Disentangling the influence of parasite genotype, host genotype and maternal environment on different stages of bacterial infection in Daphnia magna

Individuals naturally vary in the severity of infectious disease when exposed to a parasite. Dissecting this variation into genetic and environmental components can reveal whether or not this variation depends on the host genotype, parasite genotype or a range of environmental conditions. Complicating this task, however, is that the symptoms of disease result from the combined effect of a series of events, from the initial encounter between a host and parasite, through to the activation of the host immune system and the exploitation of host resources. Here, we use the crustacean Daphnia magna and its parasite Pasteuria ramosa to show how disentangling genetic and environmental factors at different stages of infection improves our understanding of the processes shaping infectious disease. Using compatible host-parasite combinations, we experimentally exclude variation in the ability of a parasite to penetrate the host, from measures of parasite clearance, the reduction in host fecundity and the proliferation of the parasite. We show how parasite resistance consists of two components that vary in environmental sensitivity, how the maternal environment influences all measured aspects of the within-host infection process and how host-parasite interactions following the penetration of the parasite into the host have a distinct temporal component.     

Sorting of malarial antigens into vesicular compartments within the host cell cytoplasm as demonstrated by immunoelectron microscopy

A double and triple immunogold labeling technique has been applied to demonstrate that several malarial antigens of the erythrocytic stages of Plasmodium falciparum are exported from the parasite into distinct compartments within the host cell cytoplasm. Multiple species of vesicles, each with specifically packaged contents, are consistent with a sorting function of vesicular structures in the Plasmodium infected erythrocyte. During schizogony, two parasite antigens, an S-antigen and a parasitophorous vacuole membrane antigen, QF 116, become packaged into such vesicles and are transported into the erythrocyte cytoplasm. At this stage of parasite development, host cell material is taken in through the parasitophorous vacuole membrane into the vacuolar space surrounding the parasite.     

Habitat heterogeneity drives the host‐diversity‐begets‐parasite‐diversity relationship: evidence from experimental and field studies

Despite a century of research into the factors that generate and maintain biodiversity, we know remarkably little about the drivers of parasite diversity. To identify the mechanisms governing parasite diversity, we combined surveys of 8100 amphibian hosts with an outdoor experiment that tested theory developed for free‐living species. Our analyses revealed that parasite diversity increased consistently with host diversity due to habitat (i.e. host) heterogeneity, with secondary contributions from parasite colonisation and host abundance. Results of the experiment, in which host diversity was manipulated while parasite colonisation and host abundance were fixed, further reinforced this conclusion. Finally, the coefficient of host diversity on parasite diversity increased with spatial grain, which was driven by differences in their species–area curves: while host richness quickly saturated, parasite richness continued to increase with neighbourhood size. These results offer mechanistic insights into drivers of parasite diversity and provide a hierarchical framework for multi‐scale disease research.     

Host patch traits have scale-dependent effects on diversity in a stickleback parasite metacommunity

Many metacommunities are distributed across habitat patches that are themselves aggregated into groups. Perhaps the clearest example of this nested metacommunity structure comes from multi-species parasite assemblages, which occupy individual hosts that are aggregated into host populations. At both spatial scales, we expect parasite community diversity in a given patch (either individual host or population) to depend on patch characteristics that affect colonization rates and species sorting. But, are these patch effects consistent across spatial scales? Or, do different processes govern the distribution of parasite community diversity among individual hosts, versus among host patches? To answer these questions, we document the distribution of parasite richness among host individuals and among populations in a metapopulation of threespine stickleback Gasterosteus aculeatus. We find some host traits (host size, gape width) are associated with increased parasite richness at both spatial scales. Other patch characteristics affect parasite richness only among individuals (sex), or among populations (lake size, lake area, elevation and population mean heterozygosity). These results demonstrate that some rules governing parasite richness in this metacommunity are shared across scales, while others are scale-specific.     

