Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi.

This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50,066. Comparison of the deduced amino acid sequence of LipDH from T. cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively. An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd1 of T. cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N-terminus of the mature protein. This system allowed the synthesis of 1000 U T. cruzi LipDH/1 bacterial cell culture. The recombinant protein was purified to homogeneity by (NH4)2SO4-precipitation and affinity chromatography on 5' AMP-Sepharose. The K(m) values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite. LipDH is present in all major developmental stages of T. cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite. In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason T. cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystallization of the protein.

Schistosomiasis in the southern region of Oman: vector snails and serological identification of patients in several locations.

The snail Biomphalaria arabica is apparently ubiquitous in the south of Oman (Dhofar province). Snails bred in the laboratory were susceptible to infection with miracidia of Schistosoma mansoni (Puerto Rican strain). The snail Bulinus wrighti, a potential intermediate host of S. haematobium, was found for the first time in Dhofar. Human sera from five localities had antibodies against adult worm antigens and in particular against Sm31/32. The prevalence of seropositive patients was 28% of 47 farm workers, 12% of 99 out-patients from a clinic and 1% of 389 children from four localities. Autochthonous transmission of schistosomiasis in Dhofar is discussed.

Efficacy of praziquantel treatment of schistosomiasis in a non-endemic country: a follow-up of parasitological, clinical and immunological parameters.

Schistosomiasis patients were immigrants to Czechoslovakia from Angola and Yemen. Most of them had light or moderate infections and felt subjectively healthy. They received treatment with praziquantel (two doses with a total of 40 mg/kg) and were followed up for several years. In nine of 13 patients, Schistosoma haematobium or S. mansoni eggs with undamaged miracidia were detected in biopsies from the bladder or the rectum one year or later after treatment. Granulomatous reactions in the rectum and bladder lesions of stage 1 including thickened bladder walls persisted in most of the patients. Antibody levels against adult S. mansoni worm antigen remained elevated for at least two years after therapy in some patients and declined in others. Among the nine patients, for whom pre- and post-treatment sera were available, the changes in relative levels of antibodies did not strictly correlate with the continued presence of schistosome eggs in, or their absence from, biopsies. We discuss the results obtained with sensitive diagnostic techniques in the absence of subjectively perceived disease.

Schistosoma mansoni and S. japonicum: decline of antibodies against diagnostic adult worm antigens (Sm31/32) following praziquantel treatment of mice.

Mice were infected with 10, 100 or 200 cercariae of Schistosoma mansoni or S. japonicum and treated between one and two months later with two doses of praziquantel. At this time, the animals had high levels of antibodies against the adult worm proteins Sm31/32 (schistosome cathepsin B and haemoglobinase). Antibody levels were followed up for about one more year by Western blots and ELISA using purified Sm31/32. Among the 34 surviving mice perfused at the end of the experiments, 14 had between 1 and 4 and one mouse had 8 residual stunted worms. In most mice without detectable worms, anti-Sm31/32 antibodies started to drop within a few months after therapy. In other animals of this group and in those harbouring residual worms, the decrease in titers was not observed or less pronounced. Thus, the presence of even one stunted worm was sufficient to induce continued high titres of antibodies. Decreasing levels of antibody against Sm31/32, however, reflected complete elimination of schistosomes.