Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Characterization of the fetuin-binding fraction of Neospora caninum tachyzoites and its potential involvement in host-parasite interactions

Terminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction of Neospora caninum tachyzoites and bradyzoites with Vero cell monolayers. Thus, Neospora extracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of approximately 65 kDa, subsequently named NcFBP (Neospora caninum fetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140 kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction of Neospora caninum tachyzoites contains components that could be potentially involved in host-parasite interactions.

Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host-parasite interactions

Neospora caninum is an apicomplexan parasite which has emerged as an important cause of bovine abortion worldwide. Abortion is usually triggered by reactivation of dormant bradyzoites during pregnancy and subsequent congenital infection of the foetus, where the central nervous system appears to be most frequently affected. We here report on an organotypic tissue culture model for Neospora infection which can be used to study certain aspects of the cerebral phase of neosporosis within the context of a three-dimensionally organised neuronal network. Organotypic slice cultures of rat cortical tissue were infected with N. caninum tachyzoites, and the kinetics of parasite proliferation, as well as the proliferation-inhibitory effect of interferon-gamma (IFN-gamma), were monitored by either immunofluorescence, transmission electron microscopy, and a quantitative PCR-assay using the LightCycler instrument, respectively. In addition, the neuronal cytoskeletal elements, namely glial acidic protein filaments as well as actin microfilament bundles were shown to be largely colocalising with the pseudocyst periphery. This organotypic culture model for cerebral neosporosis provides a system, which is useful to study the proliferation, ultrastructural characteristics, development, and the interactions of N. caninum within the context of neuronal tissue, which at the same time can be modulated and influenced under controlled conditions, and will be useful in the future to gain more information on the cerebral phase of neosporosis.

The cytoskeleton and motor proteins of human schistosomes and their roles in surface maintenance and host-parasite interactions

Schistosomes are parasitic blood flukes, responsible for significant human disease in tropical and developing nations. Here we review information on the organization of the cytoskeleton and associated motor proteins of schistosomes, with particular reference to the organization of the syncytial tegument, a unique cellular adaptation of these and other neodermatan flatworms. Extensive EST databases show that the molecular constituents of the cytoskeleton and associated molecular systems are likely to be similar to those of other eukaryotes, although there are potentially some molecules unique to schistosomes and platyhelminths. The biology of some components, particular those contributing to host-parasite interactions as well as chemotherapy and immunotherapy are discussed. Unresolved questions in relation to the structure and function of the tegument relate to dynamic organization of the syncytial layer. (C) 2004 Wiley Periodicals, Inc.

Venom proteins from polydnavirus-producing endoparasitoids: Their role in host-parasite interactions

Endoporasitoid wasps have evolved various mechanisms to ensure successful development of their progeny, including co-injection of a cocktail of maternal secretions into the host hemocoel, including venom, calyx fluid, and polydnoviruses. The components of each type of secretion may influence host physiology and development independently or in a synergistic fashion. For example, venom fluid consists of several peptides and proteins that promote expression of polydnavirus genes in addition to other activities, such as inhibition of prophenoloxidase activation, inhibition of hemocytes spreading and aggregation, and inhibition of development. This review provides a brief overview of advances and prospects in the study of venom proteins from polydnavirus-producing endoparositoid wasps with a special emphasis on the role of C. rubecula venom proteins in host-parositoid interactions.

Cancer and life-history traits: lessons from host-parasite interactions

Despite important differences between infectious diseases and cancers, tumour development (neoplasia) can nonetheless be closely compared to infectious disease because of the similarity of their effects on the body. On this basis, we predict that many of the life-history (LH) responses observed in the context of host-parasite interactions should also be relevant in the context of cancer. Parasites are thought to affect LH traits of their hosts because of strong selective pressures like direct and indirect mortality effects favouring, for example, early maturation and reproduction. Cancer can similarly also affect LH traits by imposing direct costs and/or indirectly by triggering plastic adjustments and evolutionary responses. Here, we discuss how and why a LH focus is a potentially productive but under-exploited research direction for cancer research, by focusing our attention on similarities between infectious disease and cancer with respect to their effects on LH traits and their evolution. We raise the possibility that LH adjustments can occur in response to cancer via maternal/paternal effects and that these changes can be heritable to (adaptively) modify the LH traits of their offspring. We conclude that LH adjustments can potentially influence the transgenerational persistence of inherited oncogenic mutations in populations.

