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P Overath

Publications and source records attributed to P Overath.

At least 19 recordsLinked to original sources
PubMed2001

The surface structure of trypanosomes in relation to their molecular phylogeny.

Molecular phylogenetic analysis using genes coding for ribosomal RNA and proteins suggests that trypanosomes are monophyletic. Salivarian trypanosomes showing antigenic variation of the variant surface glycoprotein (VSG) diverged from non-Salivarian trypanosomes some 200-300 million years ago. Representatives of the non-Salivarian group, the mammalian parasite, Trypanosoma cruzi, and the fresh-water fish trypanosome, T. carassii, are characterised by surfaces dominated by carbohydrate-rich mucin-like glycoproteins, which are not subject to antigenetic variation. It is suggested that this latter surface structure is typical for non-Salivarian trypanosomes as well as members of the other Kinetoplastid suborder, the Bodonina. This would imply that at some point in time in the evolution of the Salivaria the highly abundant and comparatively poorly immunogenetic mucin-like molecules must have been replaced for equally abundant but highly immunogenic VSG-like molecules. While the selective advantage for such a unique transition is difficult to imagine, the subsequent diversification of VSG genes/molecules may have been comparatively straightforward because even the most limited form of antigenic variation would have extended the duration of infection in the vertebrate and thus would have increased the chance for transfer to the vector.

Animals↗
PubMed2000

Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes.

This report describes the construction of a DNA cassette for integration into a genomic small sub-unit rRNA locus of Leishmania mexicana by homologous recombination. Reporter genes encoding beta-galactosidase or green fluorescent protein and the gene conferring hygromycin resistance were integrated downstream of a RNA polymerase I-driven rRNA promoter. To ensure high expression of the marker proteins in the intracellular, amastigote stage, transgene coding sequences were followed by the intergenic region of the L. mexicana cysteine proteinase B 2.8 gene which provides processing signals required for high level expression in this life-cycle stage. Integration of the DNA cassette was also efficiently obtained in L. major. We show that either beta-galactosidase or the green fluorescent protein were abundantly, stably and uniformly expressed in promastigotes and amastigotes of both Leishmania sp. The transgenic lines allow parasite detection at high sensitivity in the tissues of infected mice and will be useful to follow infections in macrophages in culture and in animal hosts.

Animals↗
PubMed2000

Isolation and characterization of glycosylphosphatidylinositol-anchored, mucin-like surface glycoproteins from bloodstream forms of the freshwater-fish parasite Trypanosoma carassii.

Wild and farmed freshwater fishes are widely and heavily parasitized by the haemoflagellate Trypanosoma carassii. In contrast, common carp, a natural host, can effectively control experimental infections by the production of specific anti-parasite antibodies. In this study we have identified and partially characterized mucin-like glycoproteins which are expressed in high abundance [(6. 0+/-1.7)x10(6) molecules.cell(-1)] at the surface of the bloodstream trypomastigote stage of the parasite. The polypeptide backbone of these glycoproteins is dominated by threonine, glycine, serine, alanine, valine and proline residues, and is modified at its C-terminus by a glycosylphosphatidylinositol membrane anchor. On average, each polypeptide carries carbohydrate chains composed of about 200 monosaccharide units (galactose, N-acetylglucosamine, xylose, sialic acid, fucose, mannose and arabinose), which are most probably O-linked to hydroxy amino acids. The mucin-like molecules are the target of the fish's humoral immune response, but do not undergo antigenic variation akin to that observed for the variant surface glycoprotein in salivarian trypanosomes. The results are discussed with reference to the differences between natural and experimental infections, and in relation to the recently delineated molecular phylogeny of trypanosomes.

Amino Acids↗
PubMed2000

Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants.

