Membrane Remodeling During Viral Infection, Parasite Inv

Child Health And Human Development

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Abstract

This project is aimed at the understanding of the physico-chemical mechanisms of membrane remodeling during physiological and pathogenic events. During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. During apoptosis, Bax-type proteins cause permeabilization of the outer mitochondrial membrane to release mitochondrial apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and Delta-N76Bcl-xL (the Bax-like cleavage fragment of Bcl-xL) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or Delta-N76Bcl-xL. Altogether, these results are consistent with (but do not prove) a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids, in a structure of net positive monolayer curvature. The traditional method of culturing Plasmodium falciparum has been a huge success. However, plasmodia replicate poorly in erythrocyte densities greater than a hematocrit of 20%. A new method to culture the major malaria parasite was developed utilizing a hollow fiber bioreactor that preserves healthy erythrocytes at hematocrit up to 100%. Plasmodium falciparum replicated equally well at all densities studied. This method proved advantageous for large-scale preparation of parasitized erythrocytes (and potentially immunogens thereof) since high yields (~10 logs in four days, ~11 logs in a week) could be prepared with less cost and labor. Concomitantly, secreted proteins are concentrated by molecular sieving during culture, perhaps contributing to the parasitemic limit of 8-12% with 3D7 strain. Our finding that Plasmodium falciparum can replicate at packed densities suggests that this system may be useful for studying the pathogenesis of the fatal disease cerebral malaria, that features densely packed blood cells in brain microvasculature. All enveloped viruses inject their genome into the cytoplasm via fusion of their envelope membrane with the host one. This fusion in many important cases, such as influenza virus infection, follows endocytic uptake of a virus particle into a cellular vesicle which then transports the viral particle into the cell. The vesicle microstructure, e.g. its size, shapes and membrane composition, affects viral fusion efficiency. Thus our experimental strategy is to approach both the vesicle formation and transport and the fusion as sequential stages of a single process. For that purpose several experimental systems have been developed. First, the formation of individual cellular vesicles and the pinching-off of such a vesicle into the cellular interior was detected using high-resolution admittance techniques in combination with low light level fluorescent microscopy. We resolved the evolution - from initial budding to pinching-off, of single vesicles capable of carrying individual influenza virus particles. Determination of the lipid composition of such vesicles and the detection of the transport of actual virus particles in the vesicles are in progress. Second, a special experimental system was developed to resolve individual fusion events following viral particle uptake. This system allowed us to monitor the fusion of a single virus particle with a model membrane (having a controlled composition). Experiments conducted in this system revealed that acidification of the virus interior (normally going through the M2 channel in the viral membrane) is crucial for the fusion pore expansion and thus for the RNA release and ultimate infection. Briefly, the antivirus drugs amantadines and remantadime, known inhibitors of the M2 channel, do not affect fusion pore formation by the viral fusion protein hemagglutinin. But these drugs prevented fusion pore expansion. Thus another pH-sensitive viral protein besides the classical fusion protein hemagglutinin is involved in the completion of fusion.