Essential role of chitinase in the development of the filarial nematode Acanthocheilonema viteae.

Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode Acanthocheilonema viteae, a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an A. viteae phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.

Histochemical differentiation of Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit, Leucognost-SP.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae.

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.

Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein.

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

Immunogenicity of the extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae delivered by a two-phase vaccine strain of Salmonella typhimurium.

The recombinant extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae (AVSOD2) was cloned in an expression vector under control of the bacteriophage T7 promoter and the resulting plasmid pLAT7 was introduced in tha aroA attenuated Salmonella typhimurium vaccine strain SL3261:pYZ84. This vaccine strain carries a chromosomally integrated two phase expression system containing inducible T7 RNA polymerase. The recombinant AVSOD2 was efficiently expressed, constituting up to 5% of the total bacterial protein. Furthermore, the plasmid vector containing the AVSOD2 cDNA was shown to be stable over a long period of time in the vaccine strain without antibiotic selection in vitro and in vivo. Jirds which were immunised orally with the recombinant vaccine strain expressing the A. viteae EC-SOD produced a strong humoral immune response.

A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production.

Filarial nematodes are a cause of chronic debilitating diseases in the tropics. A hallmark of filariasis is the marked down-regulation and polarization of host immune responses, yet molecular constituents of parasites causing this state have remained undefined. We describe a 17-kDa antigen (Av17) of the rodent filarial parasite Acanthocheilonema viteae, which shows amino acid homologies to cystatin C, a major cysteine protease inhibitor belonging to family 2 of the cystatin superfamily. Av17 is released by filariae in vitro. Exported molecules of A. viteae worms are shown to markedly suppress mitogen-induced T cell proliferation of mice and jirds. Av17 accounts for 45.5% of this suppressive activity in the murine system. Recombinant Av17 (rAv17), expressed in Escherichia coli, exhibits biological activity as a cysteine protease inhibitor and was used to examine the immunomodulatory effects, rAv17 induces down-regulation of murine T cell responses to mitogens, to T cell receptor cross-linking by anti-CD3 antibodies and to specific antigens, and at the same time up-regulation of interleukin-10. Hence, this filarial cystatin is a likely effector molecule of immunomodulation and a potential target for antifilarial intervention.

Identification of chitinase as the immunodominant filarial antigen recognized by sera of vaccinated rodents.

Acanthocheilonema viteae is a parasitic nematode of rodents. We identified the chitinase of A. viteae infective stage larvae (L3) as the main target of the humoral immune response of jirds, which were protected against challenge infection after vaccination with irradiation attenuated L3. The cDNA of the L3 chitinase has been sequenced, and the deduced amino acid sequence shows significant homologies to chitinases of Brugia malayi microfilariae, insects, yeast, bacteria, and Streptomyces sp. The protein has been characterized by monoclonal antibodies and substrate activity gels. The chitinase of L3 may contribute to degrading the nematode cuticle during molting and thus represents a target of protective immune responses in a phase where the parasite is highly vulnerable. In addition, it has been shown that a similar enzyme exists in uterine microfilariae, which probably has a role in casting the egg shell.

Chitinase genes expressed by infective larvae of the filarial nematodes, Acanthocheilonema viteae and Onchocerca volvulus.

State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.

In vitro antifilarial evaluation of phenoxycyclohexane derivatives.

Numerous potential inhibitors of the phosphoenolpyruvate-carboxylase found in Molinema dessetae were synthesized and evaluated as inhibitors of the purified enzyme and as killers, in vitro, of M. dessetae adult females and infective larvae. Phosphoenolpyruvate analogs (or derivatives) appeared disappointing whereas phenoxycyclohexane derivatives inhibited the enzyme in a non-competitive manner and killed the parasites. The compounds P2281 and P2285, for example, had Ki values of 122 and 93 microM, respectively. In vivo tests of the most effective phenoxycyclohexanes will now be carried out to check their potential as antifilarial drugs.

Effect of 2,2'-dicarbomethoxylamino-5,5'-dibenzimidazolyl ketone on antioxidant defenses of Acanthocheilonema viteae and its laboratory host Mastomys natalensis.

The effect of the macrofilaricidal agent of 2,2'-dicarbomethoxylamino-5,5'-dibenzimidazolyl ketone (C.D.R.I. compound 82/437), on the metabolism of reactive oxygen species (ROs) in Acanthocheilonema viteae and Mastomys natalensis was measured following intraperitoneal administration at therapeutic doses. The recovered worms possessed substantially reduced levels of catalase and glutathione peroxidase (GPx), and thus were less able to detoxify H2O2. Nonetheless, the subcutaneous and adjoining muscle tissues, in which the parasites were lodged, exhibited elevated levels of antioxidant enzymes and reduced glutathione. It is concluded that compound 82/437 kills the filariid by paralysing its H2O2 detoxifying capacity without altering ROs metabolism in the tissue in which the parasite resides. Furthermore, since catalase and GPx of the liver and lungs do not show sign of inhibition, a difference appears to exist in the enzymes of the parasite and the host.

Polyamine metabolism in some helminth parasites.

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.

An investigation of fructose utilization in Acanthocheilonema viteae.

