FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2002979500> ?p ?o ?g. }

• W2002979500 endingPage "19764" @default.
• W2002979500 startingPage "19754" @default.
• W2002979500 abstract "The balance between saturated and unsaturated fatty acids plays a crucial role in determining the membrane fluidity. In the diploid fungal pathogen Candida albicans, the gene for fatty acid Δ9 desaturase, OLE1, is essential for viability. Using a reverse genetic approach, termed the fitness test, we identified a group of structurally related synthetic compounds that induce specific hypersensitivity of the OLE1+/− strain. Genetic repression of OLE1 and chemical inhibition by two selected compounds, ECC145 and ECC188, resulted in a marked decrease in the total unsaturated fatty acids and impaired hyphal development. The resulting auxotroph of both was suppressed by the exogenous monounsaturated fatty acids (16:1Δ9 and 18:1Δ9). These correlations suggest that both compounds affect the level of unsaturated fatty acids, likely by impairing Ole1p directly or indirectly. However, the residual levels of monounsaturated fatty acids (MUFAs) resulted from chemical inhibition were significantly higher than OLE1 repression, indicating even partial inhibition of MUFAs is sufficient to stop cellular proliferation. Although the essentiality of OLE1 was suppressed by MUFAs in vitro, we demonstrated that it was required for virulence in a murine model of systemic candidiasis even when the animals were supplemented with a high fat diet. Thus, the fungal fatty acid desaturase is an attractive antifungal drug target. Taking advantage of the inhibitors and the relevant conditional shut-off strains, we validated several chemical genetic interactions observed in the fitness test profiles that reveal novel genetic interactions between OLE1/unsaturated fatty acids and other cellular processes. The balance between saturated and unsaturated fatty acids plays a crucial role in determining the membrane fluidity. In the diploid fungal pathogen Candida albicans, the gene for fatty acid Δ9 desaturase, OLE1, is essential for viability. Using a reverse genetic approach, termed the fitness test, we identified a group of structurally related synthetic compounds that induce specific hypersensitivity of the OLE1+/− strain. Genetic repression of OLE1 and chemical inhibition by two selected compounds, ECC145 and ECC188, resulted in a marked decrease in the total unsaturated fatty acids and impaired hyphal development. The resulting auxotroph of both was suppressed by the exogenous monounsaturated fatty acids (16:1Δ9 and 18:1Δ9). These correlations suggest that both compounds affect the level of unsaturated fatty acids, likely by impairing Ole1p directly or indirectly. However, the residual levels of monounsaturated fatty acids (MUFAs) resulted from chemical inhibition were significantly higher than OLE1 repression, indicating even partial inhibition of MUFAs is sufficient to stop cellular proliferation. Although the essentiality of OLE1 was suppressed by MUFAs in vitro, we demonstrated that it was required for virulence in a murine model of systemic candidiasis even when the animals were supplemented with a high fat diet. Thus, the fungal fatty acid desaturase is an attractive antifungal drug target. Taking advantage of the inhibitors and the relevant conditional shut-off strains, we validated several chemical genetic interactions observed in the fitness test profiles that reveal novel genetic interactions between OLE1/unsaturated fatty acids and other cellular processes. Candida albicans is a commensal Hemiascomycete of the normal microflora of the healthy humans. However, it can cause mycoses ranging from superficial mucosal to hematogenously disseminated infections. In fact, C. albicans persists as the single most significant fungal pathogen leading to life-threatening infections in terms of the total number of cases and the mortality rate in the hospital setting (1.Pfaller M.A. Diekema D.J. Clin. Microbiol. Rev. 2007; 20: 133-163Crossref PubMed Scopus (3040) Google Scholar). Our understandings of its pathobiology as well as our efforts to identify therapeutic agents have been greatly facilitated by studying another Hemiascomycete, Saccharomyces cerevisiae. Genomic studies indicate that Hemiascomycetes represent a phylogenetically homogenous group of eukaryotes (2.Sherman D. Durrens P. Beyne E. Nikolski M. Souciet J.L. Génolevures Consortium Nucleic Acids Res. 2004; 32: D315-D318Crossref PubMed Google Scholar). However, they are highly diverse at the physiological and ecological levels. Unlike the model yeast, C. albicans is a diploid without a sexual life cycle based on meiosis and sporulation. The application of the conventional forward genetic approaches is restricted. Its unique pathobiology, however, urges that biological studies be performed directly in the pathogen. To this end, large-scale construction of mutants has been undertaken in this diploid organism (3.Noble S.M. Johnson A.D. Annu. Rev. Genet. 