Adherence of clinical isolates of Saccharomyces cerevisiae to buccal epithelial cells.

A number of isolates of Saccharomyces cerevisiae have been associated with disease in immunocompromised individuals. Such isolates display a variety of characteristics that enable colonization and persistence in the host. The aim of the work presented here was to establish whether clinical isolates of S. cerevisiae were capable of adhering to epithelial tissue. Adherence to host tissue has been shown to be crucial to the virulence of the pathogenic yeast Candida albicans, and identification of this ability in S. cerevisiae might indicate a role for adherence in tissue colonization by this emerging pathogen. Clinical S. cerevisiae isolates were found to be capable of adhering to exfoliated buccal epithelial cells (BECs) but to a lesser degree than C. albicans. In contrast to the situation evident with C. albicans, the adherence of S. cerevisiae isolates to BECs was not influenced by the carbon source in which the yeast was grown. Treatment of S. cerevisiae with trypsin or proteinase K resulted in a significant reduction in adherence ability while adherence was unaffected by treatment of cells with mannosidase, thus indicating a possible role for proteins rather than mannoproteins in the adherence of S. cerevisiae to BECs.     

Kinetics of petite mutation and thermal death in Saccharomyces cerevisiae growing at superoptimal temperatures

Mass formation of petite mutants took place in a strain of Saccharomyces cerevisiae when grown at superoptimal temperatures. After an initial period of exponential growth, a second period followed during which exponential death and net exponential petite mutation concurred with exponential growth. The specific rates of the three exponential processes were of the same order of magnitude and varied with the temperature. Net exponential petite mutation did not occur during the deathless first period of growth at superoptimal temperatures nor at any time during growth at suboptimal temperatures. Mitochondria are discussed as possible targets of thermal death in mesophilic yeasts.     

A direct study of the relative synthesis of petite and grande mitochondrial DNA in zygotes from crosses involving suppressive petite mutants of Saccharomyces cerevisiae

Summary Work in recent years has produced indirect evidence to support the view that the phenomenon of suppressiveness in yeast is the result of the ability of the petite mtDNA to out-replicate the wild-type genome. We have developed a method, based on fluorography of gels containing restriction fragments of radioactively labelled zygotic mtDNA, by which it has been possible to follow directly the incorporation of label into the two mtDNA species and hence their relative synthesis. Four petite isolates of 70%, 43%, 23% and 12% suppressiveness were tested by this method in crosses with a grande strain. Only the mtDNA from the 70% suppressive petite showed a replicative advantage over the grande mtDNA. The mtDNA from the 43% and 23% suppressive actually appeared to undergo, if anything, less replication in the zygote than the grande mtDNA. It is concluded that while some petites may exhibit suppressiveness as a result of enhanced replicative efficiency of their mtDNA, this cannot be the explanation for all suppressive petite strains.     

In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates.

Saccharomyces spp. are widely distributed in nature and may colonize the normal human gastrointestinal tract. Although Saccharomyces cerevisiae isolates have been previously considered nonpathogenic, they appear to be increasingly associated with infections in immunocompromised or otherwise debilitated patients. The antifungal susceptibility and epidemiology of S. cerevisiae are poorly defined at present. A series of 76 isolates (mostly stool surveillance and throat swab isolates) from 70 bone marrow transplant patients hospitalized at two different medical centers were characterized by antifungal susceptibility testing and restriction endonuclease analysis of chromosomal DNA. For DNA typing, digestion with NotI followed by pulsed-field gel electrophoresis was applied. Typing results revealed 62 distinct DNA types among the 76 clinical isolates. Despite this genomic diversity, clusters of identical isolates were identified among different patients hospitalized concurrently in the same unit, indicating possible nosocomial transmission. The MICs of amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole were determined by a broth microdilution method, as recommended by the National Committee for Clinical Laboratory Standards. The MICs at which 90% of the strains were inhibited were as follows: amphotericin B, 1.0 micrograms/ml; 5-fluorocytosine, 0.25 micrograms/ml; fluconazole, 8.0 micrograms/ml; and itraconazole, 1.0 micrograms/ml. The relative resistance of S. cerevisiae to fluconazole and itraconazole may promote the emergence of this species as a pathogen among immunosuppressed patients.     

