R. Rüchel

A comparison of secretory proteinases from different strains of Candida albicans

Randomly selected strains of Candida albicans were grown with bovine serum albumin (BSA) as a single nitrogen source. From all strains tested, culture supernatant contained carboxyl proteinase (E.C.3.4.23) as has been shown that with hemoglobin as a substrate and by specific inhibition with pepstatin-A. According to the separation pattern of BSA fragments, secretory proteinases from C. albicans belong to at least three groups. We have purified the partially proteolytic enzyme of strain 113 and have compared its properties with those of the totally proteolytic enzyme of strain CBS 2730. Both enzymes have virtually identical molecular weight (ca. 44,000) and cross-react immunologically; they differ in pH optimum, isoelectric point, substrate specificity, and resistance against alkali. IgG1, which is the prevalent immunoglobulin of human serum, was not cleaved by enzyme 113. Immunoglobulins A1, A2 and secretory component were cleaved by both enzymes, which points to a role of the secretory proteinases in the persistence of yeasts on mucous membranes. Differences in the course of alkaline denaturation indicate that only a fraction of strain-specific proteinases is capable to convey long-range effects in the host.

Properties of a purified proteinase from the yeast Candida albicans.

The proteinase from culture supernatants of Candida albicans strain CBS-2730 was purified virtually to homogeneity by ion-exchange chromatography and affinity chromatography. The enzyme consists of a single polypeptide chain with tryptophan at the N- and leucine at the C-terminus. Its molecular weight is approx. 45,000 and the isoelectric point is at pH 4.4. With albumin as a substrate an apparent Km was determined to be 7 . 10(-5) M. The enzyme is inhibited by pepstatin at equimolar ratio and thus is a carboxyl proteinase (EC 3.4.23.6). Other group-specific inhibitors, though, did not efficiently block the enzyme. Above pH 8.4 the enzyme undergoes alkaline denaturation which is accompanied by dimerization. The enzyme is a glycoprotein. It is stable in presence of non-ionic detergents and can be freeze-dried. The enzyme clots milk at pH 5.5 and has trypsinogen kinase activity. Among several purified proteins that have been tested as a substrate, only horse ferritin was resistant to proteolysis, while myeloma proteins of the A1- and A2-type were readily cleaved, as were two proteinase inhibitors of human serum. Antibodies against purified enzyme did not react with several commercial Candida antigen preparations; antibodies against the enzyme, though, have been detected repeatedly in sera from patients with manifest candidiasis.

Sequence of the Candida albicans gene encoding the secretory aspartate proteinase

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.

Virulence of an aspergillopepsin-deficient mutant of Aspergillus fumigatus and evidence for another aspartic proteinase linked to the fungal cell wall

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.

Secretion of acid proteinases by different species of the genus Candida.

Strains of several Candida species that are isolated from humans aside of C. albicans have been screened for secretion of acid proteinases. A third of the strains of C. tropicalis was strongly proteolytic, other strains of this species were moderately or virtually nonproteolytic; the variety of activities resembled pattern found among strains of C. albicans (32). Strains of C. parapsilosis produced 24% of the proteolytic activity of strongly proteolytic strains of C. tropicalis. The proteolytic activity of C. pseudotropicalis, C. krusei, and C. glabrata accounted for 7.5, 2.0, and 0.8% respectively. Four strains of C. guilliermondii developed strain-specific proteolysis after prolonged cultivation only. Five proteinases from strains of C. tropicalis cross reacted immunologically as did five proteinases from C. albicans. Interspecific cross reaction, though, was not observed when certain sera from rabbits were employed.

