scholarly journals Saccharomyces cerevisiae-based probiotic as novel anti-fungal and anti-inflammatory agent for therapy of vaginal candidiasis

2018 ◽  
Vol 9 (2) ◽  
pp. 219-230 ◽  
Author(s):  
E. Gabrielli ◽  
E. Pericolini ◽  
N. Ballet ◽  
E. Roselletti ◽  
S. Sabbatini ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Vaginal Candidiasis ◽  
Therapeutic Effects ◽  
Related Factors ◽  
Vaginal Epithelial Cell ◽  
Associated Proteins ◽  
Anti Fungal

Previously we demonstrated that the treatment with live Saccharomyces cerevisiae exerts beneficial therapeutic effects against vaginal candidiasis. Here, we address potential mechanisms particularly examining the probiotic capacity to modulate both fungus and host-related factors. We show that the S. cerevisiae-based probiotic markedly affects the expression of virulence traits of Candida albicans such as aspartyl proteinases (SAPs) as well as hyphae-associated proteins Hwp1 and Ece1 in the vaginal cavity. On the host side, the probiotic suppression of the influx of neutrophils caused by the fungus into the vaginas of the mice is likely related to: (1) lower production of interleukin-8; and (2) inhibition of SAPs expression. However, these neutrophils displayed reactive oxygen species hyperproduction and increased killing activity as compared to the neutrophils of placebo-treated mice. There was no evidence of any cytotoxic effect by the probiotic, either when used in vivo on vaginal epithelial cell and organ architecture, or in in vitro in human vaginal epithelium. Inactivated yeast cells did not affect any of the factors above. In summary, the data suggest that the beneficial effect exerted by this S. cerevisiae-based probiotic is the result of its interference with the expression of fungus virulence factors coupled with the modulation of the inflammatory response of the host.

scholarly journals Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2006 ◽  
Vol 6 (2) ◽  
pp. 328-336 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Paula Magnelli ◽  
Phillips W. Robbins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chain Length ◽  
Attachment Site ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Average Chain Length ◽  
Calcofluor White ◽  
Protein Factor

ABSTRACT Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4215-4229
Author(s):  
S Heidmann ◽  
B Obermaier ◽  
K Vogel ◽  
H Domdey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Sequence ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Adh1 Gene ◽  
Polyadenylation Sites ◽  
Yeast Genes

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

scholarly journals Global Phenotype Screening and Transcript Analysis Outlines the Inhibitory Mode(s) of Action of Two Amphibian-Derived, α-Helical, Cationic Peptides on Saccharomyces cerevisiae

2007 ◽  
Vol 51 (11) ◽  
pp. 3948-3959 ◽  
Author(s):  
C. Oliver Morton ◽  
Andrew Hayes ◽  
Michael Wilson ◽  
Bharat M. Rash ◽  
Stephen G. Oliver ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Inhibitory Action ◽  
Protein Targeting ◽  
Telomere Maintenance ◽  
Yeast Cells ◽  
Membrane Disruption ◽  
Dna Integrity ◽  
Cationic Peptides

ABSTRACT Dermaseptin S3(1-16) [DsS3(1-16)] and magainin 2 (Mag 2) are two unrelated, amphibian-derived cationic peptides that adopt an α-helical structure within microbial membranes and have been proposed to kill target organisms via membrane disruption. Using a combination of global deletion mutant library phenotypic screening, expression profiling, and physical techniques, we have carried out a comprehensive in vitro analysis of the inhibitory action of these two peptides on the model fungus Saccharomyces cerevisiae. Gene ontology profiling (of biological processes) was used to identify both common and unique effects of each peptide. Resistance to both peptides was conferred by genes involved in telomere maintenance, chromosome organization, and double-strand break repair, implicating a common inhibitory action of DNA damage. Crucially, each peptide also required unique genes for maintaining resistance; for example, DsS3(1-16) required genes involved in protein targeting to the vacuole, and Mag 2 required genes involved in DNA-dependent DNA replication and DNA repair. Thus, DsS3(1-16) and Mag 2 have both common and unique antifungal actions that are not simply due to membrane disruption. Physical techniques revealed that both peptides interacted with DNA in vitro but in subtly different ways, and this observation was supported by the functional genomics experiments that provided evidence that both peptides also interfered with DNA integrity differently in vivo. This implies that both peptides are able to pass through the cytoplasmic membrane of yeast cells and damage DNA, an inhibitory action that has not been previously attributed to either of these peptides.

Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (5) ◽  
pp. 2736-2743
Author(s):  
H Xu ◽  
J D Boeke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transposition Frequency ◽  
Mating Pheromones ◽  
Retroviral Replication ◽  
Viruslike Particles

The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication. We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones. When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished. Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells. These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins. In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability. We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules

1993 ◽  
Vol 13 (8) ◽  
pp. 4884-4893
Author(s):  
W Jiang ◽  
K Middleton ◽  
H J Yoon ◽  
C Fouquet ◽  
J Carbon
Keyword(s):  
Topoisomerase Ii ◽  
Yeast Cells ◽  
Affinity Column ◽  
Bacterial Cells ◽  
Binding Domains ◽  
C Terminus ◽  
Binding Factor ◽  
Associated Proteins

Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B, CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.

scholarly journals Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2736-2743 ◽  
Author(s):  
H Xu ◽  
J D Boeke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transposition Frequency ◽  
Mating Pheromones ◽  
Retroviral Replication ◽  
Viruslike Particles

The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication. We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones. When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished. Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells. These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins. In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability. We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

scholarly journals Induction of apoptosis in Saccharomyces cerevisiae results in the spontaneous maturation of tetravirus procapsids in vivo

2007 ◽  
Vol 88 (5) ◽  
pp. 1576-1582 ◽  
Author(s):  
Michele Tomasicchio ◽  
Philip Arno Venter ◽  
Karl H. J. Gordon ◽  
Terry N. Hanzlik ◽  
Rosemary Ann Dorrington
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Conformational Changes ◽  
Yeast Cells ◽  
Genomic Rnas ◽  
Virus Like Particle ◽  
Induced Apoptosis

The Tetraviridae are a family of small, non-enveloped, insect RNA viruses consisting of one or two single-stranded, positive-sense genomic RNAs encapsidated in an icosahedral capsid with T=4 symmetry. Tetravirus procapsids undergo maturation when exposed to a low pH environment in vitro. While the structural biology of the conformational changes that mediate acid-dependent maturation is well understood, little is known about the significance of acid-dependent maturation in vivo. To address this question, the capsid-coding sequence of the tetravirus Helicoverpa armigera stunt virus was expressed in Saccharomyces cerevisiae cells. Virus-like particles were shown to assemble as procapsids that matured spontaneously in vivo as the cells began to age. Growth in the presence of hydrogen peroxide or acetic acid, which induced apoptosis or programmed cell death in the yeast cells, resulted in virus-like particle maturation. The results demonstrate that assembly-dependent maturation of tetravirus procapsids in vivo is linked to the onset of apoptosis in yeast cells. We propose that the reduction in pH required for tetraviral maturation may be the result of cytosolic acidification, which is associated with the early onset of programmed cell death in infected cells.

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