Characterization of prostaglandin formation pathways and long -chain fatty acid utilization in the pathogenic fungus <italic>Cryptococcus neoformans</italic>.

Abstract

The pathogenic fungus <italic>Cyptococcus neoformans</italic> has been shown to produce bioactive fatty acids called prostaglandins which are immunomodulatory <italic> in vitro</italic>. However, how prostaglandins are made in this system is unknown because none of the enzymes thought to be required for prostaglandin synthesis appear to exist in <italic>C. neoformans</italic>. Little is known about the effect of long-chain fatty acids, precursors for prostaglandins, on <italic>C. neoformans</italic>. The objective of this project was two-fold: (1) to characterize the enzyme(s) critical for the first step in prostaglandin synthesis in <italic>C. neoformans</italic> and (2) to characterize the effect of long-chain fatty acids on the growth and development of <italic> C. neoformans</italic>. Prostaglandins were generated in cryptococcal lysates, extracted into ethyl acetate, purified using reverse-phase HPLC and measured using a prostaglandin screening assay. Cryptococcal prostaglandins were chemically and antigenic similar to commercially available prostaglandins based on solvent solubility, HPLC elution times, and EIA reactivity. However, cyclooxygenase inhibitors had no effect on prostaglandin production. Genomic analysis also could not find an enzyme homologous to either a cyclooxygenase or lipoxygenase. The polyphenolic inhibitors reveratrol, nordihydroguaiaretic acid (NDGA) and caffeic acid proved effective at inhibiting cryptococcal prostaglandin synthesis. This led to an investigation of the diphenol oxidase (laccase) CNLAC1. Removal of CNLAC1 by antibody depletion or deletion of the gene resulted in a loss of prostaglandin synthetic activity. These results demonstrate that CNLAC1 is critical for prostaglandin production in <italic>C. neoformans</italic>. To address the role of fatty acids, <italic>C. neoformans</italic> were grown in culture with the long-chain polyunsaturated fatty acids (PUFA) arachidonic, linolenic, linoleic or oleic acid at 37&deg;C or 25&deg;C with or without resveratrol. The presence of linoleic acid shut down growth at 25&deg;C. The addition of resveratrol resulted in a growth of chains which, over time, developed into hyphal <italic>C. neoformans</italic>. The addition of nutrients to long-term hyphal masses resulted in a shift from a hyphal form to a mycelial (mold-like) form and production of new <italic>C. neoformans</italic> yeast cells. These findings demonstrate that the PUFA environment in which <italic>C. neoformans </italic> is grown can drive morphologic changes in the fungus, including yeast-to-hyphal transformation.