Genome-wide association analysis of clinical vs. nonclinical origin provides insights into Saccharomyces cerevisiae pathogenesis.

Because domesticated Saccharomyces cerevisiae strains have been used to produce fermented food and beverages for centuries without apparent health implications, S. cerevisiae has always been considered a Generally Recognized As Safe (GRAS) microorganism. However, the number of reported mucosal and systemic S. cerevisiae infections in the human population has increased and fatal infections have occurred even in relatively healthy individuals. In order to gain insight into the pathogenesis of S. cerevisiae and improve our understanding of the emergence of fungal pathogens, we performed a population-based genome-wide environmental association analysis of clinical vs. nonclinical origin in S. cerevisiae. Using tiling array-based, high-density genotypes of 44 clinical and 44 nonclinical S. cerevisiae strains from diverse geographical origins and source substrates, we identified several genetic loci associated with clinical background in S. cerevisiae. Associated polymorphisms within the coding sequences of VRP1, KIC1, SBE22 and PDR5, and the 5' upstream region of YGR146C indicate the importance of pseudohyphal formation, robust cell wall maintenance and cellular detoxification for S. cerevisiae pathogenesis, and constitute good candidates for follow-up verification of virulence and virulence-related factors underlying the pathogenicity of S. cerevisiae.

Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR.

Candida albicans and Cryptococcus neoformans cause both superficial and disseminated infections in humans. Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity. C. albicans rbp1/rbp1 mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in C. albicans, C. neoformans, and Saccharomyces cerevisiae. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.

Development of Saccharomyces cerevisiae as a model pathogen. A system for the genetic identification of gene products required for survival in the mammalian host environment.

Saccharomyces cerevisiae, a close relative of the pathogenic Candida species, is an emerging opportunistic pathogen. An isogenic series of S. cerevisiae strains, derived from a human clinical isolate, were used to examine the role of evolutionarily conserved pathways in fungal survival in a mouse host. As is the case for the corresponding Candida albicans and Cryptococcus neoformans mutants, S. cerevisiae purine and pyrimidine auxotrophs were severely deficient in survival, consistent with there being evolutionary conservation of survival traits. Resistance to the antifungal drug 5-fluorocytosine was not deleterious and appeared to be slightly advantageous in vivo. Of mutants in three amino acid biosynthetic pathways, only leu2 mutants were severely deficient in vivo. Unlike the glyoxylate cycle, respiration was very important for survival; however, the mitochondrial genome made a respiration-independent contribution to survival. Mutants deficient in pseudohyphal formation were tested in vivo; flo11Delta mutants were phenotypically neutral while flo8Delta, tec1Delta, and flo8Delta tec1Delta mutants were slightly deficient. Because of its ease of genetic manipulation and the immense S. cerevisiae database, which includes the best annotated eukaryotic genome sequence, S. cerevisiae is a superb model system for the identification of gene products important for fungal survival in the mammalian host environment.

Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.

Whole genome genetic-typing in yeast using high-density oligonucleotide arrays.

Genome sequence information in combination with new technologies has allowed researchers to approach genetic problems in new ways. High-density oligonucleotide arrays were used to probe the genome content of the yeast Saccharomyces cerevisiae. We show that these arrays, containing oligonucleotides complementary to the sequenced strain of S. cerevisiae, can be used to identify open reading frames that are missing or present in higher or lower copy number in related isolates of S. cerevisiae. We apply this method to the characterization of the genome of a strain derived from a clinical isolate of S. cerevisiae. Our results show that the telomeres are the regions with the most variability between the two strains.

Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae.

Heterologous gene replacement cassettes are powerful tools for dissecting gene function in Saccharomyces cerevisiae. Their primary advantages over homologous gene replacement cassettes include reduced gene conversion (leading to efficient site-specific integration of the cassette) and greater independence of strain background. Perhaps the most widely used cassettes are the MX cassettes containing the dominant selectable kanamycin resistance gene (kanr), which confers resistance to G418 (Wach et al., 1994). One limitation of the kanMX cassettes is that they are not counterselectable and therefore not readily recyclable, which is important when constructing strains with more than one gene deletion. To address this limitation, and to expand the choices of heterologous markers, we have created two new MX cassettes by replacing the kanr ORF from plasmids pFA6-kanMX3 and pFA6-kanMX4 with the Candida albicans URA3 ORF. These plasmids, pAG60 (CaURA3MX4) and pAG61 (CaURA3MX3) are identical to the kanMX cassettes in all other respects but have the added advantage of being counterselectable and therefore readily recyclable in S. cerevisiae.

