High susceptibility of rabbit erythrocytes toward the poreforming action of staphylococcal ␣-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve this function for ␣-toxin. Clustering is required so that oligomerization, which is prerequisite for stable attachment of the toxin to the membrane, can efficiently occur. Outside these clusters, binding to phosphocholine is too transient for toxin monomers to find each other. The principle of membrane targeting in the absence of any genuine, high affinity receptor may also underlie the assembly of other lipid-inserted oligomers including cytotoxic peptides, protein toxins, and immune effector molecules.

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A defect in the pksP gene of Aspergillus fumigatus is associated with the loss of conidial pigmentation, a profound change of the conidial surface structure, and reduced virulence. The structural change of the conidial surface structure was not observed in similar A. nidulans wA mutants. Our data indicate that the pigment of both species is important for scavenging reactive oxygen species and for protection of conidia against oxidative damage.Aspergillus spp. are the predominant causative agents of invasive pulmonary aspergillosis (IPA), an often lethal infection of the immunocompromised host (4,10,13,16). Since conidia are the infectious agent in IPA, recent studies focused on the elucidation of conidial factors contributing to pathogenicity (8,17). Previously, we and others have shown that conidia lacking pigmentation due to the defective polyketide synthase gene pksP were less resistant to the attack by monocytes in vitro and showed reduced virulence in a murine animal model (8,9,17,18). During these studies, it became apparent that coincubation of human phagocytes with pksP mutant conidia resulted in a marked increase in the release of reactive oxygen species (ROS) compared with wild-type (wt) conidia (8). Since a defective pksP gene not only impaired conidial pigmentation but concomitantly resulted in profound alterations of the conidial surface (8, 9), the question arose as to whether the large amounts of ROS detected after incubation of phagocytes with pksP mutant conidia were due to a change in the activation pattern of the cells or, alternatively, reflected the lack of ROS quenching capacity caused by the loss of conidial pigment. To address this question, conidia of wt strains of both Aspergillus fumigatus and the nonpathogenic fungus Aspergillus nidulans were compared with their respective pigmentless mutant strains.The WA mutant of A. nidulans (strain WG370; wA3 bgaO biA1) lacking the conidial pigment due to a defective polyketide synthase gene (wA) was constructed by a sexual cross of appropriate parental strains (12) using standard genetic techniques (14). The wA gene product might have a function similar to that of the pksP gene product of A. fumigatus, although major differences between the pigment biosyntheses of the two Aspergillus species exist (2, 18; this study).As previously reported, conidia of the A. fumigatus wt strain showed a rough surface; i.e., they had an ornamentation which was lacking in the pksP mutant strain (8, 9) ( Fig. 1A and B). The A. nidulans wt conidia showed a similar ornamentation (Fig. 1C). In contrast to the pigmentless pksP mutant strain of A. fumigatus, however, similar pigmentless conidia of A. nidulans (wA) still exhibited the ornamentation characteristic of wild-type conidia (Fig. 1D). Taken together, the difference in surface structure between the A. fumigatus pksP mutant and the A. nidulans wA mutant further supports the assumption that different pathways exist for either conidial pigment biosynthesis or pigment deposition in the two species (1). As was noted p...

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

Antiviral activity has been demonstrated for different tannin-rich plant extracts. Since tannins of different classes and molecular weights are often found together in plant extracts and may differ in their antiviral activity, we have compared the effect against influenza A virus (IAV) of Hamamelis virginiana L. bark extract, fractions enriched in tannins of different molecular weights and individual tannins of defined structures, including pseudotannins. We demonstrate antiviral activity of the bark extract against different IAV strains, including the recently emerged H7N9, and show for the first time that a tannin-rich extract inhibits human papillomavirus (HPV) type 16 infection. As the best performing antiviral candidate, we identified a highly potent fraction against both IAV and HPV, enriched in high molecular weight condensed tannins by ultrafiltration, a simple, reproducible and easily upscalable method. This ultrafiltration concentrate and the bark extract inhibited early and, to a minor extent, later steps in the IAV life cycle and tannin-dependently inhibited HPV attachment. We observed interesting mechanistic differences between tannin structures: High molecular weight tannin containing extracts and tannic acid (1702 g/mol) inhibited both IAV receptor binding and neuraminidase activity. In contrast, low molecular weight compounds (<500 g/mol) such as gallic acid, epigallocatechin gallate or hamamelitannin inhibited neuraminidase but not hemagglutination. Average molecular weight of the compounds seemed to positively correlate with receptor binding (but not neuraminidase) inhibition. In general, neuraminidase inhibition seemed to contribute little to the antiviral activity. Importantly, antiviral use of the ultrafiltration fraction enriched in high molecular weight condensed tannins and, to a lesser extent, the unfractionated bark extract was preferable over individual isolated compounds. These results are of interest for developing and improving plant-based antivirals.