2007; 41: 193-211Crossref PubMed Scopus (84) Google Scholar).One of the genetic strategies took advantage of the diploidy of C. albicans to create a library of heterozygous mutants in which each strain contains an insertional disruption of the Tn7 transposon. A screen of that library yielded a set of genes that was defective in the yeast-to-hypha transition when one allele was disrupted (4.Uhl M.A. Biery M. Craig N. Johnson A.D. EMBO J. 2003; 22: 2668-2678Crossref PubMed Scopus (146) Google Scholar). Haploinsufficiency (reduction in growth by a loss-of-function mutation of one allele in a diploid organism; e.g. phenotypic defects associated with heterozygous deletion strains) and conditional haploinsufficiency, in particular, provide the genetic basis to identify genes whose dosages become susceptible to a defined condition and, thus, are functionally involved in the relevant biological processes. As the genome of C. albicans has been determined and annotated (5.Jones T. Federspiel N.A. Chibana H. Dungan J. Kalman S. Magee B.B. Newport G. Thorstenson Y.R. Agabian N. Magee P.T. Davis R.W. Scherer S. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7329-7334Crossref PubMed Scopus (578) Google Scholar, 6.Braun B.R. van Het Hoog M. d'Enfert C. Martchenko M. Dungan J. Kuo A. Inglis D.O. Uhl M.A. Hogues H. Berriman M. Lorenz M. Levitin A. Oberholzer U. Bachewich C. Harcus D. Marcil A. Dignard D. Iouk T. Zito R. Frangeul L. Tekaia F. Rutherford K. Wang E. Munro C.A. Bates S. Gow N.A. Hoyer L.L. Köhler G. Morschhäuser J. Newport G. Znaidi S. Raymond M. Turcotte B. Sherlock G. Costanzo M. Ihmels J. Berman J. Sanglard D. Agabian N. Mitchell A.P. Johnson A.D. Whiteway M. Nantel A. PLoS Genet. 2005; 1: 36-57Crossref PubMed Scopus (253) Google Scholar), a reverse genetic approach can be used to explore haploinsufficiency at the genome level. The resulting assay, the C. albicans fitness test (CaFT) 2The abbreviations used are: CaFTC. albicans fitness testMOAmechanism of actionSFAsaturated fatty acidUFAunsaturated fatty acidMUFAmonounsaturated fatty acidPUFApolyunsaturated fatty acidICinhibitory concentrationGRACEgene replacement and conditional expressionpTETtetracycline-repressible promoterFASfatty acid synthasedoxdoxycyclineERendoplasmic reticulumYNBDyeast nitrogen base and dextroseCSMcomplete supplement mixtureSRPsignal recognition particlesORFopen reading frame14:0myristic acid14:1myristoleic acid16:0palmitic acid16:1 (16:1Δ9)palmitoleic acid18:0stearic acid18:1 (18:1Δ9)oleic acid18:2 (18:2Δ9Δ12)linoleic acid18:3 (18:3Δ9Δ12Δ15)linolenic acid.2The abbreviations used are: CaFTC. albicans fitness testMOAmechanism of actionSFAsaturated fatty acidUFAunsaturated fatty acidMUFAmonounsaturated fatty acidPUFApolyunsaturated fatty acidICinhibitory concentrationGRACEgene replacement and conditional expressionpTETtetracycline-repressible promoterFASfatty acid synthasedoxdoxycyclineERendoplasmic reticulumYNBDyeast nitrogen base and dextroseCSMcomplete supplement mixtureSRPsignal recognition particlesORFopen reading frame14:0myristic acid14:1myristoleic acid16:0palmitic acid16:1 (16:1Δ9)palmitoleic acid18:0stearic acid18:1 (18:1Δ9)oleic acid18:2 (18:2Δ9Δ12)linoleic acid18:3 (18:3Δ9Δ12Δ15)linolenic acid., adapted from an analogous strategy first developed in S. cerevisiae (7.Giaever G. Flaherty P. Kumm J. Proctor M. Nislow C. Jaramillo D.F. Chu A.M. Jordan M.I. Arkin A.P. Davis R.W. