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The cell surface of the epimastigote form of Trypanosoma cruzi is covered by glycoconjugates rich in galactose. The parasite cannot take up galactose through its hexose transporter, suggesting that the epimerisation of UDP-glucose to UDP-galactose may be the parasite's only route to this sugar. The T. cruzi UDP-glucose 4'-epimerase is encoded by the TcGALE gene. We were unable to make a CL-Brener strain T. cruzi epimastigote TcGALE-/- null mutant, suggesting that the gene is essential. Two TcGALE+/- single-allele knockout clones displayed aberrant morphology and haploid deficiency with respect to galactose metabolism. The morphological phenotypes included shortened flagella, increased incidence of spheromastigotes, agglutination and a novel walnut-like appearance. The reduced supply of UDP-galactose was manifest in the two clones as a six- or nine-fold reduction in the expression of galactopyranose-containing cell surface mucins and negligible or two-fold reduction in the expression of galactofuranose-containing glycoinositolphospholipids. The major loss of mucins as opposed to glycoinositolphospholipids may indicate that the latter are more important for basic parasite survival in culture. The apparent haploid deficiency suggests that epimerase levels are close to limiting, at least in the epimastigote form, and might be exploited as a potential drug target.

The parasitic protozoan Trypanosoma cruzi undergoes several differentiation events during its life cycle. Some of these transitions are thought to involve activation of adenylyl cyclase via the binding of peptide ligands to the cell surface. Here we describe the characterisation of the adenylyl cyclase gene family of T. cruzi. Two complete genes and one pseudogene have been sequenced. The protein products appear to have a large extracellular domain, a single transmembrane helix and a cytosolic catalytic domain. The adenylyl cyclase genes are present on at least six chromosomes and are scattered rather than clustered. They form a large polymorphic family in which the extracellular domain is particularly variable. An Escherichia coli adenylyl cyclase mutant could be complemented by expression of the catalytic domain of the T. cruzi enzyme. The recombinant protein had adenylyl cyclase activity in vitro, which was enhanced by increasing concentrations of divalent cations (Mn2+ > Mg2+). This constitutively active recombinant protein will be a useful tool for dissecting the catalytic mechanism of adenylyl cyclase.

The genes that encode cruzipain, the major cysteine protease of Trypanosoma cruzi are known to be arranged in tandem arrays. To gain a detailed insight into how these arrays are organised at the chromosomal level we have isolated clones from a cosmid library constructed with DNA from the X10.6 strain. In this strain we found that cruzipain is encoded by two allelic clusters composed of approximately 14 and 23 tandemly repeated genes which are located on homologous chromosomes of 650 and 670 kb. With the exception of the 3'-proximal genes, the cruzipain genes were all of identical or very similar sequence. An unusual feature of the 3'-proximal genes is that they lack the sequences that encode the 130 amino acid carboxyl terminal extension which is characteristic of cruzipain. Both gene clusters are situated in a similar chromosomal environment and are flanked by sequences which have the potential to form Z-DNA. In other eukaryotes, these motifs have been associated with recombinational hotspots and have been demonstrated to enhance gene conversion. The cruzipain genes are transcribed to produce a 1.8-kb transcript which is present at the same steady-state level in each of the parasite life cycle stages. However, protein levels and activity are 4-5-times higher in the insect epimastigote stage than in the trypomastigote and amastigote stages. By implication developmental regulation of cruzipain expression occurs predominantly at the translational and/or post-translational levels.

In the genus Leishmania there has been no convincing demonstration of genetic exchange, and it has been proposed that reproduction is clonal. However, preliminary characterization of two strains of Leishmania isolated from wild animals in a zoonotic focus of cutaneous leishmaniasis in the Eastern Province of Saudi Arabia, has suggested that they may represent hybrids of Leishmania major and Leishmania arabica. Evidence presented here strongly supports this hypothesis. Isoenzyme analysis and molecular karyotyping of cloned organisms indicated that the putative hybrids are distinct from other species of Leishmania, and possess characteristics of both L. major and L. arabica. Experiments using highly specific probes demonstrated that kinetoplast minicircle DNA from the putative hybrid contained L. major-specific, but not L. arabica-specific sequences. DNA fingerprinting data obtained using 6 genomic DNA probes were consistent in all cases with a L. major/L. arabica recombinant genotype, and implied both diploidy and allelic segregation. These observations suggest that sexual reproduction may generate genetic diversity within natural Leishmania populations.

The parasitic protozoan Trypanosoma cruzi is exposed to toxic oxygen metabolites which arise from drug metabolism or immune mechanisms, in addition to those produced by endogenous processes. Identification and functional analysis of parasite enzymes which confer protection against oxidative stress is therefore of importance. To investigate the role of T. cruzi superoxide dismutase (SOD) we transfected epimastigotes with an expression vector containing a putative Fe-SOD gene homologue and achieved overexpression of enzyme activity (5–8 fold). Inhibition studies carried out on the partially purified enzyme revealed azide and H2O2 sensitivity and cyanide insensitivity, the profile expected of an Fe-isoform. Phenotypic analysis of transformed parasites showed that they were more susceptible than control cells to growth inhibition by the trypanocidal drug benznidazole and by gentian violet, an agent which can be used to decontaminate blood supplies in endemic areas. These results may reflect an imbalance in the antioxidant defences of the parasite produced as a result of overexpression of Fe-SOD.

