Growth inhibition of Candida species and Aspergillus fumigatus by statins

Abstract

Statins are a class of drugs widely used for lowering high cholesterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the synthesis of cholesterol. We studied the effects of two major statins, simvastatin and atorvastatin, on five Candida species and Aspergillus fumigatus. The statins strongly inhibited the growth of all species, except Candida krusei. Supplementation of Candida albicans and A. fumigatus with ergosterol or cholesterol in aerobic culture led to substantial recovery from the inhibition by statins, suggesting specificity of statins for the mevalonate synthesis pathway. Our findings suggest that the statins could have utility as antifungal agents and that fungal colonization could be affected in those on statin therapy.

antifungal, cholesterol, ergosterol, HMG-CoA reductase inhibition, simvastatin, atorvastatin

Introduction

Statins are the main therapeutic agents used to decrease high serum cholesterol levels and are among the most widely prescribed drugs currently on the market. They competitively inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase by binding to the active site of HMG-CoA reductase (Qiuet al,1991), an enzyme that catalyses the conversion of HMG-CoA to mevalonate and subsequently to farnesyl diphosphate. Farnesyl diphosphate is the precursor for the production of cholesterol in humans or ergosterol in plants and eukaryotic microorganisms. If the statin-binding site of fungal HMG-CoA reductase is similar to that of human HMG-CoA reductase, it might be expected that statins or their derivatives could also be used to inhibit the growth of fungal pathogens through inhibition of ergosterol synthesis. This study examines the effects of the commonly prescribed statins, simvastatin and atorvastatin, on six species of pathogenic fungi.

Materials and methods

Chemicals

Simvastatin and atorvastatin were purchased from 7Chemicals (India). Tween 80, cholesterol and ergosterol were purchased from Sigma-Aldrich. To activate simvastatin, provided in the form of a lactone prodrug, it was hydrolysed in ethanolic NaOH [15% (v/v) ethanol and 0.25% (w/v) NaOH] at 60°C for 1h (Lorenz & Parks, 1990). Stock solutions of the hydrolysed simvastatin at a concentration of 20mgmL⁻¹ were stored at −20°C.

Yeast strain and media

The 13 yeast strains used in these studies were as follows: Candida albicans JRW#5, WM1172, ATCC90028 and CBS562; Candida glabrata ATCC90300, and CBS138; Candida tropicalis ATCC750, WM213 and WM30; Candida krusei ATCC6258, and WM03,204; Candida parapsilosis ATCC22019; and Aspergillus fumigatus strain 03.209-3938. Strains were grown in YEPD (1% yeast extract, 2% peptone, 2% dextrose), YEPE (1% yeast extract, 2% peptone, 2% ethanol) or in YNB (0.67% Difco yeast nitrogen base without amino acids, 2% glucose) media. For supplementation of media, Tween 80 (1:1 in ethanol) was added to a final concentration of 0.5%. Cholesterol and ergosterol were dissolved in the Tween 80/ethanol mixture and added to a final concentration of 12, 36 or 108μgmL⁻¹.

Yeast growth

Candida and Aspergillus species in log-phase growth in YEPD media were suspended in water and then transferred to solidified media for incubation at 30°C for 2 days. Solidified media containing statins were freshly prepared before use, although statins remained active in solidified media stored at 4°C for up to 10 days.

Results

Growth inhibition of Candida and Aspergillus species

The effect of simvastatin and atorvastatin on the growth of Candida species was measured on three types of solidified media: YEPD, YEPE and YNB media. Statin concentrations were between 0 and 1000μM. The results of growth in the different media with different concentrations of statins are shown in Table 1 (and Fig. 1).

1

Growth of Candida species in the presence of simvastatin and atorvastatin

Candida species	Strain	Untreated	YEPD (μM)		Untreated	YNB (μM)		Untreated	YEPE (μM)
AVS		SVS		AVS		SVS		AVS		SVS
100	300	100	300	10	100	3	100	100	1000	100	1000
C. albicans	JRW#5	+++	−	−	−	−	+++	+++	−	++	−	+++	−	−	−	−
C. albicans	WM1172	+++	−	−	−	−	+++	+	−	+	−	+++	−	−	−	−
C. albicans	ATCC90028	+++	−	−	−	−	+++	++	−	−	−	+++	−	−	−	−
C. albicans	CBS562	+++	−	−	−	−	+++	+	−	−	−	+++	−	−	−	−
C. glabrata	ATCC90300	+++	+++	++	+++	++	+++	+++	+	++	−	+++	+++	+	++	−
C. glabrata	CBS138	+++	+++	++	−	−	+++	++	+	+	+	+++	+++	−	−	−
C. tropicalis	ATCC750	+++	+++	+++	++	+	+++	+++	+	++	−	+++	++	+	−	−
C. tropicalis	WM213	+++	+	+	−	−	+++	+++	−	+	−	+++	+	+	−	−
C. tropicalis	WM30	+++	++	++	+	−	+++	+++	+	+++	+	+++	+	+	−	−
C. krusei	ATCC6258	+++	+++	++	+++	+	+++	+++	+++	+++	−	+++	+++	++	+++	−
C. krusei	WM03204	+++	+++	+++	+++	+++	+++	+++	+++	+++	++	+++	+++	+++	+++	−
C. parapsilosis	ATCC22019	+++	−	−	−	−	NA	NA	NA	NA	NA	+++	+	−	+	−

Strains were grown with atorvastatin (AVS) or simvastatin (SVS) at the levels shown. Growth (strong, +++; moderate, ++; low, +; none, −; NA, not applicable) is shown after 2 days on YEPD, YNB or YEPE media.