The capacity of Acanthocheilonema viteae to metabolize fructose was investigated in vitro. In common with other filarial species A. viteae oxidized fructose to lactate but its rate of consumption was only 40% of the glucose-containing control value. Fructose was not incorporated into glycogen. Release of 14CO2 from [U-14C]fructose was not detected in the presence of glucose and was about 40% of the glucose-containing value under conditions where fructose was the sole hexose substrate. Fructose consumption and lactate excretion increased in proportion to the external concentration of fructose. However, worm viability was not maintained in fructose over a 120 h in vitro incubation. In the presence of fructose, protein synthesis (measured incorporation of [35S]methionine into acid-insoluble material) was reduced compared to the glucose-containing control group; but was significantly greater than the value obtained under glucose-free conditions.

Acid phosphatase activity in microfilariae of Setaria labiato-papillosa and comparison with other blood microfilariae of dog and horse origin.

Acid phosphatase activity was demonstrated in smears of Setaria labiato-papillosa microfilariae by the naphthol AS-TR-phosphate method. The staining was restricted to 3 distinct sites, corresponding to the excretory pore, the inner body and the anal pore. This staining pattern was compared with those of Dirofilaria immitis, Dirofilaria repens and a Setaria of horse origin.

Antioxidant enzymes in Acanthocheilonema viteae and effect of antifilarial agents.

Adult worms of Acanthocheilonema viteae were found to be susceptible to the reactive oxygen intermediates (ROI) generated by the xanthine-xanthine oxidase (X-XO) system. The damage caused by this system was completely abolished by superoxide dismutase (SOD) and catalase but not by mannitol. The results, therefore, suggest that superoxide anions (O2-) and hydrogen peroxide (H2O2) alone or in combination might be toxic to the filariid. A. viteae exhibited the presence of an active enzyme system to protect itself against the oxidants. SOD and catalase were present in high levels of activities and appeared to constitute the major defence system. The role of glutathione peroxidase (GPx), on the other hand, seemed less important due to the weak activities of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH). A. viteae also released SOD, catalase and GPx in the ambient medium, which appear useful in protecting the filariid against ROI generated by the host in the immediate surroundings of the parasite. Antifilarial agents, diethylcarbamazine (DEC) and 2,2'-dicarbomethoxylamino-5,5'-dibenzimidazolyl ketone (82/437) appreciably inhibited catalase and GPx of A. viteae. Inhibition of these enzymes appears to render the parasite prone to H2O2 toxicity leading to death. No adverse effect on antioxidant enzymes of liver, lungs and subcutaneous tissue of Mastomys natalensis recorded as a result of exposure to 82/437 suggests a non-toxic nature to the compound.

The acid phosphatase activity and morphological characteristics of Dipetalonema dracunculoides (Cobbold, 1870) microfilariae.

The acid phosphatase activity and some morphological characteristics of Dipetalonema dracunculoides microfilariae are described. Their morphological features are closely related to those of the pathogenic Dirofilaria immitis when Knott's technique is used for the microfilarial diagnosis. The acid phosphatase activity pattern found in Dip. dracunculoides microfilariae is clearly different from those previously described for D. immitis, D. repens and Dip. reconditum.

A nuclear magnetic resonance study of the role of phosphoenol pyruvate carboxykinase (PEPCK) in the glucose metabolism of Dipetalonema viteae.

13C-NMR has been applied to the study of the metabolism of [1-13C]glucose by macrofilariae of Dipetalonema viteae under conditions of restricted glucose supply. In a medium buffered with 13C-labelled bicarbonate, succinate labelled in the carboxyl position is formed in good yield. Quinolinic acid, a known inhibitor of phosphoenol pyruvate carboxykinase (PEPCK) has been shown to suppress the formation of labelled succinate from [1-13C]glucose. Both sets of experiments support the formation of succinate through the PEPCK-mediated carboxylation of phosphoenol pyruvate, followed by the operation of a partial tricarboxylic acid cycle.

Protein kinases in different life stages of Brugia malayi and other filarial worms.

The investigation of protein kinase activity in different life stages of a few filarial worms indicates the presence of different protein kinases and of dissimilarities between the distribution pattern of these enzymes during the development. Phosvitin turned out to be the preferred exogenous acceptor protein, when homogenates of adults of Brugia malayi, Dipetalonema viteae, Setaria cervi and Litomosoides carinii were used as source of enzyme. In contrast to adults the L3-larval and the microfilarial stages demonstrated much lower phosvitin phosphorylating activity and the preference for acidic and basic proteins as phosphate acceptors was about equal. The occurrence of cyclic AMP-dependent protein kinase was established for adults of B. malayi.

Localization of acid phosphatase in microfilariae of Loa loa and Dipetalonema perstans from Cameroun.

The histochemical pattern of acid phosphatase activity in the microfilaria of L. loa showed a diffuse red staining all over the body, with conspicuous staining of the excretory and anal vesicles, Innenkörper, amphids and phasmids. In D. perstans, the enzymatic activity was localized chiefly in four regions corresponding to the excretory and anal vesicles, amphids and phasmids of the microfilariae. A considerable loss of enzyme activity was observed in microfilariae that had been stored in a refrigerator for somedays prior to their fixation.