Unravelling the sexual developmental biology of Cystoisospora suis, a model for comparative coccidian parasite studies.

Introduction: The apicomplexan parasite Cystoisospora suis has global significance as an enteropathogen of suckling piglets. Its intricate life cycle entails a transition from an asexual phase to sexual development, ultimately leading to the formation of transmissible oocysts. Methods: To advance our understanding of the parasite's cellular development, we complemented previous transcriptome studies by delving into the proteome profiles at five distinct time points of in vitro cultivation through LC/MS-MS analysis. Results: A total of 1,324 proteins were identified in the in vitro developmental stages of C. suis, and 1,082 proteins were identified as significantly differentially expressed. Data are available via ProteomeXchange with identifier PXD045050. We performed BLAST, GO enrichment, and KEGG pathway analyses on the up- and downregulated proteins to elucidate correlated events in the C. suis life cycle. Our analyses revealed intriguing metabolic patterns in macromolecule metabolism, DNA- and RNA-related processes, proteins associated with sexual stages, and those involved in cell invasion, reflecting the adaptation of sexual stages to a nutrient-poor and potentially stressful extracellular environment, with a focus on enzymes involved in metabolism and energy production. Discussion: These findings have important implications for understanding the developmental biology of C. suis as well as other, related coccidian parasites, such as Eimeria spp. and Toxoplasma gondii. They also support the role of C. suis as a new model for the comparative biology of coccidian tissue cyst stages.

In vitro cultivation methods for coccidian parasite research.

The subclass Coccidia comprises a large group of protozoan parasites, including important pathogens of humans and animals such as Toxoplasma gondii, Neospora caninum, Eimeria spp., and Cystoisospora spp. Their life cycle includes a switch from asexual to sexual stages and is often restricted to a single host species. Current research on coccidian parasites focuses on cell biology and the underlying mechanisms of protein expression and trafficking in different life stages, host cell invasion and host-parasite interactions. Furthermore, novel anticoccidial drug targets are evaluated. Given the variety of research questions and the requirement to reduce and replace animal experimentation, in vitro cultivation of Coccidia needs to be further developed and refined to meet these requirements. For these purposes, established culture systems are constantly improved. In addition, new in vitro culture systems lately gained considerable importance in research on Coccidia. Well established and optimized in vitro cultures of monolayer cells can support the viability and development of parasite stages and even allow completion of the life cycle in vitro, as shown for Cystoisospora suis and Eimeria tenella. Furthermore, new three-dimensional cell culture models are used for propagation of Cryptosporidium spp. (close relatives of the coccidians), and the infection of three-dimensional organoids with T. gondii also gained popularity as the interaction between the parasite and host tissue can be studied in more detail. The latest advances in three-dimensional culture systems are organ-on-a-chip models, that to date have only been tested for T. gondii but promise to accelerate research in other coccidians. Lastly, the completion of the life cycle of C. suis and Cryptosporidium parvum was reported to continue in a host cell-free environment following the first occurrence of asexual stages. Such axenic cultures are becoming increasingly available and open new avenues for research on parasite life cycle stages and novel intervention strategies.

Inhibition of sexual stage-specific proteins results in reduced numbers of sexual stages and oocysts of Cystoisospora suis (Apicomplexa: Coccidia) in vitro.

