Cardiolipin (CL) is a mitochondrial inner membrane glycerophospholipid that associates with mitoc... more Cardiolipin (CL) is a mitochondrial inner membrane glycerophospholipid that associates with mitochondrial proteins to promote their activities and to facilitate protein complex and super-complex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated so far. We now report that in Trypanosoma brucei bloodstream forms, in which the ETC is truncated and composed of alternative oxidase and glycerol-3-phosphate dehydrogenase, and the mitochondrial membrane potential is generated by the hydrolytic action of the FoF1-ATP synthase, the inducible depletion of cardiolipin synthase (TbCls) is essential for parasite survival. Loss of TbCls and CL caused a rapid drop in ATP levels and a decline in the mitochondrial membrane potential. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of FoF1-...
In this study, we evaluated the efficacy, expressed as a mean weight decrease of the whole echino... more In this study, we evaluated the efficacy, expressed as a mean weight decrease of the whole echinococcal cyst mass, of novel benzimidazole salt formulations in a murineEchinococcus granulosusinfection model. BALB/c mice were intraperitoneally infected with protoscoleces ofE. granulosus(genotype G1). At 9 months post-infection, treatment with albendazole (ABZ), ricobendazole (RBZ) salt formulations, and RBZ enantiomer salts (R)-(+)-RBZ-Na and (S)-(−)-RBZ-Na formulations were initiated. Drugs were orally applied by gavage at 10 mg kg−1body weight per day during 30 days. Experimental treatments with benzimidazole sodium salts resulted in a significant reduction of the weight of cysts compared to conventional ABZ treatment, except for the (S)-(−)-RBZ-Na enantiomer formulation. Scanning electron microscopy and histological inspection revealed that treatments impacted not only the structural integrity of the parasite tissue in the germinal layer, but also induced alterations in the laminat...
Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange... more Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed pre...
SUMMARYThe impact of di-cationic pentamidine-analogues againstToxoplama gondii(Rh- and Me49-backg... more SUMMARYThe impact of di-cationic pentamidine-analogues againstToxoplama gondii(Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites ofT. gondii RhandT. gondii Me49with an IC50of 0·11 and 0·13μm, respectively. Pre-incubation of fibroblast monolayers with 1μmDB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5–6 days of drug exposure,T. gondiitachyzoites adapted to the compound and resumed proliferation up to a concentration of 1·2μm. Out of a set of 32 di-cationic compounds screened forin vitroactivity againstT. gondii,the arylimidamide DB745, exhibiting an IC50of 0·03μmand favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adaptedT. gondii(IC50=0·07μm). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adap...
We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum... more We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residu...
Self-reactive host antibodies were shown earlier to exhibit strong and specific cross-reactivity ... more Self-reactive host antibodies were shown earlier to exhibit strong and specific cross-reactivity to a particular trypanosomal antigen, protein I/6. The current study presents the molecular characterization of protein I/6. The major structural component of the cell body cytoskeleton of Trypanosoma brucei is a cagelike array of tightly connected microtubules which is in close contact to the overlaying cell membrane. Many of the unususal properties of the cytoskeleton of trypanosomes are due to the proteins associated with these microtubules. Protein I/6 was now shown to be a microtubule-associated protein, and it may be involved in crosslinking microtubules. Protein I/6 is coded for by a single gene, representing an exception rather than the rule for trypanosomal gene organization. From this single gene, two distinct mRNAs are generated through differential splicing. They differ in their polyadenylation sites, but both code for an identical polypeptide sequence of 33 kDa. Protein I/6 ...