Exploring an uncommon host-parasite interaction to unveil the uniqueness of Perkinsus sp as a model organism

We report the exploration of some unique metabolic pathways in Perkinsus olseni a marine protist parasite, responsible to significant mortalities in mollusks, especially in bivalves all around the world. In Algarve, south of Portugal carpet shell clam Ruditapes decussatus mortalities can reach up to 70%, causing social and economic losses. The objective of studying those unique pathways, is finding new therapeutic strategies capable of controlling/eliminating P. olseni proliferation in clams. In that sense metabolic pathways, were explored, and drugs affecting these cycles were tested for activity. The first step involved the identification of the genes behind those pathways, the reconstitution of the main steps, and molecular characterization of those genes and later on, the identification of possible targets within the genes studied. Metabolic cycles were screened due to the fact of not being present in host or differ in a critical way, such as the following pathways: shikimate, MEP-­‐ isoprenoids, Leloir cycle for chitin production, purine biosynthesis (unique among protists), the de novo synthesis of folates (absent in metazoa) and some unique genes like, the alternative oxidase (a branch of respiratory chain) and the hypoxia sensor HPH. All those pathways were covered and possible chemical inhibition using therapeutic drugs was tested with positive results. The relation between the common host Ruditapes decussatus and P. olseni was also explored in a dimension not possible some years ago. With the accessibility to second generation sequencers and microarray analysis platforms, genes involved in host defense or parasite virulence and resistance to the host were deciphered, allowing aiming to new targets (mechanisms and pathways), offering new possibilities for the control of Perkinsus in close environments. The thousands of genes, generated by this work, sequenced and analyzed from this commercial valuable clam and for Perkinsus olseni will be an important and value tool for the scientific community, allowing a better understanding of host-­‐parasite interactions, promoting the usage of P. olseni as an emerging model for alveolata parasites.

Host-parasite interaction between crustaceans of six fish species from the Brazilian Amazon.

Host-parasite interactions between crustaceans and six fish species (Psectrogaster falcata, Ageneiosus ucayalensis, Acestrorhynchus falcirostris, Hemiodus unimaculatus, Serrasalmus gibbus and Geophagus proximus) from a reservoir in eastern Amazon, northern Brazil, were investigated. Eight hundred and seventy-eight parasites belonging to three crustacean species, Excorallana berbicensis, Argulus chicomendesi and Ergasilus turucuyus, which parasitized the hosts? mouth, gills and tegument, were collected from 295 fish and examined. High infestation levels were caused by E. berbicensis on the body surface of the hosts. Excorallana berbicensis showed aggregate dispersion, except in S. gibbus, while E. turucuyus showed random dispersion in A. falcirostris. The host?s sex did not influence infestation by E. berbicensis, and high parasitism failed to affect the body conditions of the fish. In the case of some hosts, rainfall rates, temperature, dissolved oxygen levels and water pH affected the prevalence and abundance of E. berbicensis, the dominant parasite species. Results revealed that the environment and life-style of the hosts were determining factors in infestations by parasites. Current assay is the first report on E. berbicensis for the six hosts, as well as on A. chicomendesi for G. proximus and P. falcata.

Echinococcus multilocularis phosphoglucose isomerase (EmPGI): a glycolytic enzyme involved in metacestode growth and parasite-host cell interactions

In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.

The strategies of the Theileria parasite: a new twist in host-pathogen interactions

Theileria parasites infect and transform cells of the ruminant immune system. Continuous proliferation and survival of Theileria-transformed cells involves the well-orchestrated activation of several host-cell signalling pathways. Constitutive NF-kappa B (nuclear factor kappa B) activation is accomplished by recruiting the IKK (I kappa B kinase) complex, a central regulator of NF-kappa B pathways, to the surface of the transforming schizont, where it becomes permanently activated. Constitutive activation of the PI-3K-PKB [phosphoinositide 3-kinase-(Akt) protein kinase B] pathway is likely to be indirect and is essential for continuous proliferation. Theileria-transformed T cells express a range of anti-apoptotic proteins that can be expected to provide protection against apoptosis induced by death receptors, as well as cellular control mechanisms that are mobilised to eliminate cells that entered a cycle of uncontrolled proliferation.