Parasites of the order Coccidia (phylum: Alveolata, subphylum: Apicomplexa) have sophisticated life cycles that include a switch from asexual to sexual development, characterised by distinct cell types. During the development of gametes (gamogony), substantial changes occur at the cellular and subcellular levels, leading to cell fusion of micro- and microgametes, and the development of a zygote that forms a protective outer layer for environmental survival as an oocyst, the transmissible stage. Studies on the porcine coccidian Cystoisospora suis already identified changes in transcription profiles during different time points in the parasite's development and identified proteins with potential roles in the sexual development of this parasite. Here, we focus on three proteins that are possibly involved in the sexual development of C. suis. Enkurin and hapless protein 2 (HAP2) play important roles in signal transduction and gamete fusion during the fertilisation process, and oocyst wall forming protein 1 (OWP1) is a homologue of oocyst wall forming proteins of related parasites. We evaluated their locations in the different life cycle stages of C. suis and their inhibition by specific antibodies in vitro. Immunolocalization detected enkurin in merozoites and sporulated oocysts, HAP2 in merozoites and microgamonts, and OWP2 in merozoites, macrogamonts, oocysts and sporozoites. Up to 100% inhibition of the development of sexual stages and oocyst formation with purified chicken immunoglobulin IgY sera against recombinant enkurin, HAP2, and especially OWP1, were demonstrated. We conclude that the three investigated sexual stage-specific proteins constitute targets for in vivo intervention strategies to interrupt parasite development and transmission to susceptible hosts.

The transcriptome from asexual to sexual in vitro development of Cystoisospora suis (Apicomplexa: Coccidia).

The apicomplexan parasite Cystoisospora suis is an enteropathogen of suckling piglets with woldwide distribution. As with all coccidian parasites, its lifecycle is characterized by asexual multiplication followed by sexual development with two morphologically distinct cell types that presumably fuse to form a zygote from which the oocyst arises. However, knowledge of the sexual development of C. suis is still limited. To complement previous in vitro studies, we analysed transcriptional profiles at three different time points of development (corresponding to asexual, immature and mature sexual stages) in vitro via RNASeq. Overall, transcription of genes encoding proteins with important roles in gametes biology, oocyst wall biosynthesis, DNA replication and axonema formation as well as proteins with important roles in merozoite biology was identified. A homologue of an oocyst wall tyrosine rich protein of Toxoplasma gondii was expressed in macrogametes and oocysts of C. suis. We evaluated inhibition of sexual development in a host-free culture for C. suis by antiserum specific to this protein to evaluate whether it could be exploited as a candidate for control strategies against C. suis. Based on these data, targets can be defined for future strategies to interrupt parasite transmission during sexual development.

Sexual Development in Non-Human Parasitic Apicomplexa: Just Biology or Targets for Control?

The phylum Apicomplexa is a major group of protozoan parasites including gregarines, coccidia, haemogregarines, haemosporidia and piroplasms, with more than 6000 named species. Three of these subgroups, the coccidia, hemosporidia, and piroplasms, contain parasites that cause important diseases of humans and animals worldwide. All of them have complex life cycles involving a switch between asexual and sexual reproduction, which is key to their development. Fertilization (i.e., fusion of female and male cells) results in the formation of a zygote that undergoes meiosis, forming a new generation of asexual stages. In eukaryotes, sexual reproduction is the predominant mode of recombination and segregation of DNA. Sex is well documented in many protist groups, and together with meiosis, is frequently linked with transmission to new hosts. Apicomplexan sexual stages constitute a bottleneck in the life cycle of these parasites, as they are obligatory for the development of new transmissible stages. Consequently, the sexual stages represent attractive targets for vaccination. Detailed understanding of apicomplexan sexual biology will pave the way for the design and implementation of effective transmission-blocking strategies for parasite control. This article reviews the current knowledge on the sexual development of Apicomplexa and the progress in transmission-blocking vaccines for their control, their advantages and limitations and outstanding questions for the future.

Progression of asexual to sexual stages of Cystoisospora suis in a host cell-free environment as a model for Coccidia.

