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F Seeber

Publications and source records attributed to F Seeber.

At least 19 recordsLinked to original sources
PubMed2001

Toxoplasma gondii and MHC-restricted antigen presentation: on degradation, transport and modulation.

Resistance against Toxoplasma gondii, an obligate intracellular protozoan parasite surrounded by a parasitophorous vacuolar membrane, is mediated by the cellular arm of the immune system, namely CD8+ and CD4+ T cells. Thus, priming and activation of these cells by presentation of antigenic peptides in the context of major histocompatibility complex class I and class II molecules have to take place. This is despite the fact that the vacuolar membrane avoids fusion with the endocytic compartment and acts like a molecular sieve, restricting passive diffusion of larger molecules. This raises several cell biological and immunological questions which will be discussed in this review in the context of our current knowledge about major histocompatibility complex-restricted antigen presentation in other systems: (1) By which pathways are parasite-derived antigens presented to T cells? (2) Has the parasite evolved mechanisms to interfere with major histocompatibility complex-restricted antigen presentation in order to avoid immune recognition? (3) To what extent and by which mechanism is antigenic material, originating from the parasite, able to pass through the vacuolar membrane into the cytosol of the infected cell and is it then accessible to the antigen presentation machinery of the infected cell? (4) What are the actual antigen-presenting cells which prime specific T cells in lymphoid organs? An understanding of these mechanisms will not only provide new insights into the pathogenesis of Toxoplasma gondii and possibly other intravacuolar parasites, but will also improve vaccination strategies.

Animals↗
PubMed2000

The plastid of Toxoplasma gondii is divided by association with the centrosomes.

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

Aniline Compounds↗
PubMed2000

Apicomplexan parasites possess distinct nuclear-encoded, but apicoplast-localized, plant-type ferredoxin-NADP+ reductase and ferredoxin.

In searching for nuclear-encoded, apicoplast-localized proteins we have cloned ferredoxin-NADP(+) reductase from Toxoplasma gondii and a [2Fe-2S] ferredoxin from Plasmodium falciparum. This chloroplast-localized redox system has been extensively studied in photosynthetic organisms and is responsible for the electron transfer from photosystem I to NADP+. Besides this light-dependent reaction in nonphotosynthetic plastids (e.g. from roots), electrons can also flow in the reverse direction, from NADPH to ferredoxin, which then serves as an important reductant for various plastid-localized enzymes. These plastids possess related, but distinct, ferredoxin-NADP+ reductase and ferredoxin isoforms for this purpose. We provide phylogenetic evidence that the T. gondii reductase is similar to such nonphotosynthetic isoforms. Both the P. falciparum [2Fe-2S] ferredoxin and the T. gondii ferredoxin-NADP+ reductase possess an N-terminal bipartite transit peptide domain typical for apicoplast-localized proteins. The recombinant proteins were obtained in active form, and antibodies raised against the reductase recognized two bands on Western blots of T. gondii tachyzoite lysates, indicative of the unprocessed and native form, respectively. We propose that the role of this redox system is to provide reduced ferredoxin, which might then be used for fatty acid desaturation or other biosynthetic processes yet to be defined. Thus, the interaction of these two proteins offers an attractive target for drug intervention.

Amino Acid Sequence↗
PubMed2000

An enzyme-release assay for the assessment of the lytic activities of complement or antimicrobial peptides on extracellular Toxoplasma gondii.

A method is described which allows the evaluation of the membrane lytic activity of either complement or antimicrobial peptides against the extracellular stage of the human protozoan parasite Toxoplasma gondii. The assay is based on lacZ transgenic parasites, determining the activity of released cytoplasmic beta-galactosidase into the culture supernatant upon membrane disintegration. This method was used to evaluate the lytic activities of (i) complement which is a natural defense mechanism in infected hosts against extracellular parasites, and (ii) antimicrobial peptides which have not been evaluated against T. gondii before. The results show that the assay provides a simple and convenient way to assess the membrane lytic activity of such compounds and that T. gondii, like other protozoan parasites, is vulnerable to the membrane-lytic effect of antimicrobial peptides.

Amino Acid Sequence↗
PubMed1998

Analysis of Toxoplasma gondii stably transfected with a transmembrane variant of its major surface protein, SAG1.