An alternative model for heme biosynthesis in the malarial parasite

The malarial parasite imports an infected host's red blood cell enzymes for heme biosynthesis during the intraerythrocytic stage. This is despite all the genes of the heme-biosynthetic pathway having been identified on the parasite genome. On the basis of predictions of parasite genome-coded enzyme localization, functionality of some of these enzymes and shuttling of intermediates between different parasite compartments, a hybrid model for parasite heme biosynthesis has been proposed. However, this model does not take into account the possible role of imported host enzymes in parasite heme biosynthesis. We propose an alternative model with an extrinsic heme-biosynthetic pathway in the parasite cytosol that uses imported host enzymes, and an intrinsic pathway confined to the organellar fractions that uses the parasite-genome-encoded enzymes.     

Fate of Parasite and Host Organelle DNA during Cellular Transformation of Red Algae by Their Parasites

The transfer of a nucleus into a cytoplasm of a genetically foreign cell and its subsequent multiplication in the cytoplasm of this cell characterize most parasitic red algal species and their interactions with specific red algal hosts. Nuclei enter the host's cytoplasm upon cell fusion of parasite and host cell; here, they replicate, are spread to contiguous host cells, and ultimately are packaged into spores that reinfect other host thalli. In this study, we examined whether the proplastids and mitochondria that occur in these red algal adelphoparasites are acquired from their host or whether they are unique to the parasite and are brought into the host along with the parasite nucleus. To establish their origins and fates, plastid and mitochondrial restriction fragment length polymorphisms (RFLPs) of parasite cells were compared with those of their host plastid and mitochondrial DNA in three host and parasite pairs. For plastids, no RFLP differences were found between hosts and parasites, supporting an earlier conclusion, based on microscopic studies, that the proplastids of parasites are acquired from their hosts. For mitochondria, characteristic RFLP differences were detected between host and parasite for two of the pairs of species but not for the third. Evidence of the evolutionary difference between hosts and their parasites was shown by RFLP differences between nuclear ribosomal repeat regions.     

Recent advances in DNA-mediated gene transfer of Entamoeba histolytica

During the past few years, the introduction of DNA-mediated gene transfer into parasite research has permitted subtle studies on fundamental aspects of parasite biology. In this paper, Egbert Tannich describes the recent breakthrough of successful Entamoeba histolytica transfection, and the subsequent developments in this field.     

Pexel/VTS: a protein-export motif in erythrocytes infected with malaria parasites

The trafficking of proteins from the malaria parasite into the erythrocyte host has an important role in both the adaptation by the parasite of its immediate environment and the pathophysiology of disease. The molecular basis of these trafficking processes, particularly export from the parasitophorous vacuole (PV), is poorly understood and a matter of some controversy. In this article, we highlight two recent, independent reports that have provided new insights into protein translocation across the PV membrane, characterizing a novel signalling motif as a key signature in cataloguing the parasite secretome.     

Plasmodium's sticky fingers

The life cycle of the malaria parasite (Plasmodium) is remarkably complex. Malaria parasites must engage in highly specific and varied interactions with cell types of both the mammalian host and the mosquito vector. In this issue of Cell, report detailed molecular insights into an intimate interaction between a malaria parasite protein and its host cell receptor that enables the parasite to invade erythrocytes.     

Parasites do not adapt to elevated temperature,as evidenced from experimental evolution of a phytoplankton–fungus system

Global warming is predicted to impact the prevalence and severity of infectious diseases. However, empirical data supporting this statement usually stem from experiments in which parasite fitness and disease outcome are measured directly after temperature increase. This might exclude the possibility of parasite adaptation. To incorporate the adaptive response of parasites into predictions of disease severity in a warmer world, we undertook an experimental evolution assay in which a fungal parasite of phytoplankton was maintained at elevated or control temperatures for six months, corresponding to 100–200 parasite generations. Host cultures were maintained at the respective temperatures and provided as substrate, but were not under parasite pressure. A reciprocal infection experiment conducted after six-month serial passages revealed no evidence of parasite adaptation. In fact, parasite fitness at elevated temperatures was inferior in parasite populations reared at elevated temperatures compared with those maintained under control temperature. However, this effect was reversed after parasites were returned to control temperatures for a few (approx. 10) generations. The absence of parasite adaptation to elevated temperatures suggests that, in phytoplankton–fungus systems, disease outcome under global warming will be largely determined by both host and parasite thermal ecology.     