A blowfly strike vaccine requires an understanding of host-pathogen interactions

The phase-out of Mulesing by 2010 means the Australian wool industry requires immediate and viable alternatives for the control and prevention of blowfly strike, an economically important parasitic disease of sheep. In this review we have analysed previous research aimed toward the development of a vaccine against blowfly strike and the reasons why the approaches taken were unsuccessful at the time. Close scrutiny has provided new insight into this host-parasite interaction and identified new opportunities for the development of a vaccine. Here we propose that addressing immunosuppression together with the induction of cellular immunity is likely to result in an anti-blowfly strike vaccine, as opposed to the use of "standard" approaches aimed at inducing humoral immunity.

Emerging Infectious Disease: Host and Parasite Perspectives

Avian malaria and related haematozoa are nearly ubiquitous parasites that can impose fitness costs of variable severity and may, in some cases, cause substantial mortality in their host populations. One example of the latter, the emergence of avian malaria in the endemic avifauna of Hawaii, has become a model for understanding the consequences of human-mediated disease introduction. The drastic declines of native Hawaiian birds due to avian malaria provided the impetus for examining more closely several aspects of host-parasite interactions in this system. Host-specificity is an important character determining the extent to which a parasite may emerge. Traditional parasite classification, however, has used host information as a character in taxonomical identification, potentially obscuring the true host range of many parasites. To improve upon previous methods, I first developed molecular tools to identify parasites infecting a particular host. I then used these molecular techniques to characterize host-specificity of parasites in the genera Plasmodium and Haemoproteus. I show that parasites in the genus Plasmodium exhibit low specificity and are therefore most likely to emerge in new hosts in the future. Subsequently, I characterized the global distribution of the single lineage of P. relictum that has emerged in Hawaii. I demonstrate that this parasite has a broad host distribution worldwide, that it is likely of Old World origin and that it has been introduced to numerous islands around the world, where it may have been overlooked as a cause of decline in native birds. I also demonstrate that morphological classification of P. relictum does not capture differences among groups of parasites that appear to be reproductively isolated based on molecular evidence. Finally, I examined whether reduced immunological capacity, which has been proposed to explain the susceptibility of Hawaiian endemics, is a general feature of an "island syndrome" in isolated avifauna of the remote Pacific. I show that, over multiple time scales, changes in immune response are not uniform and that observed changes probably reflect differences in genetic diversity, parasite exposure and life history that are unique to each species.

Does timing matter? How priority effects influence the outcome of parasite interactions within hosts

In nature, hosts are exposed to an assemblage of parasite species that collectively form a complex community within the host. To date, however, our understanding of how within-host–parasite communities assemble and interact remains limited. Using a larval amphibian host (Pacific chorus frog, Pseudacris regilla) and two common trematode parasites (Ribeiroia ondatrae and Echinostoma trivolvis), we experimentally examined how the sequence of host exposure influenced parasite interactions within hosts. While there was no evidence that the parasites interacted when hosts were exposed to both parasites simultaneously, we detected evidence of both intraspecific and interspecific competition when exposures were temporally staggered. However, the strength and outcome of these priority effects depended on the sequence of addition, even after accounting for the fact that parasites added early in host development were more likely to encyst compared to parasites added later. Ribeiroia infection success was reduced by 14 % when Echinostoma was added prior to Ribeiroia, whereas no such effect was noted for Echinostoma when Ribeiroia was added first. Using a novel fluorescent-labeling technique that allowed us to track Ribeiroia infections from different exposure events, we also discovered that, similar to the interspecific interactions, early encysting parasites reduced the encystment success of later arriving parasites by 41 %, which could be mediated by host immune responses and/or competition for space. These results suggest that parasite identity interacts with host immune responses to mediate parasite interactions within the host, such that priority effects may play an important role in structuring parasite communities within hosts. This knowledge can be used to assess host–parasite interactions within natural communities in which environmental conditions can lead to heterogeneity in the timing and composition of host exposure to parasites.