Characterization and molecular cloning of a Cu/Zn superoxide dismutase from the human parasite Onchocerca volvulus.

Evidence suggests that the helminth antioxidant enzyme superoxide dismutase (SOD) may play a role in parasite's defense against the cellular immune mechanisms of the host. In order to investigate this for the human parasite Onchocerca volvulus, the enzyme activity was characterized, the release of SOD by the parasite was examined, and a complete cDNA encoding the O. volvulus SOD was identified. The SOD activity in adult O. volvulus was found to be 8.1 +/- 4.2 U/mg of protein. A Cu/Zn-containing enzyme was demonstrated by its sensitivity towards cyanide, azide, and hydrogen peroxide. Isoelectric focusing, combined with an enzyme activity assay, revealed two activities at pI 6.8 and 7.6, with both activities inhibited by KCN. Adult parasites, maintained in vitro, released SOD into the culture medium, which was detected by enzyme activity. In parallel, lactate production was measured to ensure the viability of the parasite. Oligonucleotides (based upon conserved sequences in the SOD genes of other organisms) and the polymerase chain reaction were used to identify a portion of the SOD gene from O. volvulus genomic DNA. A cDNA library was constructed in lambda unizapII and screened with the genomic polymerase chain reaction fragment. A complete cDNA encoding the Cu/Zn SOD was identified, and its nucleotide sequence was determined. Southern blot hybridization experiments indicated that the Cu/Zn SOD is encoded by a single-copy gene with at least one intron.     

Combined protective effect of a fungal Cu/Zn-containing superoxide dismutase and rimantadine hydrochloride in experimental murine influenza a virus infection

The combined protective effect of a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced by the fungus Humicula lutea (HL-SOD) strain 103, and the selective anti-influenza drug rimantadine hydrochloride (Rim) was evaluated in experimental virus infection in mice, induced with influenza virus A/Aichi/2/68 (H3N2). A combined application of HL-SOD and Rim in doses, which by themselves did not protect significantly mice against the infection, resulted in a synergistically increased protection, determined on the basis of protective indices. Lung virus titers, lung weights and consolidation and mortality rates were all decreased significantly, while survival times were prolonged.     

Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species.     

Heterologous expression and functional characterization of thioredoxin from Fasciola hepatica

The full thioredoxin coding sequence from Fasciola hepatica has been cloned into the pGEX-2T expression vector and produced in Escherichia coli as a fusion protein. The recombinant protein proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from F. hepatica. These observations suggest that this protein could participate in a redox cascade involved in the maintenance of cell homeostasis as well as in parasite protection against reactive oxygen species produced by the host.     

Characterization and differential expression of a ferritin protein from Fasciola hepatica

Ferritins are proteins that play a central role in maintaining intracellular iron balance. A cDNA clone of Fasciola hepatica (687 bp long) encoding a putative 228-amino acid polypeptide (FhFtn-1) homologous with ferritins of vertebrates and invertebrates was identified. FhFtn-1 contains a conserved motif of the ferroxidase center typical of vertebrate ferritins. Phylogenetic tree analysis showed that FhFtn-1 clusters with two ferritins of Paragonimus westermani, which suggests a common ancestry for the ferritins of these two trematodes. Recombinant FhFtn-1 protein expressed and purified from an Escherichia coli system showed iron-uptake ability. Moreover, FhFtn-1 showed strong reactivity with sera from rabbits infected with F. hepatica for 2-12 weeks, which suggests that this protein could be a potential antigen for immunodiagnosis of fascioliasis. qPCR analysis demonstrated that FhFtn-1-mRNA is expressed at significantly higher levels in adults and unembryonated eggs than in juveniles or miracidia. These results represent the first characterization of a ferritin protein from the liver fluke F. hepatica.     

Cu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury

Background  

In the nervous system, as in other organs, Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key antioxidant enzyme involved in superoxide detoxification in normal cellular metabolism and after cell injury. Although it has been suggested that immature brain has a different susceptibility to oxidative damage than adult brain, the distribution and cell-specific expression of this enzyme in immature brain and after postnatal brain damage has not been documented.     

Polyamine N-acetyltransferase from Fasciola hepatica.

A cytosolic polyamine N-acetyltransferase which catalyses polyamine and diamine acetylation has been partially purified from the liver fluke Fasciola hepatica. The enzyme has an apparent Mr of 50,000 and unlike the corresponding mammalian liver counterpart is capable of putrescine acetylation. Among the substrates tested, spermidine had the highest reaction rate but putrescine had a lower Km value. The Km values for spermidine, spermine, norspermidine, putrescine, cadaverine and 1,3-diaminopropane were 20 microM, 1.30 mM, 20 microM, 7 microM, 10 microM and 50 microM, respectively. Acetylated polyamines were also substrates for the trematode acetylase, but histones were inactive. The partially purified enzyme had no deacetylase activity. The Km for acetyl-CoA was 4.4 microM. Coenzyme A was strongly inhibitory with a Ki value of 5.3 microM. Bis(benzyl)polyamine analogue MDL 27695 was a potent competitive inhibitor of the enzyme with a Ki of 22 microM. Inhibition by 1,4-dimethyl-putrescine was non-competitive and had a Ki value of 15 microM. The trematode acetylase is highly dependent on sulfhydryl groups for its activity. As had been reported in nematodes, polyamine acetylation could represent a process by which trematodes convert excess polyamines to forms suitable for transport and excretion. On the other hand, this could be the regulatory step of a functional interconversion pathway in these parasites.     

