Strain variation and morphogenesis of yeast- and mycelial-phase Candida albicans in low-sulfate, synthetic medium.

A low-sulfate synthetic medium was developed in which pure cultures of yeast- and mycelial-phase Candida albicans could be cultivated for investigations of the molecular biology of dimorphism. The medium contained ammonium ions, phosphate buffer, salts, glucose, and biotin. Morphogenesis was found to be dependent upon the strain of C. albicans. Of six strains tested in the low-sulfate medium at 37 degrees C, three formed mixed cultures of yeasts, true mycelium and pseudomycelium, two formed pure cultures of true mycelium, and one maintained yeast growth. All six strains produced pure cultures of yeasts at 24 degrees C. The buffering capacity of the medium maintained the pH at 6.9 even at high-density cell growth. The low concentration of sulfate and the absence of amino acids in the medium provided conditions in which to radiolabel cellular constituents with [35S]sulfate. For molecular investigations, the use of two strains is suggested, one forming yeasts and one forming true mycelium in low-sulfate medium at 37 degrees C, thus providing controls for both strain variation and for molecular changes induced by environmental change but unrelated to morphogenesis.     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.     

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.     

Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium

Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed.     

Differential expression of cytoplasmic proteins during yeast bud and germ tube formation in Candida albicans

Changes in the identity and quantity of proteins synthesized during morphogenesis may result from alterations in gene expression in the dimorphic yeast Candida albicans. Stationary phase yeast cells, upon resuming growth at 25 degrees C, form budding yeast and at 37 degrees C form germ tubes. In order to identify proteins associated with morphogenesis, we compared cytoplasmic proteins synthesized during germ tube and bud formation. Proteins synthesized during this period were labeled at four intervals with either [3H]leucine or [35S]methionine and separated by two-dimensional polyacrylamide gel electrophoresis. This study shows that, of the 230 proteins resolved on each gel, 5 were specific to the yeast morphology and 2 proteins showed reduction in net synthesis in the mycelial phase. There were, however, no mycelium-specific proteins at any labeling period. The majority of proteins were common to both morphologies and showed no major shift in number during resumption of growth. The observations reported here suggest that differential gene expression occurs during morphogenesis of C. albicans.     

Speculative cell cycle in yeast-phase growth of Sporothrix schenckii: plasma membrane ultrastructure as revealed by freeze-fracture electron microscopy

The cell cycle in yeast-phase growth of Sporothrix schenckii was investigated by light microscopy and freeze-fracture electron microscopy after a 3- to 7-day cultivation on brain heart infusion agar medium at 37 degrees C. Mother yeastlike cells were able to bear daughter yeastlike cells. They were also able to produce germ tubes that had the potential to develop into pseudohyphae and hyphae. On the other hand, hyphae or pseudohyphae born from yeastlike cells were able to bear yeastlike cells directly. These results lead us to propose a hypothetical cell cycle for yeast-phase growth involving yeastlike vegetative cells, pseudohyphae, and hyphae.     

Preparation and study of an exoantigen of Histoplasma capsulatum for serological reactions.

The aim of this study was to determine the usefulness of a yeast-phase exo-antigen of Histoplasma capsulatum in standard serologic reactions. Three native strains of H.capsulatum which belong to Mycology Center collection were employed. They were maintained in their yeast-phase by weekly subcultures in 2% dextrose broth agar at 37 degrees C. After one week incubation yeast cells were suspended in distilled water containing thimerosal and phenylmethyl sulfonyl fluoride at a concentration of 1:5000. This suspension was left at room temperature for 72 h, then the supernatant was separated by centrifugation and it was lyophilized. Proteins and polysaccharides concentrations were determined. Immunodiffusion (ID) tests were carried out with an antigenic dilution containing 1.4 mg/ml of proteins. This exo-antigen was submitted to SDS-PAGE. Seven protein fractions were detected but only two of them showed antigenic activity against a pool of positive human sera; the molecular weights of these two proteins were 97 kDa and 66 kDa respectively. A metabolic antigen from the mycelial phase of H. capsulatum was used as control. A rabbit gammaglobulin anti-H. capsulatum was prepared and employed as positive control in serologic reactions. The antigenic capacity of ten batches of this exo-antigen was studied by ID and counterimmunoelectrophoresis (CIE) tests using serum samples of 20 hamsters experimentally infected by intracardiac inoculation of the yeast-phase of H. capsulatum. All tests presented positive results after three weeks of the infection. Fifty sera from patients suffering progressive histopasmosis were analyzed: ID, CIE and complement fixation (CF) tests were performed in all cases. HIV negative patients presented 7/7 (100%) positive reactions with the yeast-phase exoantigen and 5/7 (71.4%) with histoplasmin. In HIV positive patients CIE and CF were the most sensitive serologic tests, they gave positive results in 15/43 cases (34.8%) with the yeast-phase exo-antigen and in 7/43 cases (13.9%) with histoplasmin. Sera from 10 patients with paracoccidioidomycosis, aspergillosis and candidiasis respectively were studied by ID with the aim of detecting serologic cross reactions. No cross reaction was detected in these serum samples. This yeast-phase exo-antigen of H. capsulatum is more sensitive than and equally as specific as control histoplasmin.     