Species identification and virulence attributes of Saccharomyces boulardii (nom. inval.).

Saccharomyces boulardii (nom. inval.) has been used for the treatment of several types of diarrhea. Recent studies have confirmed that S. boulardii is effective in the treatment of diarrhea, in particular chronic or recurrent diarrhea, and furthermore that it is a safe and well-tolerated treatment. The aim of the present study was to identify strains of S. boulardii to the species level and assess their virulence in established murine models. Three strains of S. boulardii were obtained from commercially available products in France and Italy. The three S. boulardii strains did not form spores upon repeated testing. Therefore, classical methods used for the identification of Saccharomyces spp. could not be undertaken. Typing by using the restriction fragment length polymorphisms (RFLPs) of the PCR-amplified intergenic transcribed spacer regions (including the 5. 8S ribosomal DNA) showed that the three isolates of S. boulardii were not separable from authentic isolates of Saccharomyces cerevisiae with any of the 10 restriction endonucleases assessed, whereas 9 of the 10 recognized species of Saccharomyces could be differentiated. RFLP analysis of cellular DNA with EcoRI showed that all three strains of S. boulardii had identical patterns and were similar to other authentic S. cerevisiae isolates tested. Therefore, the commercial strains of S. boulardii available to us cannot be genotypically distinguished from S. cerevisiae. Two S. boulardii strains were tested in CD-1 and DBA/2N mouse models of systemic disease and showed intermediate virulence compared with virulent and avirulent strains of S. cerevisiae. The results of the present study show that these S. boulardii strains are asporogenous strains of the species S. cerevisiae, not representatives of a distinct and separate species, and possess moderate virulence in murine models of systemic infection. Therefore, caution should be advised in the clinical use of these strains in immunocompromised patients until further study is undertaken.

Direct allelic variation scanning of the yeast genome.

As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

Intergenic transcribed spacer PCR ribotyping for differentiation of Saccharomyces species and interspecific hybrids.

The taxonomy of the genus Saccharomyces has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of Saccharomyces, allows unknown Saccharomyces isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of Saccharomyces and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes MaeI and HaeIII could distinguish all species of Saccharomyces from each other, as well as from Candida glabrata, Candida albicans, and Blastomyces dermatitidis. The only exception to this was the inability to distinguish between Saccharomyces bayanus and S. pastorianus (S. carlsbergensis). Furthermore, interspecific hybrids resulting from the mating of sibling species of Saccharomyces were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.

Epidemiological investigation of vaginal Saccharomyces cerevisiae isolates by a genotypic method.

Saccharomyces cerevisiae is a ubiquitous, ascomycetous yeast, and vaginitis caused by this organism has been reported only very rarely. The aim of the present investigation was to assess the epidemiological relatedness of a group of vaginal and commercial S. cerevisiae isolates by a previously reported genetic typing method, which divided the isolates into two broad groups with numerous subtypes. Nineteen S. cerevisiae isolates obtained from patients suffering from vaginitis and four isolates from commercial products in the same city were analyzed. The cellular DNA from each isolate was digested with the restriction endonuclease EcoRI, and restriction fragment length polymorphisms were generated by horizontal gel electrophoresis. The results showed that although vaginal isolates did not cluster in any particular genetic subtype, multiple patients were infected with indistinguishable strains (there were nine distinct strains among 23 isolates). For two of three patients, all three with two episodes of S. cerevisiae vaginitis, different strains were isolated during the recurrence of this disease. Three other patients with indistinguishable isolates were epidemiologically related in that two were practitioners in the same clinic and the third was a patient at this clinic. We also found that one commercial strain was indistinguishable from the strain isolated from three different women at the time that they were suffering from vaginitis. The findings of the present study suggest that some S. cerevisiae strains may possess properties permitting persistence in the human host. Furthermore, person-to-person contact and the proliferation of the use of S. cerevisiae as a health-food product, in home baking, and in home brewing may be a contributing factor in human colonization and infection with this organism.

Yeast microarrays for genome wide parallel genetic and gene expression analysis.

We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.

Whole genome analysis: experimental access to all genome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR.

The recent ability to sequence whole genomes allows ready access to all genetic material. The approaches outlined here allow automated analysis of sequence for the synthesis of optimal primers in an automated multiplex oligonucleotide synthesizer (AMOS). The efficiency is such that all ORFs for an organism can be amplified by PCR. The resulting amplicons can be used directly in the construction of DNA arrays or can be cloned for a large variety of functional analyses. These tools allow a replacement of single-gene analysis with a highly efficient whole-genome analysis.