The role of moulting in parasite defence

Parasitic infections consist of a succession of steps during which hosts and parasites interact in specific manners. At each step, hosts can use diverse defence mechanisms to counteract the parasite's attempts to invade and exploit them. Of these steps, the penetration of parasites into the host is a key step for a successful infection and the epithelium is the first line of host defence. The shedding of this protective layer (moulting) is a crucial feature in the life cycle of several invertebrate and vertebrate taxa, and is generally considered to make hosts vulnerable to parasites and predators. Here, we used the crustacean Daphnia magna to test whether moulting influences the likelihood of infection by the castrating bacterium Pasteuria ramosa. This parasite is known to attach to the host cuticula before penetrating into its body. We found that the likelihood of successful parasite infection is greatly reduced if the host moults within 12 h after parasite exposure. Thus, moulting is beneficial for the host being exposed to this parasite. We further show that exposure to the parasite does not induce hosts to moult earlier. We discuss the implications of our findings for host and parasite evolution and epidemiology.     

Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis

Plasmodium falciparum is the causative agent for the most lethal form of human malaria, killing millions annually. Genetic analyses of P. falciparum have been relatively limited due to the lack of robust techniques to manipulate this parasite. Development of transfection technologies and whole genome analyses have helped in understanding the complex biology of this parasite. Even with this wealth of information functional genomics approaches are still very limited in P. falciparum due to the cumbersome and inefficient methods of genetic manipulation. This review focuses on a recently developed, highly efficient method for transposon-based mutagenesis and transgene expression in P. falciparum that will allow functional genomics studies to be performed proficiently on this deadly malaria parasite. By using a piggyBac-based transposition system, multiple random integrations have been obtained into the genome of the parasite. This technique could hence be employed to set up several biological screens in this lethal protozoan parasite that may lead to identification of novel drug targets and vaccine candidates.     

Phospholipid Biosynthesis in Synchronous Plasmodium falciparum Cultures1

The metabolism of phospholipids in synchronous Plasmodium falciparum-infected erythrocytes was studied over one cycle of 48 h by the incorporation of labeled palmitate, serine, choline, and myo-inositol into cellular lipids. The rates of incorporation of palmitate and serine into total phospholipids and of choline into phosphatidylcholine (PC) were linear with the maturation of the parasite, increasing by a factor of 2–5.6 according to the precursors. The rate of inositol incorporation into phosphatidylinositol was 9.6 times higher at the schizont stage than at the ring stage, with a marked increase in the second half of the cycle. A significant incorporation of palmitate into triglycerides also occurred during the schizont stage of the parasite. The incorporations of serine and palmitate into phosphatidylethanolamine (PE) and PC showed a net increase at approximately the twentieth hour of the cycle, while the radioactivities recovered in phosphatidylserine (PS) had already reached a maximum by this time. These findings indicate an instantaneous transformation of PS into PE and PC through a decarboxylation of PS into PE, then a methylation of PE into PC during the second half of the cycle. Although PS is a minor component of the Plasmodium parasite, our findings demonstrate the important role of this phospholipid as a precursor of PE and PC, which are major constituents of parasite phospholipids.     

Encystation of entamoeba parasites

Entamoeba histolytica is a protozoan parasite of humans, and the causitive agent of intestinal amebiasis. The disease-causing stage of the parasite is an osmotically sensitive ameboid form, which differentiates into a thick-walled cyst for transmission from person to person. The conditions within the human intestine that induce encystment of the amoeba are unknown, but studies using an amoebic parasite of reptiles are now yielding information about the molecules and host:parasite interactions involved in the process. An understanding of the amoeba's obligatory encystment pathway should provide an approach for interrupting the transmission of this parasite, for which there is currently no vaccine.     

d-Glucose concentration is the key factor facilitating liver stage maturation of Plasmodium

