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ABSTRACT1-Hydroxy-2-dodecyl-4(1H)quinolone (HDQ) was recently identified as a Toxoplasma gondiiinhibitor. We describe here two novel 1-hydroxyquinolones, which displayed 50% inhibitory concentrations 10- and 5-fold lower than that of HDQ. In a mouse model of acute toxoplasmosis, these two compounds and HDQ reduced the percentages of infected peritoneal cells and decreased the parasite loads in lungs and livers. Compound B showed a tendency toward lowering parasite loads in brains in a mouse model of toxoplasmic encephalitis.

The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) (3,3'-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (m) in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a m collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. m depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the Fo-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on the m, since m-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.

ABSTRACTThe apicomplexan parasite Toxoplasma gondiiexpresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. We recently showed by inhibition kinetics that T. gondiiNDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondiireplication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) (3,3'-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (??m) in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a ??m collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ??m depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the Fo-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on the ??m, since ??m-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.

ABSTRACTMicrosporidia are obligate intracellular pathogens which enter host cells by the discharge of a hollow tube through which the sporoplasma is extruded into the host cell. Since this invasion mechanism is very different from common entry strategies, the formation of the parasitophorous vacuole (PV) in Encephalitozoonspecies is likely to be distinct from known principles. We investigated the origin of the nascent Encephalitozoon cuniculiPV membrane with the aid of fluorescent lipid probes. When Bodipy 500/510-C12-HPC-labeled spores were used for infection, the emerging PV membrane was unlabeled, suggesting that sporoplasma-derived lipids do not significantly contribute to the formation of the PV membrane. In contrast, when raft and nonraft microdomains of the host cell plasma membrane were selectively labeled with DiIC16and Speedy DiO, both tracers were detectable in the nascent PV membrane shortly after infection, indicating that the bulk lipids of the PV membrane are host cell derived. Time-lapse fluorescence microscopy revealed that the formation of the PV membrane is a fast event (<1.3 s), which occurred simultaneously with the extrusion of the sporoplasma. The portion of the discharged tube which is in contact with the host cell was found to be coated with labeled host cell lipids, which might be an indication for a plasma membrane invagination at the contact site. To investigate the presence of pores in the E. cuniculiPV membrane, we microinjected fluorescent dyes of different sizes into infected host cells. A 0.5-kDa dextran as well as 0.8- to 1.1-kDa peptides could rapidly enter the PV, while a 10-kDa dextran was stably excluded from the PV lumen, indicating that the PV membrane possesses pores with an exclusion size of <10 kDa, which should allow metabolite exchange.

Microsporidia are obligate intracellular pathogens which enter host cells by the discharge of a hollow tube through which the sporoplasma is extruded into the host cell. Since this invasion mechanism is very different from common entry strategies, the formation of the parasitophorous vacuole (PV) in Encephalitozoon species is likely to be distinct from known principles. We investigated the origin of the nascent Encephalitozoon cuniculi PV membrane with the aid of fluorescent lipid probes. When Bodipy 500/510-C12-HPC-labeled spores were used for infection, the emerging PV membrane was unlabeled, suggesting that sporoplasma-derived lipids do not significantly contribute to the formation of the PV membrane. In contrast, when raft and nonraft microdomains of the host cell plasma membrane were selectively labeled with DiIC16and Speedy DiO, both tracers were detectable in the nascent PV membrane shortly after infection, indicating that the bulk lipids of the PV membrane are host cell derived. Time-lapse fluorescence microscopy revealed that the formation of the PV membrane is a fast event (<1.3 s), which occurred simultaneously with the extrusion of the sporoplasma. The portion of the discharged tube which is in contact with the host cell was found to be coated with labeled host cell lipids, which might be an indication for a plasma membrane invagination at the contact site. To investigate the presence of pores in the E. cuniculi PV membrane, we microinjected fluorescent dyes of different sizes into infected host cells. A 0.5-kDa dextran as well as 0.8- to 1.1-kDa peptides could rapidly enter the PV, while a 10-kDa dextran was stably excluded from the PV lumen, indicating that the PV membrane possesses pores with an exclusion size of <10 kDa, which should allow metabolite exchange.

Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites’ viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.

