Proteolysis and pathogenicity of Candida albicans strains

Summary Proteolysing strains ofCandida can be recognized in serum-protein-agar pH 5.0 (SPA pH 5.0) and can be isolated from clinical specimens on the basis of their proteolytic activity after addition of Penicillin, Streptomycin and Chloromycetin to the medium.When injected into white mice, only the proteolysing strains ofC. albicans cause extensive peritonitis and infection of all viscera. This possible association of proteolytic properties and pathogenic action ofC. albicans strains is discussed.

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Zusammenfassung Proteolysierende Stämme der GattungCandida können auf Serum-Protein-Agar pH 5,0 (SPA pH 5,0) mit Antibiotica-Zusatz aus klinischen Untersuchungsmaterialien anhand ihrer Proteolyse-Aktivität isoliert werden. Diese stammspezifische Enzym-Aktivität kann mit Hilfe der Folin-Technik bestimmt und in Beziehungen zu pathologisch-anatomischen Veränderungen im Tierexperiment gebracht werden, nachdem in der weißen Maus nur proteolysierendeC. albicans-Stämme eine ausgedehnte Peritonitis und Infektion der parenchymatösen Organe verursacht haben. Inwieweit eine Beziehung zwischen diesen Eigenschaften und der noch ungeklärten Menschen-Pathogenität vonC. albicans-Stämmen besteht, muß weiteren Untersuchungen vorbehalten bleiben.

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This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.

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Presented at the 4th meeting of the International Society for Human and Animal Mycology, New Orleans, July 31st–August 2nd, 1967.     

Deoxyribonucleic acid-deficient strains of Candida albicans.

We analyzed a series of germ tube-negative variants isolated from Candida albicans 3153A for deoxyribonucleic acid content. As analyzed by flow microfluorometry, the deoxyribonucleic acid level in these variant strains was 50% of that of the parental strain and equivalent to that of haploid Saccharomyces cerevisiae. This finding was confirmed by comparison of survival rates when exposed to the mutagens ultraviolet light, ethyl methane sulfonate, and methyl methane sulfonate. The diameter of the variant cells as compared to the diameter of the parental 3153A strain showed a relationship similar to that of the diameters of haploid versus diploid S. cerevisiae. These results indicate that those strains may be representative of the imperfect stage of C. albicans.     

Use of polymorphic short and clustered coding-region microsatellites to distinguish strains of Candida albicans

Abstract We describe the identification of polymorphic microsatellite loci in the pathogenic yeast, Candida albicans . A search for all coding-region microsatellites with more than four repeats that can be found in Candida sequences in GenBank was conducted. Nine such microsatellite sequences consisting of trinucleotide motifs were found. Three of these were perfect microsatellites while the remaining six sequences were found in one imperfect microsatellite and two compound microsatellites. Because of the close proximity of some of these repeats, all could be assayed with six PCR primer pairs. All of these microsatellite sequences were found in five nuclear genes, ZNF1, CCN1, CPH1, EFG1 , and MNT2 . Except for a single (CTT)₅ serine tract, all coded for polyglutamine tracts. Another locus with seven alleles, a region of the ERK1 protein kinase gene, was also examined, and may be a representative of a new class of highly polymorphic ‘clustered’ microsatellites. Such loci, in which several non-contiguous but closely linked microsatellites are clustered together, may be a useful source of DNA polymorphisms in microorganisms in which long microsatellite sequences are unavailable. All seven regions amplified were polymorphic, having between two and seven variable length alleles in the 11 strains of Candida albicans examined. The results of this and similar searches will facilitate epidemiological and evolutionary studies of Candida and other microorganisms.     

Simple rapid identification of Candida albicans with emphasis on the differentiation between Candida albicans and Candida stellatoidea

Summary Subcultures ofC. albicans, made from Sabouraud agar, grown at room temperature for 48 hours, were inoculated into a 10 times saline dilution of Sabouraud liquid medium and left in the incubator for 45–60 minutes at 37° C, transferred to corn meal agar plates and incubated at 37° C for 18–24 hours.Small portions of the surface agar containing the yeasts from these plates were pressed under cover glasses and examined under the oil immersion lens.Under these conditions,C. albicans cultures were observed to produce only yeast-like cells, whereasC. stellatoidea cultures contained predominantly abundant, long, thin mycelia.     

Germ-tube formation by atypical strains of Candida albicans

Atypical isolates of Candida albicans which failed to produce germ tubes in routine diagnostic procedures were examined for their ability to produce germ tubes in various media. Bovine serum was more effective than defined media for induction of germ tubes in the majority of isolates. A few strains formed appreciable germ tubes only in bovine serum with added thioglycollate or cysteine. One strain did not produce germ tubes in any medium. Germ-tube maturation appeared to be dependent upon mitochondrial RNA polymerase activity. The failure by an isolate to produce germ tubes, particularly in tests without strictly controlled conditions, does not preclude the possibility that the organism is C. albicans.     

