Aspergillus fumigatus CYP51 sequence: potential basis for fluconazole resistance.

We have cloned and sequenced the Aspergillus fumigatus CYP51 gene which encodes the target of azole antifungal agents, namely cytochrome P450 sterol 14alpha-demethylase. Since A. fumigatus is intrinsically resistant to the widely used azole fluconazole, we compared its predicted CYP51 sequence to the CYP51 sequences from fluconazole-susceptible and resistant Candida albicans. This analysis generated specific hypotheses regarding the basis for A. fumigatus fluconazole resistance; in particular, A. fumigatus residue Ile301 corresponds to C. albicans residue Thr315 which is mutated to Ala in resistant strains and is proposed to hydrogen bond with the sterol substrate.

Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei.

Infections with Candida krusei have increased in recent years as a consequence of its intrinsic resistance to fluconazole, an antifungal azole widely used in immunocompromised individuals to suppress infections due to azole-susceptible C. albicans. One established mechanism for azole resistance is drug efflux by ATP binding cassette (ABC) transporters. Since these transporters recognize structurally diverse drugs, their overexpression can lead to multidrug resistance (MDR). To identify C. krusei genes potentially involved in azole resistance, PCR was performed with primers corresponding to conserved sequences of MDR-related ABC transporters from other fungi. Two genes, ABC1 and ABC2, were identified; Southern blots suggested that both have one or two related gene copies in the C. krusei genome. ABC1 RNA was constitutively expressed at low levels in log phase cells while ABC2 RNA was undetectable. However, both genes were upregulated as cultures approached stationary phase, and this upregulation was correlated with decreased susceptibility to the lethal activity of the azole derivative miconazole. Furthermore, ABC1 was upregulated following brief treatment of C. krusei with miconazole and clotrimazole (but not other azoles), and the unrelated compounds albendazole and cycloheximide. The latter two compounds antagonized fluconazole activity versus C. krusei, supporting a role for the ABC1 transporter in azole efflux. Finally, miconazole-resistant mutants selected in vitro demonstrated increased constitutive expression of ABC1. Based on these expression data, genetic and functional characterization of the ABC1 transporter to directly test its role in C. krusei azole resistance would appear to be warranted.

The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae.

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.

Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors.

Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, but treatment failure is not uncommon especially in immunocompromised individuals. Relatedly, in vitro studies demonstrate that azoles are nonfungicidal, with continued growth at strain-dependent rates even at high azole concentrations. We hypothesized that upregulation of ERG11, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in Candida species. RNA analysis revealed that ERG11 expression in C. albicans is maximal during logarithmic-phase growth and decreases as the cells approach stationary phase. Incubation with fluconazole, however, resulted in a two- to fivefold increase in ERG11 RNA levels within 2 to 3 h, and this increase was followed by resumption of culture growth. ERG11 upregulation also occurred following treatment with other azoles (itraconazole, ketoconazole, clotrimazole, and miconazole) and was not dependent on the specific medium or pH. Within 1 h of drug removal ERG11 upregulation was reversed. Azole-dependent upregulation was not limited to ERG11: five of five ERG genes tested whose products function upstream and downstream of lanosterol demethylase in the sterol biosynthetic pathway were also upregulated. Similarly, ERG11 upregulation occurred following treatment of C. albicans cultures with terbinafine and fenpropimorph, which target other enzymes in the pathway. These data suggest a common mechanism for global ERG upregulation, e.g., in response to ergosterol depletion. Finally, azole-dependent ERG11 upregulation was demonstrated in three additional Candida species (C. tropicalis, C. glabrata, and C. krusei), indicating a conserved response to sterol biosynthesis inhibitors in opportunistic yeasts.

Antagonism of azole activity against Candida albicans following induction of multidrug resistance genes by selected antimicrobial agents.

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.

The primitive protozoon Trichomonas vaginalis contains two methionine gamma-lyase genes that encode members of the gamma-family of pyridoxal 5'-phosphate-dependent enzymes.

Methionine gamma-lyase, the enzyme that catalyzes the breakdown of methionine by an alpha,gamma-elimination reaction and is a member of the gamma-family of pyridoxal 5'-phosphate-dependent enzymes, is present in high activity in the primitive protozoan parasite Trichomonas vaginalis but is absent from mammals. Two genes, mgl1 and mgl2, encoding methionine gamma-lyase, have now been isolated from T. vaginalis. They are both single copy, encode predicted proteins (MGL1 and MGL2) of 43 kDa, have 69% sequence identity with each other, and show a high degree of sequence identity to methionine gamma-lyase from Pseudomonas putida (44%) and other related pyridoxal 5'-phosphate-dependent enzymes such as human cystathionine gamma-lyase (42%) and Escherichia coli cystathionine beta-lyase (30%). mgl1 and mgl2 have been expressed in E. coli as a fusion with a six-histidine tag and the recombinant proteins (rMGL1 and rMGL2) purified by metal-chelate affinity chromatography. rMGL1 and rMGL2 were found to have high activity toward methionine (10.4 and 0.67 mumol/min/mg of protein, respectively), homocysteine (370 and 128 mumol/min/mg of protein), cysteine (6.02 and 1.06 mumol/min/mg of protein), and O-acetylserine (3.74 and 1.51 mumol/min/mg of protein), but to be inactive toward cystathionine. Site-directed mutagenesis of an active site cysteine (C113G for MGL1 and C116G for MGL2) reduced the activity of the recombinant enzymes toward both methionine and homocysteine by approximately 80% (rMGL1) and 90% (rMGL2). In contrast, the activity of mutated rMGL2 toward cysteine and O-acetylserine was increased (to 214 and 142%, respectively), whereas that of mutated rMGL1 was reduced to 39 and 49%, respectively. These findings demonstrate the importance of this cysteine residue in the alpha,beta-elimination and alpha, gamma-elimination reactions catalyzed by trichomonad methionine gamma-lyase.

Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae.

The bacteriocin haemocin is produced by most type b strains of Haemophilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains. An H. influenzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragment capable of conferring haemocin immunity on a haemocin-susceptible H. influenzae mutant was selected by using partially purified haemocin. Deletional and site-directed mutagenesis localized the haemocin immunity gene to the 3' open reading frame (ORF) within this chromosomal fragment. Subcloning of this ORF demonstrated that it was sufficient to confer haemocin immunity on wild-type haemocin-susceptible H. influenzae strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcl, encodes a 105-amino-acid protein with an estimated molecular mass of 12.6 kDa. Primer extension analysis revealed a putative transcriptional start site 34 bp upstream of the start codon, and the presence of a promoter immediately upstream of hmcI was confirmed by cloning the gene into a promoterless chloramphenicol acetyltransferase vector. To characterize the hmcI gene product, a His-HmcI fusion protein was constructed.

In vitro susceptibilities of the AIDS-associated microsporidian Encephalitozoon intestinalis to albendazole, its sulfoxide metabolite, and 12 additional benzimidazole derivatives.

Recent reports have described the successful treatment of Encephalitozoon intestinalis infection in AIDS patients with albendazole. However, this compound is rapidly metabolized in vivo to albendazole sulfoxide, and furthermore it is only 1 of about 15 commercially developed benzimidazole derivatives. To compare the activities of albendazole, albendazole sulfoxide, and other benzimidazoles, an in vitro system involving infection of green monkey kidney cell (E6) monolayers with E. intestinalis spores was developed. After 14 days, the effects of benzimidazoles on spore production were determined. Ten of fourteen derivatives tested, including albendazole, were inhibitory at concentrations of 1 to 10 ng/ml. Derivatives modified at the 1 or 2 position were less active. Albendazole sulfoxide was 1.7-fold more inhibitory than albendazole but significantly less toxic to E6 cells, a finding that explains the clinical efficacy of this compound. Potential alternatives to albendazole are discussed. No albendazole-resistant E. intestinalis mutants were obtained following in vitro selection.

Phylogenetic analysis of beta-tubulin sequences from amitochondrial protozoa.

It has been proposed that certain extant anaerobic protozoa are descended from organisms that diverged early in eukaryotic evolution prior to the acquisition of mitochondria. Among these are the extracellular parasites Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica, and the obligately intracellular microsporidia. Phylogenetic analysis of rRNA sequences from these amitochondrial organisms suggests that G. lamblia, T. vaginalis, and microsporidia are near the base of the eukaryotic tree, while E. histolytica clusters with mitochondria-containing species. However, since eukaryotes likely evolved by symbiotic associations, it is important to analyze other sequences which may have independent origins. Unlike ribosomes, microtubules appear to be unique to eukaryotes. Complete gene sequences for the beta-tubulin subunit of microtubules from T. vaginalis, E. histolytica, and the microsporidian Encephalitozoon hellem have recently been determined. Phylogenetic relationships among these, G. lamblia, and 20 additional beta-tubulins were analyzed by distance matrix and parsimony methods, using alpha- and gamma-tubulin outgroups. All analyses placed the E. histolytica sequence at the base of the beta-tubulin evolutionary tree. Similar results were obtained for E. histolytica alpha-tubulin using a less representative set of sequences. In contrast, the E. hellem sequence branched considerably higher, within the lineage containing animal and fungal beta-tubulins. Possible explanations are considered for these unexpected differences between the beta-tubulin and rRNA trees.

Site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin: interaction between residue 167 and benzimidazole compounds.

Benzimidazoles are widely used as anthelmintic agents and systemic fungicides. In susceptible organisms, benzimidazoles bind to beta-tubulin and block microtubule polymerization. To further characterize this interaction, site-directed mutagenesis followed by gene replacement was used to change Saccharomyces cerevisiae beta-tubulin residue Phe-167 to Tyr. Consistent with previous studies, this mutation resulted in at least 3-4-fold decreased sensitivity to the benzimidazole derivatives carbendazim and nocodazole. The Tyr-167 mutant was cold sensitive, implying a direct effect on benzimidazole binding rather than a nonspecific increase in microtubule stability. Surprisingly, the mutant had 8-fold increased sensitivity to the derivative benomyl, which is structurally identical to carbendazim except at position 1. This suggests that residue 167 interacts with benzimidazoles in the vicinity of the 1-position.

