Gene Expression in the Microfilariae of Brugia pahangi

Emes, Richard David (2000) Gene Expression in the Microfilariae of Brugia pahangi. PhD thesis, University of Glasgow.

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Abstract

Filarial nematodes have a complex developmental life cycle involving mammalian and mosquito hosts. The microfilariae (mf) which circulate in the blood stream are developmentally blocked prior to transfer to the mosquito. The switch between the different environments must be accompanied by changes in gene expression to allow development within the new host. The aim of this study was to isolate mf genes from the mf of B. pahangi that are differentially expressed within the two hosts. To this end a mammalian-derived mf cDNA library was constructed and differentially screened using mammalian and mosquito-derived mf cDNA probes. Due to the difficulty of obtaining in vivo parasites from the mosquito, the cDNA probes were produced by RT-PCR from mf culture in vitro under either mammalian or mosquito like conditions. Nine independent cDNA clones were isolated, of which five hybridised more strongly to the mammalian-derived mf probe and four to the vector-derived mf probe. Analysis of the cDNAs nucleotide sequence revealed that five of the clones were homologous to ribosomal protein mRNAs previously characterised from other species, one cDNA corresponded to the B. pahangi heat shock protein 90 mRNA and three cDNAs represented novel genes of unknown function. A more detailed molecular analysis was conducted on two of the cDNAs of unknown function, Bp-vmc-2 (B. pahangi-vector-derived mf cDNA-2) and Bp-mmc- 1 (B. pahangi-mammalian-derived mf cDNA-1). Analysis of the expression of the mRNAs by semi-quantitative RT-PCR showed that Bp-vmc-2 was expressed at varying levels throughout the life-cycle of B. pahangi. The mRNA abundance of Bp- vmc-2 increased as the parasite matured in the mosquito host and reached peak expression in infective L3 parasites. In contrast Bp-mmc-1 was exclusive to the mf stage and was not expressed by mf developing in utero. An antiserum raised to the recombinant protein localised MMC-1 throughout the body of the mf, but it was not present on the surface of the sheath or cuticle of the parasite. Western blotting using MMC-1 antiserum, reacted specifically with mf extracts, confirming the specificity of temporal expression. Analysis of immune responses to the recombinant protein showed that significant amounts of IL-5 were produced by T-cells in response to stimulation with MMC-1. Human serum from patients infected with B. malayi recognised MMC-1 and the predominant reactive immunoglobulin subclasses were IgGl and IgG3, which have been associated with disease pathology in other studies.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Eileen Devaney
Keywords: Genetics, Parasitology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-76152
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:34
Last Modified: 19 Nov 2019 16:34
URI: http://theses.gla.ac.uk/id/eprint/76152

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