Effects of T-2 toxin on induction of petite mutants and mitochondrial function in Saccharomyces cerevisiae

Summary The influence of the trichothecene mycotoxin T-2 on the mitochondria of Saccharomyces cerevisiae was studied. T-2 is a cytotoxic molecule inhibiting growth and macromolecular synthesis in S. cerevisiae. At low concentrations, T-2 toxin arrested yeast growth on glycerol medium and at higher concentrations, it arrested growth on glucose medium. The toxin was not capable itself of inducing petite mutations. Its inhibitory effect on the growth of petite strains, of both chromosomally isogenic and nonisogenic strains was less than that of grande strains. One exception to this was equally low susceptibility of psi ⁺ SUP4-3 strain in both rho ⁺ and rho ^– state. T-2 toxin was also capable of retarding the petite inducing activity of the mutagen, ethidium bromide. T-2 toxin inhibited the polymerization of P-ribosylaminoimidazole in an ade2 strain of S. cerevisiae. These results show that T-2 toxin is capable of interfering with the activity of the mitochondria in addition to its well studied effects on cytoplasmic protein synthesis.     

In vitro activity of five antifungal agents against clinical isolates of Saccharomyces cerevisiae.

We evaluated the in vitro activity of fluconazole, itraconazole, ketoconazole, 5-fluorocytosine and amphotericin B against 30 clinical isolates of Saccharomyces cerevisiae by a broth microdilution method, following the NCCLS recommendation. Testing was performed either in RPMI-1640 or yeast nitrogen base (YNB). YNB supported the growth of all isolates tested, while results in RPMI-1640 were not obtained for six isolates (20%). The MIC of all three azoles in YNB were one or two dilutions higher than those obtained in RPMI-1640 (P=0.0001 for fluconazole and itraconazole, P=0.03 for ketoconazole). Elevated MICs were observed for all three azoles, while all the isolates were susceptible to 5-fluorocytosine and amphotericin B. All MIC values were confirmed by spectrophotometric reading. Six strains of S. cerevisiae isolated from the faeces and consecutive blood cultures from an AIDS patient over a 7-month period were typed by electrophoretic karyotyping (EK). EK showed the maintenance of the same karyotype over time suggesting that the faecal isolate changed from a colonizing to infection-causing strain. The relative resistance of S. cerevisiae to azole drugs as well as its ability to cause widespread infections may promote the emergence of this species as a pathogen in immunosuppressed patients.     

Suppression of temperature sensitive mutations in oncogene-related CDC genes in Saccharomyces cerevisiae by catabolite repression resistance and cytoplasmic petite mutations

Summary The start cell division control genes CDC36 and CDC28 have been reported to contain a certain sequence homology to tissue oncogenes (ets and some protein kinase encoding oncogenes respectively). Here we report that temperature sensitive mutations in these genes are suppressed in cytoplasmic petite mutants and catabolite repression resistant mutants.     

Molecular cloning of the PEL1 gene of Saccharomyces cerevisiae that is essential for the viability of petite mutants

The PEL1 gene of Saccharomyces cerevisiae is essential for the cell viability of mitochondrial petite mutants, for the ability to utilize glycerol and ethanol on synthetic medium, and for cell growth at higher temperatures. By tetrad analysis the gene was assigned to chromosome III, centromere proximal of LEU2. The PEL1 gene has been isolated and cloned by the complementation of a pel1 mutation. The molecular analysis of the chromosomal insert carrying PEL1 revealed that this gene corresponds to the YCL4W open reading frame on the complete DNA sequence of chromosome III. The putative Pel1 protein is characterized by a low molecular weight of approximately 17 kDa, a low codon adaptation index, and a high leucine content.     

α-factor enhancement of hybrid formation by protoplast fusion in Saccharomyces cerevisiae II

Summary When Mat a cells are treated with -factor prior to being protoplasted and fused, the frequency of karyogamy is higher than in unarrested controls.     

