Activité microbienne du lysozyme : de la bactérie au système modèle

Plusieurs équipes de recherche rennaises utilisent des expériences aux interfaces fluides planes pour connaître et comprendre les propriétés de biomolécules variées. Lors d’une journée d’échanges, des scientifiques de l'UMR STLO et d’autres équipes contribueront à dresser le panorama des thématiques concernées et montrer le potentiel de ces expériences.Une journée d'échanges scientifiques est organisée par l'Institut de Physique de Rennes, le 22 janvier 2015. Elle vise à illustrer le potentiel et à construire une vision d'ensemble des expériences aux interfaces planes, dans des domaines scientifiques aussi variés que la physique des mousses et émulsions, les technologies de transformation des aliments, la digestion néonatale des lipides laitiers ou l'interaction de protéines avec les membranes biologiques. L'opportunité de montrer que les expériences aux interfaces planes livrent des informations abondantes et pertinentes.absen

Films de Langmuir: modèle membranaire des bactéries, analyse par microscopie à force atomique

International audienc

Activité antimicrobienne du lysozyme natif/chauffé : de la bactérie aux systèmes modèles

National audienc

Antimicrobial activity of proteins against E. coli. Investigation using interfacial technics

National audienceThe identification and/or design of novel antibacterial agents are hot topics, because of the increasing bacterial resistance against antibiotics. Some natural proteins and peptides can be considered promising antimicrobials because acting through different and complementary mechanisms. Lysozyme and ovotransferrin are two proteins known for its activity against bacteria. We are interested on its ability to disturb the outer and/or the inner membranes.Indeed, the membrane permeabilization is an interesting mechanism because it causes bacterial cell death, while limiting the development of bacterial resistance. The understanding of the mechanisms by which different protein can have different antibacterial activity could help goal-oriented screening and design of new antimicrobial compounds. For this propose, we develop a strategy based on the use of lipid monolayer to decompose the mechanism of the antimicrobial activity. In this presentation, we will present results

Evidence by AFM-imaging of morphological differences between E. coli K12 cells treated with native and dry-heated lysozyme

National audienceResearch in the domain of antimicrobial molecules is nowadays a hot topic because of the increasing bacterial resistance against antibiotics; moreover, consumers demand for natural food preservatives is growing. Proteins and peptides from different origins, such as hen egg white lysozyme, seem promising candidates for the discovery of novel and natural antimicrobial compounds. The understanding of the mechanism by which these compounds are active could help goal oriented screening and design of antimicrobial peptides and proteins. Membrane permeabilization is an interesting mechanism because it causes bacterial cell death, while limiting the development of bacterial resistance [1, 2, 3]. Several tools can be used to investigate membrane perturbations such as spectrophotometric methods using mutant E. coli ML - 35p, fluorescent probes and AFM -imaging [4, 5, 6].Lysozyme is a major natural antimicrobial molecule widely used for food and pharmaceutical applications.This small protein (14400 Da) is especially known for its capacity to hydrolyze the peptidoglycan of Gram -positive bacteria [3, 7].On the other hand, because of the outer membrane of Gram - negative bacteria,lysozyme is almost inactive against these microorganisms. However, some structural modifications of lysozyme are efficient to increase and broaden the antimicrobial activity of lysozyme; thus modified lysozyme would operate by disturbing the bacterial membrane [2, 8 ]°. Dry -heating of lysozyme (80°C for 7 days) could be an interesting way to increase the lysozyme antimicrobial activity , since this process makes the protein much more tensio-active than the native form [9] .[br/] Native and dry - heated lysozymes have been compared for their activity against E. coli K12, a model Gram - negative bacteria. Bacteria growth has been measured for 24 h in Luria Broth containing 0.5 g/L NaCl at 37 °C, in the presence of native or dry - heated lysozyme with concentrations ranging from 0.05 g/L to 3.7 g/L. The inner and outer membrane permeabilization has been measured using the mutant E. coli ML - 35p. The morphologic al characteristics of the bacteria have been investigated by AFM after 24 h of contact with native or dry - heated lysozyme.[br/] E. coli K12 growth is disturbed in the presence of both native and dry -heated lysozyme, but the inhibition is larger at high concentration (3.7 g/L) of dry heated lysozymeSimilarly, permeabilization of the out er and inner membranes was observed with both native and dry -heated lysozyme , in a concentration - dependant way. On the contrary, AFM - imaging enables to distinguish between bacterial cells which were non - treated , treated with native lysozyme, or with dry - heated lysozyme.The size of non - treated cells is about 2 μm long x 500 nm diameter; the surface is quite smooth and regular, and flagella can be observed (figure 1). When treated with 0.25 g/L native lysozyme, only small differences such as higher rigidity of cells are observed, compared to the non - treated cells (figure 2) ; similar images are obtained with 3.7 g/L native lysozyme . But when treated with 0.25 g/L dry - heated lysozyme, bacteria cells appear strongly disturbed , with especially irregular cell surface, local depressions, and disturbance of cell division ; besides well defined bacteria cells, cell debris are also observed . Equivalent images are obtained after incubation with 3.7 g/L dry - heated lysozyme . This underlines that AF M is a relevant and efficient tool to investigate antibacterial activity of native and modified lysozymes , since it highlights differences that are not easily detectable with usual microbiological and biochemical method

Exploring the mechanisms of membrane insertion of native and dry-heated lysozyme: use of [i]E. coli[/i] lipopolysaccharide monolayers