The course of malaria infection in mammals begins with transmission of Plasmodium sporozoites into the skin by Anopheles mosquitoes, followed by migration of the sporozoites to the liver. As no symptoms present until hepatic merozoites are released and until they infect erythrocytes in the blood vessels, sporozoites and liver-stage (LS) parasites are promising targets for anti-malaria drugs aiming to prevent mosquito-to-mammal transmission. In vitro LS parasite development system is useful in the screening of candidate drugs on LS parasite development and the elucidation of its underlying molecular mechanisms, which remain unclear. Using rodent malaria parasites (Plasmodium berghei) as a model, this study aimed to develop an optimal in vitro LS culture system for the full maturation of the LS parasite into the hepatic merozoite, the next infective stage in parasite development. As the development of this system required measurement of maturation, a novel quantitative index of LS parasite maturation based on the expression pattern of liver-specific protein 2 (LISP2) was first developed. The use of this index for comparing the effect of incubation in different culture media on LS maturation revealed that the d-glucose concentration of the culture medium is the key factor promoting parasite development in hepatocytes and that a d-glucose concentration of 2000 mg/L/day is the threshold concentration at which the maturation of P. berghei into infective hepatic merozoites is achieved. These findings can be utilized to optimize a human malaria LS culture system for drug discovery.     

Temperature-dependent costs of parasitism and maintenance of polymorphism under genotype-by-environment interactions

The maintenance of genetic variation for infection-related traits is often attributed to coevolution between hosts and parasites, but it can also be maintained by environmental variation if the relative fitness of different genotypes changes with environmental variation. To gain insight into how infection-related traits are sensitive to environmental variation, we exposed a single host genotype of the freshwater crustacean Daphnia magna to four parasite isolates (which we assume to represent different genotypes) of its naturally co-occurring parasite Pasteuria ramosa at 15, 20 and 25 degrees C. We found that the cost to the host of becoming infected varied with temperature, but the magnitude of this cost did not depend on the parasite isolate. Temperature influenced parasite fitness traits; we found parasite genotype-by-environment (G x E) interactions for parasite transmission stage production, suggesting the potential for temperature variation to maintain genetic variation in this trait. Finally, we tested for temperature-dependent relationships between host and parasite fitness traits that form a key component of models of virulence evolution, and we found them to be stable across temperatures.     

Trypanosoma vivax--out of Africa

Trypanosoma vivax is a blood parasite of ruminants that was introduced into Latin America in cattle imported from Africa, possibly in the late 19th century. The parasite has now spread to ten of the 13 countries of the South American continent, often resulting in a severe wasting disease and death. Here, we review the current state of knowledge about this parasite and the problems faced by animal health agencies in controlling the disease.     

The virulence–transmission relationship in an obligate killer holds under diverse epidemiological and ecological conditions,but where is the tradeoff?

Parasite virulence is a leading theme in evolutionary biology. Modeling the course of virulence evolution holds the promise of providing practical insights into the management of infectious diseases and the implementation of vaccination strategies. A key element of virulence modeling is a tradeoff between parasite transmission rate and host lifespan. This assumption is crucial for predicting the level of optimal virulence. Here, I test this assumption using the water flea Daphnia magna and its castrating and obligate‐killing bacterium Pasteuria ramosa. I found that the virulence–transmission relationship holds under diverse epidemiological and ecological conditions. In particular, parasite genotype, absolute and relative parasite dose, and within‐host competition in multiple infections did not significantly affect the observed trend. Interestingly, the relationship between virulence and parasite transmission in this system is best explained by a model that includes a cubic term. Under this relationship, parasite transmission initially peaks and saturates at an intermediate level of virulence, but then it further increases as virulence decreases, surpassing the previous peak. My findings also highlight the problem of using parasite‐induced host mortality as a “one‐size‐fits‐all” measure of virulence for horizontally transmitted parasites, without considering the onset and duration of parasite transmission as well as other equally virulent effects of parasites (e.g., host castration). Therefore, mathematical models may be required to predict whether these particular characteristics of horizontally transmitted parasites can direct virulence evolution into directions not envisaged by existing models.     

The nucleosome (histone-DNA complex) is the TLR9-specific immunostimulatory component of Plasmodium falciparum that activates DCs

The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs) by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-), TLR9(-/-) and MyD88(-/-) mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex) represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.