Ronald L. Cihlar

The Two-Component Signal Transduction Protein Chk1p Regulates Quorum Sensing in Candida albicans

ABSTRACT Regulation of hyphal morphogenesis in Candida albicans can occur through quorum sensing (QS). A QS signal, farnesol, is produced during high-density growth and inhibits morphogenesis. However, the signal transduction pathway that regulates QS is unknown. Here, we show that a C. albicans mutant lacking Chk1p but not either the Sln1p or the Nik1p histidine kinase is refractory to the inhibitory effect of farnesol both in cell suspension and during the formation of a biofilm. This study is the first to demonstrate a role for a two-component signal transduction protein in QS by a eukaryotic organism.

Inhibitory effect of cerulenin and sodium butyrate on germination of Candida albicans.

Candida albicans germination in liquid medium was inhibition by the antilipogenic agent cerulenin and the fatty acid sodium butyrate. Although these inhibitors prevented germ tube emergence at concentrations of 1 microgram/ml and 20 mM, respectively, neither significantly affected cell viability as judged by trypan blue staining or the rate of protein biosynthesis throughout the time course of the experiments. Cerulenin treatment resulted in inhibition of lipid biosynthesis, but lipid biosynthetic capabilities remained unaltered in sodium butyrate-supplemented cultures. Because each inhibitor blocks germination by different mechanisms, their utility in distinguishing events directly correlated to germination was examined. In this context, chitin synthase activity was inhibited by both compounds, confirming the importance of chitin biosynthesis in C. albicans germination. Images

Requirement for the Candida albicans FAS2 gene for infection in a rat model of oropharyngeal candidiasis

The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/deletion of the FAS2 gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CAI4 (delta ura3::imm434/delta ura3::imm434) were sequentially disrupted with a cassette that included a portion of FAS2 from which a 984 bp fragment containing the FAS condensing reaction domain was deleted and replaced with hisG-URA3-hisG sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFD1 and CFD3) exhibited an approximately 20% reduction in activity, when compared to wild-type. In addition, fatty acid synthase activity was abolished in a FAS2 null mutant strain (CFD2), and growth of CFD2 occurred only when the growth medium was supplemented with Tween 40 and certain fatty acids. Strain CFD2 was avirulent in the rat model, indicating that fatty acid synthase activity is required for C. albicans oropharyngeal infection. Strains with a single FAS2 allele disruption colonized the oral cavity, but the number of cells recovered from infected animals was approximately fivefold less than for the parental strain. The results suggest that FAS may be exploited as a possible target for the development of new antifungal agents.

Comparing micellar, hemolytic, and antibacterial properties of di- and tricarboxyl dendritic amphiphiles