Cestode worms, commonly also known as 'flat&a... more Cestode worms, commonly also known as 'flat' worms or tapeworms, are an important class of endoparasitic organisms. In order to complete their life cycle, they infect intermediate and definitive hosts in succession, through oral ingestion of eggs or larvae, respectively. Serious disease in humans or other mammalian hosts is mostly caused by the larval stages. Echinococcus spp. and Taenia spp. have been extensively investigated in the laboratory due to the fact that they represent important veterinary medical challenges and also cause grave diseases in humans. In contrast, Hymenolepis spp. and Mesocestoides spp. infections are relatively rare in humans, but these parasites have been extensively studied because their life cycle stages can be easily cultured in vitro, and can also be conveniently maintained in laboratory animal hosts. Thus they are more easily experimentally accessible, and represent important models for investigating the various aspects of cestode biology. This review will focus on in vitro and in vivo models which have been developed for studies on the host-parasite relationship during infection with Echinococcus, Taenia, Hymenolepis, Mesocestoides and Spirometra, and will cover the use of these models to investigate the morphology and ultrastructure of respective genera, the immunological relationship with the host and the development of vaccination approaches, as well as applications of these models for studies on parasite metabolism, physiology and gene expression. In addition, the use of these models in the development of chemotherapeutic measures against cestode infections is reviewed.
Neospora caninum is an apicomplexan parasite which invades many different cell types and tissues.... more Neospora caninum is an apicomplexan parasite which invades many different cell types and tissues. It causes neosporosis, namely stillbirth and abortion in cattle and neuromuscular disease in dogs, and has been found in several other animal species. N. caninum is closely related to Toxoplasma gondii, and controversial opinions exist with respect to its phylogenetical status. Initially, two stages of N. caninum had been identified, namely asexually proliferating tachyzoites and bradyzoites. The sexually produced stage of this parasite, oocysts containing sporozoites, has been found only recently. In order to answer the many open questions regarding its basic biology and its relationship with the host, a number of diagnostic tools have been developed. These techniques are based on the detection of antibodies against parasites in body fluids, the direct visualization of the parasite within tissue samples by immunohistochemistry, or the specific amplification of parasite DNA by PCR. Other studies have been aiming at the identification of specific antigenic components of N. caninum, and the molecular and functional characterization of these antigens with respect to the cell biology of the parasite. Clearly, molecular approaches will also be used increasingly to elucidate the immunological and pathogenetic events during infection, but also to prepare potential new immunotherapeutic tools for future vaccination against N. caninum infection.
The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mic... more The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mice by intranasal or intraperitoneal routes and challenge infection with Neospora caninum tachyzoites were investigated. Nanogels were chitosan based, with an alginate or alginate-mannose surface. None of the mice receiving recNcPDI intraperitoneal (i.p.) (without nanogels) survived, whereas intranasal (i.n.) application protected 9 of 10 mice from disease. Association of recNcPDI with nanogels improved survival of i.p. vaccinated mice, but nanogels without recNcPDI gave similar protection levels. When nanogels were inoculated via the i.n. route, 80% of the mice were protected. Association of recNcPDI with the alginate-coated nanogels protected all mice against disease. Quantification of the cerebral parasite burden showed a significant reduction of parasite numbers in most experimental groups vaccinated i.n., except those vaccinated with alginate-mannose nanogels with or without recNcPDI....
The tachyzoites and the tissue cysts containing bradyzoites of Neospora caninum and Toxoplasma go... more The tachyzoites and the tissue cysts containing bradyzoites of Neospora caninum and Toxoplasma gondii, respectively, are difficult to distinguish morphologically. Specific antigens have been identified in T. gondii tachyzoites and bradyzoites, some of which are stage-specifically expressed, and different functions have been attributed to some of them. A tachyzoite stage-specifically expressed surface protein is the major surface antigen 1 (SAG1) which has been shown to be involved in host cell attachment and invasion. Previously we have identified a cell surface-associated glycoprotein (p36) in N. caninum tachyzoites. The full length coding sequence of the cDNA coding for p36 was determined, and analysis of the deduced amino acid sequence demonstrated that p36 is closely related to SAG1. p36 is encoded by a single copy gene which produces a transcript of 1.4 kb. Immunogold labeling of resin-embedded parasites using polyclonal antibodies affinity-purified on a recombinant p36 fusion protein expressed in Escherichia coli showed that this protein is located exclusively on the tachyzoite cell surface. As SAG1 in T. gondii, p36 is expressed in the tachyzoite stage, but is absent from bradyzoites. p36 is recognized by antibodies present in sera of cows experimentally infected with N. caninum tachyzoites.
