Munday, Jane Claire
Studies on oligopeptidase B of Leishmania major.
PhD thesis, University of Glasgow.
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Peptidases of Leishmania are acknowledged virulence factors. It is hypothesised that peptidases are crucial for the survival of Leishmania in its hosts and that many could be potential targets for new antileishmanial drugs. As such, the investigation of peptidase activity in live Leishmania promastigotes was proposed as a valuable approach by which to increase knowledge on particular peptidases. In order to complete this investigation, it was decided to use short peptidyl fluorogenic substrates, which only fluoresce once the bond linking the peptide to the fluorescent moiety is cleaved. These allow detection of peptidase activity by quantifying the release of the fluorescent moiety.
Detection of peptidase activity in live Leishmania using the fluorogenic substrate Bz-R-AMC proved fruitful, enabling study of the activity of the serine peptidase oligopeptidase B (OPB) in live L. major promastigotes. OPB is a member of the Family S9 peptidases, the prolyl-oligopeptidases, which are taxonomically restricted to plants, bacteria and trypanosomatid flagellates. In African and American trypanosomes, OPB has been shown to have important roles: OPB is a virulence factor in Trypanosoma cruzi, mediating entry into host cells, and OPB is released into the serum by African trypanosomes, where it cleaves host blood factors.
In this study, the inhibition profile of L. major OPB has been determined and OPB has been localised to the cytosol, the site of hydrolysis of Bz-R-AMC. Immunoprecipitation of OPB confirmed that OPB was the sole peptidase responsible for the hydrolysis of Bz-R-AMC and anti-OPB antibodies were found to inhibit the hydrolysis of Bz-R-AMC. Inhibitors of OPB could also kill Leishmania promastigotes, suggesting OPB could be a valuable drug target.
However, genetic manipulation of OPB was successful, with mutants over-expressing OPB and opb null mutants produced. OPB is thus not essential for the growth of promastigote L. major, though the opb null mutants did have a defect in metacyclogenesis, in survival in macrophages and a reduced ability to induce lesions on the footpads of mice. A role in amastigote differentiation or survival in macrophages was also suggested. OPB is thus likely to be a virulence factor, though not essential, and thus not suitable as a primary drug target.
A number of avenues require further investigation, including the need for re-expression of OPB in the opb null mutants to confirm that lack of OPB is indeed responsible for the phenotypic deficiencies of the null mutants. Other important areas requiring attention are investigation of the role of OPB in amastigote differentiation or survival, investigation of the reported release of OPB by promastigotes, and identification of the physiological substrate of OPB.
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