Dataset Information

Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.

ABSTRACT: Nucleoside hydrolase (NH) (EC 3.2.2.3) is an essential enzyme in the purine-pyrimidine salvage pathway utilised by many protozoan parasites and may be a useful drug target. However, the search for NH inhibitors has been hampered by the lack of suitable in vitro screens. We have constructed a Saccharomyces cerevisiae strain that requires expression of the Leishmania major nucleoside hydrolase (LmNH) enzyme for growth and that may be suitable as a screen for NH inhibitors. The gene encoding LmNH was amplified using polymerase chain reaction with L. major genomic DNA as the template, cloned into an expression vector and used to transform a yeast mutant unable to grow on uridine as the sole source of uracil. Expression of LmNH yielded an ca. 35.6kDa protein, which was shown to be functional as the mutant strain was able to grow on medium containing uridine as the sole source of uracil. Importantly, this work has resulted in a strain that can be used to screen compounds as potential inhibitors of this essential Leishmania enzyme.

SUBMITTER: Miller TK

PROVIDER: S-EPMC1847609 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.

Miller Tamara K TK Patel Champa C Selitrennikoff Claude P CP

International journal of antimicrobial agents 20061201 1

Nucleoside hydrolase (NH) (EC 3.2.2.3) is an essential enzyme in the purine-pyrimidine salvage pathway utilised by many protozoan parasites and may be a useful drug target. However, the search for NH inhibitors has been hampered by the lack of suitable in vitro screens. We have constructed a Saccharomyces cerevisiae strain that requires expression of the Leishmania major nucleoside hydrolase (LmNH) enzyme for growth and that may be suitable as a screen for NH inhibitors. The gene encoding LmNH w ...[more]

PMID: 17141482

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Project description:Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani nucleoside hydrolase (NH36) induced a main CD4(+) T cell driven protective response against L. chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1-103), central domain (F2 aminoacids 104-198), and C-terminal domain (F3 amino acids 199-314) in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48 and 64%, and the parasite load in footpads to 82.6 and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR) against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4(+) and CD8(+) T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8(+) mediated immune responses.

Dataset Information

Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.

Publications

Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.

Similar Datasets

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