Thomas D. Edlind

A Naturally Occurring Proline-to-Alanine Amino Acid Change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis Accounts for Reduced Echinocandin Susceptibility

ABSTRACT Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced Vmax values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.

Azole Resistance in Candida glabrata: Coordinate Upregulation of Multidrug Transporters and Evidence for a Pdr1-Like Transcription Factor

ABSTRACT Candida glabrata has emerged as a common cause of fungal infection. This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented. Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target. Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C. glabrata these include CDR1 and PDH1. By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6- to 15-fold upregulation of CDR1 compared to susceptible strains. In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three- to fivefold, while ERG11 expression was minimally changed. Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants). Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2- to 20-fold. Effects of antifungal treatment on gene expression in susceptible C. glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4- to 12-fold. These findings suggest that these transporter genes are regulated by a common mechanism. In support of this, a mutation associated with laboratory resistance was identified in the C. glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae. The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation. Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1.

Pdr1 regulates multidrug resistance in Candida glabrata: gene disruption and genome‐wide expression studies

Candida glabrata emerged in the last decade as a common cause of mucosal and invasive fungal infection, in large part due to its intrinsic or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CDR1 and PDH1. We previously reported that azole‐resistant mutants (MIC ≥ 64 μg ml−1) of strain 66032 (MIC = 16 μg ml−1) similarly show coordinate CDR1‐PDH1 upregulation, and in one of these (F15) a putative gain‐of‐function mutation was identified in the single homologue of Saccharomyces cerevisiae transcription factors Pdr1–Pdr3. Here we show that disruption of C. glabrata PDR1 conferred equivalent fluconazole hypersensitivity (MIC = 2 μg ml−1) to both F15 and 66032 and eliminated both constitutive and fluconazole‐induced CDR1‐PDH1 expression. Reintroduction of wild‐type or F15 PDR1 fully reversed these effects; together these results demonstrate a role for this gene in both acquired and intrinsic azole resistance. CDR1 disruption had a partial effect, reducing fluconazole trailing in both strains while restoring wild‐type susceptibility (MIC = 16 μg ml−1) to F15. In an azole‐resistant clinical isolate, PDR1 disruption reduced azole MICs eight‐ to 64‐fold with no effect on sensitivity to other antifungals. To extend this analysis, C. glabrata microarrays were generated and used to analyse genome‐wide expression in F15 relative to its parent. Homologues of 10 S. cerevisiae genes previously shown to be Pdr1–Pdr3 targets were upregulated (YOR1, RTA1, RSB1, RPN4, YLR346c and YMR102c along with CDR1, PDH1 and PDR1 itself) or downregulated (PDR12); roles for these genes include small molecule transport and transcriptional regulation. However, expression of 99 additional genes was specifically altered in C. glabrata F15; their roles include transport (e.g. QDR2, YBT1), lipid metabolism (ATF2, ARE1), cell stress (HSP12, CTA1), DNA repair (YIM1, MEC3) and cell wall function (MKC7, MNT3). These azole resistance‐associated changes could affect C. glabrata tissue‐specific virulence; in support of this, we detected differences in F15 oxidant, alcohol and weak acid sensitivities. C. glabrata provides a promising model for studying the genetic basis of multidrug resistance and its impact on virulence.

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

ABSTRACT 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 ofERG11, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in Candidaspecies. 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, ERG11upregulation occurred following treatment of C. albicanscultures with terbinafine and fenpropimorph, which target other enzymes in the pathway. These data suggest a common mechanism for globalERG 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.

Candida albicans and Candida glabrata Clinical Isolates Exhibiting Reduced Echinocandin Susceptibility

ABSTRACT A recognized hotspot for mutations conferring reduced echinocandin susceptibility (RES) is residue S645 of Candida albicans Gsc1(Fks1). We report that the mutation F641Y is associated with RES in a C. albicans isolate. The analogous Fks2 residue is mutated F to V in a Candida glabrata RES isolate; the introduction of this mutation into susceptible C. glabrata confirmed its role in RES. Y641-equivalent Fks residues were identified in intrinsically RES Fusarium species and Candida guilliermondii.