A mixture of well-defined recombinant antigens together with an adjuvant that preferentially stimulates specific gamma interferon (IFN-gamma)-secreting helper type 1 CD4(+) T cells (Th1 cells) presents a rational option for a vaccine against leishmaniasis. The potential of this approach was investigated in murine infections with Leishmania mexicana, which are characterized by the absence of a parasite-specific Th1 response and uncontrolled parasite proliferation. A mixture of three antigens (glycoprotein 63, cysteine proteinases, and a membrane-bound acid phosphatase), which are all expressed in amastigotes, the mammalian stage of the parasite, were used for the immunization of C57BL/6 mice in combination with six adjuvants (interleukin 12 [IL-12], Detox, 4'-monophosphoryl lipid A, QS-21, Mycobacterium bovis BCG, and Corynebacterium parvum). All six vaccine formulations containing the mixture of recombinant antigens were protective against challenge infections with promastigotes, the insect stage of the parasite, in that mice controlled and healed infections but developed transient and, in certain cases, accentuated disease. The most effective adjuvants were IL-12 followed by Detox. Further studies using these two adjuvants showed that a similar protective effect was observed with a mixture of the corresponding native proteins, and mice which had controlled the infection showed a preponderance of IFN-gamma-secreting CD4(+) T cells in the lymph nodes draining the lesion. Using the recombinant proteins individually, it is shown that the relatively abundant cysteine proteinases and glycoprotein 63, but not the acid phosphatase, are able to elicit a protective response. The results are discussed in comparison to previous studies with subunit vaccines and with respect to cell biological aspects of antigen presentation in Leishmania-infected macrophages.

Adjuvants, Immunologic↗
PubMed2000

Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells.

The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densely packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75% of the two GPI-anchored proteins but only 13% of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% and the tubular cluster/translucent vesicle complex 3.6% of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.

Acid Phosphatase↗
PubMed1999

Antigen presentation by macrophages harboring intravesicular pathogens.

Resting macrophages can be host cells for the replication of several protozoan parasites and bacteria. Upon activation, infected cells mobilize potent microbicidal mechanisms that eliminate the intracellular pathogen. This transition from a resting to an activated state is mediated by the interaction with specific T cells that recognize pathogen-derived peptides complexed to major histocompatibility complex (MHC) molecules at the surface of host cells. In this review, Peter Overath and Toni Aebischer discuss antigen presentation in infected macrophages from a cell biological point of view, a perspective that has important implications for the design of subunit vaccines.

Animals↗
PubMed1999

Freshwater fish trypanosomes: definition of two types, host control by antibodies and lack of antigenic variation.

Haemoflagellates of the genus Trypanosoma are prevalent in freshwater fishes and are transmitted by leeches as vectors. As demonstrated by sequence comparisons of nuclear small subunit rRNA genes, trypanosomes isolated from several fish species at different localities can be divided into at least 2 closely related types, designated Type A and Type B. A clone derived from a Type A isolate from carp (Cyprinus carpio) was used to study the anti-parasite immune response in specified pathogen-free outbred carp. Infection leads to an initial rise in parasitaemia in the blood followed by a sharp decline in all fish (acute phase). Thereafter, in some carp, parasites become undetectable both in the blood and in internal organs while, in others, low numbers can be found in the blood for up to 1 year (chronic phase). Fish that have controlled an acute infection with the clone are not only protected against an homologous challenge infection, but also against the infection with parasite lines derived from carp in the chronic phase of infection. Passive immunization experiments with IgM purified from serum of recovered carp indicate that the infection is controlled by antibodies. The anti-parasite antibody level in recovered carp remains high for many months although the parasitaemia is controlled at very low levels and the half life of IgM, t1/2 = 22.5 days, is comparatively short. The effective control of trypanosomes in laboratory infections is in contrast to the high prevalence in natural and farmed freshwater fish populations.

Animals↗
PubMed1998

Structure of a filamentous phosphoglycoprotein polymer: the secreted acid phosphatase of Leishmania mexicana.