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 793-798Crossref PubMed Scopus (414) Google Scholar, 8.Lum P.Y. Armour C.D. Stepaniants S.B. Cavet G. Wolf M.K. Butler J.S. Hinshaw J.C. Garnier P. Prestwich G.D. Leonardson A. Garrett-Engele P. Rush C.M. Bard M. Schimmack G. Phillips J.W. Roberts C.J. Shoemaker D.D. Cell. 2004; 116: 121-137Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), consists of ∼2900 heterozygous deletion strains (∼45% genome coverage) in a single pool that can be screened for phenotypic variations in parallel. Because each strain was double-bar-coded at the deleted locus, the relative variations in growth (hence, the term of fitness test) of all the strains in response to chemical perturbations could be determined en masse by DNA microarrays (9.Xu D. Jiang B. Ketela T. Lemieux S. Veillette K. Martel N. Davison J. Sillaots S. Trosok S. Bachewich C. Bussey H. Youngman P. Roemer T. PLoS Pathog. 2007; 3: e92Crossref PubMed Scopus (193) Google Scholar).It has been demonstrated in both fungi that specific and significant responses (hypersensitivity and resistance) elicited by antiproliferative compounds are restricted to only small sets of heterozygous deletion strains (7.Giaever G. Flaherty P. Kumm J. Proctor M. Nislow C. Jaramillo D.F. Chu A.M. Jordan M.I. Arkin A.P. Davis R.W. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 793-798Crossref PubMed Scopus (414) Google Scholar, 8.Lum P.Y. Armour C.D. Stepaniants S.B. Cavet G. Wolf M.K. Butler J.S. Hinshaw J.C. Garnier P. Prestwich G.D. Leonardson A. Garrett-Engele P. Rush C.M. Bard M. Schimmack G. Phillips J.W. Roberts C.J. Shoemaker D.D. Cell. 2004; 116: 121-137Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 9.Xu D. Jiang B. Ketela T. Lemieux S. Veillette K. Martel N. Davison J. Sillaots S. Trosok S. Bachewich C. Bussey H. Youngman P. Roemer T. PLoS Pathog. 2007; 3: e92Crossref PubMed Scopus (193) Google Scholar). Usually, they contain strains corresponding to the targets and other factors that are directly associated with or involved in the targets or aspects of the mechanisms of action (MOAs). For example, fluconazole, the therapeutic drug used to treat fungal infections, induces specific hypersensitivity of heterozygotes for ERG11, NCP1, and CDR1, corresponding to the target, the co-factor of Erg11p, and the efflux pump for the drug, respectively (9.Xu D. Jiang B. Ketela T. Lemieux S. Veillette K. Martel N. Davison J. Sillaots S. Trosok S. Bachewich C. Bussey H. Youngman P. Roemer T. PLoS Pathog. 2007; 3: e92Crossref PubMed Scopus (193) Google Scholar). In most cases the fitness test profiles are highly indicative of the biological activities of the compounds tested, as the hypersensitive and resistant strains reveal aspects of MOAs of antiproliferative compounds that are susceptible to a 50% loss of gene dosage. Indeed, CaFT profiling was used to determine the MOAs of novel antifungal synthetic compounds (9.Xu D. Jiang B. Ketela T. Lemieux S. Veillette K. Martel N. Davison J. Sillaots S. Trosok S. Bachewich C. Bussey H. Youngman P. Roemer T. PLoS Pathog. 2007; 3: e92Crossref PubMed Scopus (193) Google Scholar, 10.Rodriguez-Suarez R. Xu D. Veillette K. Davison J. Sillaots S. Kauffman S. Hu W. Bowman J. Martel N. Trosok S. Wang H. Zhang L. Huang L.Y. Li Y. Rahkhoodaee F. Ransom T. Gauvin D. Douglas C. Youngman P. Becker J. Jiang B. Roemer T. Chem. Biol. 2007; 14: 1163-1175Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and natural products (11.Jiang B. Xu D. Allocco J. Parish C. Davison J. Veillette K. Sillaots S. Hu W. Rodriguez-Suarez R. Trosok S. Zhang L. Li Y. Rahkhoodaee F. Ransom T. Martel N. Wang H. Gauvin D. Wiltsie J. Wisniewski D. Salowe S. Kahn J.N. Hsu M.J. Giacobbe R. Abruzzo G. Flattery A. Gill C. Youngman P. Wilson K. Bills G. Platas G. Pelaez F. Diez M.T. Kauffman S. Becker J. Harris G. Liberator P. Roemer T. Chem. Biol. 2008; 15: 363-374Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 12.Ondeyka J. Harris G. Zink D. Basilio A. Vicente F. Bills G. Platas G. Collado J. Gonzáez A. de la Cruz M. Martin J. Kahn J.N. Galuska S. Giacobbe R. Abruzzo G. Hickey E. Liberator P. Jiang B. Xu D. Roemer T. Singh S.B. J. Nat. Prod. 2009; 72: 136-141Crossref PubMed Scopus (42) Google Scholar, 13.Herath K. Harris G. Jayasuriya H. Zink D. Smith S. Vicente F. Bills G. Collado J. González A. Jiang B. Kahn J.N. Galuska S. Giacobbe R. Abruzzo G. Hickey E. Liberator P. Xu D. Roemer T. Singh S.B. Bioorg. Med. Chem. 2009; 17: 1361-1369Crossref PubMed Scopus (31) Google Scholar). In this report we present the identification of structurally related small-molecule inhibitors that elicited significant hypersensitivity of the OLE1+/− strain