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Background: The trehalose synthetic pathway is present in bacteria, fungi, plants and invertebrate animals, but is absent in vertebrates. This disaccharide mainly functions as a stress protectant against desiccation, heat, cold and oxidation. Genes involved in trehalose synthesis have been observed in apicomplexan parasites, but little was known about these enzymes. Study on trehalose synthesis in apicomplexans would not only shed new light into the evolution of this pathway, but also provide data for exploring this pathway as novel drug target., Methodology/principal Findings: We have observed the presence of the trehalose synthetic pathway in Cryptosporidium and other apicomplexans and alveolates. Two key enzymes (trehalose 6-phosphate synthase [T6PS; EC 2.4.1.15] and trehalose phosphatase [TPase; EC 3.1.3.12] are present as Class II bifunctional proteins (T6PS-TPase) in the majority of apicomplexans with the exception of Plasmodium species. The enzyme for synthesizing the precursor (UDP-glucose) is homologous to dual-substrate UDP-galactose/glucose pyrophosphorylases (UGGPases), rather than the "classic" UDP-glucose pyrophosphorylase (UGPase). Phylogenetic recontructions indicate that both T6PS-TPases and UGGPases in apicomplexans and other alveolates are evolutionarily affiliated with stramenopiles and plants. The expression level of T6PS-TPase in C. parvum is highly elevated in the late intracellular developmental stage prior to or during the production of oocysts, implying that trehalose may be important in oocysts as a protectant against environmental stresses. Finally, trehalose has been detected in C. parvum oocysts, thus confirming the trehalose synthetic activity in this parasite., Conclusions/significance: A trehalose synthetic pathway is described in the majority of apicomplexan parasites including Cryptosporidium and the presence of trehalose was confirmed in the C. parvum oocyst. Key enzymes in the pathway (i.e., T6PS-TPase and UGGPase) are plant-type and absent in humans and animals, and may potentially serve as novel drug targets in the apicomplexans.

We have performed a whole-genome-sequence survey for the gregarine, Ascogregarina taiwanensis and herein describe both features unique to this early diverging apicomplexan and properties that unite it with Cryptosporidium, the Coccidia, and the Apicomplexa. Phylogenetic trees inferred from a concatenated protein sequence comprised of 10,750 amino acid positions, as well as the large subunit rRNA genes, robustly support phylogenetic affinity of Ascogregarina with Cryptosporidium at the base of the apicomplexan clade. Unlike Cryptosporidium, Ascogregarina possesses numerous mitochondrion-associated pathways and proteins, including enzymes within the Krebs cycle and a cytochrome-based respiratory chain. Ascogregarina further differs in the capacity for de novo synthesis of pyrimidines and amino acids. Ascogregarina shares with Cryptosporidium a Type I fatty acid synthase and likely a polyketide synthase. Cryptosporidium and Ascogregarina possess a large repertoire of multidomain surface proteins that align it with Toxoplasma and are proposed to be involved in coccidian-like functions. Four families of retrotransposable elements were identified, and thus, retroelements are present in Ascogregarina and Eimeria but not in other apicomplexans that have been analyzed. The sum observations suggest that Ascogregarina and Cryptosporidium share numerous molecular similarities, not only including coccidian-like features to the exclusion of Haemosporidia and Piroplasmida but also differ from each other significantly in their metabolic capacity.

The lead enzymes of polyamine biosynthesis, i.e. ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), were not detected in Toxoplasma gondii [the limit of detection for ODC and ADC was 5 pmol min(-1) (mg protein)(-1)], indicating that T. gondii lacks a forward-directed polyamine biosynthetic pathway, and is therefore a polyamine auxotroph. The biochemical results were supported by results obtained from data-mining the T. gondii genome. However, it was possible to demonstrate the presence of a highly active backconversion pathway that formed spermidine from spermine, and putrescine from spermidine, via the combined action of spermidine/spermine N(1)-acetyltransferase (SSAT) or spermidine N(1)-acetyltransferase (SAT) and polyamine oxidase (PAO). With spermine as the substrate, T. gondii SSAT had a specific activity of 1.84 nmol min(-1) (mg protein)(-1), and an apparent K(m) for spermine of 180 mM; with spermidine as the substrate, the SAT had a specific activity of 3.95 nmol min(-1) (mg protein)(-1), and a K(m) for spermidine of 240 mM. T. gondii PAO had a specific activity of 10.6 nmol min(-1) (mg protein)(-1), and a K(m) for acetylspermine of 36 mM. Furthermore, the results demonstrated that T. gondii SSAT was 50 % inhibited by 30 mM di(ethyl)norspermine. The parasite actively transported arginine and ornithine, which were converted via the arginine dihydrolase pathway to citrulline and carbamoyl phosphate, resulting in the formation of ATP via carbamate kinase. The lack of polyamine biosynthesis by T. gondii is contrasted with polyamine metabolism by other apicomplexans.

