Nutritional stress proteins in Candida albicans.

Starving cells of Candida albicans synthesize at least seven proteins that represent nutritional-stress proteins (NSP). Such NSPs are formed by both germination-competent and germination-deficient strains of C. albicans. Heat-shock proteins (HSP) are not formed by starving cells. Germination-competent cells synthesize specific sets of proteins when incubated in a starvation medium that contains the germ-tube-inducing substances N-acetyl-D-glucosamine or L-proline. Both sets of induced proteins were also synthesized by a germination-deficient strain of C. albicans.

Ethanol tolerance and the induction of stress proteins by ethanol in Candida albicans.

Ethanol is one of the products of the metabolism of glucose by Candida albicans. The amount produced is directly related to the concentration of glucose in the medium. The fungus utilizes ethanol as a sole source of carbon but is relatively intolerant of ethanol in its environment. Ethanol induces germ tube formation by blastoconidia of C. albicans. Germination was not seen under fermentation conditions even though the amount of ethanol produced was in the range form stress proteins that are similar to heat shock proteins. The possibility that stress proteins may regulate germ tube formation by C. albicans is discussed.

Phenotypic characteristics of a slow-growing, nongerminating variant of Candida albicans.

Some of the phenotypic characteristics of a slow-growing, nongerminating variant of a commonly studied strain of Candida albicans are described. The variant arose as a chance isolate. The rate of occurrence was about 0 X 1% and the reversion rate was about 1 per 10(6) cells. The colony size was typically smaller than that of the parent and the yeast cells tended not to separate from one another so that catenulate strands of cells (pseudohyphae) were formed. Under standard conditions the generation time of the small-colony variant in liquid shake cultures was about twice that of the parental strain. Growth of the variant was suppressed by antimycin A, indicating that the small colony form was not the consequence of a defect in the cytochrome system. The colony size of the variant was not influenced by chlorobenzotriazole, which suggested that adenine metabolism was not involved in the small-colony phenotype. The pseudohyphal growth pattern was not relieved by high concentrations of utilizable carbohydrates, which means the catenulate microscopic appearance of the yeast cells was not simply an exaggeration of the normal growth pattern of isolates of C. albicans but more probably represented the growth of a cell-cycle mutant defective at the cell separation step. The cytoplasmic proteins of the variant and the parent were very similar though some unique peptides were displayed by each.

Heat shock and heat stroke proteins observed during germination of the blastoconidia of Candida albicans.

Cytoplasmic proteins extracted from germinating yeast cells of Candida albicans were analyzed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Similar extracts from a recently isolated nongerminating variant were compared with those from the parent. Five proteins (18, 22, 40, 68, and 70 kilodaltons [kd]) behaved as heat-shock proteins in that they appeared or were greatly increased in amount within 20 min of a temperature shift from 23 to 37 degrees C. Three of the five (40, 68, and 70 kd) were undetected in cells incubated at 23 degrees C, appeared within 20 min of temperature shift, and were no longer detected after 120 min at 37 degrees C, whereas two of the five (18 and 22 kd) were present in small amounts at 23 degrees C, increased greatly after shift, and persisted for 120 min at the elevated temperature. Two temperature-repressed (heat-stroke) proteins (30 and 88 kd) were also observed. The same heat-shock and heat-stroke proteins were also found in the nongerminating variant. The differences in proteins expressed by blastoconidia and by germlings appeared to be related to the heat-shock response.

Proline uptake in Candida albicans.

L-Proline entered both mycelial and yeast cells of Candida albicans by an active transport system of high specificity at low (less than 0.1 mM) external concentrations of substrate. The apparent Km value of this system was 0.1 mM for both types of cells, while the V value was 4 nmol min-1 (mg dry wt)-1 for mycelial cells and 1.4 nmol min-1 (mg dry wt)-1 for yeast cells. At L-proline concentrations greater than 0.1 mM, the amino acid appeared to enter both morphological forms by diffusion as well as active transport. As saturation was approached diffusion became increasingly important. The higher uptake rate of mycelial cells seemed not to be the result of an inducible system. The optimal pH and temperature for transport of L-proline were 7.0 and 37 degrees C, respectively. Sodium azide and the proline analogues sarcosine and L-azetidine-2-carboxylic acid inhibited L-proline uptake, while L-thiazolidine-4-carboxylic acid was less effective. The active transport system was highly specific for L-proline since neither ammonium ions, which inhibit the general amino acid transport system of fungi, nor 16 different amino acids interfered substantially with uptake.

