A nonpermeant biotin derivative gains access to the parasitophorous vacuole in Plasmodium falciparum-infected erythrocytes permeabilized with streptolysin O

In its host erythrocyte, the malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole, the membrane of which forms a barrier between the host cell cytosol and the parasite surface. The vacuole is a unique compartment because it contains specific proteins that are believed to be involved in cell biological functions essential for parasite survival. As a prerequisite for the characterization of the vacuolar proteome, we have developed an experimental approach that allows the selective biotinylation of soluble vacuolar proteins. This approach utilizes nonpermeant biotin derivatives that can be introduced into infected erythrocytes after selective permeabilization of the erythrocyte membrane with the pore-forming protein streptolysin O. The derivatives gain access to the vacuolar lumen but not to the parasite cytosol, thus providing supportive evidence for the existence of nonselective pores within the vacuolar membrane that have been postulated based on electrophysiological studies. Soluble vacuolar proteins that are biotin-labeled can be isolated by affinity chromatography using streptavidin-agarose.     

A NEW BASIDIOMYCETOUS SEPTAL TYPE: THE MULTIPERFORATE SEPTUM IN KRIEGERIA ERIOPHORI

Septa from metabasidia and clamp connections of the heterobasidiomycetous sedge parasite Kriegeria eriophori were studied with light and electron microscopy. Septa from clamp connections subtending probasidia were reconstructed from serial sections. Septa at clamp connections are perforated by multiple, simple pores, while metabasidial septa possess single, central swellings which probably represent pores occluded by wall material. The occurrence of multiperforate septa in the fungi is reviewed. The septal morphology of K. eriophori is compared to that of simple-septate heterobasidiomycetes, and the systematic and functional implications are discussed.     

Symplasmic Sieve Element Continuity between Orobanche and its Host*

Electron microscopical investigations of primary haustoria of Orobanche crenata parasitizing the roots of the highly compatible host, Vicia narbonensis, reveal an uninterrupted phloem system connecting both partners. Individual sieve elements belonging to the host and parasite could be identified by the cell markers: plastids, mitochondria and P-protein, which in the present system turned out to have species-specific fine structure. Sieve pores of normal structure interconnect the sieve elements of host and parasite. They originate from interspecific plasmodesmata.     

Electroporation in dense cell suspension--theoretical and experimental analysis of ion diffusion and cell permeabilization

Electroporation is a process where increased permeability of cells exposed to an electric field is observed. It is used in many biomedical applications including electrogene transfection and electrochemotherapy. Although the increased permeability of the membrane is believed to be the result of pores due to an induced transmembrane voltage U(m), the exact molecular mechanisms are not fully explained. In this study we analyze transient conductivity changes during the electric pulses and increased membrane permeability for ions and molecules after the pulses in order to determine which parameters affect stabilization of pores, and to analyze the relation between transient pores and long-lived transport pores. By quantifying ion diffusion, fraction of transport pores f(per) was obtained. A simple model, which assumes a quadratic dependence of f(per) on E in the area where U(m)>U(c) very accurately describes experimental values, suggesting that f(per) increases with higher electric field due to larger permeabilized area and due to higher energy available for pore formation. The fraction of transport pores increases also with the number of pulses N, which suggest that each pulse contributes to formation of more and/or larger stable transport pores, whereas the number of transient pores does not depend on N.     

Therapeutic Efficacy of Stable Analogues of Vasoactive Intestinal Peptide against Pathogens

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide recently identified as a potential antimicrobial peptide. To overcome the metabolic limitations of VIP, we modified the native peptide sequence and generated two stable synthetic analogues (VIP51 and VIP51(6–30)) with better antimicrobial profiles. Herein we investigate the effects of both VIP analogues on cell viability, membrane integrity, and ultrastructure of various bacterial strains and Leishmania species. We found that the two VIP derivatives kill various non-pathogenic and pathogenic Gram-positive and Gram-negative bacteria as well as the parasite Leishmania major through a mechanism that depends on the interaction with certain components of the microbial surface, the formation of pores, and the disruption of the surface membrane. The cytotoxicity of the VIP derivatives is specific for pathogens, because they do not affect the viability of mammalian cells. Docking simulations indicate that the chemical changes made in the analogues are critical to increase their antimicrobial activities. Consequently, we found that the native VIP is less potent as an antibacterial and fails as a leishmanicidal. Noteworthy from a therapeutic point of view is that treatment with both derivatives increases the survival and reduces bacterial load and inflammation in mice with polymicrobial sepsis. Moreover, treatment with VIP51(6–30) is very effective at reducing lesion size and parasite burden in a model of cutaneous leishmaniasis. These results indicate that the VIP analogues emerge as attractive alternatives for treating drug-resistant infectious diseases and provide key insights into a rational design of novel agents against these pathogens.     

