Medically important yeasts of the genus Candida secrete aspartic proteinase
s (Saps), which are of particular interest as virulence factors. Like Candi
da albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p)
in a medium containing bovine serum albumin (BSA) as the sole source of ni
trogen. Using the gene SAPT1 as a probe and under low-stringency hybridizat
ion conditions, three new closely related gene sequences, SAPT2 to SAPT4, e
ncoding secreted proteinases were cloned from a C. tropicalis lambda EMBL3
genomic library. All bands identified by Southern blotting of EcoRI-digeste
d C. tropicalis genomic DNA with SAPT1 could be assigned to a specific SAP
gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain
only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2
p and Sapt3p, which have not yet been detected in C. tropicalis cultures in
vitro, were produced as active recombinant enzymes with the methylotrophic
yeast Pichia pastoris as an expression system. As expected, reverse transc
riptase PCR experiments revealed a strong SAPT1 signal with RNA extracted f
rom cells grown in BSA medium. However, a weak signal was obtained with all
other SAPT genes under several renditions tested, showing that these SAPT
genes could be expressed at a basic level. Together, these experiments sugg
est that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced und
er conditions yet to be described in vitro or during infection.