Sordarin inhibits fungal protein synthesis by blocking translocation differently to fusidic acid.
(73/5658)
Sordarin derivatives are selective inhibitors of fungal protein synthesis, which specifically impair elongation factor 2 (EF-2) function. We have studied the effect of sordarin on the ribosome-dependent GTPase activity of EF-2 from Candida albicans in the absence of any other component of the translation system. The effect of sordarin turned out to be dependent both on the ratio of ribosomes to EF-2 and on the nature of the ribosomes. When the amount of EF-2 exceeded that of ribosomes sordarin inhibited the GTPase activity following an inverted bell-shaped dose-response curve, whereas when EF-2 and ribosomes were in equimolar concentrations sordarin yielded a typical sigmoidal dose-dependent inhibition. However, when ricin-treated ribosomes were used, sordarin stimulated the hydrolysis of GTP. These results were compared with those obtained with fusidic acid, showing that both drugs act in a different manner. All these data are consistent with sordarin blocking the elongation cycle at the initial steps of translocation, prior to GTP hydrolysis. In agreement with this conclusion, sordarin prevented the formation of peptidyl-[(3)H]puromycin on polysomes from Candida albicans. (+info)
A conserved domain of yeast RNA triphosphatase flanking the catalytic core regulates self-association and interaction with the guanylyltransferase component of the mRNA capping apparatus.
(74/5658)
The 549-amino acid yeast RNA triphosphatase Cet1p catalyzes the first step in mRNA cap formation. Cet1p consists of three domains as follows: (i) a 230-amino acid N-terminal segment that is dispensable for catalysis in vitro and for Cet1p function in vivo; (ii) a protease-sensitive segment from residues 230 to 275 that is dispensable for catalysis but essential for Cet1p function in vivo; and (iii) a catalytic domain from residues 275 to 539. Sedimentation analysis indicates that purified Cet1(231-549)p is a homodimer. Cet1(231-549)p binds in vitro to the yeast RNA guanylyltransferase Ceg1p to form a 7.1 S complex that we surmise to be a trimer consisting of two molecules of Cet1(231-549)p and one molecule of Ceg1p. The more extensively truncated protein Cet1(276-549)p, which cannot support cell growth, sediments as a monomer and does not interact with Ceg1p. An intermediate deletion protein Cet1(246-549)p, which supports cell growth only when overexpressed, sediments principally as a discrete salt-stable 11.5 S homo-oligomeric complex. These data implicate the segment of Ceg1p from residues 230 to 275 in regulating self-association and in binding to Ceg1p. Genetic data support the existence of a Ceg1p-binding domain flanking the catalytic domain of Cet1p, to wit: (i) the ts growth phenotype of 2mu CET1(246-549) is suppressed by overexpression of Ceg1p; (ii) a ts alanine cluster mutation CET1(201-549)/K250A-W251A is suppressed by overexpression of Ceg1p; and (iii) 15 other cet-ts alleles with missense changes mapping elsewhere in the protein are not suppressed by Ceg1p overexpression. Finally, we show that the in vivo function of Cet1(275-549)p is completely restored by fusion to the guanylyltransferase domain of the mouse capping enzyme. We hypothesize that the need for Ceg1p binding by yeast RNA triphosphatase can by bypassed when the triphosphatase catalytic domain is delivered to the RNA polymerase II elongation complex by linkage in cis to the mammalian guanylyltransferase. (+info)
Antagonism of azole activity against Candida albicans following induction of multidrug resistance genes by selected antimicrobial agents.
(75/5658)
Antifungal azoles (e.g., fluconazole) are widely used for prophylaxis or treatment of Candida albicans infections in immunocompromised individuals, such as those with AIDS. These individuals are frequently treated with a variety of additional antimicrobial agents. Potential interactions between three azoles and 16 unrelated drugs (antiviral, antibacterial, antifungal, and antiprotozoal agents) were examined in vitro. Two compounds, tested at concentrations achievable in serum, demonstrated an antagonistic effect on azole activity against C. albicans. At fluconazole concentrations two to four times the 50% inhibitory concentration, C. albicans growth (relative to treatment with fluconazole alone) increased 3- to 18-fold in the presence of albendazole (2 microg/ml) or sulfadiazine (50 microg/ml). Antagonism (3- to 78-fold) of ketoconazole and itraconazole activity by these compounds was also observed. Since azole resistance has been correlated with overexpression of genes encoding efflux proteins, we hypothesized that antagonism results from drug-induced overexpression of these same genes. Indeed, brief incubation of C. albicans with albendazole or sulfadiazine resulted in a 3-to->10-fold increase in RNAs encoding multidrug transporter Cdr1p or Cdr2p. Zidovudine, trimethoprim, and isoniazid, which were not antagonistic with azoles, did not induce these RNAs. Fluphenazine, a known substrate for Cdr1p and Cdr2p, strongly induced their RNAs and, consistent with our hypothesis, strongly antagonized azole activity. Finally, antagonism was shown to require a functional Cdr1p. The possibility that azole activity against C. albicans is antagonized in vivo as well as in vitro in the presence of albendazole and sulfadiazine warrants investigation. Drug-induced overexpression of efflux proteins represents a new and potentially general mechanism for drug antagonism. (+info)
A novel injectable water-soluble amphotericin B-arabinogalactan conjugate.
