Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>Pre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen <it>P. aeruginosa</it>. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against <it>P. aeruginosa</it>.</p> <p>Results</p> <p>We show here that the cementoin domain adopts an α-helical conformation both by circular dichroism and nuclear magnetic resonance analyses in the presence of membrane mimetics, a characteristic shared with a large number of linear polycationic antimicrobial peptides. However, pre-elafin/trappin-2 and its domains display only weak lytic properties, as assessed by scanning electron micrography, outer and inner membrane depolarization studies with <it>P. aeruginosa </it>and leakage of liposome-entrapped calcein. Confocal microscopy of fluorescein-labeled pre-elafin/trappin-2 suggests that this protein possesses the ability to translocate across membranes. This correlates with the finding that pre-elafin/trappin-2 and elafin bind to DNA <it>in vitro </it>and attenuate the expression of some <it>P. aeruginosa </it>virulence factors, namely the biofilm formation and the secretion of pyoverdine.</p> <p>Conclusions</p> <p>The N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides. However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against <it>P. aeruginosa</it>. Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some <it>P.aeruginosa </it>virulence factors, possibly through acting on intracellular targets.</p

Interaction de la créatine kinase mitochondriale avec des membranes biomimétiques (liposomes, monocouches de Langmuir)

La créatine kinase mitochondriale (CKmt) est une protéine octamérique qui, in vivo, est associée à la face externe de la membrane mitochondriale interne. La liaison de l'enzyme, via des lysines C-terminales, à des liposomes contenant des phospholipides acides affecte la fluidité membranaire et entraîne une modification conformationnelle de la CKmt. Nous avons étudié par la technique de Langmuir couplée à la microscopie à l'angle de Brewster le comportement interfacial de la protéine en l'absence et en présence de phospholipides. En l'absence de monocouche, elle s'adsorbe spontanément à l'interface air/eau et doit donc posséder des zones hydrophobes accessibles. En présence de monocouches constituées de phospholipides acides ou zwitterioniques, à chaînes saturées ou insaturées, les isothermes ont un profil différent selon la présence ou non de charges négatives et la pression de surface au moment de l'injection. La présence de phospholipides acides facilite l'adsorption de la protéineLYON1-BU.Sciences (692662101) / SudocSudocFranceF

From undefined red smear cheese consortia to minimal model communities both exhibiting similar anti-listerial activity on a cheese-like matrix.

International audienceStarting from one undefined cheese smear consortium exhibiting anti-listerial activity (signal) at 15 °C, 50 yeasts and 39 bacteria were identified by partial rDNA sequencing. Construction of microbial communities was done either by addition or by erosion approach with the aim to obtain minimal communities having similar signal to that of the initial smear. The signal of these microbial communities was monitored in cheese microcosm for 14 days under ripening conditions. In the addition scheme, strains having significant signals were mixed step by step. Five-member communities, obtained by addition of a Gram negative bacterium to two yeasts and two Gram positive bacteria, enhanced the signal dramatically contrary to six-member communities including two Gram negative bacteria. In the erosion approach, a progressive reduction of 89 initial strains was performed. While intermediate communities (89, 44 and 22 members) exhibited a lower signal than initial smear consortium, eleven- and six-member communities gave a signal almost as efficient. It was noteworthy that the final minimal model communities obtained by erosion and addition approaches both had anti-listerial activity while consisting of different strains. In conclusion, some minimal model communities can have higher anti-listerial effectiveness than individual strains or the initial 89 micro-organisms from smear. Thus, microbial interactions are involved in the production and modulation of anti-listerial signals in cheese surface communities

Human Pre-Elafin Inhibits a Pseudomonas aeruginosa-Secreted Peptidase and Prevents Its Proliferation in Complex Media▿

Pseudomonas aeruginosa is a life-threatening opportunist human pathogen frequently associated with lung inflammatory diseases, namely, cystic fibrosis. Like other species, this gram-negative bacteria is increasingly drug resistant. During the past decade, intensive research efforts have been focused on the identification of natural innate defense molecules with broad antimicrobial activities, collectively known as antimicrobial peptides. Human pre-elafin, best characterized as a potent inhibitor of neutrophil elastase with anti-inflammatory properties, was also shown to possess antimicrobial activity against both gram-positive and gram-negative bacteria, including P. aeruginosa. Its mode of action was, however, not known. Using full-length pre-elafin, each domain separately, and mutated variants of pre-elafin with attenuated antipeptidase activity toward neutrophil elastase, we report here that both pre-elafin domains contribute, through distinct mechanisms, to its antibacterial activity against Pseudomonas aeruginosa. Most importantly, we demonstrate that the whey acidic protein (WAP) domain specifically inhibits a secreted peptidase with the characteristics of arginyl peptidase (protease IV). This is the first demonstration that a human WAP-motif protein inhibits a secreted peptidase to prevent bacterial growth in vitro. Since several WAP-motif proteins from various species demonstrate antimicrobial function with variable activities toward bacterial species, we suggest that this mechanism may be more common than initially anticipated