in the fitness test and demonstrate that the two selected compounds reduce the level of unsaturated fatty acids and block hyphal growth.The balance between saturated (SFA) and unsaturated (UFA) fatty acids is a critical determining factor of membrane fluidity important for the function and integrity of the membrane systems of the cell. In the yeast S. cerevisiae, the fatty acid Δ9 desaturase (ScOle1p) is located at the endoplasmic reticulum (ER) surface and converts coenzyme A-bound SFAs (palmitic (16:0) and stearic (18:0) acids) to monounsaturated fatty acids (MUFAs, palmitoleic (16:1) and oleic (18:1) acids) by introducing double-bonds at carbon 9 in the carbon chains (14.Stukey J.E. McDonough V.M. Martin C.E. J. Biol. Chem. 1989; 264: 16537-16544Abstract Full Text PDF PubMed Google Scholar, 15.Mitchell A.G. Martin C.E. J. Biol. Chem. 1995; 270: 29766-29772Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The C. albicans Ole1p was shown to play the same role (16.Krishnamurthy S. Plaine A. Albert J. Prasad T. Prasad R. Ernst J.F. Microbiology. 2004; 150: 1991-2003Crossref PubMed Scopus (39) Google Scholar). However, although 16:1 is the predominant UFA in S. cerevisiae, the pathogen produces, in addition, polyunsaturated fatty acids (PUFAs, linoleic (18:2) and linolenic (18:3) acids) through the sequential formation of extra double bonds in 18:1 via Δ12 and Δ15 desaturases (16.Krishnamurthy S. Plaine A. Albert J. Prasad T. Prasad R. Ernst J.F. Microbiology. 2004; 150: 1991-2003Crossref PubMed Scopus (39) Google Scholar, 17.Mishra P. Bolard J. Prasad R. Biochim. Biophys. Acta. 1992; 1127: 1-14Crossref PubMed Scopus (44) Google Scholar).In yeast, the level of unsaturated fatty acids is elaborately controlled. For example, multiple transcription regulatory elements have been identified in the promoter of ScOLE1. Its transcription is repressed by UFAs (which also regulates the stability of ScOLE1 mRNA) but activated by hypoxia and low temperature via the ER-bound transcription factors ScSpt23p and ScMga2p (18.Martin C.E. Oh C.S. Jiang Y. Biochim. Biophys. Acta. 2007; 1771: 271-285Crossref PubMed Scopus (158) Google Scholar). Both factors are, in turn, activated by ubiquitin/proteasome-dependent ER-associated degradation, a process that is also involved in regulating the stability of ScOle1p (19.Braun S. Matuschewski K. Rape M. Thoms S. Jentsch S. EMBO J. 2002; 21: 521-615Crossref Scopus (290) Google Scholar). Moreover, the UFA/SFA balance is important for other cellular processes. For example, a temperature-sensitive mutant of ScOLE1, identified as mdm2, resulted in a 2.5-fold decrease of monounsaturated fatty acids at the nonpermissive temperatures and caused the fragmentation of reticular network of mitochondria and accumulation of defective mitochondria in the mother cell during division (20.Stewart L.C. Yaffe M.P. J. Cell Biol. 1991; 115: 1249-1257Crossref PubMed Scopus (78) Google Scholar). Similarly, low levels of unsaturated fatty acids affect the nuclear envelope structure and, to a less extent, vesicular traffic (21.Schneiter R. Kohlwein S.D. Cell. 1997; 88: 431-434Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In C. albicans, diminished levels of OLE1 block hyphal development and formation of chlamydospores under the normoxic conditions (16.Krishnamurthy S. Plaine A. Albert J. Prasad T. Prasad R. Ernst J.F. Microbiology. 2004; 150: 1991-2003Crossref PubMed Scopus (39) Google Scholar).Our results show that the effects of chemical inhibition by two selected compounds correlate with genetic repression of OLE1 (in a conditional shut-off strain), suggesting that Ole1p be the target. However, it is possible that their affects on Ole1p is indirect, as neither compound was tested for direct inhibition of the Δ9 desaturase activity in vitro. Nonetheless, we provide evidence that partial depletion of monounsaturated fatty acids is sufficient to be antiproliferative and demonstrate the essentiality of OLE1 in a murine model of systemic candidiasis.DiscussionIn this study we applied chemical genetic profiling in C. albicans to identify a series of structurally related synthetic compounds that induced significant and specific hypersensitivity of the heterozygous deletion strain for OLE1, encoding the fungal fatty acid Δ9 desaturase (Fig. 1). The fitness test results suggest that these compounds affect unsaturated