Transposon mutant CgTn201S contains a disrupted CgERG1 gene.
One of the transposon mutants, CgTn201S, was unable to grow on a MIN agar plate containing fluconazole at 100 μg/ml, whereas host strain Cg1660 was able to grow. Genomic DNA from CgTn201S was digested with BglII, self-ligated, and electroporated into E. coli; and the chloramphenicol-resistant clones were analyzed. DNA contiguous to Tn5<CmURA3> was sequenced. A search of the GenBank database with the BLASTX algorithm found sequence homology with S. cerevisiae ERG1, which encodes a squalene epoxidase of 496 amino acids. The full sequence of the CgERG1 ORF was obtained from the Génolevures Consortium. Tn5<Cm URA3> was found to be inserted at the codon for amino acid 476 of CgERG1 (data not shown).
Southern blot analysis with purified 2.9-kb Tn5<Cm URA3> as a probe detected a single 6.5-kb band in CgTn201S as well as in the rescued plasmid, pTn201S, while there was no signal in the parental strain, Cg1660 (Fig. ). A 2.2-kb CgERG1 probe detected 3.6- and 6.5-kb fragments in Cg1660 but hybridized only to the 6.5-kb fragments in CgTn201S (Fig. ). The 3.6-kb band was absent from CgTn201S. Together, Southern blotting and DNA sequence analyses indicated that CgERG1 is the gene targeted by the transposon in CgTn201S.
Increased expression of CgERG1 and CgERG11 in mutant CgTn201S.
To investigate whether the expression of CgERG1 was affected by the insertion of Tn5<Cm URA3>, the CgERG1 gene was used as a probe for Northern blot analysis. The CgERG1 probe detected a transcript with an expected size of about 1.5 kb in parental strain Cg1660 (Fig. ). Although no 1.5-kb transcript was found in mutant CgTn201S, the probe detected abundant transcripts of approximately 2 kb. The alteration in transcript size was likely due to a read-through transcript which included part of Tn5<Cm URA3>, which had been inserted into the 3′ end of the CgERG1 ORF (Fig. ). To investigate the possibility of feedback regulation on the ergosterol biosynthetic pathway due to the defect in CgERG1, CgERG11 encoding an alpha-C-14-demethylase was also used as a probe for Northern blot analysis. The results show that the expression of CgERG11 is also up regulated in CgTn201S (Fig. ).
FIG. 3. Expression of CgERG1 and CgERG11. Ten micrograms of total RNA from Cg1660 and CgTn201S was used for Northern blotting analysis. The membranes were hybridized with the 2.2-kb CgERG1 (A) or the 0.9-kb CgERG11 (B) probe. The sizes of the putative transcripts (more ...) Accumulation of squalene and reduced ergosterol synthesis in CgTn201S.
Sterol analysis of aerobically grown cultures showed that the level of accumulation of the sterol precursor, squalene, increased dramatically in CgTn201S (Table ). Squalene is the substrate of squalene epoxidase, encoded by ERG1. While squalene constitutes only 1.6% of the total extractable sterols in Cg1660, squalene comprises 66.6% of the total extractable sterols in CgTn201S. The accumulation of squalene in CgTn201S suggested that squalene epoxidation in ergosterol synthesis was impaired due to the CgERG1 mutation in CgTn201S. The synthesis of ergosterol, which constituted 32.9% of the total extractable sterols in CgTn210S, was reduced but not abolished (63.7%) in Cg1660.
Differences in levels of cholesterol uptake and sterol compositions of C. glabrata and C. albicans strains grown aerobically
Sterol supplements restored the growth defect of CgTn201S under conditions of low oxygen tension.
While CgTn201S grew slightly more slowly than Cg1660 under aerobic conditions, growth under conditions of low oxygen tension (hypoxia) required the presence of bovine serum or cholesterol (Fig. ). In contrast, both parental strain Cg1660 and a standard laboratory strain, NCCLS84, grew slowly but well under conditions of low oxygen tension.
FIG. 4. Effect of sterol on growth of CgTn201S under conditions of low oxygen tension. Two C. glabrata wild-type strains, Cg1660 and NCCLS84, as well as Cgerg1 mutant CgTn201S, were grown aerobically or under conditions of low oxygen tension (hypoxic). (A) YPD (more ...) CgTn201 had more efficient cholesterol uptake than wild-type C. glabrata and C. albicans strains.