Cysteine transport and sulfite reductase activity in a germination-defective mutant of Histoplasma capsulatum.

A mutant of the dimorphic, zoopathogenic fungus Histoplasma capsulatum, defective in the ability of its blastospores to germinate, was used to show that the nutritional requirement for cysteine and the expression of sulfite reductase activity are temperature dependent and not related to the growth form the fungus, whereas the kinetics of cysteine transport depend on both temperature and the form of the fungus.

Mutants of Arthroderma benhamiae.

The object of these studies was to isolate conditionally lethal mutants of Arthroderma benhamiae whose vegetative growth was temperature sensitive (ts). Three sorts of mutants were derived from nitrosoguanidine treatment of the microconidia of A. benhamiae: (i) ts "pleomorphic", (ii) ts "non-pleomorphic", and (iii) slow growers (sg) which were not temperature sensitive. The growth of wild type A. benhamiae, as measured by colony diameters, was 6 mm/day at 37 degrees C (chosen as non-permissive for screening mutants) and 5 mm/day at 25 degrees C (chosen as permissive for screening mutants). Growth of mutants of the first sort was virtually stopped at 37 degrees C but their growth at 25 degrees C was more rapid than that of wild type. The colony texture of these mutants was downy and their mycelium sterile. They failed to mate with parents of opposite mating type. These mutants were considered to be ts "pleomorphic". The second sort of mutants were ts "non-pleomorphic". Two isolates of this kind were recovered. They grew normally at 25 degrees C but their growth at 37 degrees C was reduced 50--70% of that of wild type. The nature of the temperature sensitive defect has not been identified. The third sort of mutant occurred very frequently. These isolates were slow growers (sg) regardless of temperature of incubation and their response to the respiratory inhibitors antimycin and salicyl hydroxamic acid suggested a defect in mitochondrial ribosome assembly and deficiencies in cytochromes not unlike those observed in the poky mutants of Neurospora. Two of the sg-mutants (sg 2 and sg3) produced abundant cleistothecia with asci and ascospores when back crossed to parents of opposite mating type. The sg 5 mutant produced cleistothecia but no ascospores. The ergosterol content of the sg 2 mutant was nearly the same as that of wild type while the ergosterol content of sg 3 was somewhat reduced and that of sg 5 was markedly reduced as compared to that of wild type. Thus the ergosterol content seems to play some role in sexual reproduction of A. benhamiae. The pattern of sensitivity to amphotericin B also reflected differences in the sterol content of the mutants, i.e., the two mutants with some alteration in their ergosterol content, sg 3 and sg 5 were more resistant to the antibiotic. During the course of these studies a number of variants were observed that produced different sorts and degrees of pigmentation of the reverse of their colonies. One stable variant of this kind gave rise to colonies with a red reverse when incubated at 37 degrees C and a yellow reverse when incubated at 25 degrees C. When plates were shifted from one temperature to another the next wave of growth was the color specified by the temperature.

Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.

Germination of Candida albicans induced by proline.

Blastospores of Candida albicans germinated in proline-biotin-buffer medium incubated at 37 C. Certain other amino acids in the glatamate, asparate, and pyruvate families also fostered germinaton but generally to a lesser extent than did proline. L-Cysteine, D-proline, and certain structural analogues of L-proline inhibited proline-stimualted germination. The concentration of phosphate and glucose was crucial to amino acid-stimulated germination of C. albicans. Clinical isolates and stock cultures varied in their response to the germ tube-inducing activity of proline or other amino acids. The proline-buffer medium cannot be used in a diagnostic test for production of germ tubes by isolates of yeasts.