Cardiolipin (CL) is a mitochondrial inner membrane glycerophospholipid that associates with mitoc... more Cardiolipin (CL) is a mitochondrial inner membrane glycerophospholipid that associates with mitochondrial proteins to promote their activities and to facilitate protein complex and super-complex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated so far. We now report that in Trypanosoma brucei bloodstream forms, in which the ETC is truncated and composed of alternative oxidase and glycerol-3-phosphate dehydrogenase, and the mitochondrial membrane potential is generated by the hydrolytic action of the FoF1-ATP synthase, the inducible depletion of cardiolipin synthase (TbCls) is essential for parasite survival. Loss of TbCls and CL caused a rapid drop in ATP levels and a decline in the mitochondrial membrane potential. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of FoF1-...
In this study, we evaluated the efficacy, expressed as a mean weight decrease of the whole echino... more In this study, we evaluated the efficacy, expressed as a mean weight decrease of the whole echinococcal cyst mass, of novel benzimidazole salt formulations in a murineEchinococcus granulosusinfection model. BALB/c mice were intraperitoneally infected with protoscoleces ofE. granulosus(genotype G1). At 9 months post-infection, treatment with albendazole (ABZ), ricobendazole (RBZ) salt formulations, and RBZ enantiomer salts (R)-(+)-RBZ-Na and (S)-(−)-RBZ-Na formulations were initiated. Drugs were orally applied by gavage at 10 mg kg−1body weight per day during 30 days. Experimental treatments with benzimidazole sodium salts resulted in a significant reduction of the weight of cysts compared to conventional ABZ treatment, except for the (S)-(−)-RBZ-Na enantiomer formulation. Scanning electron microscopy and histological inspection revealed that treatments impacted not only the structural integrity of the parasite tissue in the germinal layer, but also induced alterations in the laminat...
Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange... more Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed pre...
SUMMARYThe impact of di-cationic pentamidine-analogues againstToxoplama gondii(Rh- and Me49-backg... more SUMMARYThe impact of di-cationic pentamidine-analogues againstToxoplama gondii(Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites ofT. gondii RhandT. gondii Me49with an IC50of 0·11 and 0·13μm, respectively. Pre-incubation of fibroblast monolayers with 1μmDB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5–6 days of drug exposure,T. gondiitachyzoites adapted to the compound and resumed proliferation up to a concentration of 1·2μm. Out of a set of 32 di-cationic compounds screened forin vitroactivity againstT. gondii,the arylimidamide DB745, exhibiting an IC50of 0·03μmand favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adaptedT. gondii(IC50=0·07μm). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adap...
We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum... more We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residu...
Self-reactive host antibodies were shown earlier to exhibit strong and specific cross-reactivity ... more Self-reactive host antibodies were shown earlier to exhibit strong and specific cross-reactivity to a particular trypanosomal antigen, protein I/6. The current study presents the molecular characterization of protein I/6. The major structural component of the cell body cytoskeleton of Trypanosoma brucei is a cagelike array of tightly connected microtubules which is in close contact to the overlaying cell membrane. Many of the unususal properties of the cytoskeleton of trypanosomes are due to the proteins associated with these microtubules. Protein I/6 was now shown to be a microtubule-associated protein, and it may be involved in crosslinking microtubules. Protein I/6 is coded for by a single gene, representing an exception rather than the rule for trypanosomal gene organization. From this single gene, two distinct mRNAs are generated through differential splicing. They differ in their polyadenylation sites, but both code for an identical polypeptide sequence of 33 kDa. Protein I/6 ...