Coccidia display a characteristic life cycle, where the parasites switch between asexual and sexual development, resulting in an environmental stage, the oocyst. The entero-pathogenic Cystoisospora suis, a coccidian parasite of swine and close relative to Toxoplasma gondii, undergoes development in one host-cycle. Despite the well-described intracellular development of Coccidia, the C. suis life cycle can progress in an in vitro, host cell-free system after initial intracellular development of merozoites. A novel host cell-free cultivation method was developed by transferring purified merozoites from cell culture supernatant (dpi 6) to culture medium and incubating them for 5 days to induce their progression to sexually differentiated stages. The development of sexual stages in the absence of host cells was verified by morphological studies, flow cytometry and the transcription analysis of three genes linked to sexual stages (HAP2, OWP and TyRP). The host cell-free culture permits the sexual development (and with this, the complete life cycle progression from sporozoites to oocysts) of C. suis in vitro and provides a new tool for detailed research on the development of C. suis and possibly other Coccidia. This will also be useful for the evaluation of novel drug or vaccine targets in these parasites.

Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi.

BACKGROUND: Chagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes. METHODOLOGY/PRINCIPAL FINDINGS: To obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication. CONCLUSION/SIGNIFICANCE: This research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action.

Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication.

Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi.

Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.

Characterization of Cystoisospora suis sexual stages in vitro.

BACKGROUND: The porcine coccidium Cystoisospora suis is characterized by a complex life-cycle during which asexual multiplication is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Genes related to the sexual stages and cell cycle progression were previously identified in related Apicomplexa. Dynein light chain type 1 and male gamete fusion factor HAP2 are restricted to microgametes. Tyrosine-rich proteins and oocyst wall proteins are a part of the oocyst wall. The Rad51/Dmc1-like protein and Nima-related protein kinases are associated with the cell cycle and fertilization process. Here, the sexual stages of C. suis were characterized in vitro morphologically and for temporal expression changes of the mentioned genes to gain insight into this poorly known phase of coccidian development. METHODS: Sexual stages of C. suis developing in vitro in porcine intestinal epithelial cells were examined by light and electron microscopy. The transcriptional levels of genes related to merozoite multiplication and sexual development were evaluated by quantitative real-time PCR at different time points of cultivation. Transcription levels were compared for parasites in culture supernatants at 6-9 days of cultivation (doc) and intracellular parasites at 6-15 doc. RESULTS: Sexual stage of C. suis was detected during 8-11 doc in vitro. Microgamonts (16.8 ± 0.9 µm) and macrogamonts (16.6 ± 1.1 µm) are very similar in shape and size. Microgametes had a round body (3.5 ± 0.5 µm) and two flagella (11.2 ± 0.5 µm). Macrogametes were spherical with a diameter of 12.1 ± 0.5 µm. Merozoite gene transcription peaked on 10 doc and then declined. Genes related to the sexual stages and cell cycle showed an upregulation with a peak on 13 doc, after which they declined. CONCLUSIONS: The present study linked gene expression changes to the detailed morphological description of C. suis sexual development in vitro, including fertilization, meiosis and oocyst formation in this unique model for coccidian parasites. Following this process at the cellular and molecular level will elucidate details on potential bottlenecks of C. suis development (applicable for coccidian parasites in general) which could be exploited as a novel target for control.

Characterization and functionality of two members of the SPFH protein superfamily, prohibitin 1 and 2 in Leishmania major.

BACKGROUND: Leishmaniasis, a disease caused by parasites of the genus Leishmania, infects roughly 12 million people worldwide, with about two million new cases per year. Prohibitins (PHBs) are highly conserved proteins belonging to the stomatin-prohibitin flotillin-HflC/K (SPFH) protein superfamily. In this study, we examine the potential functions of two proteins of Leishmania major, PHB1 and PHB2, as well as how they might help protect the protozoan against oxidative stress. RESULTS: By immunolocalization in the parasite cells, PHB1 appeared in the mitochondria and plasma membrane, whereas PHB2 was grouped in the nucleus. When Leishmania cells were under oxidative stress, PHB1 migrates towards the plasma membrane and the paraxial rod, while PHB2 remained in the nucleus and near the kinetoplast. PHB1 presented higher mRNA levels than PHB2 in the amastigotes and the infective metacyclic forms. The mRNA expression of both prohibitins was affected by the presence of the Fe3+ ion. PHBs inhibited the Fenton reaction, where reactive oxygen species could nick DNA, implying that they play a crucial role in controlling oxidative stress. CONCLUSIONS: Here, we propose that PHBs may help to protect membranes and DNA against superoxide ions, thus enhancing the survival capacity of the protozoan by controlling the ROS within the phagosome of the macrophages where the parasite multiplies.