Application of DNA typing methods and genetic analysis to epidemiology and taxonomy of Saccharomyces isolates.

We have previously described differences in phenotype and virulence among clinical and nonclinical isolates of Saccharomyces. To further characterize these isolates, a comparison of restriction fragment length polymorphism (RFLP) patterns and genetic analysis were done. The cellular DNA of each of 49 clinical and 11 nonclinical isolates of Saccharomyces was digested with the endonuclease EcoRI, and the resultant fragments were separated by electrophoresis. Sixty isolates were grouped on the basis of the presence (group B) or absence (group A) of a 3-kb band. Group A contained 43 isolates (35 clinical and 8 nonclinical isolates) in 31 discernible subgroups, and group B had 17 isolates (14 clinical and 3 nonclinical isolates) in 10 subgroups. Interestingly, six of eight known vaginal isolates were group B, with four of those six being identical. Virulence of isolates was associated with membership in group A (P = 0.03). Comparison of known members of sibling species within the genus Saccharomyces, which cannot be distinguished by standard biochemical tests, showed that S. paradoxus, S. bayanus, and S. cerevisiae could be differentiated by RFLP analysis. Genetic analysis of the isolates forming viable spores showed that most group A isolates were diploid and members of the species S. cerevisiae. Those group A and B isolates unable to form viable spores may be diploid hybrids between Saccharomyces species. The group B isolates that formed viable spores were tetraploid and may also be interspecific hybrids. Overall, clinical isolates of Saccharomyces were very heterogeneous and exhibited little clonality. RFLP pattern analysis could be a useful method of demonstrating transmission in patients with infection or between environmental sources and patients.

MOP2 (SLA2) affects the abundance of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The abundance of yeast plasma membrane H(+)-ATPase on the cell surface is tightly regulated. Modifier of pma1 (mop) mutants were isolated as enhancers of the mutant phenotypes of pma1 mutants. mop2 mutations reduce the abundance and activity of Pma1 protein on the plasma membrane without affecting the abundance of other prominent plasma membrane proteins. The MOP2 gene encodes a 108-kDa protein that has previously been identified both as a gene affecting the yeast cytoskeleton (SLA2) (Holtzman, D.A., Yang, S., and Drubin, D. G. (1993) J. Cell Biol. 122, 635-644) and as a gene affecting endocytosis (END4) (Raths, S., Roher, J., Crausaz, F., and Riezman, H. (1993) J. Cell Biol. 120, 55-65). In some strains, MOP2 (SLA2) is essential for cell viability; in others, a deletion mutant is temperature sensitive for growth. mop2 mutations do not reduce the transcription of PMA1 nor do they lead to the accumulation of Pma1 protein in any intracellular compartment. An epitope-tagged MOP2 protein behaves as a plasma membrane-associated protein whose abundance is proportional to its level of gene expression. Over-expression of MOP2 relieved the toxicity caused by the over-expression of PMA1 from a high copy plasmid; conversely, the growth of mop2 strains was inhibited by the presence of a single extra copy of PMA1. We conclude that MOP2 (SLA2) encodes a plasma membrane-associated protein that is required for the accumulation and/or maintenance of plasma membrane H(+)-ATPase on the cell surface.

Pathogenicity of Saccharomyces cerevisiae in complement factor five-deficient mice.

We have previously determined the relative virulence of isolates of Saccharomyces cerevisiae on the basis of differences in proliferation and resistance to clearance in CD-1 mice. These infections were not fatal. To further characterize S. cerevisiae pathogenesis, we studied a virulent clinical isolate, YJM128, and an avirulent nonclinical isolate, Y55, in C5-deficient mice. DBA/2N mice were infected intravenously with YJM128 or Y55, and temporal burdens of yeast cells in various organs were determined. After infection with 10(7) CFU, Y55 increased by 13-fold and YJM128 increased by 20-fold in the brain from day 0 to 3. In addition, YJM128 increased by 4-fold in the kidneys, whereas Y55 decreased by 16-fold. Both isolates declined in number in other organs. In all studies, 90% of mice infected with 10(7) CFU of YJM128 died between days 2 and 7, whereas no mice infected with equivalent numbers of Y55 died. No mice died after infection with 10(6) CFU of Y55 or YJM128. The importance of C5 was confirmed by studies using B10.D2/oSnJ (C5-) mice and their congenic C5+ counterparts. Again, the C5- mice were most susceptible to infection with S. cerevisiae, with 63% infected with YJM128 dying by day 7; no C5+ mice died. No Y55-infected mice died, and mean burdens in the brain at day 14 were sevenfold lower in C5+ mice than in C5- mice. Seven of 10 other S. cerevisiae isolates were also more virulent in DBA/2N than CD-1 mice, causing > or = 40% mortality. These data indicate that C5 is a critical factor in host resistance against S. cerevisiae infections and further confirm the pathogenic potential of some isolates of S. cerevisiae.

