Pneumococci traverse eukaryotic cells within vacuoles without intracytoplasmic multiplication. The plateletactivating factor receptor (PAFr) has been suggested as a portal of entry. Pneumococci colocalized with PAFr on endothelial cells and PAFr ؊/؊ mice showed a substantially impaired ability to support bacterial translocation, particularly from blood to brain. Pneumococci-induced colocalization of PAFr and ␤-arrestin 1 at the plasma membrane of endothelial cells and PAFr-mediated pneumococcal uptake in transfected COS cells were greatly increased by cotransfection with the scaffold/adapter protein ␤-arrestin 1. Activation of extracellular signal-regulated kinase kinases was required for uptake and was limited to the cytoplasmic compartment, consistent with activation by ␤-arrestin rather than PAFr. Uptake of the pneumococcal vacuole involved clathrin, and half the bacteria proceeded into vacuoles marked by Rab5 and later Rab7, the classical route to the lysosome. Overexpression of ␤-arrestin in endothelial cells decreased colocalization with Rab7. We conclude that the association of ␤-arrestin with the PAFr contributes to successful translocation of pneumococci.

The current model of innate immune recognition of Gram-positive bacteria suggests that the bacterial cell wall interacts with host recognition proteins such as TLRs and Nod proteins. We describe an additional recognition system mediated by the platelet-activating factor receptor (PAFr) and directed to the pathogen-associated molecular pattern phosphorylcholine that results in the uptake of bacterial components into host cells. Intravascular choline-containing cell walls bound to endothelial cells and caused rapid lethality in wild-type, Tlr2−/−, and Nod2−/− mice but not in Pafr−/− mice. The cell wall exited the vasculature into the heart and brain, accumulating within endothelial cells, cardiomyocytes, and neurons in a PAFr-dependent way. Physiological consequences of the cell wall/PAFr interaction were cell specific, being noninflammatory in endothelial cells and neurons but causing a rapid loss of cardiomyocyte contractility that contributed to death. Thus, PAFr shepherds phosphorylcholine-containing bacterial components such as the cell wall into host cells from where the response ranges from quiescence to severe pathophysiology.

Sendai virus nucleocapsids have been observed by electron microscopy to coexist in three different helical pitch conformations, 5.3, 6.8, and 37.5 nm. The 5.3and 6.8-nm conformations are present both in uranyl acetate negatively stained preparations and in tantalum-tungsten metal-shadowed preparations, whereas the 37.5-nm conformation, which has not been previously reported, is present only in the shadowed preparations. The 5.3-nm pitch conformation appears to be a mixture of two discrete structural states, with a small difference in the twist of the structure between the two. We have used image reconstruction techniques on an averaged data set from eight negatively stained nucleocapsids to produce a three-dimensional reconstruction at 2.4-nm resolution of the structure in one of the 5.3-nm pitch states. There are 13.07 nucleocapsid protein (NP) subunits in each turn of the helix in this state. The helical repeat is 79.5 nm, containing 196 subunits in 15 turns of the left-handed 5.3-nm helix. The arrangement of subunits produces a 5.0-nm-diameter hollow core which forms an internal helical groove. The RNA accounts for about 3% of the mass of the nucleocapsid, and so its location is not conspicuous in the reconstruction. Because the RNA remains associated with the NP subunits during mRNA transcription and genome replication, structural transitions in the nucleocapsid may determine the accessibility of the genome to polymerases. Alternatively, the large hollow core and internal helical groove we have reconstructed may allow access to the RNA even in the tightly coiled 5.3-nm pitch conformation.

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While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.