Homologous dicarboxyl dendritic amphiphiles-RCONHC(CH(3))(CH(2)CH(2)COOH)(2), 4(n); and ROCONHC(CH(3))(CH(2)CH(2)COOH)(2), 5(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were synthesized. Critical micelle concentrations (CMCs) in aqueous triethanolamine solutions and at pH 7.4 were measured along with hemolytic activity (effective concentrations, EC(10)) in phosphate-buffered saline (PBS). LogCMC showed a linear dependence on chain length (n); the longest chain in each series had the lowest CMC-in triethanolamine: 4(21), 180μM and 5(22), 74μM and at pH 7.4: 4(21), 78μM and 5(22), 33μM. These two series, 4(n) and 5(n), and three series of homologous tricarboxyl dendritic amphiphiles-RCONHC(CH(2)CH(2)COOH)(3), 1(n); ROCONHC(CH(2)CH(2)COOH)(3), 2(n); RNHCONHC(CH(2)CH(2)COOH)(3), 3(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were tested for growth inhibition of Staphylococcus aureus strain ATCC 6358 and methicillin-resistant S. aureus (MRSA) strain ATCC 43330 by microdilution in 0.1-strength brain heart infusion broth (BHIB). Amphiphiles 4(19), 4(21), 5(18), and 5(20) showed the strongest antibacterial activity (2.2-3.4μg/mL) against S. aureus (vancomycin, MIC=0.25μg/mL). These four plus 1(21), 2(20), 2(22), and 3(20) exhibited the strongest antibacterial activity (1.7-6.8μg/mL) against MRSA (vancomycin, MIC=0.25μg/mL). The MICs of these amphiphiles against six clinical MRSA were similar to those against the ATCC strain. In PBS, EC(10)s of the most active homologues ranged from 7 to 18μg/mL and 18 to 220μg/mL for di- and tricarboxyl dendritic amphiphiles, respectively. To assess the potential safety of using dendritic amphiphiles as drugs, measurements of micellar and hemolytic properties were conducted in the same medium (full-strength BHIB) that was used for antibacterial activity. The CMCs (9-36μg/mL, ∼18-72μM) of ten amphiphiles were measured by microdilution (log2 progression) with dye-covered beads. The EC(10)s were similar to those in PBS. The MICs of most amphiphiles (14-72μg/mL) and vancomycin (1.1-2.2μg/mL) against both S. aureus and MRSA increased significantly compared to the MICs measured in 0.1-strength BHIB. The one exception, 5(18), had an MIC against S. aureus of 1.1μg/mL compared to vancomycin (2.2μg/mL). With CMC (9-18μg/mL) and EC(10) (16μg/mL) values higher than the MIC, 5(18) was discovered as a lead for further development.

Disruption studies of a Candida albicans gene, ELF1 : a member of the ATP-binding cassette family

A 3.6 kb gene (ELF1) with homology to the ATP-binding cassette (ABC) gene family has been isolated from genomic libraries of Candida albicans. Members of this gene family include both membrane transport proteins which confer a drug-resistance phenotype, and proteins whose functions are associated with protein translation. ELF1 (Elongation Like Factor) showed greatest homology with a Saccharomyces cerevisiae ORF (YPL226W), whose function is unknown, and lower homology with fungal elongation factor 3 (EF-3) genes. In comparison, homology with a gene conferring a drug-resistant phenotype (CDR1) was low. To understand the function of ELF1 in C. albicans, gene-knockout experiments were conducted using the hisG-URA3-hisG disruption cassette. Both single-copy (heterozygote) and double-disrupted strains in ELF1 were isolated. Phenotypically, the disrupted strains grew more slowly than wild-type and produced a mixture of large, irregular cells and apparently normal cells.

Isolation and sequence of the Candida albicans FAS1 gene.

The gene (FAS1) encoding the beta-subunit of fatty-acid synthase (FAS) of Candida albicans has been isolated and analyzed. The gene was isolated on the basis of homology to the Saccharomyces cerevisiae FAS1 gene. Sequence analysis showed that the gene contained an intron-free open reading frame of 6111 bp encoding a protein of 2037 amino acids (aa) (227 916 Da). C. albicans FAS1 and its product exhibit a high degree of overall sequence relatedness to their counterparts in S. cerevisiae, with identities of 68 and 63% at the nucleotide (nt) and aa level, respectively. In addition, the beta-subunits of both organisms contain the catalytic domains in an identical sequential order. Northern blots demonstrated that the gene encodes a single mRNA of approx. 6.1 kb, and that changes in transcript levels are not a prerequisite of the yeast-to-hyphal transition. Southern blot analysis of C. albicans chromosomes separated by pulsed-field gel electrophoresis showed that FAS1 resides on chromosome 5.

Conditions for optimal Candida biofilm development in microtiter plates.