Cholesterol is not synthesized by fungi, but both wild-type C. glabrata strains tested, Cg1660 and NCCLS84, were able to take up cholesterol from bovine serum-supplemented medium under aerobic conditions, with the levels of uptake constituting 9.5 and 21.2% of total extractable sterols, respectively (Table ). By comparison, two wild-type strains of C. albicans, BWP17 and CAI4, showed negligible cholesterol uptake under the same conditions. Strain CgTn201S was the most efficient at taking up cholesterol from serum-containing medium, with the levels of uptake constituting 34% of the total extractable sterols.
Altered drug susceptibility in CgTn201.
The susceptibilities to fluconazole, terbinafine, itraconazole, and amphotericin B were assessed in Cg1660, CgTn201S, and the CgERG1-complemented strain (CgTn201C) by using either MIN or RPMI medium (Table ). CgTn201S was remarkably more susceptible to the sterol synthesis inhibitors fluconazole, itraconazole, and terbinafine; but there was a slight decrease in susceptibility to the sterol-binding antifungal amphotericin B. Complementation of CgTn201S with the CgERG1 gene restored the levels of susceptibility of the CgTn201C strain to the wild-type levels. As ergosterol constituted only 32.9% of the total extractable sterols in CgTn201S, whereas it constituted 63.7% in Cg1660 (Table ), it is possible that fewer membrane ergosterol targets for amphotericin B led to the slight reduction in susceptibility to amphotericin B.
Drug susceptibilities of C. glabrata strains
Sterol supplements reduced the fluconazole susceptibility of CgTn201S.
As determined by Etest, the fluconazole MIC for Cg1660 without sterol was approximately 48 μg/ml under aerobic conditions, while the fluconazole MIC for CgTn201S was dramatically reduced to approximately 4 μg/ml and a distinct inhibition zone was detected, in contrast to the fuzzy inhibition zone detected for strain Cg1660 (Fig. ). Addition of ethanol-Tween 80 without sterol slightly increased the fluconazole susceptibilities of both Cg1660 and CgTn201S. Sterol supplementation reduced the fluconazole susceptibility of CgTn201S. No inhibition zone around fluconazole strips was observed with CgTn201S or Cg1660 under conditions of low oxygen tension and with exogenous ergosterol or cholesterol.
FIG. 5. Ergosterol or cholesterol reduced the fluconazole susceptibility of CgTn201S. Plates were incubated aerobically or under conditions of low oxygen tension at 30°C for 2 days. The fluconazole susceptibilities of Cg1660 and CgTn201S were analyzed (more ...) CgTn201S had increased levels of uptake of rhodamine 6G and [3H]fluconazole.
Reduced ergosterol synthesis in CgTn201S could lead to an altered membrane composition and altered permeability. Therefore, the levels of both rhodamine 6G and [3H]fluconazole accumulation were analyzed to determine uptake efficiency. The accumulation of rhodamine 6G, as determined by fluorescence cytometry, was higher in strain CgTn201S than in strain Cg1660, with geometric mean values of 26 and 13.9 arbitrary units, respectively (Fig. ). In addition, four separate experiments demonstrated that CgTn201S had much higher levels of [3H]fluconazole uptake than Cg1660, with the increases ranging from 5.4-fold (Fig. ) to 7.5-fold (data not shown). The increase in the level of [3H]fluconazole uptake in CgTn201S was consistent with the finding that CgTn201S had higher levels of rhodamine 6G uptake.
FIG. 6. Rhodamine 6G accumulation by Cg1660 and CgTn201S as determined by FACS analysis. The geometric mean fluorescence (in parentheses) is given in arbitrary units. Curves on the left are for unstained strains; curves on the right are for the strains after (more ...)
FIG. 7. [3H]fluconazole uptake with and without ergosterol (erg) or growth under conditions of low oxygen tension (−O2). Bars indicate standard errors of the mean. (A) The level of uptake by wild-type strain Cg1660 was low under all conditions. Note that (more ...) CgERG1 complementation but not ergosterol supplementation reduced the levels of [3H]fluconazole uptake in CgTn201S.
Ergosterol supplementation was used to investigate the possibility that membrane integrity could be restored to reduce the levels of [3H]fluconazole uptake. Neither Cg1660 nor CgTn201S showed significant differences in the levels of [3H]fluconazole accumulation with exogenous ergosterol under aerobic conditions or under low oxygen tension (Fig. ). In contrast, the CgERG1-complemented strain, CgTn201C, had a 3.5-fold reduction in the level of [3H]fluconazole accumulation (Fig. ). These results may indicate a quantitative difference between the amount of ergosterol needed to restore growth under conditions of low oxygen tension and the amount needed to restore drug transport.