Glutathione reductase: A cytoplasmic antioxidant enzyme and a potential target for phenothiazinium dyes in Neospora caninum.

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 µM, 11 ± 2 µM, 0.7 ± 0.1 µM, and 0.9 ± 0.2 µM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.

Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening.

Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.

Functional characterisation of the actin-depolymerising factor from the apicomplexan Neospora caninum (NcADF).

Neospora caninum is an apicomplexan parasite that causes infectious abortion in cows. As an obligate intracellular parasite, N. caninum requires a host cell environment to survive and replicate. The locomotion and invasion mechanisms of apicomplexan parasites are centred on the actin-myosin system to propel the parasite forwards and into the host cell. The functions of actin, an intrinsically dynamic protein, are modulated by actin-binding proteins (ABPs). Actin-depolymerising factor (ADF) is a ubiquitous ABP responsible for accelerating actin turnover in eukaryotic cells and is one of the few known conserved ABPs from apicomplexan parasites. Apicomplexan ADFs have nonconventional properties compared with ADF/cofilins from higher eukaryotes. In the present paper, we characterised the ADF from N. caninum (NcADF) using computational and in vitro biochemical approaches to investigate its function in rabbit muscle actin dynamics. Our predicted computational tertiary structure of NcADF demonstrated a conserved structure and phylogeny with respect to other ADF/cofilins, although certain differences in filamentous actin (F-actin) binding sites were present. The activity of recombinant NcADF on heterologous actin was regulated in part by pH and the presence of inorganic phosphate. In addition, our data suggest a comparatively weak disassembly of F-actin by NcADF. Taken together, the data presented herein represent a contribution to the field towards the understanding of the role of ADF in N. caninum and a comparative analysis of ABPs in the phylum Apicomplexa.

Neospora caninum ROP16 play an important role in the pathogenicity by phosphorylating host cell STAT3.

Neospora caninum is a common cause of abortions in cattle and nervous system dysfunctions in dogs. Our analysis shows that NcROP16 and TgROP16 have similar structures and may have similar functions. To our surprise, we found that similar to the T. gondii RH strain, the N. caninum Nc-1 strain could phosphorylate STAT3Y705, but in contrast to T. gondii, N. caninum Nc-1 could not phosphorylate STAT6Y641. We constructed a gene-knockout plasmid and screened ΔNcROP16 strains at the gene, protein and transcription levels. Plaque assays, invasion assays and intracellular proliferation tests indicated that the ΔNcROP16 strain phenotypes had changed, resulting in smaller plaques and slower intracellular growth. A virulence analysis showed that the cerebral loads of the parasite in mice infected with the ΔNcROP16 strain were significantly reduced compared to the loads in mice infected with the Nc-1 strain. In contrast, the overexpression of ROP16 led to the largest number of parasites observed in the mouse brains. Similarly, the overexpression of ROP16 caused the most powerful virulence in mice. In addition, NcROP16 takes part in the STAT3 signaling pathway in different host cells. This occurs by the secretion of NcROP16 into the host cell, where it phosphorylates STAT3, and phosphorylated STAT3 then migrates to the cell nucleus. NcROP16 can enter the host nucleus and continuously phosphorylate STAT3, resulting in the induction of host cell apoptosis. The parasites engineered to over express the NcROP16 induce the increased transcription of apoptotic-related genes, such as Fas, FasL and Bax and enhanced ANA1 cell apoptosis. The results show that NcROP16 is a key virulence factor in N. caninum, promoting the host cell apoptosis and enhancing the pathogenicity of the parasites for the host by phosphorylating STAT3.

Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites.

We present the effects of two novel bumped kinase inhibitors, BKI-1517 and BKI-1553, against Neospora caninum tachyzoites in vitro and in experimentally infected pregnant mice. These compounds inhibited tachyzoite proliferation of a transgenic beta-galactosidase reporter strain cultured in human foreskin fibroblasts with 50% inhibitory concentrations (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 µM, respectively. As assessed by an alamarBlue assay, fibroblast IC50s were above 20 µM; however, morphological changes occurred in cultures treated with >5 µM BKI-1517 after prolonged exposure (>6 days). Treatment of intracellular tachyzoites with 5 µM BKI-1553 for 6 days inhibited endodyogeny by interfering with the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 µM BKI-1517 did not form large complexes and showed much more evidence of cell death. However, after a treatment duration of 10 days in vitro, both compounds failed to completely prevent the regrowth of parasites from culture. BALB/c mice experimentally infected with N. caninum Spain7 (Nc-Spain7) and then treated during 6 days with BKI-1517 or BKI-1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by BKI-1517 when applied at 50 mg/kg of body weight/day. At 20 mg/kg/day, BKI-1517 significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. BKI-1553 was less detrimental to fertility and also provided significant but clearly less pronounced protection of dams and offspring. These results demonstrate that, when judiciously applied, this compound class protects offspring from vertical transmission and disease.