National audienceAntibiotic resistance causes public health problems and stimulates research for novelantimicrobials. Particular attention is given to molecules that limit drug resistancedevelopment.1Hen egg white lysozyme acting on the bacterial cell envelope through itsphysico-chemical properties is thus a good candidate.2,3However, its antimicrobial effectcaused by membrane permeabilization on Gram-negative bacteria remains limited. But somephysico-chemical modifications of the lysozyme can modify its membrane activity, increasinglysozyme antimicrobial properties against E. coli ; dry-heating is able to induce suchmodifications. 4 Especially, we previously highlighted that native (N-L) and dry-heatedlysozyme (DH-L) disrupt the outer membrane of E. coli , but in different ways.3,4 The mode of insertion into the bacterial outer membrane and molecular interactions remains unknown.This was thus investigated using an E. coli lipopolysaccharide monolayer (LPSM) membranemodel, mimicking the outer leaflet of the bacterial outer membrane. The interactions betweenlysozyme and LPSM were studied by tensiometry, ellipsometry, atomic force microscopy(AFM) and Brewster angle microscopy (BAM). Both N-L and DH-L are able to insert into aLPSM. As expected, electrostatic interactions between the negatively charged LPSM and bothpositively charged forms of lysozyme were observed. Furthermore, we could establish that N-L and DH-L insertion into the LPSM depends on the presence of the polysaccharide moieties.These polysaccharide chains might increase the space between the lipid headgroups, enablinglysozyme insertion. Moreover, dry-heating increases the lysozyme affinity for the LPSM.Microscopic observations (BAM and AFM) show that the LPSM reorganizes and reorients inthe presence of DH-L, in contrast to N-L. Dry-heating thus improves the lysozyme insertion,which might explain the increased activity on the outer membrane of E. coli , resulting in ahigher antimicrobial effe

Exploring the mechanisms of membrane insertion of native and dry-heated lysozyme: use of E. coli lipopolysaccharide monolayers

National audienceAntibiotic resistance causes public health problems and stimulates research for novelantimicrobials. Particular attention is given to molecules that limit drug resistancedevelopment.1 Hen egg white lysozyme acting on the bacterial cell envelope through itsphysico-chemical properties is thus a good candidate.2,3 However, its antimicrobial effectcaused by membrane permeabilization on Gram-negative bacteria remains limited. But somephysico-chemical modifications of the lysozyme can modify its membrane activity, increasinglysozyme antimicrobial properties against E. coli; dry-heating is able to induce suchmodifications.4 Especially, we previously highlighted that native (N-L) and dry-heatedlysozyme (DH-L) disrupt the outer membrane of E. coli, but in different ways.3,4 The mode ofinsertion into the bacterial outer membrane and molecular interactions remains unknown.This was thus investigated using an E. coli lipopolysaccharide monolayer (LPSM) membranemodel, mimicking the outer leaflet of the bacterial outer membrane. The interactions betweenlysozyme and LPSM were studied by tensiometry, ellipsometry, atomic force microscopy(AFM) and Brewster angle microscopy (BAM). Both N-L and DH-L are able to insert into aLPSM. As expected, electrostatic interactions between the negatively charged LPSM and bothpositively charged forms of lysozyme were observed. Furthermore, we could establish that NLand DH-L insertion into the LPSM depends on the presence of the polysaccharide moieties.These polysaccharide chains might increase the space between the lipid headgroups, enablinglysozyme insertion. Moreover, dry-heating increases the lysozyme affinity for the LPSM.Microscopic observations (BAM and AFM) show that the LPSM reorganizes and reorients inthe presence of DH-L, in contrast to N-L. Dry-heating thus improves the lysozyme insertion,which might explain the increased activity on the outer membrane of E. coli, resulting in ahigher antimicrobial effect

Dry heating of lysozyme: strong effects regarding antimicrobial activity

International audienceFor food preservation as well as for medical applications, because of increasing bacterial resistance towards antibiotics, novel and natural antimicrobial molecules are request. The widely studied lysozyme is a promising candidate to develop such molecules. This protein is well known and already used for its enzymatic activity against Gram-positive bacteria, but it also presents some activity against Gram-negative bacteria, supposedly due to its capability to disturb bacteria membrane. Otherwise, molecules that provoke bacterial membrane disruption are generally positively charged, amphipathic, and hydrophobic, all characteristics which can be modified. Especially, we previously showed that dry heating is a safe and easy process resulting in slight chemical modifications of lysozyme, with strong consequences regarding the charge, the hydrophobicity, and finally the interfacial properties of the protein. The question then arose: could lysozyme dry heating be an opportunity to create a new efficient antimicrobial? We actually demonstrated that the activity of dry heated lysozyme (DH-L) against Escherichia coli was higher than that of native lysozyme (N-L). Using optical microscopy and atomic force microscopy (AFM), strong morphological modifications of the bacteria were observed, consistently with the higher membrane permeabilization when E. coli cells were treated with DH-L: either larger pores or more pores in the outer membrane, as well as more ion channels in the cytoplasmic membrane were obtained with DH-L as compared to N-L. Using a lipopolysaccharide (LPS) monolayer and a phospholipid (PL) mixture monolayer as models of the E. coli outer and cytoplasmic membranes, respectively, the interaction between lysozyme and these monolayer models has been investigated by biophysics techniques such as tensiometry, ellipsometry, Brewster angle microscopy and AFM. We could thus established that dry heating increases lysozyme affinity for the model monolayers and its insertion capacity; the resulting reorganization of the model monolayers was also more drastic. Finally, using similar investigations with each of the lysozyme isoforms produced by dry heating, we could show that the most positive, flexible and hydrophobic isoform shows the highest antimicrobial activity. However, it is noticeable that the lysozyme isoforms mixture, I;e; DH-L was the most efficient against E. coli, suggesting synergetic cooperation between lysozyme isoforms

Antimicrobial activity of lysozyme isoforms: Key molecular features

International audienceIncreasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms