RESUMEN
Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.
Asunto(s)
Poliaminas/metabolismo , Xenorhabdus/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Vías Biosintéticas , Familia de Multigenes , Poliaminas/química , Xenorhabdus/química , Xenorhabdus/genéticaRESUMEN
Caspase-8 is involved in a number of cellular functions, with the most well established being the control of cell death. Yet caspase-8 is unique among the caspases in that it acts as an environmental sensor, transducing a range of signals to cells, modulating responses that extend far beyond simple survival. Ranging from the control of apoptosis and necroptosis and gene regulation to cell adhesion and migration, caspase-8 uses proteolytic and non-proteolytic functions to alter cell behavior. Novel interacting partners provide mechanisms for caspase-8 to position itself at signaling nodes that affect a variety of signaling pathways. Here, we examine the catalytic and noncatalytic modes of action by which caspase-8 influences cell adhesion and migration. The mechanisms vary from post-cleavage remodeling of the cytoskeleton to signaling elements that control focal adhesion turnover. This is facilitated by caspase-8 interaction with a host of cell proteins ranging from the proteases caspase-3 and calpain-2 to adaptor proteins such as p85 and Crk, to the Src family of tyrosine kinases.
Asunto(s)
Caspasa 8/metabolismo , Animales , Movimiento Celular , Humanos , FosforilaciónRESUMEN
Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Asunto(s)
Productos Biológicos/química , Vías Biosintéticas/genética , Metabolómica/métodos , HumanosRESUMEN
Caspase-8 is a cysteine protease that plays an essential role in apoptosis. Consistently with its canonical proapoptotic function, cancer cells may genetically or epigenetically downregulate its expression. Unexpectedly, Caspase-8 is often retained in cancer, suggesting the presence of alternative mechanisms that may be exploited by cancer cells to their own benefit. In this regard, we reported that Src tyrosine kinase, which is aberrantly activated in many tumors, promotes Caspase-8 phosphorylation on Tyrosine 380 (Y380) preventing its full activation. Here, we investigated the significance of Caspase-8 expression and of its phosphorylation on Y380 in glioblastoma, a brain tumor where both Caspase-8 expression and Src activity are often aberrantly upregulated. Transcriptomic analyses identified inflammatory response as a major target of Caspase-8, and in particular, NFκB signaling as one of the most affected pathways. More importantly, we could show that Src-dependent phosphorylation of Caspase-8 on Y380 drives the assembly of a multiprotein complex that triggers NFκB activation, thereby inducing the expression of inflammatory and pro-angiogenic factors. Remarkably, phosphorylation on Y380 sustains neoangiogenesis and resistance to radiotherapy. In summary, our work identifies a novel interplay between Src kinase and Caspase-8 that allows cancer cells to hijack Caspase-8 to sustain tumor growth.
Asunto(s)
Caspasa 8 , Glioblastoma , Familia-src Quinasas , Humanos , Apoptosis , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Glioblastoma/genética , Fosforilación , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismoRESUMEN
Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.
Asunto(s)
Caspasa 8/química , Precursores Enzimáticos/química , Secuencia de Aminoácidos , Caspasa 8/metabolismo , Caspasas/química , Caspasas/metabolismo , Catálisis , Dimerización , Activación Enzimática , Precursores Enzimáticos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Especificidad por SustratoRESUMEN
Maximizing more ecosystem functions may require more species. This relationship results from imperfect correlations among ecosystem functions because species contribute differently to each function. These correlations among species contributions to functions and the extent of interspecific competition are crucial when determining how many species are necessary to maximize additional functionality.
Asunto(s)
Biodiversidad , EcosistemaRESUMEN
In an effort to find a suitable genetic background for efficient cellulolytic secretion, genetically diverse strains were transformed to produce core fungal cellulases namely, ß-glucosidase (BGLI), endoglucanase (EGII) and cellobiohydrolase (CBHI) in various combinations and expression configurations. The secreted enzyme activity levels, gene copy number, substrate specificities, as well as hydrolysis and fermentation yields of the transformants were analysed. The effectiveness of the partially cellulolytic yeast transformants to convert two different pre-treated corn residues, namely corn cob and corn husk was then explored. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants produced up to 1.34 fold higher glucose concentrations (g/L) than a control composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted ratio of 1:15 (cellulase activity unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments and yielded 4.05â¯g/L ethanol in fermentations. We demonstrate that the choice of optimal genetic background and cellulase activity secretion ratio can improve cellulosic ethanol production by consolidated bioprocessing yeast strains.