Optimal killing for obligate killers: the evolution of life histories and virulence of semelparous parasites.

Many viral, bacterial and protozoan parasites of invertebrates first propagate inside their host without releasing any transmission stages and then kill their host to release all transmission stages at once. Life history and the evolution of virulence of these obligately killing parasites are modelled, assuming that within-host growth is density dependent. We find that the parasite should kill the host when its per capita growth rate falls to the level of the host mortality rate. The parasite should kill its host later when the carrying capacity, K, is higher, but should kill it earlier when the parasite-independent host mortality increases or when the parasite has a higher birth rate. When K(t), for parasite growth, is not constant over the duration of an infection, but increases with time, the parasite should kill the host around the stage when the growth rate of the carrying capacity decelerates strongly. In case that K(t) relates to host body size, this deceleration in growth is around host maturation.     

The curse of the pharaoh hypothesis.

The ''curse of the pharaoh'' has been used as a metaphor for the hypothesis that higher parasite propagule survival selects for higher virulence. Indeed, the mysterious death of Lord Carnavon after entering the tomb of the Egyptian pharaoh Tutankhamen could potentially be explained by an infection with a highly virulent and very long-lived pathogen. In this paper, I investigate whether parasite virulence increases with high propagule survival. In this respect, I derive an analytic expression of the evolutionarily stable level of parasite virulence as a function of propagule survival rate when the host-parasite system has reached a stable ecological equilibrium. This result shows that, if multiple infection occurs, higher propagule survival generally increases parasite virulence. This effect is enhanced when parasite dispersal coevolves with parasite virulence. In a more general perspective, the model shows the importance of taking into account the combination of direct and indirect effects (which I call inclusive effects) of higher transmission ability on the evolution of parasite virulence. The recognition of these effects has several practical implications for virulence management.     

How can immunopathology shape the evolution of parasite virulence?

Immunopathology (immune-mediated pathology) is a ubiquitous cause of disease during infection, but how will parasite exploitation strategies evolve in its presence? Immunopathology can act to increase parasite fitness if it increases transmission rate, but can equally act to decrease parasite fitness if it increases host mortality. The focus here is on understanding how immunopathology, mediated through different immune mechanisms, can influence parasite fitness and how experimental manipulations of the immune system can be carried out to examine this. A better understanding of how parasite fitness scales with, or responds to, immunopathology is crucial to understanding the nature of selection acting on parasite virulence traits and will allow more informed predictions to be made regarding the trajectory of parasite virulence evolution.     

Rapid change in parasite infection traits over the course of an epidemic in a wild host–parasite population

By combining a field study with controlled laboratory experimentation, we examined how infection traits of the sterilizing bacterium, Pasteuria ramosa, changed over the course of a growing season in a natural population of its crustacean host Daphnia magna. The number of parasite transmission spores per infected host increased ten‐fold over the course of the season, concomitant with a decline in the density of infected hosts. Plausible explanations for this variation include changes in environmental conditions, changes in host quality, or that parasite migration or natural selection caused a genetic change in the parasite population. We sought to distinguish some of these possibilities in a laboratory experiment. Thus, we preserved field‐collected parasite spores throughout the season, and later exposed a set of hosts to a fixed dose of these spores under controlled laboratory conditions. Parasites collected late in the season were more infectious and grew more rapidly than parasites collected early in the season. This result is compatible with the hypothesis that the observed increase in infectivity in the field was due to genetic change, i.e. evolution in the P. ramosa population.     