DNA-damaging agents stimulate the formation of directed reciprocal translocations in Saccharomyces cerevisiae

DNA-damaging agents can stimulate the formation of directed reciprocal translocations of Saccharomyces cerevisiae containing his3 recombinational substrates to generate chromosomal rearrangements. Such agents were compared with those that can stimulate sister- chromatid recombination. We show that chemicals and environmental agents that produce a variety of DNA lesions, including bulky adduct, thymidine dimers, interstrand cross-links, double-strand breaks and alkylated bases, can stimulate recombination to yield reciprocal translocations. Of the agents teted, only the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguinidine (MNNG), and a bifunctional agent that causes bulky DNA adducts, 4-nitroquinoline-N-oxide (4-NQO), significantly stimulate sister-chromatid recombination in our assay. Factors that contribute to the stimulation of interchromosomal recombination include strain genetic background and ploidy.     

Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains

The ability of 15 Escherichia coli strains to form biofilms on polystirene plates was studied. The strains were serotyped, and their phenotypic expression of surface virulence factors (VFs), and antibiotic susceptibility was also determined. Moreover, 30 VFs-associated genes were analysed, including 15 adhesins (papC, papG and its three alleles, sfa/focDE, sfaS, focG, afa/draBC, iha, bmaE, gafD, nfaE, fimH, fimAvMT78, agn43, F9 fimbriae and type 3 fimbriae-encoding gene clusters), four toxins (hlyA, cnf1, sat and tsh), four siderophore (iron, fyuA, iutA and iucD), five proctetins/invasion-encoding genes (kpsM II, kpsMT III, K1 kps variant- neuC, traT and ibeA), and the pathogenicity island malX and cvaC. Morphological appearance and thickness of biofilms of two strong and three weak biofilm producers were also studied by confocal laser scanning microscopy (CLSM). Seven strains were classified as strong biofilm producers and the remaining eight strains were regarded as weak biofilm producers. Mannose-resistant haemagglutination was the only phenotypically expressed surface virulence factor more frequently found in the strong biofilm group. Five virulence-associated genes were more common (p<0.05) in strong biofilm producers: papC and papG alleles, sfa/focDE, focG, hlyA and cnf1. CLSM images showed irregular biofilms with projections at the top mainly in strong biofilm.     

Cytoplasmic mixing in Saccharomyces cerevisiae: Studies on a grande X neutral petite mating and implications for the mechanism of neutrality

Summary We have studied zygotic cytoplasmic mixing in two neutral petite by grande matings. Cytoplasmic mixing in zygotes and zygotic buds was followed histochemically and genetically. Both techniques showed that in most zygotes rapid cytoplasmic mixing occurred after early zygote formation and before first bud completion. The progeny mitochondrial genotypes produced from each cross were compatible with a model assuming random segregation of petite and grande inputs between the zygote and its first bud.While both crosses produced only grande zygotic colonies and grande diploid progeny when assayed by classical genetic methods, a class of events producing petite zygotes, retaining petite mitochondrial DNA, with grande first diploid buds was discovered. The results show that the expression of neutrality in our strains requires efficient cytoplasmic mixing during zygote maturation. The results suggest that the expression of mitochondrial genome neutrality requires competition or interaction between petite and grande parental mitochondrial DNA molecules. The possible nature of these phenomena is discussed.     

Some aspects of the phenylacetylcarbinol biosynthesis by Saccharomyces cerevisiae

The biosynthesis of phenylacetylcarbinol (PAC) by Saccharomyces cerevisiae in media containing benzaldehyde and fermentable sugars was studied under different experimental conditions. In shaking cultures with small volumes of medium a sigmoidal production curve for PAC was obtained. During the first and second part of the fermentation period (5 to 10 hours), the number of living yeast cells increased. In the third period (from the 10th to the 20th hour), the number of living yeast cells decreased rapidly. In agitated batch cultures the PAC produced during the first 5 hours of fermentation decreased only slowly on further incubation. In agitated but non aerated batch cultures, the PAC production increased rapidly, but the yields were much lower than in aerated batches. Acetone dried cells of baker's yeast produced PAC from benzaldehyde but no benzylalcohol. Besides PAC and acetylbenzoyl, a new biosynthetic product, trans-cinnam-aldehyde was determined.     

RAD59 and RAD1 cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails.     

Investigation of Batten disease with the yeast Saccharomyces cerevisiae

The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.     

Correlation between antibiotic resistance, phage-like particle presence, and virulence in Rhodococcus equi human isolates.