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress.

BACKGROUND: NTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided. METHODS: Herein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points. RESULTS: We describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress. CONCLUSIONS: The present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies.

The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites.

Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

A transgenic Neospora caninum strain based on mutations of the dihydrofolate reductase-thymidylate synthase gene.

Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (ß-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii.

Vaccination of mice with chitosan nanogel-associated recombinant NcPDI against challenge infection with Neospora caninum tachyzoites.

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. For i.p. vaccinated groups, no significant differences in cerebral infection densities were measured, but there was a reduction in the groups vaccinated with recNcPDI associated with both types of nanogels. Analysis of the immune responses of infected mice indicated that association of recNcPDI with nanogels altered the patterns of cytokine mRNA expression profiles, but had no major impact on the antibody subtype responses. Nevertheless, this did not necessarily relate to the protection.

Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii.

The nucleoside triphosphate hydrolases that are produced by Neospora caninum (NcNTPase) and Toxoplasma gondii (TgNTPase-I) have a different physiological function from the ubiquitous ecto-ATPases. The recombinant enzymes were crystallized at 293 K using polyethylene glycol 3350 as a precipitant and X-ray diffraction data sets were collected for NcNTPase (to 2.8 Šresolution) and TgNTPase-I (to 3.1 Šresolution) at 100 K using synchrotron radiation. The crystals of NcNTPase and TgNTPase-I belonged to the orthorhombic space group I222 (unit-cell parameters a = 93.6, b = 140.8, c = 301.1 Å) and the monoclinic space group P2(1) (unit-cell parameters a = 87.1, b = 123.5, c = 120.2 Å, ß = 96.6°), respectively, with two NcNTPase (V(M) = 3.7 Å(3) Da(-1)) and four TgNTPase-I (V(M) = 2.7 Å(3) Da(-1)) molecules per asymmetric unit. SAD phasing trials using a data set (λ = 0.97904 Å) collected from a crystal of selenomethionylated NcNTPase gave an initial electron-density map of sufficient quality to build a molecular model of NcNTPase.

Spatial analysis of Neospora caninum distribution in dairy cattle from Sweden.

The national herd prevalence and spatial distribution of Neospora caninum infected dairy herds in Sweden were investigated. The study was based on a bulk milk survey comprising samples from 2,978 herds. Test-positive herds were found in all parts of Sweden and the overall prevalence of test-positive herds was 8.3% (95% confidence interval = 7.3-9.3%). The presence of spatial autocorrelation was tested using the Moran's I test. Possible clusters of test-positive herds were identified by applying the local indicator of spatial association (LISA) test statistic and the spatial scan statistic. Analysis based on data aggregated by postal code areas as well as analysis based on exact coordinates identified significant clusters of high prevalence in the middle parts of Sweden and low prevalence in the south. This was not expected considering the results from other European studies of N. caninum in cattle. However, the findings are supported by the distribution of previously known case herds.

Neospora caninum: functional inhibition of protein disulfide isomerase by the broad-spectrum anti-parasitic drug nitazoxanide and other thiazolides.

Nitazoxanide (NTZ) and several NTZ-derivatives (thiazolides) have been shown to exhibit considerable anti-Neospora caninum tachyzoite activity in vitro. We coupled tizoxanide (TIZ), the deacetylated metabolite, to epoxy-agarose-resin and performed affinity chromatography with N. caninum tachyzoite extracts. Two main protein bands of 52 and 43kDa were isolated. The 52kDa protein was readily recognized by antibodies directed against NcPDI, and mass spectrometry confirmed its identity. Poly-histidine-tagged NcPDI-cDNA was expressed in Escherichia coli and recombinant NcPDI (recNcPDI) was purified by Co2+-affinity chromatography. By applying an enzyme assay based on the measurement of insulin crosslinking activity, recNcPDI exhibited properties reminiscent for PDIs, and its activity was impaired upon the addition of classical PDI inhibitors such as bacitracin (1-2mM), para-chloromercuribenzoic acid (0.1-1mM) and tocinoic acid (0.1-1mM). RecNcPDI-mediated insulin crosslinking was inhibited by NTZ (5-100 microM) in a dose-dependent manner. In addition, the enzymatic activity of recNcPDI was inhibited by those thiazolides that also affected parasite proliferation. Thus, thiazolides readily interfere with NcPDI, and possibly also with PDIs from other microorganisms susceptible to thiazolides.

Characterization of the fetuin-binding fraction of Neospora caninum tachyzoites and its potential involvement in host-parasite interactions.

Terminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction of Neospora caninum tachyzoites and bradyzoites with Vero cell monolayers. Thus, Neospora extracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of approximately 65 kDa, subsequently named NcFBP (Neospora caninum fetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140 kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction of Neospora caninum tachyzoites contains components that could be potentially involved in host-parasite interactions.