ABSTRACTMany apicomplexan parasites, such as Toxoplasma gondiiand Plasmodiumspecies, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites’ viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondiito either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondiigenome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondiigenome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.

ABSTRACTBoth apicomplexan parasites Toxoplasma gondiiand Plasmodium falciparumlack type I NADH dehydrogenases (complex I) but instead carry alternative (type II) NADH dehydrogenases, which are absent in mammalian cells and are thus considered promising antimicrobial drug targets. The quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) was recently described as a high-affinity inhibitor of fungal alternative NADH dehydrogenases in enzymatic assays, probably by interfering with the ubiquinol binding site of the enzyme. We describe here that HDQ effectively inhibits the replication rates of P. falciparumand T. gondiiin tissue culture. The 50% inhibitory concentration (IC50) of HDQ for T. gondiiwas determined to be 2.4 ± 0.3 nM with a growth assay based on vacuole sizes and 3.7 ± 1.4 nM with a growth assay based on beta-galactosidase activity. Quantification of the P. falciparumreplication rate using a fluorometric assay revealed an IC50of 14.0 ± 1.9 nM. An important feature of the HDQ structure is the length of the alkyl side chain at position 2. Derivatives with alkyl side chains of C6, C8, C12(HDQ), and C14all displayed excellent anti-T. gondiiactivity, while a C5derivative completely failed to inhibit parasite replication. A combined treatment of T. gondii-infected cells with HDQ and the antimalarial agent atovaquone, which blocks the ubiquinol oxidation site of cytochrome bin complex III, resulted in synergism, with a calculated fractional inhibitory concentration of 0.16 nM. Interference of the mitochondrial ubiquinone/ubiquinol cycle at two different locations thus appears to be a highly effective strategy for inhibiting parasite replication. HDQ and its derivatives, particularly in combination with atovaquone, represent promising compounds with a high potential for antimalarial and antitoxoplasmal therapy.

ABSTRACTMicrosporidia of the genus Encephalitozoondevelop inside a parasitophorous vacuole (PV) of unknown origin. Using colocalization studies, the PV was found to be absent from the endocytic pathway markers early endosomal autoantigen 1, transferrin receptor, and lysosome-associated membrane protein 1 and for the endoplasmic reticulum marker calnexin. The nonfusiogenic characteristic of the PV appears to be acquired as early as 1 min postinfection and is not reversed by drug treatment with albendazole or fumagillin.

Infection with the apicomplexan parasite Toxoplasma gondii results in a significant alteration of the host-cell transcriptional profile. We have previously shown that the transferrin receptor (TfR) is specifically up-regulated in T. gondii-infected human fibroblasts but not in host cells infected with the bacterial pathogens Salmonella Typhimurium and Chlamydia trachomatis. In this report, we describe the prerequisites and physiological conditions that are associated with this pathogen-specific gene induction. Band-shift assays revealed that T. gondii infection resulted in increased activity in the iron response protein IRP1, which, in this state, stabilizes TfR mRNA from degradation. Although T. gondii depends on host-cell iron as demonstrated by sensitivity to deferoxamine, a parasite-induced iron starvation is not responsible for TfR up-regulation. The increased iron availability due to treatment with holotransferrin and FeNTA did not prevent TfR induction nor was the transferrin-independent iron-transporter NRAMP2 up-regulated in infected host cells. In addition, inhibition of parasite replication by drug treatment did not prevent TfR up-regulation. Instead, TfR induction was sensitive to cycloheximide and could be induced by treatment with conditioned media from infected human fibroblasts. Together our findings suggest that the T. gondii-specific TfR up-regulation is not due to a direct interaction of parasitic factors with the iron-uptake machinery of the host cell but is instead mediated indirectly as a result of secreted host cell- or parasite-derived factors.

Abstract We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.

ABSTRACTThe invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninumtachyzoites which, due to its homology to MIC1 in Toxoplasma gondii(TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.

ABSTRACTMicrosporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in Encephalitozoon cuniculiwith the aid of a monoclonal antibody, and the corresponding gene, SWP1, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements. SWP1is a single-copy gene that is also present in the genomes ofEncephalitozoon intestinalisand Encephalitozoon hellemas demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.