Entamoeba histolytica encodes a highly divergent beta-tubulin.

The microtubules of the amitochondrial parasite Entamoeba histolytica are atypical in certain respects. Consistent with this, we report that E. histolytica encodes the most divergent beta-tubulin identified to date, with only 54% to 58% identity to beta-tubulins from various species. A similarly divergent beta-tubulin is encoded by the related Entamoeba invadens; single gene copies appear to be present in both organisms. The Entamoeba sequences were compared with a database of 101 beta-tubulins, including the highly divergent sequence from another amitochondrial protozoan, Trichomonas vaginalis. A total of 81 residues were universally conserved, and 76 residues varied only once. Correlations with previous studies indicate that microtubule function is altered when most, but not all, conserved residues are mutated.

A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping.

Oligonucleotide primers complementary to conserved regions of the 16S and 23S ribosomal RNA genes were used to amplify the 16S-23S intergenic spacer region of bacterial pathogens. The amplification patterns produced were compared for their potential use in molecular epidemiologic analysis. This method, polymerase chain reaction (PCR) ribotyping, was applied to isolates of Staphylococcus aureus, Enterococcus faecium, Escherichia coli, and Enterobacter species. Length polymorphisms in the amplified DNA distinguished unrelated strains of all bacteria. The banding patterns of 3 S. aureus isolates from the blood of 1 patient on 3 consecutive days were identical. Plasmid analysis, biotyping, and antibiograms were also obtained on the Enterobacter isolates. All three of these methods showed considerable variability after in vitro passage of bacteria, but PCR ribotypes remained stable. Results demonstrate the utility of the conserved primers for PCR ribotyping, a widely applicable method for the molecular epidemiology of genetically diverse bacteria.

Comparisons of ribosomal RNA sequences from amitochondrial protozoa: implications for processing, mRNA binding and paromomycin susceptibility.

The amitochondrial (a-mt) protozoa include four groups of organisms that are of interest as important human parasites and as probable descendents of the earliest branches of eukaryotic evolution. These organisms have not been directly compared in terms of structure and function of a specific molecule. We sequenced portions of their rRNA-encoding genes coding for the internal transcribed spacers (ITS1 and 2) and adjoining small subunit (SS), 5.8S and large subunit (LS) rRNAs. Included are sites for RNA processing, mRNA interaction and aminoglycoside binding, as well as potential protein-encoding genes. The ITS of all a-mt protozoa examined are relatively short, but otherwise diverse. They include one or two predominant nucleotides (A in Entamoeba and Trichomonas, T in Encephalitozoon and C in Giardia) and have minimal potential secondary structure, which may form the basis for the preferential processing of ITS sequences. The mechanism employed by a-mt protozoa to bind mRNA may be unique, since Giardia, Trichomonas and Entamoeba mRNAs have usually short 5' non-coding regions. In bacteria, the 3' terminus of the SS rRNA is involved in mRNA binding; analysis of Entamoeba and Trichomonas mRNA 5' non-coding sequences suggests an analogous mechanism involving potential base pairing to the loop of the terminal SS rRNA hairpin. Giardia sensitivity to paromomycin was previously correlated with the presence of a C:G bp near the decoding region of SS rRNA. This bp is also present in Entamoeba and Trichomonas, consistent with their susceptibility. Its absence in Encephalitozoon and other microsporidia predicts paromomycin resistance, and suggests a distinct evolutionary origin for this group.

Antiprotozoal activities of benzimidazoles and correlations with beta-tubulin sequence.

Benzimidazoles have been widely used since the 1960s as anthelmintic agents in veterinary and human medicine and as antifungal agents in agriculture. More recently, selected benzimidazole derivatives were shown to be active in vitro against two protozoan parasites, Trichomonas vaginalis and Giardia lamblia, and clinical studies with AIDS patients have suggested that microsporidia are susceptible as well. Here, we first present in vitro susceptibility data for T. vaginalis and G. lamblia using an expanded set of benzimidazole derivatives. Both parasites were highly susceptible to four derivatives, including mebendazole, flubendazole, and fenbendazole (50% inhibitory concentrations of 0.005 to 0.16 microgram/ml). These derivatives also had lethal activity that was time dependent: 90% of T. vaginalis cells failed to recover following a 20-h exposure to mebendazole at 0.17 microgram/ml. G. lamblia, but not T. vaginalis, was highly susceptible to five additional derivatives. Next, we examined in vitro activity of benzimidazoles against additional protozoan parasites: little or no activity was observed against Entamoeba histolytica, Leishmania major, and Acanthamoeba polyphaga. Since the microtubule protein beta-tubulin has been identified as the benzimidazole target in helminths and fungi, potential correlations between benzimidazole activity and beta-tubulin sequence were examined. This analysis included partial sequences (residues 108 to 259) from the organisms mentioned above, as well as the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi and the sporozoan Cryptosporidium parvum. beta-tubulin residues Glu-198 and, in particular, Phe-200 are strong predictors of benzimidazole susceptibility; both are present in Encephalitozoon spp. but absent in C. parvum.