Identification of a protein disulfide isomerase of Neospora caninum in excretory-secretory products and its IgA binding and enzymatic activities.

A protein disulfide isomerase of Neospora caninum (NcPDI) with a molecular weight of 50kDa was identified in tachyzoite lysate and excretory-secretory (ES) products. The IgA antibody in 58.0% of the individual cattle tear samples recognized the NcPDI, which suggests that the PDI-specific antibody may be involved in defense against parasites. In addition, PDI-specific inhibitors and NcPDI antiserum showed inhibitory effects on the growth of N. caninum tachyzoites. Furthermore, the purified recombinant NcPDI demonstrated biological activities in vitro by catalysis and refolding of reduced RNase A and assisted in the recovery of native lysozyme. These findings indicate that NcPDI possesses PDI-specific enzymatic activity and could be a putative target for chemotherapy for neosporosis.

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction.

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.

The identification of a sequence related to apicomplexan enolase from Sarcocystis neurona.

Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by Sarcocystis neurona, an apicomplexan parasite. S. neurona is also associated with EPM-like diseases in marine and small mammals. The mechanisms of transmission and ability to infect a wide host range remain obscure; therefore, characterization of essential proteins may provide evolutionary information allowing the development of novel chemotherapeutics that target non-mammalian biochemical pathways. In the current study, two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry were combined to characterize and identify an enolase protein from S. neurona based on peptide homology to the Toxoplasma gondii protein. Enolase is thought to be a vestigial, non-photosynthetic protein resulting from an evolutionary endosymbiosis event of an apicomplexan ancestor with green algae. Enolase has also been suggested to play a role in parasite stage conversion for T. gondii. Characterization of this protein in S. neurona and comparison to other protozoans indicate a biochemical similarity of S. neurona enolase to other tissue-cyst forming coccidians that cause encephalitis.

Cloning, expression, and characterization of iron-containing superoxide dismutase from Neospora caninum.

A gene encoding superoxide dismutase (SOD) from Neospora caninum, a causative agent of neosporosis, has been cloned and its gene product functionally expressed and characterized. The gene had an open reading frame of 606 bp and deduced 201 amino acids. Sequence analysis showed that the gene had conserved metal-binding residues and conserved amino acid residues that were found in Fe-SODs. Comparison of the deduced amino acid sequence of the enzyme with previously reported Fe-SOD amino acid sequences of the other parasitic protozoans revealed significant high homology. The coding region of the N. caninum Fe-SOD was cloned and functionally expressed in Escherichia coli. Enzyme activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide and potassium cyanide, and the enzyme showed similar biochemical properties with typical Fe-SODs of other parasitic protozoans. Southern blot analysis showed that the SOD gene appears to be present as a single-copy gene in N. caninum genome. Semiquantitative reverse transcription-polymerase chain reaction and immunoblot using antiserum raised against the purified recombinant protein showed that Fe-SOD is expressed in both developmental stages of N. caninum, i.e., in bradyzoites and tachyzoites. In an immunofluorescence assay, the enzyme was localized on the cell surface of N. caninum tachyzoites. These results suggest that Fe-SOD might be essential for the intracellular survival of N. caninum and may play an important role in the pathogenesis of the parasite by protecting the parasite from oxidative killing.

Characterization of Neospora caninum protease, NcSUB1 (NC-P65), with rabbit anti-N54.

NcSUB1 (formerly known as NC-p65) is the first molecularly described proteolytic enzyme of the intracellular protozoan parasite Neospora caninum. This report describes the characterization of a rabbit anti-N54, which is an antiserum generated against an internal fragment of NcSUB1 (amino acids 649-783). In immunofluorescence studies rabbit and-N54 labeled the apical end of the fixed parasite. By immuno-gold electron microscopy, the antibody bound primarily to the microneme organelles of the parasite. Analysis of secreted parasitic proteins indicated that a protein of molecular weight 65 kDa (reduced) or 55 kDa (nonreduced) was recognized bythe antibody. The same secreted proteins were affinity purified with rabbit anti-N54-coupled resins and were shown to contain major proteolytic activity by zymography. Thus, rabbit anti-N54 is the first antibody developed for N. caninum that binds to themicroneme proteins and recognizes a major secreted enzyme.

Sequence evidence for an altered genetic code in the Neospora caninum plastid.

The plastid DNA of Neospora caninum encodes a homologue of the rpoB gene, which is believed to encode a subunit of a bacterial or chloroplast-like RNA polymerase. The predicted protein product of the N. caninum rpoB gene has three in-frame UGA codons which appear to encode tryptophan residues rather than act as stop codons. Based on the nucleotide sequence of a portion of the ssrRNA gene of the N. caninum plastid, a model for suppression of UGA termination in this plastid is presented.