The obligate intracellular parasite Toxoplasma gondiiis able to persist lifelong in its hosts by differentiating from the replicative tachyzoite stage into cyst forming latent bradyzoites. Beside the clinical relevance of stage conversion and its importance for pathogenesis and prevention of toxoplasmic encephalitis, reversible stage differentiation in T. gondiiis an interesting model system of protozoan differentiation in general. In recent years a variety of molecular techniques have been devel9ped for T. gondii, including transfection systems and the development of many selectable markers. Together with tissue culture models in which stage differentiation from tachyzoites to bradyzoites can be induced these techniques provide the tools for a molecular dissection of the differentiation pathways. Three aspects of stage conversion are highlighted in this review, including the alteration of the parasite surface, alterations in parasite metabolism and the induction of genes associated with stress response.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Examination of the arylsulfatase A gene in a patient suffering from late infantile metachromatic leukodystrophy revealed an 11-bp deletion in exon 8. Although this allele produces normal amounts of ASA mRNA, no arylsulfatase A cross-reacting material could be detected in cultured fibroblasts from the patient. The patient was found to be a compound heterozygote, the other allele is also known to generate no ASA polypeptides. This patient is another example where absence of ASA polypeptides correlates with the severe late infantile form of metachromatic leukodystrophy.

Plasmid vectors that incorporate sequence elements from the dehydrofolate reductase–thymidylate synthase (DHFR-TS) locus ofToxoplasma gondiiintegrate into the parasite genome with remarkably high frequency (>1% of transfected parasites). These vectors may—but need not—include mutantDHFR–TSalleles that confer pyrimethamine resistance to transgenic parsites. Large genomic constructs integrate at the endogenous locus by homologous recombination, but cDNA-derived sequences lacking long stretches of contiguous genomic DNA (due to intron excision) typically integrate into chromosomal DNA by nonhomologous recombination. Nonhomologous integration occurs effectively at random; and coupled with the high frequency of transformation, this allows a large fraction of the parasite genome to be tagged in a single electroporation cuvette. Genomic tagging permits insertional mutagenesis studies conceptually analogous to transposon mutagenesis in bacteria, yeast,Drosophila,etc. In theory (and, thus far, in practice), this allows identification of any gene whose inactivation is not lethal to the haploid tachyzoite form ofT. gondiiand for which a suitable selection or screen is available. Transformation vectors can be engineered to facilitate rescue of the tagged locus and to include a variety of reporters or selectable markers. Genetic strategies are also possible, using reporters whose function can be assayed by metabolic, visual, or immunological screens to “trap” genes that are activated (or inactivated) under various conditions of interest.

Objectives. Helicobacter pylori (H. pylori) isolates resistant to clarithromycin and quinolones are increasing worldwide. Data regarding the magnitude of H. pylori resistance are limited in developing countries. Here, we report the prevalence of mutations conferring resistance to clarithromycin and fluoroquinolones among dyspeptic patients attending a tertiary hospital, Tanzania. Methods. Between August 2014 and August 2016, patients undergoing upper gastrointestinal endoscopy at the Bugando Medical Centre were enrolled. Biopsies were taken for polymerase chain reaction (PCR) and sequencing to detect mutations conferring resistance to clarithromycin and fluoroquinolones. Results. A total of 208 nonrepetitive biopsies were examined of which 188 (90.4%) tested positive for H. pylori specific 23S rRNA PCR. Clarithromycin resistance mutations were detected in 54/188 (28.7%) of patients tested. The most frequently detected mutation was A2143G (30) followed by A2142G (20). Out of 131 nonrepetitive biopsies tested for fluoroquinolones resistance mutations, 77/131 (58.8%) were positive, with N87I (20) mutation being the most frequently detected mutation followed by A92T mutation which was detected in 16 samples. Conclusion. A significant proportion of dyspeptic patients attending tertiary hospital in Tanzania are infected with H. pylori strains harbouring clarithromycin or fluoroquinolones resistance mutations. Detection of more than 50% of strains with fluoroquinolones resistance mutations makes the H. pylori second line treatment questionable in our setting. There is a need of surveillance of H. pylori resistance patterns in Tanzania to provide data that can guide empirical treatment to reduce associated morbidity of H. pylori infections. The correlation between A92T fluoroquinolone mutation and phenotypic resistance requires further investigations.