Immunoelectrophoresis to detect differences between strains of Candida albicans

Summary Aqueous extracts of two parental strains ofCandida albicans and two mutants obtained from these strains were studied through immunoelectrophoresis and differential staining for protein and polysaccharide fractions after electrophoresis. Differences in antibody-antigen reactions, and protein and polysaccharide fractions could be detected between these strains. These differences reflected changes in synthesizing ability and genetic information inherent in these yeast cells.     

Mannan synthetase activity in three strains of Candida albicans

Abstract Mannan synthetase activity has been investigated in Candida albicans , strain 4918, as well as in two relatively avirulent, cerulenin-resistant mutant derivative strains, 4918-2 and 4918-10. In addition, investigations pertaining to the effects of the agents, cerulenin and sodium butyrate, on the level of mannan synthetase activity during the yeast to hyphal transition of these strains have been performed. The results show that mannan synthetase activity in yeast cells of both mutant strains is consistently higher than that observed in the parental strain. Similarly, the profile of enzyme activity exhibited by the mutant strains as morphogenesis proceeds differs from that of the wild-type. Sodium butyrate has no significant effect on enzyme activity in these strains, but the presence of cerulenin results in alterations in mannan synthethase activity during morphogenesis of strain 4918.     

Candida albicans strain differentiation in complete denture wearers

Strain differentiation of 66 clinical isolates of Candida albicans obtained from healthy dentate and complete denture wearers was performed. Resistogram method based on differences in the resistance of C. albicans isolates to sodium selenite, boric acid, cetrimide, sodium periodate and silver nitrate was used for strain differentiation. Of the 32 potential strains that can be distinguished, 14 different resistogram strains of C. albicans were found among the 66 isolates tested. Strain-C--was the most predominant (24.3% of total isolates), while strain A-CDE was the least predominant (1.5%). The results showed no particular association of certain strains with Candida infections in complete denture wearers. Sensitivity to antifungal agents showed that isolates from different strains were most sensitive to amphotericin B and nystatin and least sensitive to miconazole.     

Hydrogen peroxide induces hyphal differentiation in Candida albicans

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H₂O₂ in Candida albicans. This finding is confirmed by the changing intracellular concentration of H₂O₂. In order to induce the same level of differentiation, low concentrations of exogenous H₂O₂ are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.     

Adhesion of Candida albicans mutant strains to host tissue

Adhesion of Candida albicans to host cells is believed to represent a fungal virulence factor and a significant step in the development of candidiasis. As C. albicans strains may differ in their in vitro adhesion ability we initiated a study to investigate whether mutant strains differ in this respect from their parent wild-type. We assessed the in vitro adhesion of C. albicans CBS562 and two mutants obtained by mutagenesis with N′-nitrosoguanidine: a histidine auxotroph, SAG5, derived from CBS562, and a respiratory-deficient strain (a petite mutant), SAR1, derived from SAG5. The adhesion was tested in vitro using two target cell systems: (1) exfoliated human buccal epithelial cells (BEC); and (2) human keratinocyte tissue line cells (HaCaT cells). Adhesion to BEC was evaluated microscopically and that to HaCaT cells by a direct ELISA technique. The results indicated a 54% reduction in adhesion to BEC for SAG5 and 30% for SAR1 as compared to the wild-type, and a 25% reduction in adhesion to HaCaT cells for SAG5 and 20% for SAR1. To verify whether the prototrophy restores the adhesion ability, we complemented the his-negative auxotroph by transforming the strain with the HIS4 gene. Then we assayed the adhesion to BEC of the complemented his-negative mutant in comparison to that of the wild-type, the his-negative mutant (SAG5) and the plasmid-cured transformant. The adhesion values of the complemented his-negative strain were similar to those of the wild-type, whereas the values of the plasmid-cured strain were similar to those of SAG5.     