Asunto(s)
Celulasa/metabolismo , Expresión Génica , Levaduras/enzimología , Levaduras/metabolismo , Zea mays/metabolismo , Biotransformación , Celulasa/genética , Diploidia , Fermentación , Dosificación de Gen , Hidrólisis , Levaduras/genéticaRESUMEN
NcMIC1 is a 460 amino acid Neospora caninum microneme protein implicated in host cell adhesion and invasion processes. In this study, we assessed the potential protectivity of NcMIC1-based vaccination against experimental N. caninum infection in mice, employing both recombinant antigen vaccines and DNA vaccines. Recombinant NcMIC1 (recNcMIC1) was expressed in Escherichia coli as gluthatione-S-transferase-fusion protein. The corresponding NcMIC1 cDNA was cloned into the pcDNA3.1 expression plasmid (pcDNA-MIC1), and expression was checked in transfected Vero cells. Mice (10 animals/group) were vaccinated either with recNcMIC1 antigen suspended in Ribi-adjuvant (3 intraperitoneal injections), pcDNA-NcMIC1 (3 intramuscular injections), or pcDNA-NcMIC1 (twice intramuscularly), followed by 1 intraperitoneal recNcMIC1 antigen boost. Control groups included corresponding treatments with adjuvant, pcDNA3.1 without insert, and PBS (= infection control). All vaccinated and control groups were then challenged intraperitoneally with 2 x 10(6) N. caninum tachyzoites. Animals were inspected daily for a period of 3 wk postinfection (PI). At day 21, all animals were killed and assessed for infection. Before day 21 PI, clinical signs such as walking disorders, rounded back, apathy, and paralysis occurred in infection controls (50% of the mice), pcDNA and adjuvant controls (20% each), and the combined pcDNA-NcMIC1/recNcMIC1-treated group (30%). No clinical symptoms were observed in the recNcMIC1 and pcDNA-NcMIC1 vaccinated groups. All mice were positive for cerebral N. caninum infection as assessed by PCR of brain tissue. However, quantitative real-time PCR revealed that the infection intensity was significantly reduced in the group vaccinated with recNcMIC1 antigen. Immunohistochemistry confirmed these findings. In contrast, the infection intensity was highest in the group vaccinated with the pcDNA-NcMIC1/recNcMIC1 combination, indicating that the sequential application of the DNA vaccine and recombinant antigen had a deleterious effect. Serological analysis showed that only recNcMIC1-immunized animals generated detectable antibody levels recognizing native NcMIC1. Thus, of all protocols applied here, only recNcMIC1 vaccination appears to be suited to reduce cerebral infection in mice challenged with N. caninum tachyzoites.
Asunto(s)
Encefalopatías/prevención & control , Coccidiosis/prevención & control , Neospora/inmunología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Encéfalo/parasitología , Encefalopatías/inmunología , Encefalopatías/parasitología , Chlorocebus aethiops , Coccidiosis/inmunología , Femenino , Inmunización Secundaria , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/inmunología , Vacunación , Vacunas de ADN , Vacunas Sintéticas , Células VeroRESUMEN
Neospora caninum and Toxoplasma gondii are characterised by a very low host cell specificity, thus they are able to infect a wide range of different cells in vivo and in vitro. Infection of the host cell by tachyzoites is a process which is preceded by adhesion onto the host cell surface. The receptors on the host cell surface which would allow N. caninum to establish a physical interaction have not been investigated so far. Here we report the role of host cell surface proteoglycans as receptors for the adhesion of N. caninum tachyzoites to Vero cell monolayers. We found that N. caninum tachyzoites, similar to T. gondii tachyzoites, can bind to sulphated proteoglycans which naturally occur on the surface of mammalian cells, including heparin/heparan sulphate, chondroitin sulphates, as well as to the artificially sulphated glycosaminoglycan dextran sulphate. Although removal of heparan sulphate from the host cell surface results in decreased adhesion of T. gondii tachyzoites, binding of N. caninum tachyzoites is not affected by this treatment. Conversely, enzymatic removal of chondroitin sulphate A, B and C decreases N. caninum adhesion but does not affect T. gondii binding to Vero cells. Thus, T. gondii and N. caninum tachyzoites exhibit differential adhesive properties with regard to host cell surface glycosaminoglycans. Additional experiments employing Triton X-100 solubilised NcSRS2 and NcMIC3 showed that NcSRS2 binds to the host cell surface, but not through those sulphated glycosaminoglycans investigated in this study. In contrast, NcMIC3 binding to the host cell surface is dramatically influenced by these modifications. Further experiments showed that the NcMIC3 adhesive motif comprised of four consecutive epidermal growth factor-like domains expressed as a recombinant protein exhibits a high binding activity for sulphated glycosaminoglycans. These results suggest that host cell surface proteoglycan interaction of N. caninum differs from that observed for T. gondii, and that the epidermal growth factor-like adhesive motif in NcMIC3 could be involved in this process.
Asunto(s)
Adhesión Celular/fisiología , Coccidiosis/parasitología , Neospora/fisiología , Proteoglicanos/fisiología , Proteínas Protozoarias/fisiología , Animales , Anticuerpos Antiprotozoarios/análisis , Chlorocebus aethiops , Ensayo de Inmunoadsorción Enzimática , Glicosaminoglicanos/farmacología , Interacciones Huésped-Parásitos , Toxoplasma/metabolismo , Células VeroRESUMEN
Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.
