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Poster De Conférence Année : 2013

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

Résumé

Research 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 methods
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Dates et versions

hal-01209467 , version 1 (02-10-2015)

Identifiants

  • HAL Id : hal-01209467 , version 1
  • PRODINRA : 201702

Citer

Mélanie Derde, Francoise Nau, Valérie Lechevalier-Datin, Catherine Guérin-Dubiard, Sophie Jan, et al.. Evidence by AFM-imaging of morphological differences between E. coli K12 cells treated with native and dry-heated lysozyme. Forum des Microscopies à Sonde Locale, Mar 2013, Spa, Belgium. , 2013. ⟨hal-01209467⟩
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