Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.

BACKGROUND: Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. RESULTS: We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (DeltaLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of DeltaLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving DeltaLicpa::CPA) was sufficient to complement the reduced infectivity of both DeltaLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone DeltaLicpaC1::CPA compared with the CPA-deficient mutant DeltaLicpaC1. CONCLUSION: The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the DeltaLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.

Tight control of transcription in Toxoplasma gondii using an alternative tet repressor.

Fusion of yellow fluorescent protein (YFP) to the N-terminus of the Escherichia coli Tn10 tet repressor (TetR) created a functional YFP-TetR repressor with the capacity of 88-fold repression of transcription when expressed in Toxoplasma gondii. As a test promoter we used the T. gondii ribosomal protein RPS13 promoter for which we provide experimental evidence of having a single major transcriptional start site, a condition favourable to the design of inducible expression systems. Integration of four tet operator (tetO) elements, 23-43 bp upstream of the RPS13 transcriptional start site, resulted in maximal repression of transcription (88-fold). Moreover, integration of these four tetO elements reduced the promoter activity only 20% in comparison with the wildtype promoter. Regulation was six-fold higher compared with an inducible expression system employing wildtype TetR. Importantly, only 0.1 microg/ml tetracycline was required for maximal induction demonstrating a higher affinity of tetracycline for YFP-TetR than for wildtype TetR which required 1 microg/ml tetracycline for maximal induction. The use of 0.1 microg/ml tetracycline allows prolonged continuous culturing of T. gondii for which levels of 1 microg/ml tetracycline are toxic. Our results show that YFP-TetR is superior to TetR for transcriptional regulation in T. gondii and we expect that its improved characteristics will be exploitable in other parasites or higher eukaryotes.

Novel baculovirus-derived p67 subunit vaccines efficacious against East Coast fever in cattle.

Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67deltaSS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculovirus envelope protein GP64. Both GFP:p67deltaSS and GP64:p67C induced antibodies with high ELISA titers that neutralized T. parva sporozoites with high efficiency. Upon challenge, a correlation was observed between the in vitro neutralizing capacity and the reduction in severe ECF for individual animals. A protection level upto 85% was obtained. This level of protection was achieved with only two inoculations of 100 microg per dose, which is a major improvement over previous recombinant p67 products.

De novo ribosome biosynthesis is transcriptionally regulated in Eimeria tenella, dependent on its life cycle stage.

Protozoan parasites go through various developmental stages during their parasitic life, which requires the expression of different genes. To identify stage specific gene products in Eimeria tenella, a differential screening was performed comparing the intracellular schizont stage with the extracellular oocyst stage. De novo transcripts of 18S-5.8S-26S rRNA transcription units and of two ribosomal proteins (RPL5 and RPL23) were specifically identified in schizonts and were undetectable in oocysts. The stage specific transcription of pre-rRNAs (prior to processing) was confirmed with Northern blot analysis. Since the E. tenella genome contains a repeated gene cluster with an estimated 140 large rRNA transcription units, they all might be similarly regulated. Specific expression of RPL5 and RPL23 in E. tenella schizonts was also confirmed by Northern blotting. Furthermore, an analysis of the E. tenella EST database with 26,705 ESTs showed that 9.5% of all merozoite ESTs and only 0.2% of the sporozoite ESTs encoded ribosomal proteins (RPs). These ESTs encoded 69 different RPs, suggesting that most and possibly all RPs are differentially transcribed in E. tenella. Analysis of EST data from other Coccidia, such as Toxoplasma gondii, indicated a similar stage dependent transcription of RP genes. We conclude that ribosome biosynthesis is transcriptionally regulated in E. tenella and other Coccidia, such that rapidly growing parasite stages utilize much of their resources to de novo biosynthesis of ribosomes, and that "dormant" oocyst stages do not synthesize new ribosomes. The 50- to 100-fold reduction in transcription of RPs together with the reduced rRNA transcription prevents that unnecessary new ribosomes are synthesized in oocysts.

Avian coccidiosis: a disturbed host-parasite relationship to be restored.

The co-evolution of Eimeria and its host the domestic chicken has resulted in a delicate balance of mutual understanding and respect. This balance has been broken by the complete change of the environment in which the parasite was able to reproduce to such an extent that the host, stressed and weakened by heat, crowding and concurrent infections could not combat the shear numbers of organisms. The use of drugs to control the situation has been shown to only temporarily create relief. Resistance widely developed by the flexible genome of the parasite returned new drugs at a greater speed than they had been developed. Improved hygienic measures, better facility management and good understanding of epidemiology of the parasites spreading and proliferation seem the first and most promising set of tools to control the balance. Reduction of stock density may only provide any relief if this is done at a factor of 10 or higher and this is not a realistic measure in relation to the profit. Free-range chickens are an alternative if only animal welfare is at stake. However, in terms of prevalence of parasitic infections, such as coccidia, helminthes or ectoparasites, chickens do not seem to be better off (Permin et al., 2002). Immunological surveillance and the development of safe, effective and economical vaccines are further refinements that can be used to restore the relationship between parasite and host. Several live vaccines are effective and applied, but certainly have drawbacks in safety and production. New technology such as recombinant vectors together with a better understanding of the cell biology of the parasite from biological and genomic information should provide improved vaccines for the future. The strong genetically determined characteristics involved in the induction and maintenance of a sustainable protective immune response might turn out to be of decisive importance for the success of these strategies. The consequences for the physiology of the parasite remain to be understood.