The insect stage of the protozoan parasite Leishmania mexicana secretes a filamentous acid phosphatase (secreted acid phosphatase, SAP), a polymeric phosphoglycoprotein. The wild-type (wt) SAP filament is a copolymer composed of two related gene products SAP1 and SAP2, which are identical in the enzymatically active NH2-terminal domain and the COOH-terminal domain, but differ in the length of a highly glycosylated Ser/Thr-rich repeat region (32 amino acids and 383 amino acids, respectively) which is located between these domains. When expressed separately, full length SAP1, SAP2, or the NH2-terminal domain alone, are able to assemble into filaments. The Ser/Thr-rich region is the exclusive target for a novel type of O-glycosylation via phosphoserines. By using glycerol spraying/low-angle rotary metal shadowing and labelling with monoclonal antibodies it is demonstrated that the repetitive region adopts an extended conformation forming side arms which project radially from the filament core and terminate with the COOH-terminal domain. The length of the side arms of SAP1 and SAP2 (20 nm and 90 nm, respectively) corresponds to the predicted length of the Ser/Thr-rich repeat region of SAP1 and SAP2. Mass determination by scanning electron microscopy (STEM) shows that one morphologically defined globular particle of the filament core is a polypeptide dimer. We propose a model for the filament core, in which the globular NH2-terminal SAP domains form one strand composed of polypeptide dimers or two tightly associated strands of monomers which may twist into a double helix, similar to actin filaments. The highly O-glycosylated side arms project from the filament core conferring an overall bottle-brush-like appearance. The L. mexicana SAP is compared to SAPs secreted by the closely related species L. amazonensis and L. donovani.

Acid Phosphatase↗
PubMed1998

The molecular phylogeny of trypanosomes: evidence for an early divergence of the Salivaria.

Chronic infections with trypanosomes dwelling extracellularly in the blood and tissues of their hosts are observed in all vertebrate classes. We present here a molecular phylogenetic reconstruction of trypanosome evolution based on nucleotide sequences of small subunit rRNA genes. The evolutionary tree suggests an ancient split into one branch containing all Salivarian trypanosomes and a branch containing all non-Salivarian lineages. The latter branch splits into a clade containing bird, reptilian and Stercorarian trypanosomes infecting mammals and a clade with a branch of fish trypanosomes and a branch of reptilian/amphibian lineages. The branching order of the non-Salivarian trypanosomes supports host-parasite cospeciation scenarios, but also suggests host switches, e.g. between bird and reptilian trypanosomes. The tree is discussed in relation to the modes of adaptation that allow trypanosomes to infect immunocompetent vertebrates. Most importantly, the early divergence of the Salivarian lineages suggests that the presence of a dense proteinaceous surface coat that is subject to antigenic variation is a unique invention of this group of parasites.

Animals↗
PubMed1998

Cultivation of bloodstream forms of Trypanosoma carassii, a common parasite of freshwater fish.

Trypanosoma carassii (syn. T. danilewskyi) is a widespread parasite of carp and other cyprinid as well as some noncyprinid freshwater fish. It lives extracellularly in the blood and tissues of its hosts, causing chronic infections. In this paper the isolation of T. carassii from fish blood and the propagation and cloning of bloodstream forms in vitro are described. By several criteria, cultured and fish-derived trypomastigotes are indistinguishable. The culture system should be useful for the biochemical characterization of this trypanosome and its interaction with the fish immune system.

Animals↗
PubMed1997

Secreted proteophosphoglycan of Leishmania mexicana amastigotes activates complement by triggering the mannan binding lectin pathway.

Cutaneous lesions induced by infection of mice with the protozoan parasite, Leishmania mexicana, contain abundant amounts of a high molecular mass proteophosphoglycan (PPG), which is secreted by the amastigote stage residing in phagolysosomes of macrophages and can then be released into the tissue upon rupture of the infected cells. Amastigote PPG forms sausage-shaped but soluble particles and belongs to a novel class of serine-rich proteins that are extensively O-glycosylated by phosphooligosaccharides capped by mannooligosaccharides. The purified molecule is shown here to efficiently activate complement (C) and deplete hemolytic activity of normal serum and may prevent the opsonization of L. mexicana amastigotes. Complement activation is Ca2+ dependent but does not depend on antibodies or the complement component C1. PPG binds to serum mannan binding protein (MBP), thus activating the MBP-associated serine protease, P100. Subsequently, the C cascade is triggered through C4 leading to covalent modification probably of carbohydrate hydroxyls of PPG by C3 fragments. Thus, PPG is able to activate C via the mannan binding lectin pathway which is unusual for secreted, soluble products of microbial origin. The proteophosphoglycan-induced complement activation is postulated to contribute to the lesion development and pathology caused by the parasite.