Detection of Weakly Expressed Trypanosoma cruzi Membrane Proteins Using High-Performance Probes.

Membrane proteins in trypanosomatids are, in general, weakly expressed and confirmation of their subcellular localization frequently requires their overexpression with epitope tags. However, overexpression can lead to mislocalization of the probes. Viswanathan et al. (Nat. Methods, 2015, 12:568) described high performance tags for localization of weakly expressed proteins. We report here the use of these protein tags, named "spaghetti monster," for CRISPR/Cas9-mediated C-terminal endogenous tagging of Trypanosoma cruzi to localize two weakly expressed transient receptor potential channels to acidic compartments. The results indicate that this method will improve the detection of membrane proteins in T. cruzi.

An Intracellular Ammonium Transporter Is Necessary for Replication, Differentiation, and Resistance to Starvation and Osmotic Stress in Trypanosoma cruzi.

Trypanosoma cruzi, the etiologic agent of Chagas disease, undergoes drastic metabolic changes when it transits between a vector and mammalian hosts. Amino acid catabolism leads to the production of ammonium (NH4+), which needs to be detoxified. However, T. cruzi does not possess a urea cycle, and it is unknown how intracellular levels of ammonium are controlled. In this work, we identified an intracellular ammonium transporter of T. cruzi (TcAMT) that localizes to acidic compartments (reservosomes, lysosomes). TcAMT has 11 transmembrane domains and possesses all conserved and functionally important amino acid residues that form the pore in other ammonium transporters. Functional expression in Xenopus oocytes followed by a two-electrode voltage clamp showed an inward current that is NH4+ dependent at a resting membrane potential (Vh ) lower than -120 mV and is not pH dependent, suggesting that TcAMT is not an NH4+/H+ cotransporter but an NH4+ or NH3/H+ transporter. Ablation of TcAMT by clustered regularly interspaced short palindromic repeat analysis with Cas9 (CRISPR-Cas9) resulted in significant defects in epimastigote and amastigote replication, differentiation, and resistance to starvation and osmotic stress. IMPORTANCETrypanosoma cruzi is an important human and animal pathogen and the etiologic agent of Chagas disease. The parasite undergoes drastic changes in its metabolism during its life cycle. Amino acid consumption becomes important in the infective stages and leads to the production of ammonia (NH3), which needs to be detoxified. We report here the identification of an ammonium (NH4+) transporter that localizes to acidic compartments and is important for replication, differentiation, and resistance to starvation and osmotic stress.

A Riboswitch-based Inducible Gene Expression System for Trypanosoma brucei.

Generation of conditional mutants in Trypanosoma brucei can be done by the use of RNA interference (RNAi). However, RNAi frequently produces off target effects. Here, we present an alternative strategy in which the glmS ribozyme is inserted in the C-terminal region of one allele of a GOI and effectively knocks it down in response to the presence of glucosamine in the culture medium. Using several endogenous genes, we show that the glmS ribozyme cleaves the mRNA in vivo leading to reduction in mRNA and protein expression following glucosamine treatment in both T. brucei procyclic and bloodstream forms. Glucosamine-induced ribozyme activation can be rapidly reversed by removing the inducer. In summary, the glmS ribozyme could be used as a tool to study essential genes in T. brucei.

Functionalized immunostimulating complexes with protein A via lipid vinyl sulfones to deliver cancer drugs to trastuzumab-resistant HER2-overexpressing breast cancer cells.