Saccharomyces cerevisiae virulence phenotype as determined with CD-1 mice is associated with the ability to grow at 42 degrees C and form pseudohyphae.

Saccharomyces cerevisiae isolates have been shown previously to exhibit a high degree of variation in their ability to proliferate and persist in CD-1 mice (K.V. Clemons, J.H. McCusker, R. W. Davis, and D.A. Stevens, J. Infect. Dis. 169:859-867, 1994). Isolate origin was not a firm predictor of virulence phenotype, since the virulence phenotypes of clinical and nonclinical isolates ranged from virulent to avirulent and from intermediate to avirulent, respectively. Therefore, it was important to determine if there was any association between putative virulence traits and virulence that might help explain the variation in virulence phenotypes. S. cerevisiae isolates spanning a range of virulence phenotypes in experimental infections were examined for putative virulence traits: the ability to grow at supraoptimal temperatures (42, 39, and 37 degrees C), gelatin liquefaction, casein utilization, and pseudohyphal formation. Gelatin liquefaction appeared to be unrelated to pseudohyphal formation on casein or to virulence. Significant differences in the ability to grow at 39 and 42 degrees C were observed when the virulent and intermediate classes were compared with the avirulent class. Less extreme but still significant differences in pseudohyphal formation were observed when the virulent and intermediate classes were compared with the avirulent class. Therefore, two virulence traits, similar to those identified in other pathogenic fungi, the ability to grow at elevated temperatures and pseudohyphal formation, have been identified in S. cerevisiae.

Genetic characterization of pathogenic Saccharomyces cerevisiae isolates.

Saccharomyces cerevisiae isolates from human patients have been genetically analyzed. Some of the characteristics of these isolates are very different from laboratory and industrial strains of S. cerevisiae and, for this reason, stringent genetic tests have been used to confirm their identity as S. cerevisiae. Most of these clinical isolates are able to grow at 42 degrees, a temperature that completely inhibits the growth of most other S. cerevisiae strains. This property can be considered a virulence trait and may help explain the presence of these isolates in human hosts. The ability to grow at 42 degrees is shown to be polygenic with primarily additive effects between loci. S. cerevisiae will be a useful model for the evolution and genetic analysis of fungal virulence and the study of polygenic traits.

Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae.

Although considered nonpathogenic, Saccharomyces cerevisiae is being encountered more frequently in the clinical setting. To assess pathogenic potential, 13 clinical isolates, 10 nonclinical isolates, and 5 constructed strains of S. cerevisiae were analyzed. All were S. cerevisiae by biochemical profiles, sporulation, or genetic evidence. Intravenous inoculation of yeasts into CD-1 mice showed that some clinical isolates proliferated in the brain (5-fold) but nonclinical isolates were cleared (1000-fold) by day 7 after infection. Comparison of burdens with those of YJM128 (clinical) and Y55 (laboratory strain) revealed three virulence groupings: virulent, those greater than or equal to YJM128 (5 clinical and 2 genetic constructs); intermediate virulent, those less than YJM128 and greater than Y55 (5 clinical, 3 genetic constructs, and 4 nonclinical); and avirulent, those less than or equal to Y55 (1 clinical and 6 nonclinical). Genetic crosses indicated that virulence was a dominant trait. Growth of various isolates at 37 degrees C and 39 degrees C indicated that temperature is associated with but not solely responsible for differences in virulence. These data demonstrate that some clinical isolates of S. cerevisiae can proliferate and resist clearance in vivo and support the potential of S. cerevisiae as a cause of clinical disease.

Genomic mismatch scanning: a new approach to genetic linkage mapping.

Genomic mismatch scanning (GMS) is a new method of genetic linkage analysis that does not require conventional polymorphic markers or gel electrophoresis. GMS is ideally suited to affected-relative-pair mapping. DNA fragments from all regions of identity-by-descent between two relatives are isolated based on their ability to form extensive mismatch-free hybrid molecules. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step. Here we demonstrate the practicality of GMS in a model organism, Saccharomyces cerevisiae. GMS is likely to be applicable to other organisms, including humans, and may be of particular value in mapping complex genetic traits.

The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae.

The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.