Mitochondrial creatin kinase interaction with heterogeneous monolayers : Effect on lipid lateral organization

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Mitochondrial creatine kinase interaction with heterogeneous monolayers : effect on lipid lateral organization

International audienceOur study highlights the tight relationship between protein binding to monolayers and the phase-state of the phospholipids. Interaction of mitochondrial creatine kinase with phospholipidic membranes was analysed using a two-phase monolayer system containing anionic phospholipids under chain mismatch conditions. Monolayers were made up of mixtures of DMPC/DPPG or DPPC/DMPG containing 40% negatively charged phospholipids which is approximately the negative charge content of the mitochondrial inner membrane. Langmuir isotherms of these monolayers showed that they underwent a phase transition from a liquid expanded state to a liquid-condensed phase at about 2 mN/m and 5 mN/m respectively. Interface morphology modifications caused by injection of mtCK under these monolayers at low or high surface pressure were monitored by Brewster angle microscopy. This work provides evidence that the presence at the air/water interface of discrete domains with increased charge density, may lead to difference in partition of soluble proteins such as mtCK, interacting with the lipid monolayer. Conversely these proteins may help to organize charged phospholipid domains in a membrane

Reduced growth of Listeria monocytogenes in two model cheese microcosms is not associated with individual microbial strains

Two model antilisterial microbial communities consisting of two yeasts, two Gram positive and two Gram negative bacteria, and originating from Livarot cheese smear were previously designed. They were used in the present study to analyse the impact of microbial population dynamics on growth of Listeria monocytogenes in cheese microcosm. Specific culture media and PCR primers were developed for simultaneous culture-dependent and real-time PCR quantification of strains belonging to Marinomonas sp., Paenibacillus sp., Staphylococcus equorum, Arthrobacter arilaitensis, Pseudomonas putida, Serratia liquefaciens, Candida natalensis, and Geotrichum candidum, in cheese microcosms. All strains were enumerated after 3, 5, 8 and 14 days at 15 C. They established well at high counts in all cheese microcosms. Growth dynamics for all strains in presence of L. monocytogenes WSLC 1685 were compared to those of microbial communities obtained by omitting in turn one of the six members of the initial community. The growth of the microbial strains was neither markedly disturbed by Listeria presence nor by the removal of each strain in turn. Furthermore, these communities had a significant reducing effect on growth of L. monocytogenes independently of pH, as confirmed by mathematical modelling. A barrier effect was observed, that could be explained by specific competition for nutrients

Mitochondrial sub-cellular localization of cAMP-specific phosphodiesterase 8A in ovarian follicular cells

Cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that plays a central role in endocrine tissue function, particularly in the synthesis of steroid hormones. The intracellular concentration of cAMP is regulated through its synthesis by cyclases and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Although the expression and activity of PDEs impact the specifcity and the amplitude of the cAMP response, it is becoming increasingly clear that the subcellular localization of PDE emphasizes the spatial regulation of the cell signalling processes that are essential for normal cellular function. We frst examined the expression of PDE8A in porcine ovarian cells. PDE8A is expressed in granulosa cells, cumulus cells and oocytes. Second, we assessed the mitochondrial sub-cellular localization of PDE8A. Using western blotting with isolated mitochondrial fractions from granulosa cells and cumulus-oocyte complexes revealed immuno-reactive bands. PDE assay of isolated mitochondrial fractions from granulosa cells measured specifc PDE8 cAMP-PDE activity as PF-04957325-sensitive. The immune-reactive PDE8A signal and MitoTracker labelling colocalized supporting mitochondrial sub-cellular localization of PDE8A, which was confrmed using immuno-electron microscopy. Finally, the efect of PDE8 on progesterone production was assessed during the in-vitro maturation of cumulus-oocyte complexes. Using PF-04957325, we observed a signifcant increase (P<0.05) in progesterone secretion with follicle-stimulating hormone (FSH). Active mitochondria stained with MitoTracker orange CMTMRos were also increased by the specifc PDE8 inhibitor supporting its functional regulation. In conclusion, we propose the occurrence of mitochondrial sub-cellular localization of PDE8A in porcine granulosa cells and cumulus cells. This suggests that there is potential for new strategies for ovarian stimulation and artifcial reproductive technologies, as well as the possibility for using new media to improve the quality of oocytes

Mitochondrial creatine kinase adsorption to biomimetic membranes : a Langmuir monolayer study.

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Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese

International audienceThe diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts