A dual labelling method for measuring uptake of low molecular weight compounds into the pathogenic yeast Candida albicans.

In contrast to other eukaryotic cells the pathogenic yeast Candida albicans is resistant to many structurally unrelated metabolic inhibitors. Reduced permeability due to the cell wall and/or altered plasma membrane composition is thought to be at least partly responsible for this phenomenon. To study the uptake of low molecular weight compounds into C. albicans we developed a dual labelling method. Intact cells, metabolically inactivated cells, spheroplasts or membrane fragments of C. albicans were incubated with various [14C]-labelled compound in the presence of [3H]-labelled water. After separation of cells and supernatant isotope ratios [3H]/[14C] were determined. Quotients of the isotope ratios from cells and supernatant, called enrichment coefficients, were calculated under all four conditions. The enrichment coefficients indicated whether a compound can enter C. albicans cells, is trapped within the cell wall, is enriched in the lipophilic membrane compartment, is actively accumulated or actively exported by multidrug resistance carriers. We used six structurally unrelated compounds to test our method. We found no evidence for a general impermeability of C. albicans.

[1]  K. Ziegelbauer,et al.  Molecular Mode of Action of the Antifungal β-Amino Acid BAY 10-8888 , 1998, Antimicrobial Agents and Chemotherapy.

[2]  M. Raymond,et al.  AP1-mediated Multidrug Resistance in Saccharomyces cerevisiae Requires FLR1 Encoding a Transporter of the Major Facilitator Superfamily* , 1997, The Journal of Biological Chemistry.

[3]  D. Sanglard,et al.  Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. , 1997, Microbiology.

[4]  S. Fridkin,et al.  Epidemiology of nosocomial fungal infections , 1996, Clinical microbiology reviews.

[5]  D. Sanglard,et al.  Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors , 1996, Antimicrobial agents and chemotherapy.

[6]  A. Markham,et al.  Ciprofloxacin. An updated review of its pharmacology, therapeutic efficacy and tolerability. , 1996, Drugs.

[7]  F. Odds.  Epidemiological shifts in opportunistic and nosocomial Candida infections: mycological aspects. , 1996, International journal of antimicrobial agents.

[8]  K. Kuchler,et al.  Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters , 1995, Antimicrobial agents and chemotherapy.

[9]  L. Hutwagner,et al.  Opportunistic candidiasis: an epidemic of the 1980s. , 1995, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[10]  M. Pfaller,et al.  Epidemiology of candidiasis. , 1995, The Journal of hospital infection.

[11]  D. Hirata,et al.  The physiological roles of membrane ergosterol as revealed by the phenotypes of syr1/erg3 null mutant of Saccharomyces cerevisiae. , 1995, Bioscience, biotechnology and biochemistry.

[12]  U. Pleiss,et al.  Synthesis of N-[4-[2-(2,4-dimethylphenoxy)phenyl]-2-thiazolyl]-hexahydro-2-pyrimidinimine, [14C]BAY w 6341 , 1995 .

[13]  A. Goffeau,et al.  Solubilization and characterization of the overexpressed PDR5 multidrug resistance nucleotide triphosphatase of yeast. , 1994, The Journal of biological chemistry.

[14]  N. Georgopapadakou,et al.  Human mycoses: drugs and targets for emerging pathogens. , 1994, Science.

[15]  J. Becker,et al.  Candida albicans gene encoding resistance to benomyl and methotrexate is a multidrug resistance gene , 1994, Antimicrobial Agents and Chemotherapy.

[16]  K. Kuchler,et al.  Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance. , 1994, The Journal of biological chemistry.

[17]  A. Goffeau,et al.  PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1. , 1994, The Journal of biological chemistry.

[18]  P. Troke,et al.  Fluconazole resistance in Candida glabrata , 1993, Antimicrobial Agents and Chemotherapy.

[19]  K. Schlemmer,et al.  A new structural analogue antagonist of peptido-leukotrienes: The discovery of BAY x7195 , 1993 .

[20]  H. Nikaido,et al.  Penetration of lipophilic agents with multiple protonation sites into bacterial cells: tetracyclines and fluoroquinolones as examples , 1993, Antimicrobial Agents and Chemotherapy.

[21]  C. Beck-Sague,et al.  Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. , 1993, The Journal of infectious diseases.

[22]  F. Odds.  Resistance of yeasts to azole-derivative antifungals. , 1993, The Journal of antimicrobial chemotherapy.

[23]  J. Davies,et al.  Natural cycloheximide resistance in yeast. The role of ribosomal protein L41. , 1993, European journal of biochemistry.

[24]  R. Hector,et al.  Compounds active against cell walls of medically important fungi , 1993, Clinical Microbiology Reviews.

[25]  C. Nombela,et al.  A reorganized Candida albicans DNA sequence promoting homologous non‐integrative genetic transformation , 1992, Molecular microbiology.

[26]  A. Janulaitis,et al.  Cloning and sequence analysis of a Candida maltosa gene which confers resistance to cycloheximide. , 1992, Gene.

[27]  M. Nishiyama,et al.  A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P‐glycoproteins , 1992, Molecular microbiology.

[28]  S. Kawai,et al.  Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts , 1992, Journal of bacteriology.

[29]  J. A. Barnett,et al.  Passage of molecules through yeast cell walls: A brief essay‐review , 1991, Yeast.

[30]  J. Dubremetz,et al.  Secretion of glycoproteins through the cell wall of Candida albicans. , 1989, European journal of cell biology.

[31]  K. Struhl,et al.  ATR1, a Saccharomyces cerevisiae gene encoding a transmembrane protein required for aminotriazole resistance , 1988, Molecular and cellular biology.

[32]  R. Prasad.  Nutrient transport in Candida albicans, a pathogenic yeast , 1987, Yeast.

[33]  W. Merz,et al.  Incidence of polyene-resistant yeasts recovered from clinical specimens , 1980, Antimicrobial Agents and Chemotherapy.

[34]  J. Vry,et al.  BAY X 3702 : NEUROPROTECTANT 5-HT1A AGONIST , 1997 .

[35]  N. Russell,et al.  Sterols in Candida albicans mutants resistant to polyene or azole antifungals, and of a double mutant C. albicans 6.4. , 1987, Critical reviews in microbiology.

[36]  E. F. Gale,et al.  Nature and development of phenotypic resistance to amphotericin B in Candida albicans. , 1986, Advances in Microbial Physiology.

[37]  H. Rottenberg.  The measurement of membrane potential and deltapH in cells, organelles, and vesicles. , 1979, Methods in enzymology.

[38]  F. Odds,et al.  Candida and candidosis , 1979 .

[39]  E. Svalland.  [Fungal infections]. , 1966, Den Norske tannlaegeforenings tidende.