fatty acids, likely by impairing Ole1p. Two representative compounds were selected for characterization of mechanism of action. The conditional shut-off strains provided a genetic means to examine the level of cellular fatty acids (Table 2), and the effect on hyphal growth (Fig. 4) when OLE1 expression was repressed. The overall correlations between genetic repression and chemical inhibition (Table 2 and Fig. 4), and suppression of both by the exogenous monounsaturated fatty acids (FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5) supported the MOA hypothesis. The significant reduction in total unsaturated fatty acids and the concomitant increase in total saturated fatty acids (Table 2) are clear indications that both ECC145 and ECC188 inhibit UFA biosynthesis. The shared chemical structures and the CaFT profiles (Fig. 1) suggest that other OLE1 compounds act in a similar manner. They provide another example that illustrates the predicative power of the fitness test in studying mechanisms of action of unknown compounds; thus, its application in antifungal drug discovery (9.Xu D. Jiang B. Ketela T. Lemieux S. Veillette K. Martel N. Davison J. Sillaots S. Trosok S. Bachewich C. Bussey H. Youngman P. Roemer T. PLoS Pathog. 2007; 3: e92Crossref PubMed Scopus (193) Google Scholar, 10.Rodriguez-Suarez R. Xu D. Veillette K. Davison J. Sillaots S. Kauffman S. Hu W. Bowman J. Martel N. Trosok S. Wang H. Zhang L. Huang L.Y. Li Y. Rahkhoodaee F. Ransom T. Gauvin D. Douglas C. Youngman P. Becker J. Jiang B. Roemer T. Chem. Biol. 2007; 14: 1163-1175Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11.Jiang B. Xu D. Allocco J. Parish C. Davison J. Veillette K. Sillaots S. Hu W. Rodriguez-Suarez R. Trosok S. Zhang L. Li Y. Rahkhoodaee F. Ransom T. Martel N. Wang H. Gauvin D. Wiltsie J. Wisniewski D. Salowe S. Kahn J.N. Hsu M.J. Giacobbe R. Abruzzo G. Flattery A. Gill C. Youngman P. Wilson K. Bills G. Platas G. Pelaez F. Diez M.T. Kauffman S. Becker J. Harris G. Liberator P. Roemer T. Chem. Biol. 2008; 15: 363-374Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 12.Ondeyka J. Harris G. Zink D. Basilio A. Vicente F. Bills G. Platas G. Collado J. Gonzáez A. de la Cruz M. Martin J. Kahn J.N. Galuska S. Giacobbe R. Abruzzo G. Hickey E. Liberator P. Jiang B. Xu D. Roemer T. Singh S.B. J. Nat. Prod. 2009; 72: 136-141Crossref PubMed Scopus (42) Google Scholar, 13.Herath K. Harris G. Jayasuriya H. Zink D. Smith S. Vicente F. Bills G. Collado J. González A. Jiang B. Kahn J.N. Galuska S. Giacobbe R. Abruzzo G. Hickey E. Liberator P. Xu D. Roemer T. Singh S.B. Bioorg. Med. Chem. 2009; 17: 1361-1369Crossref PubMed Scopus (31) Google Scholar). However, the question remains of whether the effects of ECC145 and ECC188 are because of direct inhibition of Ole1p, as neither was tested for direct inhibition of the Δ9 desaturase activity in vitro.Fatty Acid Biosynthesis in C. albicansFatty acids are a fundamental constituent of the cellular membrane systems that are involved in diverse biological processes. Their biosynthesis is energy-consuming and, thus, subject to elaborate regulation in response to physiological conditions and environmental cues. In particular, any imbalance between saturated and unsaturated fatty acids could have a deleterious consequence, as in the case of the pTET-FAS1 strain exposed to 18:0, 16:1, and 18:1 under the non-repressing conditions (Fig. 2). Most likely, the replacement of the native promoter by a heterologous promoter escapes transcriptional regulation of FAS1 expression in the presence of excess downstream products, resulting in imbalances between not only unsaturated and saturated fatty acids, but also 16:0 and 18:0; our results (Table 2) and others (16.Krishnamurthy S. Plaine A. Albert J. Prasad T. Prasad R. Ernst J.F. Microbiology. 2004; 150: 1991-2003Crossref PubMed Scopus (39) Google Scholar, 17.Mishra P. Bolard J. Prasad R. Biochim. Biophys. Acta. 1992; 1127: 1-14Crossref PubMed Scopus (44) Google Scholar, 28.Oh C.S. Martin C.E. J. Biol. Chem. 2006; 281: 7030-7039Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) indicate that the level of 16:0 is significantly higher than that of 18:0. On the other hand, the effects of exogenous 18:0 and 16:1 on the pTET-FAS2 strain were less severe (Fig. 2), and only the pTET-FAS1 strain was partially impaired in growth under the non-repressing conditions (Fig. 2B, mock). In S. cerevisiae, expression of ScFAS2 depends on the cellular level of free ScFas1p, and a responsive cis-regulatory element is in the ORF of ScFAS2 (29.Wenz P. Schwank S. Hoja U. Schüller H.J. Nucleic Acids Res. 2001; 29: 4625-4632Crossref PubMed Scopus (34) Google Scholar). As this might be the case in C. albicans (9.Xu D. Jiang B. Ketela T. Lemieux S. Veillette K. Martel N. Davison J. Sillaots S. Trosok S. Bachewich C. Bussey H. Youngman P. Roemer T. PLoS Pathog. 