Cryptosporidium parvum is one of the major species causing mild to severe cryptosporidiosis in humans and animals. We have previously observed that 2‐deoxy‐d‐glucose (2DG) could inhibit both the enzyme activity of C. parvum hexokinase (CpHK) and the parasite growth in vitro. However, the action and fate of 2DG in C. parvum was not fully investigated. In the present study, we showed that, although 2DG could be phosphorylated by CpHK to form 2DG‐6‐phosphate (2DG6P), the anti‐cryptosporidial activity of 2DG was mainly attributed to the action of 2DG on CpHK, rather than the action of 2DG or 2DG6P on the downstream enzyme glucose‐6‐phosphate isomerase (CpGPI) nor 2DG6P on CpHK. These observations further supported the hypothesis that CpHK could serve as a drug target in the parasite. We also screened 1,200 small molecules consisting of marketed drugs against CpHK, from which four drugs were identified as CpHK inhibitors with micromolar level of anti‐cryptospordial activities at concentrations nontoxic to the host cells (i.e. hexachlorphene, thimerosal, alexidine dihydrochloride, and ebselen with EC50 = 0.53, 1.77, 8.1 and 165 μM, respectively). The anti‐CpHK activity of the four existing drugs provided us new reagents for studying the enzyme properties of the parasite hexokinase. [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum is unable to synthesize fatty acids de novo, but possesses three long-chain fatty acyl-CoA synthetase (Cp ACS) isoforms for activating fatty acids. We have recently shown that these enzymes could be targeted to kill the parasite in vitro and in vivo. Here, we demonstrated that the Cp ACS genes were differentially expressed during the parasite life cycle, and their proteins were localized to different subcellular structures by immunofluorescence and immuno-electron microscopies. Among them, Cp ACS1 displayed as an apical protein in sporozoites and merozoites, but no or little presence during the intracellular merogony until the release of merozoites, suggesting that Cp ACS1 probably functioned mainly during the parasite invasion and/or early stage of intracellular development. Both Cp ACS2 and Cp ACS3 proteins were present in all parasite life cycle stages, in which Cp ACS2 was present in the parasite and the parasitophorous vacuole membranes ( PVM), whereas Cp ACS3 was mainly present in the parasite plasma membranes with little presence in the PVM. These observations suggest that Cp ACS2 and Cp ACS3 may participate in scavenging and transport of fatty acids across the PVM and the parasite cytoplasmic membranes, respectively. [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum may cause virtually untreatable infections in AIDS patients, and is recently identified as one of the top four diarrheal pathogens in children in developing countries. Cryptosporidium differs from other apicomplexans (e.g., Plasmodium and Toxoplasma ) by lacking many metabolic pathways including the Krebs cycle and cytochrome-based respiratory chain, thus relying mainly on glycolysis for ATP production. Here we report the molecular and biochemical characterizations of a hexokinase in C. parvum (CpHK). Our phylogenetic reconstructions indicated that apicomplexan hexokinases including CpHK were highly divergent from those of humans and animals (i.e., at the base of the eukaryotic clade). CpHK displays unique kinetic features that differ from those in mammals and Toxoplasma gondii (TgHK) in the preference towards various hexoses and its capacity to use ATP and other NTPs. CpHK also displays substrate inhibition by ATP. Moreover, 2-deoxy-D-glucose (2DG) could not only inhibit the CpHK activity, but also the parasite growth in vitro at concentrations nontoxic to host cells (IC 50 = 0.54 mM). While the exact action of 2-deoxy-D-glucose on the parasite is subject to further verification, our data suggest that CpHK and the glycolytic pathway may be explored for developing anti-cryptosporidial therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Eimeria tenella, an apicomplexan parasite in chickens, possesses an apicoplast and its associated metabolic pathways including the Type II fatty acid synthesis (FAS II). Malonyl-CoA:acyl-carry protein transacylase (MCAT) encoded by the fabD gene is one of the essential enzymes in the FAS II system. In the present study, the entire E. tenella MCAT gene (EtfabD) was cloned and sequenced. Immunolabeling located this protein in the apicoplast organelle in coccidial sporozoites. Functional replacement of the fabD gene with amber mutation of E. coli temperature-sensitive LA2-89 strain by E. tenella EtMCAT demonstrated that EcFabD and EtMCAT perform the same biochemical function. The recombinant EtMCAT protein was expressed and its general biochemical features were also determined. An alkaloid natural product corytuberine (CAS: 517-56-6) could specifically inhibit the EtMCAT activity (IC50 =16.47μM), but the inhibition of parasite growth in vitro by corytuberine was very weak (the predicted MIC50 =0.65mM). [Copyright &y& Elsevier]