(76/5658)
New, stable, highly water-soluble, nontoxic polysaccharide conjugates of amphotericin B (AmB) are described. AmB was conjugated by a Schiff-base reaction with oxidized arabinogalactan (AG). AG is a highly branched natural polysaccharide with unusual water solubility (70% in water). A high yield of active AmB was obtained with the conjugates which were similarly highly water soluble and which could be appropriately formulated for injection. They showed comparable MICs for Candida albicans and Cryptococcus neoformans (MICs, 0.1 to 0.2 microg/ml). The reduced AmB conjugate, which was synthesized at pH 11 for 48 h at 37 degrees C, was nonhemolytic and was much safer than conventional micellar AmB-deoxycholate. It was the least toxic AmB-AG conjugate among those tested with mice (maximal tolerated dose, 50 mg/kg of body weight), and histopathology indicated no damage to the liver or kidneys. This conjugate, similarly to the liposomal formulation (AmBisome), was more effective than AmB-deoxycholate in prolonging survival. It was more effective than both the liposomal and the deoxycholate formulations in eradicating yeast cells from target organs. The overall results suggest that after further development of the AmB-AG conjugate, it may be a potent agent in the treatment of fungal infections. (+info)
Effects of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on in vitro activities of secreted aspartyl proteinases of Candida albicans isolates from HIV-infected patients.
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The effects of therapeutically relevant concentrations of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on the in vitro proteinase activity of Candida albicans were investigated with isolates from HIV-infected and uninfected patients with oral candidiasis. After exposure to the HIV proteinase inhibitors, proteinase activity was significantly reduced in a dose-dependent manner. These inhibitory effects, which were similar to that of pepstatin A, and the reduced virulence phenotype in experimental candidiasis after application of saquinavir indicate the usefulness of these HIV proteinase inhibitors as potential anticandidal agents. (+info)
Differential susceptibility of two species of macaques to experimental vaginal candidiasis.
(78/5658)
Vulvovaginal candidiasis (VVC) caused by Candida albicans is a significant problem in women of childbearing age. Unfortunately, protective host defense mechanisms against VVC are poorly understood. Although rodent models of experimental vaginal candidiasis have been useful, several differences from humans limit the correlation of experimental data. The purpose of the present study was to examine two species of macaques as an alternative model of experimental vaginitis. Screening of pig-tailed and rhesus macaques demonstrated that each had mucosal Candida colonization and prior immune sensitization to C. albicans. Vaginal-associated immunity (cytokines, antibodies, and innate resistance) was also detected in cervicovaginal lavage fluid from both species. Nevertheless, intravaginal inoculation of C. albicans into both species, either untreated or under estrogen-treated conditions, resulted in vaginal infection in rhesus, but not pig-tailed, macaques. Several estrogen-dependent changes in the rhesus immune status coincided with susceptibility to infection. Taken together, these results suggest that pig-tailed and rhesus macaques may be useful in studying pathogenesis and immunity associated with C. albicans vaginitis. (+info)
Relationship between Ureaplasma urealyticum vaginal colonization and polymorphism in the interleukin-1 receptor antagonist gene.
(79/5658)
The relationship between polymorphisms in the interleukin-1 receptor antagonist (IL-1ra) gene and microbial vaginal colonization was examined in 88 asymptomatic women of reproductive age. Alleles of the intron 2 region of the IL-1ra gene were identified by polymerase chain reaction (PCR). PCR was also used to detect Ureaplasma urealyticum, Mycoplasma hominis, and Candida albicans; bacterial vaginosis (BV) was identified by clinical criteria. Among the 31 women with vaginal U. urealyticum, only 3 (9.7%) were homozygous for allele 2 of the IL-1ra gene; 21 (36.8%) of the 57 women who were negative for this organism were positive for allele 2 (P=.006). Only 7 women were positive for M. hominis; none were allele 2 homozygotes as opposed to 24 (29.6%) of the 81 women negative for M. hominis. There was no relation between C. albicans or BV and any IL-1ra allele. Reduced susceptibility to vaginal colonization with mycoplasmas may be associated with homozygosity of the 2 allele of the IL-1ra gene. (+info)
The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis.
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The Candida albicans CHS4 gene encoding chitin synthase 4 has been isolated using the Saccharomyces cerevisiae CHS4/SKT5 gene as a probe. The gene contains a 2061 bp open reading frame capable of encoding a protein of 687 amino acids (76053 Da). No intron was observed in the gene. Disruption of CHS4 in C. albicans yielded a Calcofluor-resistant phenotype, indicating that Chs4p contributes to chitin biosynthesis. Consistent with this, overexpression of Chs4p under the regulation of the ScGAL1 promoter enhanced chitin synthase 3 activity in S. cerevisiae 7- to 38-fold. In addition, chs3 and chs4 null mutants were significantly defective in Calcofluor white staining and their chitin content was 10% of that of the parental strain. Chs4p of C. albicans and S. cerevisiae showed 61% identity in the C-terminal half of the proteins and that region of C. albicans Chs4p complemented the Chs4p function of a mutant of S. cerevisiae resistant to Calcofluor white. Therefore, it appears that Chs4p is involved in chitin synthase 3 activity by combining with Chs3p to interact synergistically in chitin biosynthesis. (+info)