Chitin synthase in the filarial parasite, Brugia malayi

Abstract

Fragments of putative chitin synthase ( chs) genes from two filarial species ( Brugia malayi and Dirofilaria immitis) were amplified by PCR using degenerate primers. The full genomic and cDNA sequences were obtained for the B. malayi chs gene ( Bm- chs-1); the predicted amino acid sequence is highly similar, over a large region, to two CHS sequences of the nematode Caenorhabditis elegans and also to two insect CHS sequences. Bm- chs- 1 is abundantly transcribed in B. malayi adult females, independent of their fertilization status, but is also expressed in males and microfilariae. Oocytes and early embryos contain large amounts of Bm-chs-1 transcript by in situ hybridization, but later stage embryos within the maternal uterus show little or no Bm-chs-1 transcript. No specific hybridization could be demonstrated in maternal somatic tissues. Polyclonal antibodies were raised against a peptide expressed from a recombinant cDNA fragment of Bm-chs-1; immunostaining detected CHS protein in oocytes and early to midstage embryos. These studies characterize a gene that is likely to be essential to oogenesis and embryonic development in a parasitic nematode. Because chitin synthesis and eggshell formation begin after fertilization, the presence of CHS protein in early oocytes suggests that the enzyme must be activated as a result of fertilization. These studies also demonstrate that chitin synthesis may not be restricted to eggshell formation in nematodes, as the Bm-chs-1 gene is transcribed in life cycle stages other than adult females.