Cestode worms, commonly also known as 'flat&a... more Cestode worms, commonly also known as 'flat' worms or tapeworms, are an important class of endoparasitic organisms. In order to complete their life cycle, they infect intermediate and definitive hosts in succession, through oral ingestion of eggs or larvae, respectively. Serious disease in humans or other mammalian hosts is mostly caused by the larval stages. Echinococcus spp. and Taenia spp. have been extensively investigated in the laboratory due to the fact that they represent important veterinary medical challenges and also cause grave diseases in humans. In contrast, Hymenolepis spp. and Mesocestoides spp. infections are relatively rare in humans, but these parasites have been extensively studied because their life cycle stages can be easily cultured in vitro, and can also be conveniently maintained in laboratory animal hosts. Thus they are more easily experimentally accessible, and represent important models for investigating the various aspects of cestode biology. This review will focus on in vitro and in vivo models which have been developed for studies on the host-parasite relationship during infection with Echinococcus, Taenia, Hymenolepis, Mesocestoides and Spirometra, and will cover the use of these models to investigate the morphology and ultrastructure of respective genera, the immunological relationship with the host and the development of vaccination approaches, as well as applications of these models for studies on parasite metabolism, physiology and gene expression. In addition, the use of these models in the development of chemotherapeutic measures against cestode infections is reviewed.
Neospora caninum is an apicomplexan parasite which invades many different cell types and tissues.... more Neospora caninum is an apicomplexan parasite which invades many different cell types and tissues. It causes neosporosis, namely stillbirth and abortion in cattle and neuromuscular disease in dogs, and has been found in several other animal species. N. caninum is closely related to Toxoplasma gondii, and controversial opinions exist with respect to its phylogenetical status. Initially, two stages of N. caninum had been identified, namely asexually proliferating tachyzoites and bradyzoites. The sexually produced stage of this parasite, oocysts containing sporozoites, has been found only recently. In order to answer the many open questions regarding its basic biology and its relationship with the host, a number of diagnostic tools have been developed. These techniques are based on the detection of antibodies against parasites in body fluids, the direct visualization of the parasite within tissue samples by immunohistochemistry, or the specific amplification of parasite DNA by PCR. Other studies have been aiming at the identification of specific antigenic components of N. caninum, and the molecular and functional characterization of these antigens with respect to the cell biology of the parasite. Clearly, molecular approaches will also be used increasingly to elucidate the immunological and pathogenetic events during infection, but also to prepare potential new immunotherapeutic tools for future vaccination against N. caninum infection.
The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mic... more The effects of nanogel encapsulation of recombinant NcPDI (recNcPDI) following vaccination of mice by intranasal or intraperitoneal routes and challenge infection with Neospora caninum tachyzoites were investigated. Nanogels were chitosan based, with an alginate or alginate-mannose surface. None of the mice receiving recNcPDI intraperitoneal (i.p.) (without nanogels) survived, whereas intranasal (i.n.) application protected 9 of 10 mice from disease. Association of recNcPDI with nanogels improved survival of i.p. vaccinated mice, but nanogels without recNcPDI gave similar protection levels. When nanogels were inoculated via the i.n. route, 80% of the mice were protected. Association of recNcPDI with the alginate-coated nanogels protected all mice against disease. Quantification of the cerebral parasite burden showed a significant reduction of parasite numbers in most experimental groups vaccinated i.n., except those vaccinated with alginate-mannose nanogels with or without recNcPDI....
The tachyzoites and the tissue cysts containing bradyzoites of Neospora caninum and Toxoplasma go... more The tachyzoites and the tissue cysts containing bradyzoites of Neospora caninum and Toxoplasma gondii, respectively, are difficult to distinguish morphologically. Specific antigens have been identified in T. gondii tachyzoites and bradyzoites, some of which are stage-specifically expressed, and different functions have been attributed to some of them. A tachyzoite stage-specifically expressed surface protein is the major surface antigen 1 (SAG1) which has been shown to be involved in host cell attachment and invasion. Previously we have identified a cell surface-associated glycoprotein (p36) in N. caninum tachyzoites. The full length coding sequence of the cDNA coding for p36 was determined, and analysis of the deduced amino acid sequence demonstrated that p36 is closely related to SAG1. p36 is encoded by a single copy gene which produces a transcript of 1.4 kb. Immunogold labeling of resin-embedded parasites using polyclonal antibodies affinity-purified on a recombinant p36 fusion protein expressed in Escherichia coli showed that this protein is located exclusively on the tachyzoite cell surface. As SAG1 in T. gondii, p36 is expressed in the tachyzoite stage, but is absent from bradyzoites. p36 is recognized by antibodies present in sera of cows experimentally infected with N. caninum tachyzoites.
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Papers by A. Hemphill