Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase

Cryptococcus neoformans, the causative agent of cryptococcosis, produces la rge amounts of mannitol in culture and in infected mammalian hosts. Althoug h there is considerable indirect evidence that mannitol synthesis may be re quired for wild-type stress tolerance and virulence in C. neoformans, this hypothesis has not been tested directly. It has keen proposed that mannitol -1-phosphate dehydrogenase (MPD) is required for fungal mannitol synthesis, but no MPD-deficient fungal mutants or cDNAs or genes encoding fungal MPDs have been described. Therefore, C, neoformans was purified from a 148 kDa homotetramer of 36 kDa subunits that catalysed the reaction mannitol l-phos phate + NAD <-> fructose 6-phosphate + NADH. Partial peptide sequences were used to isolate the corresponding cDNA and gene, and the deduced MPD prote in was found to be homologous to the zinc-containing long-chain alcohol/pol yol dehydrogenases. Lysates of Saccharomyces cerevisiae transformed with th e cDNA of interest (but not vector-transformed controls) contained MPD cata lytic activity. Lastly, Northern analyses demonstrated MPD mRNA in glucose- and mannitol-grown C. neoformans cells. Thus, MPD has been purified and ch aracterized from C. neoformans, and the corresponding cDNA and gene (MPD1) cloned and sequenced. Availability of C. neoformans MPD1 should permit dire ct testing of the hypotheses that (i) MPD is required for mannitol biosynth esis and (ii) the ability to synthesize mannitol is essential for wild-type stress tolerance and virulence.