Histone Deacetylase Inhibitors Enhance Candida albicans Sensitivity to Azoles and Related Antifungals: Correlation with Reduction in CDR and ERG Upregulation

ABSTRACT Histone acetylation and deacetylation play important roles in eukaryotic gene regulation. Several histone deacetylase (HDA) inhibitors have been characterized, including trichostatin A (TSA), apicidin, and sodium butyrate. We tested their effects on Candida albicans in vitro growth, heat sensitivity, and germ tube formation; minimal effects were observed. However, there was a dramatic effect of TSA on C. albicans sensitivity to the azoles fluconazole, itraconazole, and miconazole. Similar effects were observed with other HDA inhibitors and with the antifungals terbinafine and fenpropimorph, which target, as do azoles, enzymes in the ergosterol biosynthetic pathway. In contrast, HDA inhibitors had minimal effect on the activities of amphotericin B, flucytosine, and echinocandin, which have unrelated targets. Specifically, addition of 3 μg of TSA/ml lowered the itraconazole MIC for five susceptible C. albicans isolates an average of 2.7-fold at 24 h, but this increased to >200-fold at 48 h. Thus, the primary effect of TSA was a reduction in azole trailing. TSA also enhanced itraconazole activity against Candida parapsilosis and Candida tropicalis but had no effect with four less related yeast species. To examine the molecular basis for these effects, we studied expression of ERG genes (encoding azole and terbinafine targets) and CDR/MDR1 genes (encoding multidrug transporters) in C. albicans cells treated with fluconazole or terbinafine with or without TSA. Both antifungals induced to various levels the expression of ERG1, ERG11, CDR1, and CDR2; addition of TSA reduced this upregulation 50 to 100%. This most likely explains the inhibition of azole and terbinafine trailing by TSA and, more generally, provides evidence that trailing is mediated by upregulation of target enzymes and multidrug transporters.

Ribotype analysis of Pseudomonas cepacia from cystic fibrosis treatment centers

orbital lesions: surface coil MR imaging. Radiology 1985;156:669-74. 4. Sencer S, Coulter-Knoff A, Day D, Joker J, .Thompson T, Burke B. Splenic hemangioma with thrombocytopenia in a newborn. Pediatrics 1987;79:960-6. 5. Schwartz AC, Weaver RG, Bloomfield R. Cavernous hemangioma of the retina, cutaneous angiomas, and intracranial vascular lesion by computed tomography and nuclear magnetic resonance imaging. Am J Ophthalmol 1984;98:483-7. 6. Cammarata C, Han JS, Haaga JR, Alfidi R J, Kaufman B. Cerebral venous angiomas imaged by MR. Radiology 1985;155:639-43. 7. Front D, Royal HD, Israel O, et al. Scintigraphy of hepatic hemangiomas: the value of technetium-99m-labeled red blood cells--concise communication. J Nucl Med 1981;22:684-7. 8. Engel MA, Marks DS, Sandier MA, et al. Differentiation of focal intrahepatic lesions with technetium-99m-red blood cell imaging. Radiology 1983;146:777-83. 9. Rabinowitz SA, McKusick KA, Strauss HW. Tc-99m red blood cell scintigraphy in evaluating focal liver lesions. A JR. 1984;143:63-8. 10. Brant WE, Floyd JL, Jackson DE, Gilliland JD. The radiological evaluation of hepatic caver/ous hemangioma. JAMA 1987;257:2471-4. 11. Miller JH. Technetium-99m-labeled red blood cells in the evaluation of hemangiomas of the liver in infants and children. J Nucl Med 1987;28:1412-8.