We have genetically engineered Toxoplasma gondii so that its major surface antigen SAG1 is anchored by a human transmembrane domain (SAG1-TM) instead of its natural GPI anchor (SAG1-GPI) in order to initiate studies to address the function of this protein anchor in parasitic protozoa as well as to get insights into the functional role of SAG1. Our results show that SAG1-TM is correctly folded (at least as judged by the presence of conformationally dependent epitopes) and targeted to the surface of the parasite, indicating that the GPI anchor does not determine its localization nor overall three-dimensional structure. No significant difference was seen in any aspect of the growth of the SAG1-TM mutant. However, compared to the natural SAG1-GPI, SAG1-TM does not form strong associations with itself and/or other molecules in high molecular weight complexes suggesting that allowing such complexes to form may be one role of the GPI anchor. The in vitro half-life of SAG1-TM of extracellular parasites is significantly lower than that of SAG1-GPI suggesting a stabilizing function of the glycolipid anchor against degradation and/or membrane release. Antibodies to SAG1 are shed from SAG1-TM parasites as they invade, just as they are stripped from SAG1-GPI bearing parasites. The stripping, therefore, is unlikely to be driven by the action of lipases.

Amino Acid Sequence↗
PubMed1997

Localization of T and B cell stimulating domains of the immunodominant 33-kDa protein of Onchocerca volvulus (Ov33).

The localization of T and B cell epitopes on a well characterized 33-kDa protein of the filarial nematode Onchocerca volvulus (Ov33) was studied using peripheral blood mononuclear cells (PBMC) and sera from a total of 52 onchocerciasis patients with the generalized form of infection. A proportion of the PBMC samples proliferated in response to recombinant Ov33-GST fusion protein and to fusion free Ov33-6xHis. Proliferative responses of patient PBMC to seven truncated Ov33-6xHis polypeptides and to three synthetic peptides revealed at least one major and two minor T cell epitopes in the protein. The dominant T cell stimulating domain was localized between amino acids 113 and 143. ELISA studies with the Ov33-GST fusion protein revealed that patient sera contained Ov33-specific IgG1, IgG4, IgE, and IgM antibodies. Analysis of the IgG4 response with 10 truncated Ov33 polypeptides identified four B cell stimulating domains in the N-terminal, central, and C-terminal region of the molecule. The B cell domain recognized by the majority of sera was localized between amino acids 113 and 143. The data indicate that this region of the protein is the major T and B cell stimulating domain of Ov33 and might be relevant for vaccine development and for improved immunodiagnosis of onchocerciasis.

Adolescent↗
PubMed1997

Differences in cytokine responses to Onchocerca volvulus extract and recombinant Ov33 and OvL3-1 proteins in exposed subjects with various parasitologic and clinical states.

Subjects with generalized onchocerciasis (GEN), with the sowdah form, and with exposure but without onchocerciasis (endemic normal/putatively immune; EN/PI) were studied for cytokine responses to Onchocerca volvulus extract (OvAg) and recombinant Ov33 and OvL3-1 proteins. Higher levels of cytokines were produced in response to OvAgs in sowdah and EN/PI than in GEN subjects. Peripheral blood mononuclear cells did not produce interferon-gamma in response to antigens. OvAg induced interleukin (IL)-5, IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and soluble IL-2 receptor. EN/PI and sowdah persons produced significantly more IL-5 and IL-2 than GEN subjects, and EN/PI subjects had significantly higher GM-CSF levels than GEN persons. The low IL-5 and GM-CSF levels in GEN subjects were increased by addition of exogenous IL-2. Ov33 and OvL3-1 stimulated production of IL-10 and less IL-5 and IL-2. The study groups did not show a strict Th2-like cytokine response.

Animals↗
PubMed1997

Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro.

A microtiter assay for drug evaluation has been developed with a strain of Toxoplasma gondii that expresses bacterial beta-galactosidase. By using chlorophenol red-beta-D-galactopyranoside (CPRG) as the substrate for beta-galactosidase, the efficacy of a drug against the parasite can be determined with a colorimetric readout. Drugs known to have activity against T. gondii (specifically, pyrimethamine, sulfadiazine, atovaquone, and clindamycin) were tested, and efficacies were determined by CPRG cleavage. The 50% inhibitory concentrations determined by the CPRG-based colorimetric assay were similar to those determined by the traditional radiolabelled uracil incorporation assay. Since CPRG is nontoxic to the parasite, viable drug-treated parasites can be obtained at the conclusion of the assay for further evaluation if desired. This assay provides a high-throughput and nonradioactive alternative for the identification of anti-T. gondii compounds.

Animals↗
PubMed1997

Reactivation of chronic toxoplasmosis: is there a link to strain-specific differences in the parasite?