In Trypanosoma cruzi a cell surface enzyme with trans-sialidase (TS) activity has been implicated as an important factor in establishing infection. The enzyme is encoded by genes belonging to a large super-family which on the basis of sequence has been subdivided into 4 groups. TS mediates the transfer of sialic acid residues from host glycoconjugates to acceptor molecules on the parasite surface. To study the organisation of the TS genes we isolated several distinct cosmids from a library constructed with DNA from the T. cruzi X10.6 clone. In these cosmids, the TS genes (group I) were present either as single copies or as a direct tandem repeat. A common feature of the cosmids was the presence of a related group III gene located 10–12kb downstream of the TS gene(s) and arranged in the same orientation. In several of the cosmids we also identified a mucin-like glycoprotein gene located between the group I and group III genes. The mucin-like genes are part of a large polymorphic family and contour clamped homogeneous electric field electrophoresis (CHEFE) analysis showed that they were linked to members of the TS super-family at multiple sites in the X10.6 genome. Screening of a second cosmid library made with DNA from the CL-Brener clone confirmed this multiple linkage suggesting that it is a common feature of the species. This genetic organisation may have important functional significance since the mucin-like glycoproteins are the major cell surface acceptors of sialic acid.

Development of new chemotherapeutic agents against Chagas’ disease is a major WHO objective [I]. Drugs currently in use, such as nifurtimox, often have serious side effects and can fail to eradicate parasitemia [2]. The trypanocidal effects of nifurtimox are thought to be mediated through the generation of nitro-radicals which promote the formation of toxic oxygen metabolites within the parasite [3,4]. Oxidative stress resulting from the generation of the superoxide radical has also been implicated as a mechanism of action of gentian violet, an agent employed to prevent transmission of Chagas’ disease by blood transfusion [5,6]. Thus, identification and functional analysis of Trypanosoma cruzi enzymes involved in antioxidant defence could be of importance in the context of improved chemotherapeutic approaches.

To extend the range of genetic tools available for the functional analysis of trypanosomatid genes we have constructed a cosmid shuttle vector (pcosTL) which facilitates the introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani. The vector contains several features to simplify library construction and insert mapping and transformed cells can be selected on the basis of G418 resistance. To evaluate the vector and to determine the fidelity of replication we first constructed cosmid libraries and isolated clones containing the T. cruzi major cysteine protease genes (a tandemly repeated array) and the L. donovani trypanothione reductase gene (a single copy gene). T. cruzi and L. donovani cells transfected with their respective cosmids were characterised by the presence of multiple copies of cosmid DNA and by a considerable over-expression of the corresponding enzyme activity. Rearrangements or deletions of insert sequences were not detected. These findings and the observation that cosmid DNA can be rescued unaltered from transformed parasites suggest that the pcosTL vector will be ideally suited for studies involving functional complementation.

Leishmania donovani (HU3 strain) metacyclic promastigotes generated in vitro were used to construct a cDNA library in the bacteriophage vector λgt10. A cDNA clone (Lmet 2), was isolated by differential screening with metacyclic-derived or log-phase promastigote-derived cDNA. The clone insert was comprised predominantly of four copies of an imperfect 60-bp repeat motif, which was represented in the genome by multiple tandem repeats distributed among at least six chromosomes. The corresponding mRNA transcript was a developmentally regulated 12-kb doublet. The Lmet 2 sequence was entirely specific to L. donovani donovani, L. donovani (East Africa), L. donovani infantum and L. donovani chagasi , even when genomic Southern blots and slot blots of other Leishmania species were washed at low stringencies. Twenty-two strains of L. donovani were clearly detected by radiolabelled Lmet 2 cDNA probe, with signals of approximately equal intensity, irrespective of geographical origin, which encompassed widely dispersed endemic regions. The probe could detect DNA from fewer than 100 organisms and identified small numbers of promastigotes in infected sand flies. Amastigotes were also detected in impression smears of organs from infected hamsters. The Lmet 2 probe is likely to be a valuable reagent for clinical diagnosis and epidemiological investigations.

A lambda gt11 expression library was constructed using mRNA from the promastigote form of Leishmania donovani (African isolate MHOM/ET/67/HU3). The library was screened with serum obtained from a patient who contracted visceral leishmaniasis in the Sudan. Several cDNA clones which expressed beta-galactosidase/L. donovani antigen fusion proteins were isolated. One of these clones corresponded to a 60 kDa membrane-associated antigen. By a Western blot assay antibodies against the fusion protein were detected universally in the sera of visceral leishmaniasis patients. They were not detected in sera from patients with cutaneous leishmaniasis or other parasitic protozoan infections. The gene coding for this antigen was specific to the genus Leishmania as judged by DNA hybridisation, and its chromosomal location was conserved. A 20 kb mRNA was detected on Northern blots of promastigote RNA.