Animals↗
PubMed1996

Gene cloning and cellular localization of a membrane-bound acid phosphatase of Leishmania mexicana.

In a previous publication, we described the purification of a membrane-bound acid phosphatase of Leishmania mexicana as a heterogeneously N-glycosylated protein of an apparent molecular mass of 70000-72000 expressed in both the promastigote and the amastigote stage of the parasite [19]. Screening of a genomic DNA library of L. mexicana with degenerate oligonucleotides designed according to the NH2-terminus of the protein led to the cloning of the lmmbap gene, which is present in one copy per haploid genome. The open reading frame predicts a protein of 516 amino acids composed of a signal sequence, a large hydrophilic region, a trans-membrane alpha-helix and a short cytoplasmic tail. The sequence of the hydrophilic region is homologous to acid phosphatases from other organisms. While in wild-type promastigotes, the acid phosphatase is located in the endosomal/lysosomal compartment between the flagellar pocket and the nucleus, overexpression leads to its abundant exposure on the cell surface. In cells transfected with a construct lacking the region corresponding to the trans-membrane and the cytoplasmic parts, the resulting altered acid phosphatase is efficiently secreted into the culture medium. The potential of this system for studies on membrane trafficking in kinetoplastid organisms is discussed.

Acid Phosphatase↗
PubMed1996

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface.

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.

Animals↗
PubMed1995

Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase.

The insect stage of the protozoan parasite Leishmania mexicana secretes a phosphomonoesterase in the form of a filamentous complex. The polypeptide subunits of this polymer are modified by phosphoglycans and/or oligomannosyl residues linked to phosphoserine. Based on peptide sequence data of a predominant 100 kDa protein of the filamentous complex, two tandemly arranged, single copy genes, lmsap1 and lmsap2, were cloned and sequenced. lmsap1 predicts a protein with features characteristic of acid phosphatases and a remarkable serine- and threonine-rich region of 32 amino acids close to the C-terminus. In the otherwise identical lmsap2 product, this region is extended to 383 amino acids and is composed of short Ser/Thr-rich repeats. Deletion analysis demonstrates that lmsap1 encodes the major 100 kDa protein of the complex while a minor 200 kDa component is derived from the lmsap2 gene. Null mutants of either gene retain the ability to secrete acid phosphatase filaments, while a deletion of both genes results in Leishmania defective in enzyme formation. The Ser/Thr-rich domains are the targets for phosphoglycan modifications as shown by the expression of secreted fusion proteins composed of these C-terminal regions and the N-terminal domain of a lysosomal acid phosphatase.

Acid Phosphatase↗
PubMed1995

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites.

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (Th1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-gamma do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-gamma and tumor necrosis factor-alpha. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.

Animals↗
PubMed1995

Genomic organization of an invariant surface glycoprotein gene family of Trypanosoma brucei.

The genomic organization of a gene family for the invariant surface glycoprotein, ISG75 (invariant surface glycoprotein with a molecular mass of 75 kDa), from Trypanosoma brucei is described. In T. brucei strain 427 ISG75 genes are present in tandem arrays at two loci, A and B, containing 5 and 2 copies, respectively. At the 3'-end of locus A, a single gene was identified that encodes a structural isoform of ISG75. This isoform contains a unique amino-terminal domain, whereas the rest of the protein is nearly identical to the polypeptides encoded by the other genes. This isoform is transcribed into a stable mRNA, but the expression of the derived polypeptide was below the detection limit. The ISG75 gene clusters are present on chromosomal bands 9' and 10, supporting the hypothesis of Gottesdiener et al. [25] that these bands contain allelic chromosomes. The total number of ISG75 genes is strain dependent, but at least one copy of the unique isoform is present in every variant tested.

Amino Acid Sequence↗