1. Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes? ? Since there is no effective vaccine for malaria, and because the causal agent Plasmodium falciparum is resistant to many drugs, there is interest in developing inhibitors that target cysteine and serine proteases. These drugs are known to interfere with the parasitic asexual life cycle by trapping maturing parasites in clusters and diminishing de novo infection of erythrocytes. A deeper understanding of the precise role proteases play in the pathophysiology of this disease is critically needed, since despite decades of research there remains controversy about their function in the malarial erythrocyte cycle, particularly in parasite release. Two recent reports identified two parasite proteases that may mediate the cascade of final cycle events: cysteine protease dipeptidyl peptidase 3 (DPAP3) and subtilisin-family serine protease PfSUB1, but the precise time and place of their action within the release process remains enigmatic. ? ? Pursuing the dual goal of deciphering the mechanism of malaria parasite release from erythrocytes and the role of proteases in this process, and taking into account the extreme fragility of late stage infected erythrocytes we developed a new approach for the differential labeling of the membrane of live infected erythrocytes and a quantitative parasite release assay coupled with the morphological analysis of live infected cells undergoing a cycle transition. ? ? By studying the inactivation of malaria parasite culture by cysteine protease inhibition using confocal microscopy of living cells, and electron microscopy of high-pressure frozen and freeze-substituted cells, we report the precise step in the release of malaria parasites from erythrocytes that is likely regulated by cysteine proteases: the opening of the erythrocyte membrane, liberating parasites for the next round of infection. Inhibition of cysteine proteases within the last few minutes of cycle does not affect rupture of the parasitophorus vacuole but irreversibly blocks the subsequent rupture of the host cell membrane, locking in resident parasites, which die within a few hours of captivity. This irreversible inactivation of mature parasites inside host cells makes plasmodial cysteine proteases attractive targets for anti-malarials, as parasite-specific cysteine protease inhibitors may significantly augment multi-target drug cocktails. ? ? In summary, treating live cells with inhibitors of cysteine protease prevents parasite release from infected erythrocytes at the end of the cycle, probably because cysteine proteases suddenly enter the erythrocyte cytoplasm once the parasitophorus vacuole membrane is disrupted. Once active in the compartment facing the plasma membrane of the erythrocyte, these proteases act to disrupt it. In the presence of protease inhibitor, parasites degrade and die within clusters. It is unlikely that E 64 has a direct toxic effect on parasites. Our finding that clustered parasites locked inside erythrocyte in E64-containing medium have approximately the same life span as released parasites that did not invade red cells in normal medium in vitro is argues against this scenario. Our working hypothesis is that E 64 toxicity is executed through a fatal prolongation of the otherwise normal transient cluster stage, when separated parasites enter erythrocyte cytoplasm as a result of the breaking of the vacuolar membrane. Combined with a short live span of mature parasites, this cluster stage prolongation leads to the irreversible blockage of the plasmodial erythrocyte cycle. The withdrawal of irreversible and reversible cysteine protease inhibitors do not restart parasite release from clusters as previously suggested, but permits schizonts to release parasites at future times. Thus, a scheduling of dosage informed by these studies may be important for testing protease inhibitors as antimalarial drugs. Hopes for antimalarial protease inhibitor drugs are augmented by the finding that relatively low concentrations of a reversible inhibitor, calpeptin, had potent inhibitory effect on parasite release. ? ? 2. Progressive ordering with decreasing temperature of the phospholipids of influenza virus.? ? The influenza virus has played a pivotal role in the development of the raft hypothesis, starting with early studies inferring ordered domains using spin-probes and fluorescence suggesting that an ordered lipid domain is selected in-toto as the envelope during budding from the plasma membrane. These lipids are either selected at the time of budding or pre-selected as the pre-envelope suggested by clusters of the viral envelope protein hemagglutinin (HA) seen in immunoelectron microscopy. ? ? The substantial line broadening of 1H MAS NMR lipid hydrocarbon chain resonances from lipid immobilization in ordered phases (lo or so) enables determination of the fraction of ordered lipids in model and biomembranes. We find that phospholipids in viral membranes form ordered lipid phases over a wide range of physiologically relevant temperatures. We have evidence that both lo- and so phases coexist with the liquid disordered phase. At 37oC lipids with broadened spectra (the sum of lo- and so phases) represent a minute fraction of the membrane, but at 4oC almost the entire membrane is in the ordered state. Since envelope budding occurs at physiological temperature, these data rule out the hypothesis that the influenza envelope is created entirely from an ordered lipid domain. Since the density of envelope protein is near close-packed, a majority of the lipid subtending the envelope protein microdomain of the envelope has liquid disordered properties at physiological temperatures. Thus phase behavior can not simply explain protein clustering during viral assembly at 37oC. ? ? At physiological temperature and higher, there is little physical evidence by MAS NMR for ordered lipid, and our assay for the chief function of the object, fusion to cell membranes, continues unabated without detectable ordered lipid in the host or target membrane. Thus the study of phase properties of complex, biologically relevant lipid mixtures must be done at the appropriate temperatures. The reason that the viral envelope lipid composition is set to be near an apparent lipid phase boundary with respect to temperature is an interesting question. It may be instructive to return to the finding that the lipid composition of the influenza virus is quite different from that of another virus that buds from the same cells, vesicular stomatitis virus (VSV). Both of these viruses are enveloped and both bud from the plasma membrane, albeit from different locations. The difference in location relates to their mode of host-to-host transmission, apical budding for aerial transmission of influenza, and serosal budding for transmission through animal bite for VSV (and the other rhabdoviridea pathogenic genus, rabies). Virus would be at room temperature during aerial transmission but not during transmission by animal bite; the ordered phases we documented here may be important for stability. Indeed, a recent report shows that airborne transmission of influenza virus by guinea pigs is increased at lower temperatures, a result predicted by our proposal that the progressive ordering of lipids we demonstrate here by lower temperatures is important during the low-temperature stages of the influenza life cycle.
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