Warmer temperatures reduce the vectorial capacity of malaria mosquitoes

The development rate of parasites and pathogens within vectors typically increases with temperature. Accordingly, transmission intensity is generally assumed to be higher under warmer conditions. However, development is only one component of parasite/pathogen life history and there has been little research exploring the temperature sensitivity of other traits that contribute to transmission intensity. Here, using a rodent malaria, we show that vector competence (the maximum proportion of infectious mosquitoes, which implicitly includes parasite survival across the incubation period) tails off at higher temperatures, even though parasite development rate increases. We also show that the standard measure of the parasite incubation period (i.e. time until the first mosquitoes within a cohort become infectious following an infected blood-meal) is incomplete because parasite development follows a cumulative distribution, which itself varies with temperature. Including these effects in a simple model dramatically alters estimates of transmission intensity and reduces the optimum temperature for transmission. These results highlight the need to understand the interactive effects of environmental temperature on multiple host-disease life-history traits and challenge the assumptions of many current disease models that ignore this complexity.     

Pore-forming properties of iturin A, a lipopeptide antibiotic

The addition of iturin A, a lipopeptide antibiotic extracted from Bacillus subtilis, to a bimolecular lipid membrane (BLM) increases dramatically its electrical conductance. For very low concentration of iturin A, discrete conductance steps are observed which are assigned to the formation of conducting pores. The characteristics of these pores depend on the lipid content of the BLM and they change with time. Cholesterol considerably increases the lifetimes of open states. The pores are slightly anion versus cation selective. These first observations unable us to briefly discuss the pore-forming properties of lipopeptides.     

Cellular interactions of Plasmodium liver stage with its host mammalian cell

The Plasmodium liver forms are bridgehead stages between the mosquito sporozoite stages and mammalian blood stages that instigate the malaria disease. In hepatocytes, Plasmodium achieves one of the fastest growth rates among eukaryotic cells. However, nothing is known about host hepatic cell interactions, e.g. nutrient scavenging and/or subversion of cellular functions necessary for Plasmodium development and replication. Plasmodium usually invades hepatocytes by establishing a parasitophorous vacuole wherein it undergoes multiple nuclear division cycles. We show that Plasmodium preferentially develops in the host juxtanuclear region. By comparison with the parasitophorous vacuole of other apicomplexan parasites which associate with diverse host organelles, the Plasmodium parasitophorous vacuole only forms an association with the host endoplasmic reticulum. Intrahepatic Plasmodium actively modifies the permeability of its vacuole to allow the transfer of a large variety of molecules from the host cytosol to the vacuolar space through open channels. In contrast with malaria blood stages, the pores within the parasitophorous vacuole membrane of the liver stage display a smaller size as they restrict the passage of solutes to less than 855Da. These pores are stably maintained during parasite karyokinesis until complete cellularisation. Host-derived cholesterol accumulated at the parasitophorous vacuole membrane may modulate the channel activity. These observations define the parasitophorous vacuole of the Plasmodium liver stage as a dynamic and highly permeable compartment that can ensure the sustained supply of host molecules to support parasite growth in the nutrient-rich environment of liver cells.     

The influence of red cell mechanical properties on flow through single capillary-sized pores

The resistive pulse technique was used to study the influence of specific mechanical properties of the red cell on its ability to enter and flow through single capillary-sized pores with diameters of 3.6, 5.0 and 6.3 micron and lengths of 11 micron. A two-fold increase in membrane shear elasticity resulted in a 40 percent increase in the cell's transit time through a 3.6 micron pore but produced no change in transit time through a 6.3 micron pore. A two-fold increase in membrane shear viscosity produced a 40 percent increase in transit time through the 3.6 micron pore and small but significant increases in transit times through the larger pores. Osmotically dehydrated cells showed no increase in transit time through a 6.3 micron pore, but showed increases in transit times of 50 to 70 percent through 5.0 and 3.6 micron pores. Dense red cells showed increased transit times through both 5.0 micron and 6.0 micron pores. These results indicate that for cells with normal geometric properties, the membrane's shear viscosity and elasticity only influence the cell's transit through pores of 5 micron or less in diameter. However, alterations in the cell's geometric properties can extend the influence of membrane shear properties to larger diameter pores.     

