Biomedical subjects

Boris Striepen

Publications and source records attributed to Boris Striepen.

8 recordsLinked to original sources

PubMed2025

Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection.

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium, a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual life cycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection-selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

Cryptosporidium parvum↗

PubMed2003

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development.

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.

Amino Acid Sequence↗

PubMed2003

High-throughput growth assay for Toxoplasma gondii using yellow fluorescent protein.

A high-throughput growth assay for the protozoan parasite Toxoplasma gondii was developed based on a highly fluorescent transgenic parasite line. These parasites are stably transfected with a tandem yellow fluorescent protein (YFP) and are 1,000 times more fluorescent than the wild type. Parasites were inoculated in optical-bottom 384-well culture plates containing a confluent monolayer of host cells, and growth was monitored by using a fluorescence plate reader. The signal was linearly correlated with parasite numbers over a wide array. Direct comparison of the YFP growth assay with the beta-galactosidase growth assay by using parasites expressing both reporters demonstrated that the assays' sensitivities were comparable but that the accuracy of the YFP assay was higher, especially at higher numbers of parasites per well. Determination of the 50%-inhibitory concentrations of three known growth-inhibiting drugs (cytochalasin D, pyrimethamine, and clindamycin) resulted in values comparable to published data. The delayed parasite death kinetics of clindamycin could be measured without modification of the assay, making this assay very versatile. Additionally, the temperature-dependent effect of pyrimethamine was assayed in both wild-type and engineered drug-resistant parasites. Lastly, the development of mycophenolic acid resistance after transfection of a resistance gene in T. gondii was followed. In conclusion, the YFP growth assay limits pipetting steps to a minimum, is highly versatile and amendable to automation, and should enable rapid screening of compounds to fulfill the need for more efficient and less toxic antiparasitic drugs.

Animals↗

PubMed2002

Isolation and characterization of TgVP1, a type I vacuolar H+-translocating pyrophosphatase from Toxoplasma gondii. The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor.

Here we report the isolation and characterization of a type I vacuolar-type H(+)-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K(+)-activated pyrophosphate (PP(i)) hydrolysis and PP(i)-energized H(+) translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 microm had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I(50) for the inhibition of TgVP1 activity in vitro (0.9 microm) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.

Amino Acid Sequence↗

PubMed2002

Genetic complementation in apicomplexan parasites.

A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from epsilon-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.

Animals↗

PubMed2002

Mining the Plasmodium genome database to define organellar function: what does the apicoplast do?

Apicomplexan species constitute a diverse group of parasitic protozoa, which are responsible for a wide range of diseases in many organisms. Despite differences in the diseases they cause, these parasites share an underlying biology, from the genetic controls used to differentiate through the complex parasite life cycle, to the basic biochemical pathways employed for intracellular survival, to the distinctive cell biology necessary for host cell attachment and invasion. Different parasites lend themselves to the study of different aspects of parasite biology: Eimeria for biochemical studies, Toxoplasma for molecular genetic and cell biological investigation, etc. The Plasmodium falciparum Genome Project contributes the first large-scale genomic sequence for an apicomplexan parasite. The Plasmodium Genome Database (http://PlasmoDB.org) has been designed to permit individual investigators to ask their own questions, even prior to formal release of the reference P. falciparum genome sequence. As a case in point, PlasmoDB has been exploited to identify metabolic pathways associated with the apicomplexan plastid, or 'apicoplast' - an essential organelle derived by secondary endosymbiosis of an alga, and retention of the algal plastid.

Animals↗

PubMed2002

Expression variance, biochemical and immunological properties of Toxoplasma gondii dense granule protein GRA7.

During intracellular stay, Toxoplasma gondii secretes dense granule proteins (GRA) which remodel the parasitophorous vacuole and are considered functional in parasite-host interrelation. Comparative analysis of parasites from mouse-virulent strain BK and an in vitro attenuated variant revealed that the level of GRA7 expression correlates with T. gondii virulence: proteome analysis and quantitation by immunoblot demonstrated a massive decrease in GRA7 steady-state synthesis parallel to the loss of virulence. Properties of GRA7 that are pertinent to its membrane targeting and to GRA7-directed immune resistance were studied in detail. GRA7 is exclusively membrane-associated in both parasites and infected host cells as demonstrated by subcellular fractionations. Triton X-114 partitioning of isolated parasites substantiated that GRA7 is an integral membrane protein, the hydrophobic stretch from amino acid 181 to 202 providing a possible membrane anchor. A fraction enriched for membranous material from infected host cells contained additional forms of GRA7 with reduced mobility in gel electrophoresis, indicating that the protein is modified after exocytosis from the parasite. By flow cytometric analysis, GRA7 was detected on the surface of intact host cells. An intracellular origin of surface-associated GRA7 seems likely since GRA7 released from extracellular parasites failed to label the host cell surface. Consistent with a role at a parasite-host interface, GRA7 proved to be a target antigen of the intracerebral immune response as evidenced by the presence of GRA7-specific antibodies in mouse cerebrospinal fluid during chronic infection.

Animals↗

PubMed2002

Daughter cell assembly in the protozoan parasite Toxoplasma gondii.

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii. Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites.

Animals↗