Respiratory metabolism of normal and divisionless strains of Candida albicans

Respiration of a normal strain of Candida albicans was compared with that of a divisionless mutant which has a biochemical lesion such that metabolically generated hydrogen "spills over," during growth, for non-specific dye reduction. This waste is not at expense of growth, since both strains grow at essentially similar rates, nor at expense of respiration, since the mutant reduces oxygen more rapidly than the normal strain. Respiration in both strains is qualitatively similar, and seemingly unique among highly aerobic organisms in that it is not mediated by cytochrome oxidase. In resting cells of both strains, respiration is not only resistant to, but markedly stimulated by, high concentrations of cyanide, carbon monoxide, and azide. In contrast, growth of these yeasts is inhibited by low concentrations of cyanide and azide. Cytochrome oxidase could not be detected in cell-free preparations; reduced cytochrome c was not oxidized by such preparations. Cytochrome bands could not be observed in thick cell suspensions treated with reducing agents. However, incorporation of superoptimal levels of zinc and iron into the culture medium resulted in growth of cells possessing distinct cytochrome bands; respiration of these cells remained insensitive to cyanide, monoxide, and azide, and the bands were maintained in a reduced form on oxygenation. In the divisionless yeast, tetrazolium dyes compete with oxygen for reduction; this is not the case in the normal strain. The firmness with which hydrogen transfer is channeled in the latter for reduction of disulfide bonds (of importance in the division mechanism) and of oxygen, is contrasted with the lack of such control in the mutant.     

Spontaneous variability in a population of Candida albicans strains]

The spontaneous variability of the populations of C. albicans strains of different genesis in the morphological properties of their colonies and in the potential of the activity of their extracellular proteolytic and phospholipid enzymes has been studied. The isolated types of colonies, differing in their morphology, have the phenotypic character of variability. Different populations of strains exhibited variability in the activity of enzymes, depending on morphological variants isolated from these populations. Selected morphological variants with high potential of their proteolytic enzymes retained stability in this property for 5 generations and can be used in medical practice for the isolation of C. albicans antigens.     

Molecular analysis of cyp51 from fluconazole-resistant Candida albicans strains

The target enzyme for fluconazole is sterol 14α-demethylase, a cytochrome P450 encoded by cyp51. One mechanism of fluconazole resistance likely to occur in Candida albicans is through an altered target site. To test this hypothesis DNA sequencing of the cyp51 coding sequence from 19 fluconazole-resistant and 19 fluconazole-sensitive C. albicans was undertaken. A number of point mutations were identified in the resistant isolates which were not present in the sensitive ones: F105L (five), E266D (five), K287R (one), G448G (one), G450E (one), G464S (three) and V488I (one). These alterations are discussed in the light of a molecular model of the enzyme regarding potential roles in resistance. It was also demonstrated that sequence-specific primers can be employed to identify polymorphisms which may be associated with resistance; diagnostic tests for resistant strains will prove of value in combating this serious clinical problem.     

The Candida albicans vacuole is required for differentiation and efficient macrophage killing

Yeast-hypha differentiation is believed to be necessary for the normal progression of Candida albicans infections. The emergence and extension of a germ tube from a parental yeast cell are accompanied by dynamic changes in vacuole size and morphology. Although vacuolar function is required during this process, it is unclear if it is vacuolar expansion or some other vacuolar function that is important. We previously described a C. albicans vps11Delta mutant which lacked a recognizable vacuole compartment and with defects in multiple vacuolar functions. These include sensitivities to stress, reduced proteolytic activities, and severe defects in filamentation. Herein we utilize a partially functional VPS11 allele (vps11hr) to help define which vacuolar functions are required for differentiation and which influence interaction with macrophages. Mutant strains harboring this allele are not osmotically or temperature sensitive and have normal levels of secreted aspartyl protease and carboxypeptidase Y activity but have a fragmented vacuole morphology. Moreover, this mutant is defective in filamentation, suggesting that the major role the vacuole plays in yeast-hypha differentiation may relate directly to its morphology. The results of this study support the hypothesis that vacuole expansion is required during germ tube emergence. Both vps11 mutants were severely attenuated in their ability to kill a macrophage cell line. The viability of the vps11delta mutant was significantly reduced during macrophage interaction compared to that in the control strains, while the vps11hr mutant was unaffected. This implies some vacuolar functions are required for Candida survival within the macrophage, while additional vacuolar functions are required to inflict injury on the macrophage.     

Physicochemical modification of the excretion product of Saccharomyces cerevisiae killer strains results in fungicidal activity against Candida albicans and Tricophyton mentagrophytes

It is known that certain yeast strains, so called 'killers', can produce and excrete proteinaceous toxins that can induce death of other sensitive strains. We obtained a stable fungicidal factor (SKF) through concentration and stabilization of the excretion product of certain killer strains of Saccharomyces cerevisiae (K1 and K2). The isolated proteinaceous complex exhibited activity at broad ranges of pH (4-7.5) and temperatures (20-37.5 degrees C). It was significantly lethal against Candida albicans and Tricophyton mentagrophytes. SKF showed stability and activity after storage, with a mean half-life of 6 months at 4 degrees C or at -20 degrees C.     

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.