The impact of host genetic diversity on virus evolution and emergence

Accumulating evidence indicates that biodiversity has an important impact on parasite evolution and emergence. The vast majority of studies in this area have only considered the diversity of species within an environment as an overall measure of biodiversity, overlooking the role of genetic diversity within a particular host species. Although theoretical models propose that host genetic diversity in part shapes that of the infecting parasite population, and hence modulates the risk of parasite emergence, this effect has seldom been tested empirically. Using Rabies virus (RABV) as a model parasite, we provide evidence that greater host genetic diversity increases both parasite genetic diversity and the likelihood of a host being a donor in RABV cross‐species transmission events. We conclude that host genetic diversity may be an important determinant of parasite evolution and emergence.     

Aggregation and species coexistence of ectoparasites of marine fishes.

Interspecific interaction may lead to species exclusion but there are several ways in which species can coexist. One way is by reducing the overall intensity of competition via aggregated utilisation of fragmented resources. Known as the 'aggregation model of coexistence', this system assumes saturation and an equilibrium number of species per community. In this study we tested the effects of interspecific aggregation on the level of intraspecific aggregation among ectoparasites of marine fishes (36 communities of gill and head ectoparasite species). If parasite species are distributed in a way that interspecific aggregation is reduced relative to intraspecific aggregation then species coexistence is facilitated. We found a positive relationship between parasite species richness and fish body size, controlling for host phylogeny. A positive relationship between infracommunity species richness and total parasite species richness was also found, providing no evidence for saturation. This result supports the view that infracommunities of parasites are not saturated by local parasite residents. The observed lack of saturation implies that we are far from a full exploitation of the fish resource by parasites. Ectoparasites were aggregated at both population and species levels. However, only half of the ectoparasite communities were dominated by negative interspecific aggregation. We found that infracommunity parasite species richness was positively correlated with the level of intraspecific aggregation versus interspecific aggregation. This means that intraspecific aggregation increases compared with interspecific aggregation when total parasite species richness increases, controlling fish size and phylogeny. This supports one assumption of the 'aggregation model of coexistence', which predicts that interspecific interactions are reduced relative to intraspecific interactions, facilitating species coexistence.     

Oligomerization of membrane-bound Bcl-2 is involved in its pore formation induced by tBid

Both pro-apoptotic Bax and anti-apoptotic Bcl-2 are structurally homologous to the pore-forming domain of bacterial toxins. Bax proteins oligomerize in the mitochondrial outer membranes forming pores that release cytochrome c from the mitochondrial intermembrane space. Bcl-2 proteins also form pores that, however, are much smaller than the Bax pore. It is unknown whether Bcl-2 forms monomeric or oligomeric pores. Here, we characterized the Bcl-2 pore formation in liposomes using biophysical and biochemical techniques. The results show that the Bcl-2 pore enlarges as the concentration of Bcl-2 increases, suggesting that the pore is formed by Bcl-2 oligomers. As expected from oligomerization-mediated pore-formation, the small pores are formed earlier than the large ones. Bcl-2 oligomers form pores faster than the monomer, indicating that the oligomerization constitutes an intermediate step of the pore formation. A Bcl-2 mutant with higher affinity for oligomerization forms pores faster than wild type Bcl-2. Bcl-2 oligomers were detected in the liposomal membranes under conditions that Bcl-2 forms pores, and the extent of oligomerization was positively correlated with the pore-forming activity. Therefore, Bcl-2 oligomerizes in membranes forming pores, but the extent of oligomerization and the size of the resulting pores are much smaller than that of Bax, supporting the model that Bcl-2 is a defective Bax.     

Immunity,antigenic heterogeneity,and aggregation of helminth parasites

Empirical studies of helminth parasites reveal that the distribution of parasite burdens in their host populations is highly aggregated. This aggregation is fundamental to the ecology and epidemiology of helminth parasites. Results from a stochastic model predict that aggregation of helminth parasites is inversely related to the intensity of host immunity. Aggregation also decreases with antigenic heterogeneity and increases with heterogeneity in transmissibility among parasite strains. It is also found that the degree of aggregation is greater when immunity affects parasite fecundity than when immunity acts on host susceptibility. Potential relevance of this result for assessing the influence of vaccines that target either host susceptibility or parasite fecundity on the level of aggregation and consequent effects on drug resistance and disease prevalence are discussed.     

Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.     

Assembly of macromolecular pores by immune defense systems.

Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability.