scholarly journals Genomic Approach to Identification of Mutations Affecting Caspofungin Susceptibility in Saccharomyces cerevisiae

2004 ◽  
Vol 48 (10) ◽  
pp. 3871-3876 ◽  
Author(s):  
Sarit Markovich ◽  
Aya Yekutiel ◽  
Itamar Shalit ◽  
Yona Shadkchan ◽  
Nir Osherov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Ergosterol Biosynthesis ◽  
Membrane Function ◽  
Minimum Effective Concentration ◽  
Fourfold Increase ◽  
Microdilution Method ◽  
New Genes ◽  
Broth Microdilution Method ◽  
Glucan Synthesis

ABSTRACT The antifungal agent caspofungin (CAS) specifically interferes with glucan synthesis and cell wall formation. To further study the cellular processes affected by CAS, we analyzed a Saccharomyces cerevisiae mutant collection (4,787 individual knockout mutations) to identify new genes affecting susceptibility to the drug. This collection was screened for increased CAS sensitivity (CAS-IS) or increased CAS resistance (CAS-IR). MICs were determined by the broth microdilution method. Disruption of 20 genes led to CAS-IS (four- to eightfold reductions in the MIC). Eleven of the 20 genes are involved in cell wall and membrane function, notably in the protein kinase C (PKC) integrity pathway (MID2, FKS1, SMI1, and BCK1), chitin and mannan biosynthesis (CHS3, CHS4, CHS7, and MNN10), and ergosterol biosynthesis (ERG5 and ERG6). Four of the 20 genes (TPO1, VPS65, VPS25, and CHC1) are involved in vacuole and transport functions, 3 of the 20 genes (CCR4, POP2, and NPL3) are involved in the control of transcription, and 2 of the 20 genes are of unknown function. Disruption of nine additional genes led to CAS-IR (a fourfold increase of MIC). Five of these nine genes (SLG1, ERG3, VRP1, CSG2, and CKA2) are involved in cell wall function and signal transduction, and two of the nine genes (VPS67 and SAC2) are involved in vacuole function. To assess the specificity of susceptibility to CAS, the MICs of amphotericin B, fluconazole, flucytosine, and calcofluor for the strains were tested. Seven of 20 CAS-IS strains (with disruption of FKS1, SMI1, BCK1, CHS4, ERG5, TPO1, and ILM1) and 1 of 9 CAS-IR strains (with disruption of SLG1) demonstrated selective susceptibility to CAS. To further explore the importance of PKC in CAS susceptibility, the activity of the PKC inhibitor staurosporine in combination with CAS was tested against eight Aspergillus clinical isolates by the microdilution assay. Synergistic or synergistic-to-additive activities were found against all eight isolates by use of both MIC and minimum effective concentration endpoints.

Recent Status and Advancements in the Development of Antifungal Agents: Highlights on Plant and Marine Based Antifungals

2019 ◽  
Vol 19 (10) ◽  
pp. 812-830 ◽  
Author(s):  
P. Marie Arockianathan ◽  
Monika Mishra ◽  
Rituraj Niranjan
Keyword(s):  
Cell Wall ◽  
Antifungal Agents ◽  
Fungal Diseases ◽  
Fungal Cell Wall ◽  
Fungal Resistance ◽  
Ergosterol Biosynthesis ◽  
Fungal Cell ◽  
Ergosterol Synthesis ◽  
Glucan Synthesis ◽  
Anti Fungal

The developing resistance in fungi has become a key challenge, which is being faced nowadays with the available antifungal agents in the market. Further search for novel compounds from different sources has been explored to meet this problem. The current review describes and highlights recent advancement in the antifungal drug aspects from plant and marine based sources. The current available antifungal agents act on specific targets on the fungal cell wall, like ergosterol synthesis, chitin biosynthesis, sphingolipid synthesis, glucan synthesis etc. We discuss some of the important anti-fungal agents like azole, polyene and allylamine classes that inhibit the ergosterol biosynthesis. Echinocandins inhibit β-1, 3 glucan synthesis in the fungal cell wall. The antifungals poloxins and nikkomycins inhibit fungal cell wall component chitin. Apart from these classes of drugs, several combinatorial therapies have been carried out to treat diseases due to fungal resistance. Recently, many antifungal agents derived from plant and marine sources showed potent activity. The renewed interest in plant and marine derived compounds for the fungal diseases created a new way to treat these resistant strains which are evident from the numerous literature publications in the recent years. Moreover, the compounds derived from both plant and marine sources showed promising results against fungal diseases. Altogether, this review article discusses the current antifungal agents and highlights the plant and marine based compounds as a potential promising antifungal agents.

scholarly journals Antimicrobial Activity of Exebacase (Lysin CF-301) against the Most Common Causes of Infective Endocarditis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Aubrey Watson ◽  
Jun Taek Oh ◽  
Karen Sauve ◽  
Patricia A. Bradford ◽  
Cara Cassino ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
Infective Endocarditis ◽  
Broth Microdilution ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Microdilution Method ◽  
Cell Wall Hydrolase ◽  
Broth Microdilution Method ◽  
Common Causes

ABSTRACT Exebacase, a recombinantly produced lysin (cell wall hydrolase), and comparator antibiotics were tested by the broth microdilution method against strain sets of Staphylococcus and Streptococcus spp., which are the most common causes of infective endocarditis in humans. Exebacase was active against all Staphylococcus spp. tested, including S. aureus and coagulase-negative staphylococci (MIC50/90, 0.5/1 μg/ml). Activity against Streptococcus spp. was variable, with S. pyogenes, S. agalactiae, and S. dysgalactiae (MIC50/90, 1/2 μg/ml) among the most susceptible.

scholarly journals Chitin Synthesis in Saccharomyces cerevisiae in Response to Supplementation of Growth Medium with Glucosamine and Cell Wall Stress

2003 ◽  
Vol 2 (5) ◽  
pp. 886-900 ◽  
Author(s):  
Dorota A. Bulik ◽  
Mariusz Olczak ◽  
Hector A. Lucero ◽  
Barbara C. Osmond ◽  
Phillips W. Robbins ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Growth Medium ◽  
Specific Activity ◽  
Neck Region ◽  
Wall Stress ◽  
Chitin Synthesis ◽  
Fourfold Increase ◽  
Cell Wall Stress

ABSTRACT In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.

scholarly journals Quantitation of Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans

1999 ◽  
Vol 37 (10) ◽  
pp. 3332-3337 ◽  
Author(s):  
Beth A. Arthington-Skaggs ◽  
Hoda Jradi ◽  
Tejal Desai ◽  
Christine J. Morrison
Keyword(s):  
Susceptibility Testing ◽  
Yeast Cells ◽  
Ergosterol Biosynthesis ◽  
National Committee ◽  
Broth Microdilution ◽  
End Points ◽  
Ergosterol Content ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Novel Method

MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive “trailing” of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating ergosterol production in cells grown in 0, 1, 4, 16, or 64 μg of fluconazole per ml. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane. Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm. We used this sterol quantitation method (SQM) to test 38 isolates with broth microdilution end points of ≤8 μg/ml (susceptible), 16 to 32 μg/ml (susceptible dose-dependent [SDD]), or ≥64 μg/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates showed a mean reduction in ergosterol content of 72% after exposure to 1 μg of fluconazole/ml, an 84% reduction after exposure to 4 μg/ml, and 95 and 100% reductions after exposure to 16 and 64 μg of fluconazole/ml, respectively. Ten SDD isolates showed mean ergosterol reductions of 38, 57, 73, and 99% after exposure to 1, 4, 16, and 64 μg of fluconazole/ml, respectively. In contrast, 10 resistant isolates showed mean reductions in ergosterol content of only 25, 38, 53, and 84% after exposure to the same concentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or greater inhibition of overall mean ergosterol biosynthesis compared to that in the drug-free control. Of 38 isolates which gave clear end points by the broth microdilution method, the SQM MIC was within 2 dilutions of the broth microdilution MIC for 33 (87%). The SQM also discriminated between resistant and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equivocal end points by the broth microdilution method due to trailing growth. In contrast to the broth microdilution method, the SQM determined trailing isolates to be susceptible rather than resistant, indicating that the SQM may predict clinical outcome more accurately. The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may provide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.

scholarly journals Yapsins Are a Family of Aspartyl Proteases Required for Cell Wall Integrity in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (8) ◽  
pp. 1364-1374 ◽  
Author(s):  
Damian J. Krysan ◽  
Elizabeth L. Ting ◽  
Claudia Abeijon ◽  
Lee Kroos ◽  
Robert S. Fuller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Specific Activity ◽  
Wall Stress ◽  
Cell Wall Integrity ◽  
Dependent Manner ◽  
Glucan Synthesis ◽  
Aspartyl Proteases

ABSTRACT The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Δ2Δ mutant and the quintuple yapsin mutant (5ypsΔ) undergo osmoremedial cell lysis at 37°C. The cell walls of both 5ypsΔ and yps1Δ2Δ mutants have decreased amounts of 1,3- and 1,6-β-glucan. Although there is decreased incorporation of both 1,3- and 1,6-β-glucan in the 5ypsΔ mutant in vivo, in vitro specific activity of both 1,3- and 1,6-β-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsΔ. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Δ mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.

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