A baculovirus homolog of a Cu/Zn superoxide dismutase gene

A gene with greater than 50% amino acid sequence identity to eukaryotic Cu/Zn superoxide dismutase genes (sod) was found at 19 map units in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). This gene was transcribed into 1.4- and 1.5-kb RNAs late in virus infection. The two late RNAs have coterminal 3' ends but initiate from two different start points, both of which map to the central adenine of the sequence motif ATTAAG. The late gene product was found to be a low abundance 20-kDa polypeptide which was observed only upon partial purification by chloroform/ethanol extraction. Viruses with mutations in sod were able to replicate normally both in cell culture and in insect larvae. Viruses lacking an intact sod showed no evidence of a replication disadvantage even in the presence of paraquat, an inducer of superoxide anions. A viral-encoded superoxide dismutase (SOD) activity was not detected although endogenous insect SODs appeared to be induced in insect hemolymph and in other tissues following virus infection. The function of sod in the baculovirus infection process remains unclear.     

Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni.

Our laboratories previously isolated a putative extracellular or membrane-associated Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene, designated a signal peptide-containing (SP) Cu/Zn-SOD, from Schistosoma mansoni. SOD activity was thus investigated throughout the life cycle of S. mansoni and found in all stages: eggs, miracidia, cercariae, schistosomula, lung-stage worms, and adult worms. The adult worms had the highest SOD activity (53 +/- 9 nitrite units), which was five times higher than that of eggs or miracidia and twice as high as that of 3-h-old mechanically transformed schistosomula. Cu/Zn-SOD constituted over 95% of the total SOD activity found in S. mansoni, compared with that of Mn-SOD. Most of Cu/Zn-SOD specific activity was associated with a detergent-extractable fraction of the parasite. Isoelectric focusing gel electrophoresis analysis revealed that there were four major pI variants of Cu/Zn-SOD present in the adult worms. Only two of these Cu/Zn-SOD pI variants were present in the 3-h-old mechanically transformed schistosomula. Fast protein liquid chromatography gel filtration fractionation of adult parasite extract was carried out to correlate the SP Cu/Zn-SOD with the SOD activity by using anti-SP Cu/Zn-SOD monoclonal antibodies, which separated the immunoreactive gene product and the SOD activity into different fractions. Quantitative tissue fractionation also revealed a discordant distribution of the gene product compared with that of Cu/Zn-SOD activity. These results indicated the existence of another Cu/Zn-SOD(s) in the parasite. Purification of the Cu/Zn-SOD activity from the adult worms showed that it represented the two lower-pI variants found in both adult worms and 3-h-old schistosomula. Peptide sequence analysis of the purified Cu/Zn-SOD confirmed that there is a second form of Cu/Zn-SOD in the parasite.     

Citrate synthase from the liver fluke Fasciola hepatica

Citrate synthase catalyses the first step of the Krebs’ tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K _m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.     

Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli.

A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.     

Identification, expression and in situ hybridization of an eggshell protein gene from Fasciola hepatica

The molecular basis of egg formation in the parasitic liver fluke, Fasciola hepatica, was investigated by isolating and characterizing an abundant cDNA from a female genital complex cDNA library. It was expressed in Escherichia coli as a beta-galactosidase fusion protein, which was purified and used to produce polyclonal antibodies. Using immunoblots, the antiserum recognized two soluble constituents of isolated egg shells, both significantly larger than predicted from cDNA sequencing. Using in situ hybridization, the message was detected in cells in the adult vitelline follicles. Eggshell protein mRNA expressed in E. coli will provide a source of precursor protein for further studies of parasite eggshell formation.     

Cloning and molecular characterization of Cu,Zn superoxide dismutase from Actinobacillus pleuropneumoniae.

Copper-zinc superoxide dismutases (Cu,Zn SODs), until recently considered very unusual in bacteria, are now being found in a wide range of gram-negative bacterial species. Here we report the cloning and characterization of sodC, encoding Cu,Zn SOD in Actinobacillus pleuropneumoniae, a major pathogen of pigs and the causative organism of porcine pleuropneumonia. sodC was shown to lie on a monocistronic operon, at the chromosomal locus between the genes asd (encoding aspartate semialdehyde dehydrogenase) and recF. The primary gene product was shown to have an N-terminal peptide extension functioning as a leader peptide, so that the mature Actinobacillus enzyme, like other bacterial examples, is directed to the periplasm, where it is appropriately located to dismutate exogenously generated superoxide. While the role of these secreted bacterial SODs is unknown, we speculate that in A. pleuropneumoniae the enzyme may confer survival advantage by accelerating dismutation of superoxide derived from neutrophils, a central host defense response in the course of porcine infection.     

Preparation and in vitro translation of mRNA from Fasciola hepatica

Total RNA was extracted from mature and juvenile Fasciola hepatica by homogenizing in 5.0 M guanidine thiocyanate and centrifugation through a 5.7 M CsCl cushion. Yields of 2 mg/g and 1 mg/g wet weight starting material were obtained, respectively. Messenger RNA was separated from the bulk extracted RNA by binding to oligo(dT)-cellulose. About 25% of the extracted RNA bound in both adult and juvenile cases. This material was subsequently tested in a rabbit reticulocyte cell-free translation system. Up to a 12-fold stimulation of incorporation of [35S]methionine into trichloroacetic acid-precipitable material was observed over that where no message was added. When the in vitro translation products were analysed by autoradiography of SDS-polyacrylamide gradient gels, polypeptides ranging in apparent molecular weight from about 10,000 to 100,000 were observed. Several minor differences in the electrophoretic patterns obtained from juvenile and adult mRNA were observed.