2007; 3: e92Crossref PubMed Scopus (193) Google Scholar), our results provide further evidence for the conservation of such regulatory scheme.OLE1 repression was partially suppressed by 16:1 and further enhanced by 18:1, the latter of which did not suppress by itself but impaired severely the pTET-OLE1 strain under the non-repressing conditions (Fig. 2C). 18:0 had a similar but less severe deleterious effect on this strain (Fig. 2B). Our analyses indicated that the heterologous pTET drove a lower level of OLE1 expression than the native promoter.3 This was further confirmed by two additional lines of evidence. 1) The level of monounsaturated fatty acids in this strain (MUFA/SFA ratio = 0.75) was significantly lower than that in the control strain (MUFA/SFA = 1.40); a less significant difference was observed with polyunsaturated fatty acids (Table 2). 2) The pTET-OLE1 strain was intrinsically more susceptible to both ECC145 and ECC188 than the OLE1+/− counterpart (Table 1, Fig. 3). In S. cerevisiae, transcription of ScOLE1 is repressed by unsaturated fatty acids, which also destabilizes the mRNA via its 5′-untranslated region. Saturated fatty acids, on the other hand, activate transcription of ScOLE1 without affecting the RNA stability (18.Martin C.E. Oh C.S. Jiang Y. Biochim. Biophys. Acta. 2007; 1771: 271-285Crossref PubMed Scopus (158) Google Scholar). Our results suggest that the ∼250-base pair region immediately upstream of the OLE1 ORF is important for its regulation in response to both exogenous 18:0 and 18:1. The interplay between saturated and unsaturated fatty acids was further demonstrated by partial suppression of cerulenin, an inhibitor of fatty acid synthase, by low concentrations of ECC145 and ECC188 (Fig. 6), suggesting relative growth advantage of partially restored UFA/SFA balance even when the overall fatty acid syntheses are impaired.Mechanisms of Action of ECC145 and ECC188Consistent with the MOA hypothesis generated in the fitness test, our results corroborate the chemical inhibition of both compounds with the genetic repression of OLE1 (Table 2, FIGURE 4, FIGURE 5). However, neither compound was tested for in vitro inhibition of the Δ9 desaturase activity (although the microsomal ScOle1p activity was tested in S. cerevisiae (26.Bossie M.A. Martin C.E. J. Bacteriol. 1989; 171: 6409-6413Crossref PubMed Google Scholar), none has been reported for C. albicans). We cannot, therefore, rule out the possibility that Ole1p is not the direct molecular target of these compounds and that their effects on unsaturated fatty acids are indirect.In S. cerevisiae, expression of ScOLE1 is exquisitely regulated by two homologous transcription factors, ScSpt23p and ScMga2p (18.Martin C.E. Oh C.S. Jiang Y. Biochim. Biophys. Acta. 2007; 1771: 271-285Crossref PubMed Scopus (158) Google Scholar). The only ortholog, SPT23/orf19.1751, in C. albicans has been shown to play the same role. Its repression leads to reduction in monounsaturated fatty acids, and the resulting lethality is rescued by exogenous UFAs (28.Oh C.S. Martin C.E. J. Biol. Chem. 2006; 281: 7030-7039Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The heterozygous deletion for SPT23 was not constructed for the CaFT; neither was the pTET strain. Nevertheless, the chemical inhibition of ECC145 and ECC188 is also in agreement with the terminal phenotypes of SPT23 repression as reported (28.Oh C.S. Martin C.E. J. Biol. Chem. 2006; 281: 7030-7039Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). It is, thus, possible that these compounds inhibit Spt23p, which in turn stalls the OLE1 expression. The chemical genetic interactions characterized here reflect the exclusive role the transcription factor in regulating OLE1.On the other hand, the partial suppression of ECC145 (and likely ECC1883) by exogenous MUFAs (Fig. 5) suggests that the antiproliferative activities are not restricted to inhibition of monounsaturated fatty acids. Although genetic repression of OLE1 predominantly reduced the level of monounsaturated fatty acids (MUFA/SFA = 0.06), both MUFAs and PUFAs were affected by the two compounds (MUFA/SFA ≈ PUFA/SFA ≈ 0.3, Table 2). The genes for the two polydesaturases, FAD2 (orf19.118) and FAD3 (orf19.4933), are not essential (30.Murayama S.Y. Negishi Y. Umeyama T. Kaneko A. Oura T. Niimi M. Ubukata K. Kajiwara S. Microbiology. 