Virulence of Candida albicans mutants toward larval Galleria mellonella (Insecta, Lepidoptera, Galleridae)

Culture medium affected the virulence of a strain of Candida albicans toward Galleria mellonella larvae, but the yeast growth rates in yeast extract - peptone - dextrose broth and synthetic Galleria serum were not correlated with yeast virulence. Virulent C. albicans grew rapidly in larval serum, whereas, it limited nodulation and continued development in vivo, producing toxins that damaged the hemocytes and fat body. Nonpathogenic yeast-phase cells grew slowly in larval serum but induced extensively melanized nodules in vivo and developed no further. There was no discernible relationship in 14 exo-enzymes between the virulent and avirulent yeast strains and virulence. The avirulent myosin-I-defective yeast cells were rapidly removed from the hemolymph in vivo because of lysozyme-mediated yeast agglutination and the possible binding of the yeast cells by lysozyme and apolipophorin-III. Both lysozyme and apolipophorin-III are proteins that bind beta-1,3-glucan. Finally, insects with nonpathogenic C. albicans exhibited induced immunity and were more resistant to candidiasis from the wild-type yeast cells than were noninduced insects.     

Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli.

Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130- and 135-kilodalton insecticidal proteins, respectively, of Bacillus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37 degrees C, but bipyramidal crystals practically free of contaminants were observed at 30 degrees C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

Nutrition-dependent modulations of protein synthesis in Candida albicans during germ-tube formation or maintenance of the yeast form in N-acetyl glucosamine media

Abstract Stationary phase, yeast-form cells of Candida albicans grown in glucose-yeast extract medium were shifted to N -acetylglucosamine (GlcNAc) and/or glucose medium, and the pattern of protein synthesized under conditions of a progressive decrease in the rate of total protein synthesis was analyzed by SDS-PAGE and autoradiography.
Marked temporal modulations in the rate of synthesis of some cytoplasmic proteins were detected both in cells forming germ-tubes (at 37°C) and in yeast cells (at 28°C). The major modulated components showed molecular weights of 63, 53, 48 and 34 kDa. These products could not be qualified as heat-shock or heat-stroke proteins, because analogous modulations were observed on shifting cells from 28°C to 37°C or from 28°C to 28°C. However, no marked modulations in the synthesis of specific proteins were detected when amino acids were added to the medium fostering germ-tube formation under conditions of unimpaired overall rate of protein synthesis.
It is suggested that the modulations observed in cells incubated in GlcNAc-glucose medium could represent a response to a nutritional stress.     

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.     

The influence of higher temperature on dengue-2 virus infected C6/36 mosquito cell line

Dengue-2 virus infection of C6/36 cells was studied at 28 and 37 degrees C. In infected cells maintained at 28 degrees C, syncytial development was seen on day 4 postinfection, whereas at 37 degrees C, extensive syncytial development was seen by 32 h. Extracellular virus titre was found to correlate with the cytopathic changes. Nine Dengue-2 virus specified proteins were observed in polyacrylamide analyses of cytoplasmic extracts of C6/36 infected cells. All the proteins were observed, although in varied intensities by 32 h postinoculation at 37 degrees C and only on day 4 postinoculation at 28 degrees C. The GP60 glycoprotein appeared at 32 h postinfection when the cells were maintained at 37 degrees C and became prominent only on day 5 at 28 degrees C. The results revealed that a higher temperature accelerated the onset of cytopathic effects, hastened the development of virus specified proteins, and also enhanced the titre of extracellular infectious virus. The importance of the accumulation of the envelope protein GP60 for the development of CPE was indicated.     

Detection and identification of virulence factors in Yersinia pestis using SELDI ProteinChip system

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.     

Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.