A Synoptical Approach to the Diagnosis of Candidosis, Relying on Serological Antigen and Antibody Tests, on Culture, and on Evaluation of Clinical Data/Diagnose der Candidose durch synoptische Bewertung serologischer Antigen‐ und Antikörpertests sowie von Kulturisolaten und klinischen Daten

Summary: In a prospective study from December 1985 to September 1986, we have tested 1153 sera from 516 patients suspected of candidosis. Serological data, clinical conditions and cultures indicated involvement of Candida in 190 cases. Among them, 36 patients probably suffered from invasive mucocutaneous candidosis. Deep‐seated candidosis was diagnosed in 50 patients. Nearly half of the patients of each group had been subjected to systemic antifungal therapy, which did not improve the prognosis of patients suffering from invasive mucocutaneous candidosis. However, among patients with deep‐seated candidosis, overall lethality was reduced by more than 50% when systemic antifungals were applied. Therefore, differentiation of severe candidosis into an invasive mucocutaneous type and into truly deep‐seated mycosis is diagnostically important. The latex agglutination test for detection of Candida antigen in serum (Cand‐Tec, Ramco) is suitable for excluding severe candidosis; it does not allow further differentiation. The enzyme immunoassay for detection of fungal proteinase antigen showed a higher specificity for deep‐seated mycosis; however, proteinase antigen was detected only in 50% of the suspected cases. Titers of anti‐candidal antibodies (including anti‐proteinase) did not allow differentiation of severe mycosis. They were useful for confirmation of the diagnosis, provided the patient was able to mount a regular immune response. In immuno‐compromised patients, the criteria of the antigen titers have to be adapted to the degree of neutropenia. Likewise, positive cultures and clinical presentation have to be interpreted differently in such patients. A fair degree of reliability of early diagnosis of deep‐seated candidosis is only attained by synoptical evaluation of clinical, serological, and cultural data.

Demonstration of fungal proteinase during phagocytosis of Candida albicans and Candida tropicalis

The extracellular acid proteinase of Candida albicans and Candida tropicalis was monitored in vitro during phagocytosis by murine peritoneal macrophages. Fungal blastospores were quickly ingested by the thioglycolate-elicited macrophages and the intracellular blastospores partly resisted killing and started to grow out after 6 h incubation, causing destruction of the macrophage. Proteinase antigen appeared on fungal cells after 30 min in culture medium containing 10% fetal calf serum. The antigen was detected on ingested blastoconidia and filamentous cells of C. albicans serotype A. The proteinase antigen was also expressed by blastoconidia of C. albicans serotype B but was missing on the filamentous cells of this serotype. Isolates of C. tropicalis behaved similarly to C. albicans serotype A. The acid proteolytic activity of Candida cells was confirmed by the haemoglobin test on culture supernatants. Lysates of infected and noninfected phagocytes showed a differential acid proteolytic activity; noninfected macrophages revealed rising activity, while infected macrophages showed a distinct reduction of activity. The proteolytic activity of lysates of noninfected cells is due to lysosomal cathepsin-D. Cathepsin-D was also most likely to be responsible for the declining proteolytic activity in lysates from infected phagocytes; such lysates contained increasing amounts of fungal proteinase antigen. The differential kinetics of this antigen and the total acid proteolytic activity in the lysates suggest a conflict between microbial and lysosomal hydrolases in infected phagocytes. The outcome of this conflict depends on the number and hydrolytic activity of the ingested yeasts and may be decisive in the progress of infection.

Purification and characterization of an extracellular aspartic proteinase from Aspergillus fumigatus

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.

Modulation of experimental systemic murine candidosis by intravenous pepstatin

The effect of intravenous pepstatin-A on systemic candidosis in NWNI mice was investigated. True solutions of the inhibitor proved ineffective due to a very fast clearance. Pepstatin was effective as a crystal suspension (0.69 mg in 0.1 ml saline) which produced serum inhibitory activity for greater than 29 h. From the intravenously applied suspension, pepstatin was taken up predominantly into the liver, no inhibitor being taken up by the kidneys. The suspension was protective if it was injected once before the mice were infected and repeatedly following infection. It was also effective if it was administered concomitantly with the infecting agent and thereafter. The suspension was ineffective if it was only given once before infection, and it proved to be detrimental if it was given only after infection. The results support previous findings (2), suggesting a role of fungal proteinase early in the adherence of Candida to host epithelia. Our results also suggest an inhibition of lysosomal cathepsin-D in vivo by pepstatin, which prohibits a parenteral therapeutic use of nonmodified pepstatin A.

Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus.

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.