Development of Candida spp. biofilms on medical devices such as catheters and voice prosthesis has been recognized as an increasing clinical problem. Simple device removal is often impossible, while in addition, resulting candidal infections are difficult to resolve due to their increased resistance to many antifungal agents. Susceptibility studies of clinical isolates are generally performed according to the CLSI standard, which measures planktonic cell susceptibility, but similar standards have not been designed or applied to testing of cells growing within a biofilm. As consistent biofilms from many strains are more difficult to simultaneously obtain and analyze than are independent planktonic cultures, any standard assay must address these concerns. In the present chapter, optimized conditions that promote biofilm formation within individual wells of microtiter plates are described. In addition, the method has proven useful in preparing C. albicans biofilms for investigation by a variety of microscopic and molecular techniques.

Growth response of several Candida albicans strains to inhibitory concentrations of heavy metals

Prolonged exposure of several Candida albicans strains to inhibitory concentrations of Cd, Cu, or Zn resulted in the appearance of resistant colonies at frequencies and with kinetics significantly different than expected based solely upon the predicted spontaneous mutation rate. Characteristics of the response included: (i) a delay usually of 4-10 days in the emergence of the first resistant colonies; (ii) continued accumulation of resistant colonies for a minimum of 21 days after initial exposure to selection; and (iii) final mutation frequencies ranging from 7.0 x 10(-6) to 9.8 x 10(-4). Further examination of the response of one of the strains to Cd, demonstrated that pretreatment with either ultraviolet irradiation or hydroxyurea resulted in approximately a 10-fold increase in the number of resistant colonies detected. While the distribution and identity of colony phenotypes was altered for all strains after exposure to the heavy metals, no specific morphologies could be correlated to development of resistance.

Phenotypic variation of a virulent Candida albicans strain and two spontaneous, relatively avirulent mutant strain derivatives

The effect of media and temperature of incubation on colony phenotype of Candida albicans strain 4918, and two relatively avirulent mutant strains, designated 4918-2 and 4918-10, has been investigated. In addition, the strains were characterized on the basis of morphotyping pattern. Colony phenotypes were determined for cultures grown on either Lees medium supplemented with arginine and zinc, or M63 medium supplemented with casamino acids. Incubation was at either 24 or 37 degrees C for 7 days. The results demonstrated that the predominant colony phenotype observed at 24 degrees C was different from that at 37 degrees C for all three strains, irrespective of the medium. While the growth medium influenced the specific colony phenotypes observed, as well as their categorical distribution, no significant medium effect on switching frequency was apparent. The switching repertoire of strain 4918-10 was consistently more varied than either the parental strain or 4918-2 under the conditions examined. However, categorization of the colony phenotypes shown by the three strains suggested that the pattern exhibited by strain 4918-2 was distinct from that of the other two strains. In addition, individual primary colonies of each phenotype observed were clonally plated in order to examine further the switching frequencies. The results established that all three strains were capable of high frequency switching. Other experiments demonstrated that morphotypes of all three strains were different from one another as expected from the differences in their virulence reported previously.

Purification and characterization of fatty acid synthase from Candida albicans strain 4918 and two derived spontaneous cerulenin-resistant mutants

Fatty acid synthase from three strains of Candida albicans (parental strain 4918, and two spontaneous cerulenin-resistant mutants, 4918-2 and 4918-10) has been purified and characterized. In all three cases the purification protocol included ammonium sulfate precipitation, fractionation with butyl-Toyopearl, differential centrifugation and sedimentation velocity centrifugation. Inclusion of protease inhibitors, aprotinin, leupeptin and pepstatin was a prerequisite to maximize recoveries. Polyacrylamide gel electrophoresis analysis demonstrated protocol efficacy and showed the apparent molecular mass of the two enzyme sub-units from each strain to be 195 kDa and 210 kDa. The Km (malonyl-CoA) and Vmax of each fatty acid synthase were similar. In contrast, inactivation kinetics of the respective enzymes in the presence of cerulenin showed enzyme activity from both mutants to differ significantly from the parent and from each other. Other experiments suggested in vivo cerulenin resistance of mutant strains is not solely attributable to enzyme alteration.