Membrane fluidity determines sensitivity of filamentous fungi to chitosan.

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan-resistant but not chitosan-sensitive fungi. The plasma membranes of chitosan-sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan-resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady-state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.

Hepatitis C virus core protein binding to lipid membranes: the role of domains 1 and 2.

We have analysed and identified different membrane-active regions of the Hepatitis C virus (HCV) core protein by observing the effect of 18-mer core-derived peptide libraries from two HCV strains on the integrity of different membrane model systems. In addition, we have studied the secondary structure of specific membrane-interacting peptides from the HCV core protein, both in aqueous solution and in the presence of model membrane systems. Our results show that the HCV core protein region comprising the C-terminus of domain 1 and the N-terminus of domain 2 seems to be the most active in membrane interaction, although a role in protein-protein interaction cannot be excluded. Significantly, the secondary structure of nearly all the assayed peptides changes in the presence of model membranes. These sequences most probably play a relevant part in the biological action of HCV in lipid interaction. Furthermore, these membranotropic regions could be envisaged as new possible targets, as inhibition of its interaction with the membrane could potentially lead to new vaccine strategies.

Monitoring dynamics of human adenovirus disassembly induced by mechanical fatigue.

The standard pathway for virus infection of eukaryotic cells requires disassembly of the viral shell to facilitate release of the viral genome into the host cell. Here we use mechanical fatigue, well below rupture strength, to induce stepwise disruption of individual human adenovirus particles under physiological conditions, and simultaneously monitor disassembly in real time. Our data show the sequence of dismantling events in individual mature (infectious) and immature (noninfectious) virions, starting with consecutive release of vertex structures followed by capsid cracking and core exposure. Further, our experiments demonstrate that vertex resilience depends inextricably on maturation, and establish the relevance of penton vacancies as seeding loci for virus shell disruption. The mechanical fatigue disruption route recapitulates the adenovirus disassembly pathway in vivo, as well as the stability differences between mature and immature virions.

Minimizing tip-sample forces in jumping mode atomic force microscopy in liquid.

Control and minimization of tip-sample interaction forces are imperative tasks to maximize the performance of atomic force microscopy. In particular, when imaging soft biological matter in liquids, the cantilever dragging force prevents identification of the tip-sample mechanical contact, resulting in deleterious interaction with the specimen. In this work we present an improved jumping mode procedure that allows detecting the tip-sample contact with high accuracy, thus minimizing the scanning forces (-100 pN) during the approach cycles. To illustrate this method we report images of human adenovirus and T7 bacteriophage particles which are prone to uncontrolled modifications when using conventional jumping mode.