BACKGROUND: Around 20%-30% of breast cancers overexpress the proto-oncogene human epidermal growth receptor 2 (HER2), and they are characterized by being very invasive. Therefore, many current studies are focused on testing new therapies against tumors that overexpress this receptor. In particular, there exists major interest in new strategies to fight breast cancer resistant to trastuzumab (Tmab), a humanized antibody that binds specifically to HER2 interfering with its mitogenic signaling. Our team has previously developed immunostimulating complexes (ISCOMs) as nanocapsules functionalized with lipid vinyl sulfones, which can incorporate protein A and bind to G immunoglobulins that makes them very flexible nanocarriers. METHODS AND RESULTS: The aim of this in vitro study was to synthesize and evaluate a drug delivery system based on protein A-functionalized ISCOMs to target HER2-overexpressing cells. We describe the preparation of ISCOMs, the loading with the drugs doxorubicin and paclitaxel, the binding of ISCOMs to alkyl vinyl sulfone-protein A, the coupling of Tmab, and the evaluation in both HER2-overexpressing breast cancer cells (HCC1954) and non-overexpressing cells (MCF-7) by flow cytometry and fluorescence microscopy. Results show that the uptake is dependent on the level of overexpression of HER2, and the analysis of the cell viability reveals that targeted drugs are selective toward HCC1954, whereas MCF-7 cells remain unaffected. CONCLUSION: Protein A-functionalized ISCOMs are versatile carriers that can be coupled to antibodies that act as targeting agents to deliver drugs. When coupling to Tmab and loading with paclitaxel or doxorubicin, they become efficient vehicles for the selective delivery of the drug to Tmab-resistant HER2-overexpressing breast cancer cells. These nanoparticles may pave the way for the development of novel therapies for poor prognosis resistant patients.

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo, Determined by Immunolabelling.

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.

The shell organic matrix of the crossed lamellar queen conch shell (Strombus gigas).

In molluscs, the shell organic matrix comprises a large set of biomineral-occluded proteins, glycoproteins and polysaccharides that are secreted by the calcifying mantle epithelium, and are supposed to display several functions related to the synthesis of the shell. In the present paper, we have characterized biochemically the shell matrix associated to the crossed-lamellar structure of the giant queen conch Strombus gigas. The acid-soluble (ASM) and acid-insoluble (AIM) matrices represent an extremely minor fraction of the shell. Both are constituted of polydisperse and of few discrete proteins among which three fractions, obtained by preparative SDS-PAGE and named 1P3, 2P3 and 3P3, are dominant and were further characterized. Compared to other matrices, the acid-soluble matrix is weakly glycosylated (3%) and among the discrete components, only 3P3 seems noticeably glycosylated. The monosaccharide composition of the ASM shows that mannose represents the main monosaccharide. To our knowledge, this is the first report of a high ratio of this sugar in a skeletal matrix. Furthermore, the ASM interacts with the in vitro crystallization of calcium carbonate, but this interaction is moderate. It differs from that of the isolated 1P3 fraction but is similar to that of the 2P3 and 3P3 fractions. At last, antibodies developed from the 3P3 fraction were used to localize this fraction within the shell by immunogold. This study is the first one aiming at characterizing the organic matrix associated to the crossed-lamellar structure of the queen conch shell.

Intranasal immunization of lambs with serine/threonine phosphatase 2A against gastrointestinal nematodes.