Rhodococcus equi is a gram-positive coccobacillus that appears to be emerging as a pulmonary pathogen in AIDS patients. In four human clinical isolates, two antibiotic resistance phenotypes were found to coexist: one beta-lactam resistant and the other beta-lactam susceptible. In vitro, beta-lactam-resistant mutants were obtained at a frequency of 1 x 10(-5) to 5 x 10(-5) from beta-lactam-susceptible strains on cephalothin-containing plates. Neither beta-lactamase nor plasmid DNA was detected in beta-lactam-resistant or -susceptible strains. The penicillin-binding protein patterns for the two types of strains were identical. Electron microscopy revealed that the beta-lactam-resistant strains possessed cell-surface-associated appendages and produced phage-like particles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein showed at least three additional bands of 42, 39, and 30 kDa found only in the beta-lactam-resistant strains. Testing for virulence in Swiss mice revealed that (i) phage-like-particle-producing strains had lower 50% lethal doses when injected intravenously in euthymic and nude mice than the non-phage-like-particle-producing strains did and (ii) intravenous inoculation of a sublethal dose (5 x 10(6) CFU) in nude mice led to chronic infection by the phage-like-particle-producing bacteria only. Finally, in vitro growth curves indicated that the phage-like-particle-producing strains possessed an ecological selection advantage. These results suggest that, among R. equi human isolates, the antibiotic resistance phenotype is associated with virulence and may be phage mediated.     

High frequency FLP-independent homologous DNA recombination of 2 μ plasmid in the yeast Saccharomyces cerevisiae

Summary The purpose of this work is to identify and quantitate in vivo 2 plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 DNA recombination events occurring in FLP⁺ cells is the contribution of both FLP-mediated and FLP-independent events.     

Effects of the kar gene on cytoplasmic mixing and mitochondrial genome suppressiveness,and consequences for cytoduction of petite DNA in Saccharomyces cerevisiae

Summary During a series of cytoduction experiments to transfer Saccharomyces cerevisiae mitochondrial genomes from one nuclear background to another, using the karl-1 nuclear fusion mutation, one of the five petite genomes used proved difficult to transfer. This genome, ^- F13, was highly suppressive (90%) in its original nuclear background. Molecular and genetic studies on the putative karl-1 ^–F13 cytoductant were done to discover the nature of this difficulty. They showed that while the ^–F13 was maintained in a karl-l background, zygotes from a mating with a ⁰ strain showed poor cytoplasmic mixing and therefore inefficient ^–F 13 DNA transfer into first zygotic buds. This also caused a reduction of ^–F13 suppressiveness to 20–30% in crosses with different ⁺ strains. The effect was genome specific since another highly suppressive petite in the karl-l background did not show suppressiveness reduction when crossed to ⁺. The nature of suppressiveness modulation is discussed. Since the ^–F13 genome was eventually transferred using a modification of the original scheme, the problems were not caused by the inability of the acceptor nuclear background to maintain the ^–F13 genome.     

Radioprotective effect of podophyllotoxin in Saccharomyces cerevisiae.

Recent reports showed that whole extract of Podophyllum hexandrum was radioprotective in mice. Podophyllotoxin is one of the major constituents of the whole extract of Podophyllum. In this study we report on the radioprotective action of podophyllotoxin in Saccharomyces cerevisiae yeast. Proliferating yeast cells pretreated with podophyllotoxin (2.5-5.0 microg/mL) for > or =3 hours showed a higher surviving fraction after (60)Co-gamma-irradiation (200-600 Gy) than did the irradiated cells not pretreated with podophyllotoxin. The maximum increase (2.0 times) in surviving fraction was observed in cells treated with 2.5 microg/mL podophyllotoxin, 5 hours before (60)Co-gamma-irradiation (400 Gy). Podophyllotoxin was not mutagenic or recombinogenic at radioprotective doses (2.5 microg/mL). A post-irradiation decrease in revertants and gene convertants was observed in cells treated with podophyllotoxin (2.5 microg/mL podophyllotoxin, -5 hours, 400 Gy). This study indicates that podophyllotoxin is radioprotective in yeast, and its radioprotective effects in higher eukaryotes would be worth investigating.