An isolate of Cryptococcus neoformans in which the structural gene for diphenol oxidase (CNLAC1) has been dirupted was crossed with a wild type and the F1 progency studied for virulence. A F1 strain which produced diphenol oxidase (DPO) killed mice whereas a strain which did not produce DPO did not cause lethal infection. However, complementation of the CNLAC1 deletant with CNLAC1 produced a strain which was not lethal for mice, even though the isolate produced DPO. All these strains were the same mating type (a) and had the uracil auxotrophy of the CNLAC1 deletant restored by either backcrossing or transformation. The deletant was found to be missing not only the 5' end of the gene and the URA5 gene used for positive selection, but also approximately 4kb of upstream sequences. Although no transcript has been detected from the area of the 5' deleted sequences as yet, it is possible that sequences coding for regulatory or structural genes are present in this area. The upstream deletion might have decreased virulence apart from disrupted CNLAC1 gene. Another experiemnt which supports the nonessentiality of the CNLAC1 gene for virulence is that a mutant (mel2) obtained from J.C. Edman did not produce DPO but was able to kill mice. When transformed with CNLAC1 the isolate made DPO but did not have a substantial increase in virulence. CNLAC1 was used to integratively transform Pichia pastoris. The expressed and secreted protein was partially purified on phenylsepharose and DEAE chromatography. The expressed protein was contained in a fraction containing at least two proteins, based on agar gel precipitin bands. The mixture was heavily glycosylated, containing 41% mannose. Deglycosylated enzyme was inactive. On TSK 4000 HPLC chromatography, DPO enzymatic activity was approximately 100-130 kd and had a pI of less than 4.5 on isoelectric focusing. When compared with a DPO transcript size of 1.3-1.8 kb, it seems likely that DPO is being expressed as a heavily mannosylated protein with a heterodisperse molecular weight.
