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An extracellular aspartic proteinase (Rmap) from Rhizopus microsporus var. rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA). The proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose. Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids. The preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids. The deduced amino-acid sequence of the preproenzyme was 82% homologous to an extracellular aspartic proteinase of Rhizopus niveus. Low stringency Southern blot analysis of R. microsporus DNA suggested the presence of other homologous genes. Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant. Both enzyme preparations exhibited a similar optimum of activity in the pH 2.5 region. Furthermore, Rmap was shown to activate bovine blood coagulation factor X at slightly acidic pH in vitro. Expression of the proteinase during mycosis was proven by a specific immune response of patient HA.

An aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes. PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis.

A method for the detection of Candida albicans from up to 15 ml of blood by polymerase chain reaction (PCR), based on the differential resistance of mammalian and fungal cells towards detergent was developed. The procedure essentially involved removal of the blood cells by sodium dodecyl sulfate (SDS) induced lysis, followed by DNA extraction after degradation of fungal cell walls by a recombinant beta-1,3-glucanase. The genes of two different aspartic proteinases from C. albicans, SAP1 and SAP2, with an overall homology of 77% in their nucleotide sequences, were chosen as targets for PCR. The oligonucleotide primers used were directed to strictly conserved regions similar in both genes. As the number of base pairs between the primers are different in the two genes, amplification products of 220 bp and 238 bp in length were obtained. This led to a characteristic double band in subsequent agarose gel electrophoresis. The detection limit for a nested PCR was less than 10 C. albicans cells ml-1 of seeded blood. The detection limit of conventional PCR from a blood volume in the 10 ml range was less than 100 yeasts ml-1. Preliminary trials with clinical blood specimens suggested, that conventional PCR from large blood samples, being less laborious and prone to contamination than nested PCR, could be suited for the detection of deepseated C. albicans mycosis.

A gene replacement was performed to produce mutants of Aspergillus fumigatus deficient in the aspergillopepsin PEP (E.C. 3.4.23.18). The correct replacement of the PEP gene was confirmed by PCR and Southern hybridization experiments, whereas the absence of PEP production was demonstrated by Western blots. The culture supernatant of the transformants showed no detectable acid proteinase activity, suggesting that there is only one acid proteinase secreted by the fungus. The wild-type strain and the PEP-deficient mutants invaded tissues to a similar extent and produced comparable mortality in guinea pigs. As PEP represents a third secretory proteinase of A. fumigatus and the other two proteinases also did not show significant influence on fungal invasiveness, it is probable that secreted proteinases do not contribute decisively to tissue invasion in the pathogenesis of systemic aspergillosis. However, immunofluorescence on A. fumigatus colonies using polyclonal antibodies to PEP showed a similar pattern for the wild-type and for the mutants, with a bright fluorescence on young conidiophores, on submerged mycelium and on the tips of growing aerial mycelium. Conidia and mature aerial hyphae were not fluorescent. This pattern could reflect the existence of another crossreacting aspartic proteinase (PEP2) which was found to be sensitive to pepstatin but tightly linked to the fungal cell wall.

Sporulating colonies of Aspergillus fumigatus, A. flavus, and A. niger were subjected to immunofluorescence using specific polyclonal antibodies against the aspergillopepsin PEP (EC 3.4.23.18), a secretory aspartic proteinase produced by A. fumigatus. The proteinase antigen was found mainly in developing conidiophores of aspergilli, in submerged mycelia and on the tips of growing aerial mycelia. Mature aerial hyphae and spores showed no immunofluorescence at all. Sporulating conidiophores revealed only weak activity in A. fumigatus and A. flavus. The distinct pattern of expression of the aspartic proteinase antigens suggests a role for such enzymes in the growth of hyphae and the development of conidiophores and thus for the sporulation process in aspergilli.

Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus Fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

The yeasts Candida albicans. Candida tropicalis, and Candida parapsilosis secrete aspartic proteinases which may enhance virulence. Profiles of pH-dependent irreversible denaturation of such enzymes were determined at 37 degrees C. C. albicans proteinases from both serotypes A and B maintained 50% of their activity near pH 7.25. Proteinases from C. parapsilosis and C. tropicalis lost 50% of their activity at pH 6.75 and pH 6.15, respectively. This suggests that in the infected host only proteinases of C. albicans maintain a native state for any length of time.

An aspartic proteinase (PEP) from the culture supernatant of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 24%. The procedure involved affinity chromatography on pepstatin agarose, the interaction requiring a chaotropic salt (sodium trifluoroacetate) for complete elution of the enzyme. Among 11 amino acids of the N-terminal region, nine were identical with the corresponding sequence of the aspartic proteinase aspergillopepsin A from Aspergillus niger var. awamori (previously called Aspergillus awamori). Thus PEP belongs to the aspergillopepsins, a family of closely related aspartic proteinases produced by aspergilli. Specific antibodies against PEP were detected by dot blot assay in sera of two patients with aspergillosis. In addition, PEP antigen was demonstrated by immunofluorescence in mycotic human lung, using specifically elicited antibodies from guinea-pigs. PEP had an estimated molecular mass of 38 kDa and the pI was determined at pH 4.2. PEP is therefore likely to be closely related to an acid proteinase of A. fumigatus which was originally described in 1981.

The gene encoding the Candida albicans aspartate proteinase that is secreted by cells grown in protein-containing media was cloned from a C. albicans genomic bank. The base sequence of the insert shows a 1173 bp open-reading frame and indicates an amino acid sequence typical of aspartate proteinases, with amino acid sequence homology to other enzymes of this class and a putative signal peptide consisting of 50 amino acids upstream of the active enzyme.