78 research outputs found
Novel insights into host-fungal pathogen interactions derived from live-cell imaging
Acknowledgments The authors acknowledge funding from the Wellcome Trust (080088, 086827, 075470 and 099215) including a Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377 and FP7-2007–2013 grant agreement HEALTH-F2-2010-260338–ALLFUN to NARG.Peer reviewedPublisher PD
Development of an In Vitro Model for the Multi-Parametric Quantification of the Cellular Interactions between Candida Yeasts and Phagocytes
We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes
Nanoscale Imaging and Mechanical Analysis of Fc Receptor-Mediated Macrophage Phagocytosis against Cancer Cells
Fusogenic Tilted Peptides Induce Nanoscale Holes In Supported Phosphatidylcholine Bilayers
Tilted peptides are known to insert in lipid bilayers with an oblique
orientation, thereby destabilizing membranes and facilitating membrane fusion
processes. Here, we report the first direct visualization of the interaction of
tilted peptides with lipid membranes using in situ atomic force microscopy (AFM)
imaging. Phase-separated supported
dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers
were prepared by fusion of small unilamellar vesicles and imaged in buffer
solution, in the absence and in the presence of the simian immunodeficiency virus
(SIV) peptide. The SIV peptide was shown to induce the rapid appearance of
nanometer scale bilayer holes within the DPPC gel domains, while keeping the
domain shape unaltered. We attribute this behavior to a local weakening and
destabilization of the DPPC domains due to the oblique insertion of the peptide
molecules. These results were directly correlated with the fusogenic activity of
the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By
contrast, the nontilted ApoE peptide did not promote liposome fusion and did not
induce bilayer holes but caused slight erosion of the DPPC domains. In
conclusion, this work provides the first direct evidence for the production of
stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a
behavior that we have shown to be specifically related to the tilted character of
the peptide. A molecular mechanism underlying spontaneous insertion of the SIV
peptide within lipid bilayers and the subsequent removal of bilayer patches is
proposed, and its relevance to membrane fusion processes is discussed
Nanoscale Modification Of Supported Lipid Membranes: Synergetic Effect Of Phospholipase D And Viral Fusion Peptides
peer reviewedUnderstanding the molecular bases of biomembrane fusion events is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various important diseases. In this study, we used atomic force microscopy (AFM) to address the crucial question as to whether negatively curved lipids influence the ability of a viral fusion peptide to perturb the organization of supported lipid bilayers. To this end, an original approach was developed that makes use of an AFM tip functionalized with phospholipase D (PLD) enzymes to generate in situ small amounts of negatively curved phosphatidic acid (PA) in mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers. Real-time AFM imaging revealed that this nanomodification dramatically enhanced subsequent interaction with the simian immunodeficiency virus (SIV) fusion peptide. At short incubation time, the SIV peptide induced a 1.9 nm thickness reduction of the DPPC domains, reflecting either interdigitation or fluidification of the lipids. At longer incubation time, these depressed domains transformed into elevated striated domains, protruding one to several nanometers above the bilayer surface. Two complementary experiments, i.e. addition of the peptide onto DOPC/DPPC/DOPA bilayers or onto DOPC/DPPC bilayers pretreated with a PLD solution, confirmed that both PA and SIV peptides are required to induce depressed and striated domains. Accordingly, this is the first time that a high-resolution imaging technique is used to demonstrate that negatively curved lipids affect the membrane activity of fusion peptides. We believe the nanoscale approach presented here, i.e. use of enzyme-functionalized AFM tips to modify lipid bilayers, will find exciting new applications in nanobiotechnology for the design of biomimetic surfaces
The Siv Tilted Peptide Induces Cylindrical Reverse Micelles In Supported Lipid Bilayers
Elucidation of the molecular mechanism leading to biomembrane fusion is a
challenging issue in current biomedical research in view of its involvement in
controlling cellular functions and in mediating various important diseases.
According to the generally admitted stalk mechanism described for membrane
fusion, negatively curved lipids may play a central role during the early steps
of the process. In this study, we used atomic force microscopy (AFM) to address
the crucial question of whether negatively curved lipids influence the
interaction of the simian immunodeficiency virus (SIV) fusion peptide with model
membranes. To this end,
dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers
containing 0.5 mol % dioleoylphosphatidic acid (DOPA) were incubated with the SIV
peptide and imaged in real time using AFM. After a short incubation time, we
observed a 1.9 nm reduction in the thickness of the DPPC domains, reflecting
either interdigitation or fluidization of lipids. After longer incubation times,
these depressed DPPC domains evolved into elevated domains, composed of nanorod
structures protruding several nanometers above the bilayer surface and attributed
to cylindrical reverse micelles. Such DOPC/DPPC/DOPA bilayer modifications were
never observed with nontilted peptides. Accordingly, this is the first time that
AFM reveals the formation of cylindrical reverse micelles in lipid bilayers
promoted by fusogenic peptides
Comparative study of plant protein extracts as wall materials for the improvement of the oxidative stability of sunflower oil by microencapsulation
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