Asunto(s)
Células Epidérmicas , Queratinocitos/parasitología , Estadios del Ciclo de Vida , Neospora/crecimiento & desarrollo , Óxido Nítrico/farmacología , Animales , Antígenos de Protozoos/análisis , Células Cultivadas , Chlorocebus aethiops , Quistes/química , Quistes/ultraestructura , Epidermis/anatomía & histología , Interacciones Huésped-Parásitos , Humanos , Ratones , Neospora/metabolismo , Óxido Nítrico/metabolismo , Nitroprusiato/metabolismo , Reacción en Cadena de la Polimerasa , Células VeroRESUMEN
Procaspase-8, the zymogen form of the apoptosis-initiator caspase-8, undergoes phosphorylation following integrin-mediated cell attachment to an extracellular matrix substrate. Concordant with cell attachment to fibronectin, a population of procaspase-8 becomes associated with a peripheral insoluble compartment that includes focal complexes and lamellar microfilaments. Phosphorylation of procaspase-8 both impairs its maturation to the proapoptotic form and can promote cell migration. Here we show that the cytoskeletal adaptor protein CrkL promotes caspase-8 recruitment to the peripheral spreading edge of cells, and that the catalytic domain of caspase-8 directly interacts with the SH2 domain of CrkL. We show that the interaction is abolished by shRNA-mediated silencing of Src, in Src-deficient MEFs, and by pharmacologic inhibitors of the kinase. The results provide insight into how tyrosine kinases may act to coordinate the suppression caspase-8 mediated apoptosis, while promoting cell invasion.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Caspasa 8/metabolismo , Movimiento Celular/fisiología , Proteínas Nucleares/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Western Blotting , Caspasa 8/genética , Línea Celular Tumoral , Movimiento Celular/genética , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Proteínas Nucleares/genética , Fosforilación , Familia-src Quinasas/genéticaRESUMEN
Bcl-xL is an antiapoptotic member of the Bcl-2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl-xL from a DNA-encoded chemical library using affinity-capture selections and massively parallel high-throughput sequencing of >30,000 sequence tags of library members. The most potent binder identified, compound 19/93 [(R)-3-(amido indomethacin)-4-(naphthalen-1-yl)butanoic acid], bound to Bcl-xL with a dissociation constant (K(d)) of 930 nM and was able to compete with a Bak-derived BH3 peptide, an antagonist of Bcl-xL function.
Asunto(s)
Antineoplásicos/química , ADN/química , Proteína bcl-X/antagonistas & inhibidores , Antineoplásicos/toxicidad , Apoptosis , Línea Celular Tumoral , Humanos , Análisis de Secuencia de ADN , Bibliotecas de Moléculas Pequeñas , Proteína bcl-X/metabolismoRESUMEN
The Drosophila Sterile-20 kinase Slik promotes tissue growth during development by stimulating cell proliferation and by preventing apoptosis. Proliferation within an epithelial sheet requires dynamic control of cellular architecture. Epithelial integrity fails in slik mutant imaginal discs. Cells leave the epithelium and undergo apoptosis. The abnormal behavior of slik mutant cells is due to failure to phosphorylate and activate Moesin, which leads to excess Rho1 activity. This is distinct from Slik's effects on cell proliferation, which are mediated by Raf. Thus Slik acts via distinct pathways to coordinate cell proliferation with epithelial cell behavior during tissue growth.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Proteínas de Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Actinas/metabolismo , Animales , Apoptosis/genética , Diferenciación Celular/genética , División Celular/fisiología , Supervivencia Celular/fisiología , Drosophila/genética , Proteínas de Drosophila/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Péptidos y Proteínas de Señalización Intracelular , Larva , Quinasas Quinasa Quinasa PAM , Proteínas de Microfilamentos/genética , Microvellosidades/genética , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Alas de Animales/citología , Alas de Animales/crecimiento & desarrollo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
Asunto(s)
Glicosaminoglicanos/metabolismo , Neospora/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/metabolismo , Chlorocebus aethiops , ADN Protozoario , Genes Protozoarios , Datos de Secuencia Molecular , Neospora/genética , Péptidos/genética , Péptidos/metabolismo , Proteínas Protozoarias/genética , Homología de Secuencia de Aminoácido , Sulfatos , Células VeroRESUMEN
Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.
Asunto(s)
Lactosa/metabolismo , Proteínas de la Membrana , Neospora/crecimiento & desarrollo , Neospora/patogenicidad , Proteínas Protozoarias , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Neospora/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADN , Células VeroRESUMEN
Neospora caninum is an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related to Toxoplasma gondii and Hammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genus Neospora. N. caninum exhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly, N. caninum has a particular significance as a cause of abortion in cattle. In vitro culture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages of N. caninum. Tissue culture systems include maintenance of N. caninum tachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection by N. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies on N. caninum tachyzoites and bradyzoites, and on the physical interactions between parasites and host cells.