The Attwater's prairie chicken (APC; Tympanuchus cupido attwateri Bendire. 1894) has been a federally listed endangered species since 1967. Several captive propagation programs consisting of small populations are being used to keep this species from extinction. Fecal samples were collected from APCs in April 2007 and again in August 2008 from 2 separate captive propagation facilities in Texas after clinical signs of coccidiosis were observed. One Eimeria species was observed (Eimeria attwateri), which we describe as a putative new species. Sporulated oocysts are ellipsoidal, 30.0 x 18.4 (27.4-31.3 X 16.0-22.4) urn. Oocysts have a smooth wall 0.7 um thick and lack both a micropyle and oocyst residuum, but 1 ellipsoidal polar granule is present, 2.3 x 1.9 (2.1-2.4 x 1.7- 2.0) µm. Sporocysts have a nipple-like Stieda body with a rounded opposite end and are 14.0 x 7.1 (10.2-16.8 X 6.0-9.2) µm. The sporocysts contain a sporocyst residuum usually consisting of 2-4 dispersed globules, and each sporozoite contains 2 large posterior spheroid refractile bodies 3.4 urn wide. Nucleotide sequence amplified from the 18S rDNA does not match any DNA sequence information for publicly available Eimeria species, and phylogenetic reconstructions place this species with other eimerians from Galliformes. The discovery of a potentially pathogenic species of Eimeria in captive APCs is of great importance, and managers should be aware of the potential devastating effect(s) this parasite could have on the APC conservation programs. [ABSTRACT FROM AUTHOR]

Abstract: Cryptosporidium parvum is a member of the Apicomplexa that lacks a plastid and associated nuclear-encoded genes, which has hampered its use in evolutionary comparisons with algae and eliminated a pool of potentially useful drug targets. Here we show that apicomplexan parasites possess an unusual family of class II histone deacetylase (HDAC) proteins with orthologues that are present in other chromalveolates and primitive algae. A striking feature of these HDAC proteins is the presence of ankyrin repeats in the amino-terminus that appear to be required for enzyme activity. In vitro and in vivo analyses of the C. parvum orthologue indicate that this subclass of chromatin-remodelling proteins is targeted by the anti-cancer drug suberoylanilide hydroxamic acid and that these proteins are most likely involved in the essential process of H4 histone deacetylation that coincides with DNA replication. We propose that members of this novel class of histone deacetylase can serve as promising new targets for treatments against debilitating diseases such as cryptosporidosis, toxoplasmosis and malaria. [Copyright &y& Elsevier]

Abstract: The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h =1.46, K 50 =0.751μM, V max =2.236μmolmg−1 min−1) and CoA (h =0.704, K 50 =5.627μM, V max =0.557μmolmg−1 min−1), and biphasic kinetics towards adenosine 5′-triphosphate (K m1 =3.149μM, V max1 =373.3nmolmg−1 min−1, K m2 =121.0μM, and V max2 =563.7nmolmg−1 min−1). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50 =6.64μM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure. [Copyright &y& Elsevier]

Replication protein A is a single stranded DNA-binding protein that has multiple roles in eukaryotic DNA metabolism. Typically, eukaryotic replication protein A is a stable heterotrimeric complex with three subunits of 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3). We have previously cloned and characterised an RPA1 subunit from Cryptosporidium parvum, which shares high homology with other eukaryotic replication protein A 1 proteins, but lacks an N-terminal domain. Here, we have identified a second replication protein A 1 (termed CpRPA1B) from the ongoing C. parvum genome-sequencing project. The deduced protein sequence to CpRPA1B shows only 16% sequence identity with CpRPA1, indicating that two different types of RPA1 subunits are present in C. parvum. The CpRPA1B gene predicts a 75.5 kDa peptide similar in size to those of higher eukaryotes, but in contrast to the 53.9 kDa N-terminal short-type CpRPA1 protein. However, western blot analysis suggested that, although the entire CpRPA1B open reading frame might be translated, the protein may be cleaved by posttranslational modification, similar to that observed with the replication protein A 1 gene product in Plasmodium falciparum. Indirect immunofluorescence studies indicated a diffused pattern for both proteins in sporozoites. However, differential localisation was observed with CpRPA1 to the anterior region that contains the apical-complex and CpRPA1B to the central region in/or around the nuclei of the sporozoites. Both CpRPA1 and CpRPA1B full-length open reading frames were expressed for functionality assays. The CpRPA1 and CpRPA1B recombinant proteins were expressed in bacterial Escherichia coli as maltose-binding protein fusion proteins and the entire fusion proteins were assayed for their DNA-binding properties. Studies indicate that CpRPA1B binds ssDNA of ≥5 nucleotides (dT), while CpRPA1 only binds ssDNA ≥20 nucleotides (dT). This study indicates that C. parvum possesses two different types of replication protein A large subunits (replication protein A 1), both differing significantly from their hosts. [Copyright &y& Elsevier]