Seven 3-month-old, female, helminth-free lambs were immunized intranasally with three doses (1 mg total) of a recombinant part of the catalytic region of the serine/threonine phosphatase 2A (PP2Ar) (group 1 [G1]). In addition, four lambs were used as an adjuvant control group (G2), four as unimmunized, infected controls (G3), and four as unimmunized, uninfected controls (G4). Fifteen days after the last immunization, lambs from G1, G2, and G3 were challenged with 10,000 larval stage 3 (L3) organisms in a plurispecific nematode infection composed of ca. 40% Trichostrongylus colubriformis, 40% Haemonchus contortus, and 20% Teladorsagia circumcincta. All the lambs were clinically monitored throughout the experiment. Parasitological (fecal egg output and immunological response), biopathological (packed-cell volume and leukocyte and eosinophil counts), and zootechnical (live-weight gain) analyses were conducted. On day 105 of the experiment, all the animals were slaughtered and the adult worm population in their abomasa examined. Intranasal administration of PP2Ar with bacterial walls as an adjuvant elicited a strong immune response in the immunized lambs, as evidenced by their humoral immune response. Immunized animals and animals receiving the adjuvant shed significantly (P < 0.001) fewer numbers of parasites' eggs in their feces. The immunization significantly reduced the helminth burden in the abomasa by the end of the experiment (>68%), protection being provided against both Haemonchus and Teladorsagia. Live-weight gain in the immunized lambs was similar to that in the uninfected controls versus the infected or adjuvanted animal groups. Our results suggest that heterologous immunization of ruminants by intranasal administration may be efficacious in the struggle to control gastrointestinal helminths in these livestock.

Functionalization of immunostimulating complexes (ISCOMs) with lipid vinyl sulfones and their application in immunological techniques and therapy.

BACKGROUND: Immunostimulating complexes (ISCOM)-type nanocapsules have been functionalized with lipid vinyl sulfones that anchor to them via the hydrophobic zone of their structure and can be charged with pharmacologically active molecules or macromolecules. These functionalized nanocapsules can incorporate protein A and bind to G immunoglobulins (IgGs) to make vehicles directed at the surface antigens of infectious agents, tumor cells, or receptor cells and deliver the encapsulated molecules in a highly specific way. They may be of particular use in pharmacological treatments with highly toxic molecules that should not be used in solution whenever it can be avoided. When bound to antibodies they can be used in biological processes that require the delivery or presentation of macromolecules to certain specific cells, in immunization processes for instance, or in diagnostic immunological techniques, as they are able to transport both the secondary antibodies and the reaction labels. METHODS AND RESULTS: We describe the preparation of ISCOMs, the binding to the ISCOMS of newly synthesized compounds composed of chain alkyl vinyl sulfone, and the subsequent binding of the vinyl-sulfone compounds to IgGs. Within this context, a compound deriving from cholesterol functionalized with vinyl sulfone and used together with cholesterol in varying proportions has been linked to the structure of the ISCOMs and bound to protein A-IgG. This functionalization in no way altered the form or structure of the ISCOMs and allowed the nanocapsules carrying the specific IgGs to bind to forms of Trypanosoma cruzi against which antibodies had been developed. The fact that functionalized ISCOMs containing antibodies could deliver actinomycin D directly to the parasite meant that the effective dose of the antibiotic could be reduced very significantly. CONCLUSION: We have developed ISCOM-type nanocapsules functionalized with lipid vinyl sulfone capable of anchoring to the surface of functional IgGs, which favors the recognition and transport of these nanocapsules precisely to certain kinds of cell.

Differential expression and characterization of a member of the mucin-associated surface protein family secreted by Trypanosoma cruzi.

We describe the characterization, purification, expression, and location of a 52-kDa protein secreted during interaction between the metacyclic form of Trypanosoma cruzi and its target host cell. The protein, which we have named MASP52, belongs to the family of mucin-associated surface proteins (MASPs). The highest levels of expression of both the protein and mRNA occur during the metacyclic and bloodstream trypomastigote stages, the forms that infect the vertebrate host cells. The protein is located in the plasma membrane and in the flagellar pockets of the epimastigote, metacyclic, and trypomastigote forms and is secreted into the medium at the point of contact between the parasite and the cell membrane, as well as into the host-cell cytosol during the amastigote stage. IgG antibodies specific against a synthetic peptide corresponding to the catalytic zone of MASP52 significantly reduce the parasite's capacity to infect the host cells. Furthermore, when the protein is adsorbed onto inert particles of bentonite and incubated with a nonphagocytic cell culture, the particles are able to induce endocytosis in the cells, which seems to demonstrate that MASP52 plays a role in a process whereby the trypomastigote forms of the parasite invade the host cell.