The protozoan parasite Toxoplasma gondii comprises three clonal lineages that are associated with the clinical outcome in infected individuals. Whereas group C strains are mainly found in animals, group A and B strains are associated with human disease (Howe and Sibley, 1995). An increased level of transcripts of the tachyzoite-specifically expressed gene SAG1 could be identified in group A T. gondii strains compared to group B strains. Since SAG1-mediated host-cell invasion seems to be important for parasite replication, the observed higher replication rate in group A T. gondii strains might explain the association with clinically overt symptoms at the acute stage in patients who are infected with this group of parasite strains. The presence of external stress factors, such as interferon-gamma (IFN-gamma)-mediated nitric oxide (NO) formation has been identified to stabilize the cyst stage, most likely by activation of promoter(s) which drive the expression of genes encoding bradyzoite-specific antigens. Reactivation of chronic toxoplasmosis thus might occur in the absence of external stress factors, as has been observed in AIDS patients with decreases levels of IFN-gamma. Since group B T. gondii strains might form more cysts in infected individuals due to an increased potential to convert into bradyzoites, reactivation with resulting toxoplasmic encephalitis could be a more common event in those AIDS patients who were infected with persistent cysts of this group of parasite strains.

Animals↗
PubMed1996

Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii.

We have developed several protocols for the use of beta-galactosidase (betaGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent betaGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chorophenol red-beta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing betaGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of betaGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites.

Animals↗
PubMed1995

Molecular cloning and characterization of the filarial LIM domain proteins AvL3-1 and OvL3-1.

A full-length cDNA of the filarial nematode Acanthocheilonema viteae was isolated from a cDNA library of female worms, using a partial cDNA of the OvL3-1 gene of Onchocerca volvulus as a probe. The AvL3-1 cDNA contained an open reading frame which encoded for a protein with a theoretical molecular weight of 64 kDa. The deduced protein contained a predicted signal sequence, a short repetitive motive of unknown function, and three LIM domains. The structure of the LIM domains was identical to those of zyxin, a cytoskeleton-associated protein of chicken fibroblasts, suggesting that AvL3-1 has a similar role in filarial nematodes. The sequence information was used to isolate the homologous cDNA of O. volvulus by PCR from a cDNA library of female O. volvulus, which showed an overall identity of 76.9% to AvL3-1 on the protein level. AvL3-1 was expressed in Escherichia coli and the affinity-purified fusion free protein was used to immunized jirds (Meriones unguiculatus). Immunization together with the adjuvant STP or with Freund's adjuvant induced IgG and IgM antibody responses, but no significant protection against a challenge infection with L3 of A. viteae, compared to appropriate control groups.

Amino Acid Sequence↗
PubMed1995

Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii.

This report describes the use of restriction enzyme-mediated integration (REMI) to increase the transformation frequency and allow co-transfection of several unselected constructs under the selection of a single selectable marker. We found that while BamHI (the enzyme used to originally demonstrate REMI (Schiestl, R.H. and Petes, T.D. (1991) Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Nati. Acad. Sci. USA 88, 7585-7589) increased the number of transformants by 2-5-fold over the control without added enzyme, NotI proved to be a further 29-46-times more effective in enhancing stable transformation. This simple technique was used in the transformation of three non-selective markers (two modified membrane proteins and beta-galactosidase) with a selectable construct expressing chloramphenicol acetyltransferase. Following chloramphenicol selection, four out of ten independent transformants stably acquired all four constructs with at least two expressing all four genes at the protein level. These results demonstrate that REMI may be used in the efficient stable transformation and co-transfection of this and perhaps other protozoan parasites.

Animals↗
PubMed1993

Characterization of a recombinant T cell and B cell reactive polypeptide of Onchocerca volvulus.

To identify potentially protective Ag of the filarial nematode Onchocerca volvulus on the molecular level we screened a cDNA library of O. volvulus with a human serum raised against radiation-attenuated infective larvae of O. volvulus. A cDNA clone of 218 bp (OvL3-1) was selected for further studies. It was expressed in Escherichia coli and affinity purified recombinant polypeptide was tested for its ability to stimulate in vitro PBMC from African onchocerciasis patients and PBMC from chimpanzees experimentally infected with O. volvulus. An enhanced cell proliferation by PBMC was observed in many patients after stimulation with the recombinant OvL3-1 polypeptide. In addition, some patients' PBMC responded to OvL3-1 stimulation with enhanced IL-2 production. Infected chimpanzees also showed an increase in T cell proliferation. Onchocerciasis patients had variable levels of specific antibodies directed to the recombinant polypeptide when sera were tested by ELISA. A mAb directed against the recombinant protein located the native target Ag in the muscles of the adult worm. The molecular mass of native OvL3-1 was found to be 50 kDa on immunoblots. Polymerase chain reaction analysis of RNA from different life stages of the parasite showed that OvL3-1 is transcribed in all parasite stages within the mammalian host. A homologous gene is also present in other filarial parasites. The protein corresponding to OvL3-1, therefore, represents an immunogen present during the whole life-span of the parasite, and because of its B and T cell stimulatory properties, it may be a candidate for a protective Ag in human filariasis.

Adult↗