Role of the Leishmania mexicana CPB cysteine proteinases in the host-parasite interaction

Pollock, Kevin George James (2000) Role of the Leishmania mexicana CPB cysteine proteinases in the host-parasite interaction. PhD thesis, University of Glasgow.

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Abstract

Leishmania mexicana express several cysteine proteinases, some of which appear to be good drug targets. Findings suggested that cysteine proteinases may have an immunomodulatory role within the mammal and recently it has been shown that cysteine proteinase-deficient mutants stimulate a Th1 response in susceptible mice strains compared with the Th2 phenotype normally observed in wild-type infections. To investigate further the immunomodulatory activity of leishmanial cysteine proteinases and their role in host-parasite interactions, a major cysteine proteinase of L. mexicana, CPB, was produced in recombinant form. The enzyme, CPB2.8DeltaCTE, generated without its C-terminal extension was successfully expressed in Escherichia coli and purified from inclusion bodies to yield enzyme with high specific activity. The successful method was also used for the attempted production of a full length CPB with its C-terminal extension of L. infantum in recombinant form. This proved less successful apparently because of the presence of the hydrophobic C-terminal extension. The recombinant CPB of L. mexicana was analysed for its effects on the host response to the parasites and parasite antigen. In vitro analysis entailed incubation of explanted peritoneal exudate cells with recombinant CPB2.8 to assess production of inflammatory mediators considered to be important in leishmanial infection. The enzyme did not affect the production of interleukin-12 or nitric oxide but it did reduce the level of IL-10. The vaccine potential of the enzyme was investigated by co-administration with recombinant IL-12 as a vaccine, in three mouse strains, before challenge with wild-type L. mexicana. The consequent infection and host immune responses were monitored. The enzyme was partially protective when administered with IL- 12 even in the highly susceptible BALB/c line. The activity of the enzyme was shown to be unimportant in its ability in promoting a Th 1 response rather than the exacerbative Th 2 response normally seen in wild-type infection. These results demonstrate the potential of recombinant CPB as part of a vaccine against leishmaniasis. Targeted gene deletion of the cysteine proteinases of L. mexicana has produced several null mutants including the single and double null mutants. The single null mutant (denoted by Deltacpb) is generated by deletion of the entire cpb array, which encodes the 19 CPB isoenzymes, while deletion of both the cpb array and the cpa gene generates the double null mutant (denoted by Deltacpb/cpa). The latter mutant produces a no lesion growth phenotype in BALB/c mice and has been suggested as being a potential vaccine candidate. The parts played by the cysteine proteinases were investigated further by using these mutant parasite lines in which CPB was analysed by methods of re-integration directly into the chromosome or by re-expression via a cosmid vector. In order to compare metacyclic-specific and amastigote-specific genes and see if time of cysteine proteinase expression was important in establishing infection, the single null mutant was re-integrated with either metacyclic- expressed CPB2 or amastigote-expressed CPB2.8. Furthermore, each proteinase was engineered with its native promoter or with a chimaeric promoter, to assess expression levels and to investigate if there was a direct correlation between CPB expression and virulence since metacyclic CPBs are normally not expressed in the amastigote and vice versa. Re-integration of metacyclic CPB2 with its native and chimaeric promoter, significantly increased parasite proliferation resulting in increased lesion growth, re-integration of amastigote CPB2.8, did not promote an increase in virulence as the enzyme with its native promoter but did promote virulence when the enzyme was re-integrated with the chimaeric promoter. Analysis of the humoral and cellular responses to the single null mutants, indicated a mixed Th phenotype with IFN-gamma and IL-4 being produced by ex vivo splenocytes in response to leishmanial antigen stimulus. Antibody and lesion analysis of the double null mutant-infected mice was also carried out and did not result in an increase in virulence. Indeed reintegration of either CPB2 or CPB2.8 resulted in significantly higher IgG2a production indicating more of a Th 1 response than that already seen in double null-infected mice. Re-expression of the cpb array as a cosmid vector into the single null mutant did not result in a significant increase id virulence in mice compared with that of the single null mutant alone. The methods used to analyse these immune responses have been developed to permit further examination of other leishmanial CPs and should help to elucidate the role of these important parasite molecules.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Graham H Coombs
Keywords: Parasitology, Molecular biology, Pharmacology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-72907
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72907

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