2006; 152: 1551-1558Crossref PubMed Scopus (20) Google Scholar).3 The two heterozygous deletion strains displayed no significant growth variations against any of the OLE1 compounds or ECC145 in the presence of MUFAs in the fitness test (supplemental Table S1). We cannot, however, exclude a possible synergistic effect of impairing both MUFAs and PUFAs by ECC145 or ECC188 (particularly at high concentrations).The structural variability of the putative OLE1 compounds (Fig. 1, A and B) suggests the possibility that they could be metabolized in C. albicans, which in turn generates a common product that causes the observed effects. If so, the fitness test did not ostensibly reveal this aspect of the mechanism of action. As only ∼45% of the genome was represented in the current version of the fitness test (9.Xu D. Jiang B. Ketela T. Lemieux S. Veillette K. Martel N. Davison J. Sillaots S. Trosok S. Bachewich C. Bussey H. Youngman P. Roemer T. PLoS Pathog. 2007; 3: e92Crossref PubMed Scopus (193) Google Scholar), another possibility exists that these compounds affect another cellular process(es) which becomes synthetically lethal when chemically perturbed, with a genetic impaired unsaturated fatty acid biosynthesis (as in the case of OLE1+/− strain). If so, the potential target of these compounds could be related to the so-called secondary profiles in the fitness test (Fig. 1C and see below). We noted that the fatty acid profile of the parental strain (CAI-4), used to construct all the pTET strains, is different (Table 2). In particular, the level of monounsaturated fatty acids is significantly higher than both HIS3 (the control) and OLE1 conditional shut-off strains. It remains to be determined if genetic manipulations in CAI-4 and/or the pTET strains are responsible for changes in the biosynthesis of unsaturated fatty acids and if such changes affect the susceptibility to these compounds.OLE1 as an Antifungal TargetRegardless of the exact molecular target of these compounds, our results and others (16.Krishnamurthy S. Plaine A. Albert J. Prasad T. Prasad R. Ernst J.F. Microbiology. 2004; 150: 1991-2003Crossref PubMed Scopus (39) Google Scholar, 28.Oh C.S. Martin C.E. J. Biol. Chem. 2006; 281: 7030-7039Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) suggest that an incomplete depletion of monounsaturated fatty acids is sufficient to block cellular proliferation. Although the results in Table 2 were not replicated biologically and, thus, must be interpreted with cautions, the fatty acid profiles resulted from ECC145 and ECC188 inhibition are in general agreement with each other. We note that the residual level of monounsaturated fatty acids was significantly higher in cells treated with either compound (at ∼5× minimal inhibitory concentration for 15 h, with ∼5-fold reduction in the MUFA/SFA ratio from ∼1.5 to ∼0.3) than in those depleted of OLE1 expression (∼15-fold decrease from ∼0.75 to ∼0.06) (Table 2). Using a similar promoter replacement strategy but with the MET3 promoter, Krishnamurthy et al. (16.Krishnamurthy S. Plaine A. Albert J. Prasad T. Prasad R. Ernst J.F. Microbiology. 2004; 150: 1991-2003Crossref PubMed Scopus (39) Google Scholar) demonstra" @default.
• W2002979500 created "2016-06-24" @default.
• W2002979500 creator A5004392744 @default.
• W2002979500 creator A5009121625 @default.
• W2002979500 creator A5011252897 @default.
• W2002979500 creator A5026378317 @default.
• W2002979500 creator A5026851342 @default.
• W2002979500 creator A5028007694 @default.
• W2002979500 creator A5031545809 @default.
• W2002979500 creator A5034305981 @default.
• W2002979500 creator A5042239893 @default.
• W2002979500 creator A5042774299 @default.
• W2002979500 creator A5049154015 @default.
• W2002979500 creator A5057738220 @default.
• W2002979500 creator A5066716873 @default.
• W2002979500 creator A5077010575 @default.
• W2002979500 creator A5079581525 @default.
