Molecular analysis of factors involved in chitin synthesis in Candida albicans
The Griffin.1 phage-antibody library was used to pan against Candida albicans cell walls in an attempt to produce a novel diagnostic or therapeutic agents against this human pathogenic fungus. After 6 round of selection, phage-ELISA indicated that the phage-antibody population was most enriched after pan 4. These phage-antibodies were used to perform indirect immunofluorescence in order to visualise the binding effect upon whole cells of the yeast and hyphal growth forms. A low level of fluorescence revealed poor phage-antibody binding and it was surmised that the cell wall material used in the panning process was a poor antigenic mixture for use in such an experiment. The C. albicans BN14 gene was identified which encoded a predicted 1655 amino acid protein that shared 20% overall identity to the Saccharomyces cerevisiae Bni4p, with 73% identity over the C terminal 63 amino acids. Expression analysis of CaBNI4 was performed by northern blot and transcripts of the expected size were detected in both the yeast and hyphal growth forms. The function of CaBNI4 was determined by targeted gene disruption using the 'Ura-blaster' method and this resulted in the deletion of amino acids 1120-1611. The resulting ΔCabni4/ΔCabni4 null mutant showed a reduction in chitin content of 25% for yeast cells and 40% for hyphae. Therefore CaBni4p is proposed to have a more global role in the targeting or activation of chitin synthesis and is not exclusive to involvement at the bud-neck region.