1

Rescue of statin-induced growth inhibition of Candida albicans by sterols. The treatments are: SVS, 3μM simvastatin; AVS, 10μM atorvastatin; E, ergosterol/Tween 80; and C, cholesterol/Tween80. Panel (a) shows the OD_600nm measurements after 18h culture of Candida albicans ATCC90028 with treatments as indicated. Panel (b) shows the OD_600nm measurements after 42h culture with 36μgmL⁻¹ cholesterol or ergosterol where indicated.

On rich media (YEPD), C. parapsilosis and all four C. albicans isolates were strongly inhibited by concentrations of 100μM simvastatin or atorvastatin. One C. glabrata isolate (ATCC90300) exhibited low sensitivity to 300μM atorvastatin and simvastatin, while the other (CBS138) similarly showed low sensitivity to 300μM atorvastatin but was sensitive to 100μM simvastatin. Inhibition within species appeared to be uniform as the four isolates of C. albicans, three of C. tropicalis and two of C. glabrata exhibited similar sensitivity profiles. Of the two isolates of C. krusei tested, minimal inhibition was observed; however, some sensitivity was apparent in one of these (ATCC6258) when exposed to 300μM simvastatin.

Overall, when using YEPE, the sensitivity profiles obtained at 100μM simvastatin and atorvastatin were similar to those observed when using rich media, although inhibition was found to be slightly stronger on YEPE. At concentrations of 1000μM simvastatin, all of the yeast isolates were to be strongly inhibited, whereas atorvastatin, at this same concentration, showed less effective inhibition. Of two isolates of C. krusei tested, both showed complete inhibition at 1000μM simvastatin but with no sensitivity to 1000μM atorvastatin.

When minimal medium (YNB) was used, increased levels of inhibition were observed so in Table 1 we show the results with lower concentrations of statins. All Candida isolates tested on this medium showed significant inhibition at 100μM statin levels, with the exception of the C. krusei isolates, which appeared resistant to 100μM atorvastatin. In addition, many strains exhibited inhibition by concentrations as low as 3μM simvastatin and 10μM atorvastatin. The C. parapsilosis isolate was incapable of growth on minimal media.

The effects of statins on the growth of A. fumigatus, a filamentous fungal pathogen from a different genus, were also investigated on solidified minimal media. In the absence of statins, A. fumigatus exhibited robust growth with production of conidia after 4 days at 30°C (Fig. 2). In the presence of statins, there was growth inhibition. Atorvastatin at 3μM caused substantial growth inhibition (data not shown), with no growth being observed at 10μM (Fig. 2), while simvastatin caused substantial growth inhibition at 0.1μM (data not shown) and no visible growth at 1μM (Fig. 2).

2

Aspergillus fumigatus: growth inhibition by statins and rescue of growth with sterols. Aspergillus fumigatus growth in the absence of statins and in the presence of 3μM simvastatin (SVS), 10μM atorvastatin (AVS), cholesterol/Tween 80 (C) and/or ergosterol/Tween 80 (E) as indicated. Growth after 4 days on minimal media is shown.

Rescue of A. fumigatus and C. albicans growth inhibition with sterols

As the action of the statins appears to be mediated through the sterol synthesis pathway, the action of the statins on growth inhibition was examined by testing rescue of growth inhibition with sterols. This was performed qualitatively for A. fumigatus and quantitatively for C. albicans ATCC90028. Minimal media containing statins at concentrations of 3μM simvastatin or 10μM atorvastatin (levels shown on solid media to inhibit growth) were used. For sterol treatment, the media were supplemented with ergosterol/Tween 80 or cholesterol/Tween 80 combination (using 12μgmL⁻¹ sterol). While erg1 mutants, lacking ergosterol due to a defect in squalene epoxidase, require anaerobic conditions for sterol rescue (Leberet al,1998), it has been reported that the lovastatin-treated Saccharomyces cerevisiae cells can be rescued by sterol supplementation under aerobic conditions (Lorenz & Parks, 1990). Thus, we used aerobic conditions to test sterol rescue.

After treatment with sterols for 4 days at 30°C, A. fumigatus showed recovery from statin-induced growth inhibition (Fig. 2), although growth was slower compared with that of untreated A. fumigatus. Both ergosterol and cholesterol appeared equally effective in rescuing A. fumigatus growth following treatment of simvastatin and atorvastatin.

