. These results identify fluid viscosity as a major environmental parameter affecting bacterial inactivation by HPH, as opposed to water activity and product composition, and should contribute to the development of HPH applications for the purpose of bacterial inactivation.

Inactivation of Escherichia coli by high hydrostatic pressure at different temperatures in buffer and carrot juice.

The inactivation of Escherichia coli MG1655 was studied at 256 different pressure (150-600 MPa)-temperature (5-45 degrees C) combinations under isobaric and isothermal conditions in Hepes-KOH buffer (10 mM, pH 7.0) and in fresh carrot juice. A linear relationship was found between the log10 of inactivation and holding time for all pressure-temperature combinations in carrot juice, with R2-values>or=0.91. Decimal reduction times (D-values), calculated for each pressure-temperature combination, decreased with pressure at constant temperature and with temperature at constant pressure. Further, a linear relationship was found between log10D and pressure and temperature. A first order kinetic model, describing log10D in carrot juice as a function of pressure and temperature was formulated that allows to identify process conditions (pressure, temperature, holding time) resulting in a desired level of inactivation of E. coli. For Hepes-KOH buffer, the Weibull model more accurately described the entire set of inactivation curves of E. coli MG1655 compared to the log-linear or the biphasic model. Several secondary models (first and second order polynomial and Weibull) were evaluated, but all had poor fitting capacities. When the Hepes-KOH dataset was limited to 22 of the 34 pressure-temperature combinations, a first order model was appropriate and enabled us to use the same model structure as for carrot juice, for comparative purposes. The major difference in kinetic behaviour of E. coli in buffer and in carrot juice was that inactivation rate as a function of temperature showed a minimum around 20-30 degrees C in buffer, whereas it increased with temperature over the entire studied temperature range in carrot juice.

Moderate temperatures affect Escherichia coli inactivation by high-pressure homogenization only through fluid viscosity.

The inactivation of suspensions of Escherichia coli MG1655 by high-pressure homogenization was studied over a wide range of pressures (100-300 MPa) and initial temperatures of the samples (5-50 degrees C). Bacterial inactivation was positively correlated with the applied pressure and with the initial temperature. When samples were adjusted to different concentrations of poly(ethylene glycol) to have the same viscosity at different temperatures below 45 degrees C and then homogenized at these temperatures, no difference in inactivation was observed. These observations strongly suggest, for the first time, that the influence of temperature on bacterial inactivation by high-pressure homogenization is only through its effect on fluid viscosity. At initial temperatures > or =45 degrees C, corresponding to an outlet sample temperature >65 degrees C, the level of inactivation was higher than what would be predicted on the basis of the reduced viscosity at these temperatures, suggesting that under these conditions heat starts to contribute to cellular inactivation in addition to the mechanical effects that are predominant at lower temperatures. Second-order polynomial models were proposed to describe the impact of a high-pressure homogenization treatment of E. coli MG1655 as a function of pressure and temperature or as a function of pressure and viscosity. The pressure-viscosity inactivation model provided a better quality of fit of the experimental data and furthermore is more comprehensive and versatile than the pressure-temperature model because in addition to viscosity it implicitly incorporates temperature as a variable.

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.

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.

Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria.

The purpose of this review is to describe the antibacterial properties and mode of action of lysozyme against gram-positive and gram-negative bacteria, and to provide insight in the underlying causes of bacterial resistance or sensitivity to lysozyme. Such insight improves our understanding of the role of this ubiquitous enzyme in antibacterial defense strategies in nature and provides a basis for the development and improvement of applications of this enzyme as an antibacterial agent. The bactericidal properties of lysozyme are primarily ascribed to its N-acetylmuramoylhydrolase enzymic activity, resulting in peptidoglycan hydrolysis and cell lysis. However, an increasing body of evidence supports the existence of a nonenzymic and/or nonlytic mode of action. Because gram-negative bacteria, including some major foodborne pathogens, are normally insensitive to lysozyme by virtue of their outer membrane that acts as a physical barrier preventing access of the enzyme, several strategies have been developed to extend the working spectrum of lysozyme to gram-negative bacteria. These include denaturation of lysozyme, modification of lysozyme by covalent attachment of polysaccharides, fatty acids and other compounds, attachment of C-terminal hydrophobic peptides to lysozyme by genetic modification, and the use of outer membrane permeabilizing agents such as EDTA or polycations or permeabilizing treatments such as high hydrostatic pressure treatment.

Na+-mediated piezoprotection in Rhodotorula rubra.

Sodium concentrations as low as 2 mM exerted a significant protective effect on the high-pressure inactivation (160-210 MPa) of Rhodotorula rubra at pH 6.5, but not on two other yeasts tested (Schizosaccharomyces pombe and Saccharomyces cerevisiae). A piezoprotective effect of similar magnitude was observed with Li+ (2 and 10 mM), and at elevated pH (8.0-9.0), but no effect was seen with K+, Ca2+, Mg2+, Mn2+, or NH4(+). Intracellular Na+ levels in cells exposed to low concentrations of Na+ or to pH 8.0-9.0 provided evidence for the involvement of a plasma membrane Na+/H+ antiporter and a correlation between intracellular Na+ levels and pressure resistance. The results support the hypothesis that moderate high pressure causes indirect cell death in R. rubra by inducing cytosolic acidification.