Antifungal activity in Saccharomyces cerevisiae is modulated by calcium signalling

The most important group of antifungals is the azoles (e.g. miconazole), which act by inhibiting lanosterol demethylase in the sterol biosynthesis pathway. Azole activity can be modulated through structural changes in lanosterol demethylase, altered expression of its gene ERG11, alterations in other sterol biosynthesis enzymes or altered expression of multidrug transporters. We present evidence that azole activity versus Saccharomyces cerevisiae is also modulated by Ca2+‐regulated signalling. (i) Azole activity was reduced by the addition of Ca2+. Conversely, azole activity was enhanced by the addition of Ca2+ chelator EGTA. (ii) Three structurally distinct inhibitors (fluphenazine, calmidazolium and a W‐7 analogue) of the Ca2+‐binding regulatory protein cal‐modulin enhanced azole activity. (iii) Two structurally distinct inhibitors (cyclosporin and FK506) of the Ca2+‐calmodulin‐regulated phosphatase calcineurin enhanced azole activity. (iv) Strains in which the Ca2+ binding sites of calmodulin were eliminated and strains in which the calcineurin subunit genes were disrupted demonstrated enhanced azole sensitivity; conversely, a mutant with constitutively activated calcineurin phosphatase demonstrated decreased azole sensitivity. (v) CRZ1/TCN1 encodes a transcription factor regulated by calcineurin phosphatase; its disruption enhanced azole sensitivity, whereas its overexpression decreased azole sensitivity. All the above treatments had comparable effects on the activity of terbinafine, an inhibitor of squalene epoxidase within the sterol biosynthesis pathway, but had little or no effect on the activity of drugs with unrelated targets. (vi) Treatment of S. cerevisiae with azole or terbinafine resulted in transcriptional upregulation of genes FKS2 and PMR1 known to be Ca2+ regulated. A model to explain the role of Ca2+‐regulated signalling in azole/terbinafine tolerance is proposed.

The β-tubulin gene from rat and human isolates of Pneumocystis carinii

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long‐term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short‐term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the β‐tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single β‐tubulin gene from a rat P. carinii isolate. There was 89‐91% identity at the amino acid level to β‐tubulins from filamentous fungi, but only 79‐82% identity to yeast and protozoal β‐tubulins. Also, eight introns were distributed throughout the P. cariniiβ‐tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. cariniiβ‐tubulin. The polymerase chain reaction was used to amplify a segment of P. cariniiβ‐tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the ammo‐acid level.

Cyclic AMP Signaling Pathway Modulates Susceptibility of Candida Species and Saccharomyces cerevisiae to Antifungal Azoles and Other Sterol Biosynthesis Inhibitors

ABSTRACT Azoles are widely used antifungals; however, their efficacy is compromised by fungistatic activity and selection of resistant strains during treatment. Recent studies demonstrated roles for the protein kinase C and calcium signaling pathways in modulating azole activity. Here we explored a role for the signaling pathway mediated by cyclic AMP (cAMP), which is synthesized by the regulated action of adenylate cyclase (encoded by CDC35 in Candida albicans and CYR1 in Saccharomyces cerevisiae) and cyclase-associated protein (encoded by CAP1 and SRV2, respectively). Relative to wild-type strains, C. albicans and S. cerevisiae strains mutated in these genes were hypersusceptible to fluconazole (>4- to >16-fold-decreased 48-h MIC), itraconazole (>8- to >64-fold), or miconazole (16- to >64-fold). Similarly, they were hypersusceptible to terbinafine and fenpropimorph (2- to >16-fold), which, like azoles, inhibit sterol biosynthesis. Addition of cAMP to the medium at least partially reversed the hypersusceptibility of Ca-cdc35 and Sc-cyr1-2 mutants. An inhibitor of mammalian adenylate cyclase, MDL-12330A, was tested in combination with azoles; a synergistic effect was observed against azole-susceptible and -resistant strains of C. albicans and five of six non-C. albicans Candida species. Analysis of cAMP levels after glucose induction in the presence and absence of MDL-12330A confirmed that it acts by inhibiting cAMP synthesis in yeast. RNA analysis suggested that a defect in azole-dependent upregulation of the multidrug transporter gene CDR1 contributes to the hypersusceptibility of the Ca-cdc35 mutant. Our results implicate cAMP signaling in the yeast azole response; compounds similar to MDL-12330A may be useful adjuvants in azole therapy.