Cryptosporidium parasites are known to be highly divergent from other apicomplexan species at evolutionary and biological levels. Here we provide evidence showing that the zoonotic Cryptosporidium parvum also differs from other apicomplexans, such as Toxoplasma gondii, by possessing only two tubulin-based filamentous structures, rather than an array of subpellicular microtubules. Using an affinity-purified polyclonal antibody against C. parvum β-tubulin (CpTubB), we observed a long and a short microtubule that are rigid and stable in the sporozoites and restructured during the intracellular parasite development. In asexual development (merogony), the two restructuring microtubules are present in pairs (one pair per nucleus or merozoites). In sexual developmental stages, tubulin-based structures are detectable only in microgametes, but undetectable in macrogametes. These observations indicate that C. parvum parasites use unique microtubule structures that differ from other apicomplexans as part of their cytoskeletal elements. [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum is a globally recognized zoonotic parasite of medical and veterinary importance. This parasite mainly infects intestinal epithelial cells and causes mild to severe watery diarrhea that could be deadly in patients with weakened or defect immunity. However, its molecular interactions with hosts and pathogenesis, an important part in adaptation of parasitic lifestyle, remain poorly understood. Here we report the identification and characterization of a C. parvum T-cell immunomodulatory protein homolog (CpTIPH). CpTIPH is a 901-aa single-pass type I membrane protein encoded by cgd5_830 gene that also contains a short Vibrio, Colwellia, Bradyrhizobium and Shewanella (VCBS) repeat and relatively long integrin alpha (ITGA) N-terminus domain. Immunofluorescence assay confirmed the location of CpTIPH on the cell surface of C. parvum sporozoites. In congruence with the presence of VCBS repeat and ITGA domain, CpTIPH displayed high, nanomolar binding affinity to host cell surface (i.e., Kd(App) at 16.2 to 44.7 nM on fixed HCT-8 and CHO-K1 cells, respectively). The involvement of CpTIPH in the parasite invasion is partly supported by experiments showing that an anti-CpTIPH antibody could partially block the invasion of C. parvum sporozoites into host cells. These observations provide a strong basis for further investigation of the roles of CpTIPH in parasite-host cell interactions. [ABSTRACT FROM AUTHOR]

Abstract: D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Unicellular apicomplexans possess an algal-originated plastid referred to as an apicoplast. Although apicomplexan parasites are comprised of highly diverse protists, the complete apicoplast genome sequences have only been determined from the hematozoan Plasmodium falciparum and cyst-forming coccidian Toxoplasma gondii. Here, we report the third complete sequence of apicoplast genome from the intestinal coccidian Eimeria tenella that may serve as a new drug target against coccidiosis in the livestock. The AT-rich E. tenella plastid genome is a 35-kb circular element. Its gene organization resembles more closely that of T. gondii than P. falciparum. Although the E. tenella plastid genome contains an almost identical set of genes to that found in P. falciparum and T. gondii, its encoded genes share low or moderate homologies with their counterparts in the other two apicomplexans. With the addition of this coccidian plastid genome sequence, we attempted to reexamine the apicoplast genome evolution and performed phylogenetic reconstructions using maximum likelihood and Bayesian inference (BI) methods based on a concatenated dataset of plastid-encoded rpoB, rpoC1 and rpoC2 proteins. All resulting rpo protein trees placed apicoplast as a sister to Euglena within the green lineage. On the other hand, many recent studies based on the organization of plastid genes and some nuclear-encoded plastid proteins have supported a common red algal ancestry of apicomplexan and dinoflagellate plastids. If the apicoplast indeed originated from a red ancestor, the green relationship of apicomplexan genes would probably imply that the ancestral host that gave rise to the (red) apicoplast might have already contained some primary green plastid genes. [Copyright &y& Elsevier]