• W2002979500 creator A5079841116 @default.
• W2002979500 creator A5083458592 @default.
• W2002979500 date "2009-07-01" @default.
• W2002979500 modified "2023-09-27" @default.
• W2002979500 title "Chemical Genetic Profiling and Characterization of Small-molecule Compounds That Affect the Biosynthesis of Unsaturated Fatty Acids in Candida albicans" @default.
• W2002979500 cites W1483746134 @default.
• W2002979500 cites W1503938960 @default.
• W2002979500 cites W1875164239 @default.
• W2002979500 cites W1965526938 @default.
• W2002979500 cites W1975938982 @default.
• W2002979500 cites W1980535454 @default.
• W2002979500 cites W1996109826 @default.
• W2002979500 cites W2000930832 @default.
• W2002979500 cites W2008714956 @default.
• W2002979500 cites W2037262024 @default.
• W2002979500 cites W2037264038 @default.
• W2002979500 cites W2044480313 @default.
• W2002979500 cites W2049505636 @default.
• W2002979500 cites W2060322845 @default.
• W2002979500 cites W2064806768 @default.
• W2002979500 cites W2074554547 @default.
• W2002979500 cites W2078807391 @default.
• W2002979500 cites W2081625270 @default.
• W2002979500 cites W2092806983 @default.
• W2002979500 cites W2092899154 @default.
• W2002979500 cites W2094285560 @default.
• W2002979500 cites W2100810331 @default.
• W2002979500 cites W2109785145 @default.
• W2002979500 cites W2137918904 @default.
• W2002979500 cites W2141563676 @default.
• W2002979500 cites W2143712580 @default.
• W2002979500 cites W2145319716 @default.
• W2002979500 cites W2150926065 @default.
• W2002979500 cites W2162070972 @default.
• W2002979500 cites W2163104149 @default.
• W2002979500 cites W2163138997 @default.
• W2002979500 cites W2167798305 @default.
• W2002979500 cites W2171571929 @default.
• W2002979500 doi "https://doi.org/10.1074/jbc.m109.019877" @default.
• W2002979500 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2740599" @default.
• W2002979500 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19487691" @default.
• W2002979500 hasPublicationYear "2009" @default.
• W2002979500 type Work @default.
• W2002979500 sameAs 2002979500 @default.
• W2002979500 citedByCount "68" @default.
• W2002979500 countsByYear W20029795002012 @default.
• W2002979500 countsByYear W20029795002013 @default.
• W2002979500 countsByYear W20029795002014 @default.
• W2002979500 countsByYear W20029795002015 @default.
• W2002979500 countsByYear W20029795002016 @default.
• W2002979500 countsByYear W20029795002017 @default.
• W2002979500 countsByYear W20029795002018 @default.
• W2002979500 countsByYear W20029795002020 @default.
• W2002979500 countsByYear W20029795002021 @default.
• W2002979500 countsByYear W20029795002022 @default.
• W2002979500 countsByYear W20029795002023 @default.
• W2002979500 crossrefType "journal-article" @default.
• W2002979500 hasAuthorship W2002979500A5004392744 @default.
• W2002979500 hasAuthorship W2002979500A5009121625 @default.
• W2002979500 hasAuthorship W2002979500A5011252897 @default.
• W2002979500 hasAuthorship W2002979500A5026378317 @default.
• W2002979500 hasAuthorship W2002979500A5026851342 @default.
• W2002979500 hasAuthorship W2002979500A5028007694 @default.
• W2002979500 hasAuthorship W2002979500A5031545809 @default.
• W2002979500 hasAuthorship W2002979500A5034305981 @default.
• W2002979500 hasAuthorship W2002979500A5042239893 @default.
• W2002979500 hasAuthorship W2002979500A5042774299 @default.
• W2002979500 hasAuthorship W2002979500A5049154015 @default.
• W2002979500 hasAuthorship W2002979500A5057738220 @default.
• W2002979500 hasAuthorship W2002979500A5066716873 @default.
• W2002979500 hasAuthorship W2002979500A5077010575 @default.
• W2002979500 hasAuthorship W2002979500A5079581525 @default.
• W2002979500 hasAuthorship W2002979500A5079841116 @default.
• W2002979500 hasAuthorship W2002979500A5083458592 @default.
• W2002979500 hasBestOaLocation W20029795001 @default.
• W2002979500 hasConcept C104317684 @default.
• W2002979500 hasConcept C185592680 @default.
• W2002979500 hasConcept C2780917455 @default.
• W2002979500 hasConcept C553450214 @default.
• W2002979500 hasConcept C55493867 @default.
• W2002979500 hasConcept C86803240 @default.

• next