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Fluorescent staining using optical brighteners (diaminostilbenes) affords the semispecific and rapid detection of fungal elements in clinical specimens. After yielding a first hint of mycosis, the identification of the involved fungal genus is often desirable in cases when culture proves unsuccessful. In such cases, immunohistochemistry or in situ hybridisation may further the diagnosis with respect to establish an appropriate therapy.

A case of a mixed infection due to Candida albicans and the zygomycete Absidia corymbifera in a 38-year-old, previously healthy, Caucasian male is presented. The infection developed following serial rib fractures, and ruptures of kidney, liver and biliary tract as well as a pancreatic contusion resulting from a traffic accident. During intensive care treatment the patient underwent several surgical procedures but subsequently experienced multi-organ failure and sepsis. Some weeks later, fungal growth was observed macroscopically on the patient's skin and wounds. From wound swabs C. albicans and A. corymbifera were grown. Histopathology of abdominal tissue yielded pseudohyphae and coenocytic hyphae. Although surgical debridement and antifungal treatment with amphotericin B and 5-flucytosine were started immediately, the patient died in therapy-refractory septic multi-organ failure.

Candida albicans has an advantage on other fungal opportunists, being the only fungus among the normal microbial flora on human mucosa. The potential factors of virulence of C.albicans are essentially responsible for the persistence of the yeast on mucosal surfaces. If the normal regeneration of epithelia is impeded. C.albicans may invade the mucosa. It may penetrate deeply into the host during granulocytopenia or cellular immunodeficiency, apparently using mechanisms of mimicry and the activity of secretory hydrolases.

The secretory aspartic proteinases of Candida albicans and C. tropicalis are potential factors for virulence produced during infection. By indirect immunofluorescence, we have demonstrated proteinase antigen on elements of both species in deparaffinized tissue sections derived from clinical cases of mucosal and deep-seated candidiasis. Occasionally, we observed a halo of fluorescence in the close vicinity of candidal cells, which may reflect secretion of the enzyme. In kidneys, a ring of amorphous fluorescent material surrounding candidal colonies may illustrate alkaline denaturation of secreted enzyme within a pH gradient, which is generated by the fungus. Our findings support the view that candidal proteinase may be a diagnostically relevant antigen.

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.

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.

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.

Several secretory acid proteinases from C. albicans and C. tropicalis as well as membrane extracts from two strains of C. albicans were compared for their renin-like activity and for their ability to activate blood coagulation. The highest renin-like activity as measured fluorometrically, was found in the membrane extract of C. albicans 113, it accounted for approximately a tenth of the activity of pure human renin. Activation of blood coagulation by acid Candida proteinases is due to the trypsinogen kinase effect, it was detected preferentially at the level of factor X (Stuart-Prower). Conversion of factor X at rates sufficiently rapid to be of biological significance was effected by several proteinases tested. The highest activity was found in the membrane extract of C. albicans CBS-2730 and involves a proteinase of the serine type. Both renin-like activity and conversion of factor X are examples of limited proteolysis; they are discussed with respect to strain-specific pathogenicity in opportunistic yeasts.

Double antibody immunoassay of secretory aspartic proteinases from 2 strains of the yeast Candida albicans was performed in microtest plates with rabbit antibody and peroxidase conjugated rabbit antibody. The test detects approximately 0.1 ng of proteinase and is 2 orders of magnitude more sensitive than direct measurement of enzymic activity with hemoglobin as a substrate. The immunoassay was affected both by alkaline denaturation, which is a common feature of aspartic proteinases, and by the presence of the proteinase scavenger alpha-2-macroglobulin. Conditions are described that allow for comparison of proteinases from different strains of yeast and minimize the interference of macroglobulin.

Evidence is presented for the involvement of proteinases from Candida albicans in the pathogenesis of acronecrosis that occurred in a young woman and which coincided with Candida sepsis. Secretory acid Candida proteinase by immunofluorescence was traced in the obstructed blood vessels of necrotic skin that was infested with yeast. The specificity of immunofluorescence was proven by exclusion of cross reactivity with pepsin, cathepsin-D, acid erythrocyte proteinase and porcine renin. The possible molecular mechanisms of interference of fungal proteinases are discussed with respect to the renin-angiotensin system and blood coagulation.

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.

We describe the production and characterization of three murine monoclonal antibodies (M1-M3) which are directed against different epitopes of the secretory aspartic proteinase of Candida albicans CBS 2730. All antibodies belonged to the IgM class, and they recognized denatured enzyme. Only antibody M1 was capable to react with the active proteinase. Differential reactivity was also observed with a large fragment of the proteinase of C. albicans. All antibodies recognized the corresponding proteinase of C. tropicalis 293 both in the active, and in the denatured state. Denatured porcine pepsin was also recognized by all three antibodies. However, active pepsin was recognized only by antibodies M1 and M2. The antibodies did not inhibit enzymatic activity, and they were not suited for immunofluorescence detection of proteinase on fungal cells. However, employing Western blot analysis, proteinase antigen was detected by antibody M 1 in the serum of a patient suffering from candidal pneumonia. The circulating proteinase antigen was found to be bound to patient's IgM. Implications for the use of monoclonal antibodies in the serodiagnosis of candidosis, and first experiences with other monoclonal anti-proteinase antibodies are discussed.

As we have communicated previously, the fungal opportunist Candida albicans produces an angiotensin-1 liberating proteinase and a serine proteinase that acts as a converter of the coagulation factor X in vitro (25). Both enzymes are attached to delipidated cell fragments of fungal blastospores. They copurify upon ion exchange chromatography and gel filtration. The enzymes could be separated by hydrophobic chromatography on Phenylsepharose. According to their substrate and inhibition pattern, the enzymes have been classified as an aspartyl proteinase and a chymotrypsin-like proteinase. Their partial characterization includes estimates of the molecular weight and isoelectric points.

The present knowledge on the pathogenetic relevance of extracellular and cell wall-attached proteinases from fungal pathogens is briefly reviewed. [ABSTRACT FROM AUTHOR]