To test for sterol rescue of C. albicans, growth yields were measured after 18 and 42h. Using concentrations of 3μM simvastatin or 10μM atorvastatin, growth yields were reduced to levels less than 2% of those obtained without addition of statins (Fig. 1a). When the media were supplemented with the ergosterol or cholesterol, growth levels recovered to ∼50% of the levels where no statin was present after 18h (Fig. 1a). Increasing the levels of either sterol to 108μgmL⁻¹ sterol did not lead to complete growth recovery: the greatest growth obtained was ∼70% of that with no statin (data not shown). However, we found that after extended culture, growth yields were much higher. Cells treated with 10μM atorvastatin plus cholesterol or ergosterol achieved yields equal to the levels of the untreated culture, while cells treated with 3μM simvastatin plus cholesterol or ergosterol reached yields of ∼90% of the untreated culture (Fig. 1b). This would appear to indicate that rates of uptake of cholesterol or ergosterol are too slow to permit uninhibited growth, but with slow growth the sterol needs can be met by uptake. The combination of Tween 80 with a sterol was essential: no rescue was obtained with ergosterol or cholesterol alone.

Discussion

This study is the first to report an effect of the major cholesterol-lowering drugs, simvastatin and atorvastatin, on the growth of pathogenic yeast. Both simvastatin and atorvastatin caused strong inhibition of growth in four Candida species and A. fumigatus. This growth inhibition is likely to be due to lower levels of ergosterol in the cell caused by growth in the presence of simvastatin or atorvastatin. This has been demonstrated in previous studies with the related statin, lovastatin (also known as mevinolin), where growth inhibition and lowering of ergosterol levels were observed when the yeasts Rhodotorula rubra (Baranovaet al,1996), S. cerevisiae (Lorenz & Parks, 1990) and the mould Tolypocladium inflatum (Baranovaet al,1996; Bibikovaet al,2004) were grown in its presence. We have confirmed a decrease in ergosterol levels in simvastatin-treated Candida (unpublished data). Lower ergosterol levels are expected to arise as a result of inhibition of HMG-CoA reductase, a key enzyme in mevalonate biosynthesis. It is notable that rescue with ergosterol or cholesterol of the statin-induced growth inhibition in our studies has been incomplete. This could be due to sterol uptake rates being growth limiting or due to competition. Alternatively, it should be noted that other effects might be expected, given the number of end products resulting from the mevalonate pathway or the effects on the membrane caused by ergosterol depletion. For example, reduced protein prenylation has been shown to be a major effect of lovastatin in Mucor racemosus, where reduced prenylation of Ras proteins results in apoptosis-like cell death (Roze & Linz, 1998). Experiments in rich media that contained an unspecified amount of ergosterol also demonstrated a statin-induced growth inhibition, albeit at higher statin levels, than required when ergosterol was absent.

The results indicate that statins may have a potential role as antifungal agents. In addition to the previously mentioned studies with lovastatin, Chin (1997) found that lovastatin, pravastatin and simvastatin had no antifungal activity in an agar well drug diffusion assay. However, this may be due to a lack of activity of the prodrugs lovastatin and simvastatin. We found it necessary to activate simvastatin by hydrolysis to produce the active component that normally arises by metabolic alteration. It is likely that the statins used by Chin (1997) were not being activated by hydrolysis to produce the active component. In addition, drug diffusion assays also depend on drugs being amenable to diffusion. Chin (1997) showed that fluvastatin (which is not a prodrug) was an active antifungal against Candida species and against Cryptococcus neoformans. In addition, when fluvastatin was combined with fluconazole or itraconazole, substantially synergistic antifungal activities were demonstrated (Chinet al,1997), although another study failed to find synergy (Nashet al,2002). It would appear that there may be potential benefits in new antifungal therapies when considering statins alone and in combination with existing antifungal agents.

Our work shows that simvastatin and atorvastatin, and we suggest other statins too, may be effective as antifungal agents, although our work indicates that C. krusei is refractory to these statins. It is of interest that C. krusei displays insensitivity to the azole drugs that also target ergosterol synthesis (Orozcoet al,1998). The levels of statins required for inhibition in our studies are similar to the levels used in the treatment of hypercholesterolaemia (Corsiniet al,1999). A complicating factor in treatment of systemic infections with statins, however, would be the high levels of cholesterol. For example, a typical level of serum cholesterol is 2mgmL⁻¹ (5mM) and we have demonstrated that levels as low as 12μgmL⁻¹ cholesterol can cause a 50% reduction in the statin-induced growth inhibition. Where there is an absence of competing cholesterol, statins may be able to provide more benefit.

The widespread use of statins in the human population and their differential effects on fungal species may suggest that statins could alter the normal pattern of fungal colonization. Furthermore, if statin treatments led to lower ergosterol levels in fungi carried by a statin-treated individual, would this lead to altered sensitivity to the many antifungal drugs (e.g. amphotericin, fluconazole) that target ergosterol or its production? We consider that our studies suggest a need for further investigations to answer these questions.

Acknowledgements

The provision of Aspergillus fumigatus and Candida strains by Dr Wieland Meyer and Dr John Warmington is gratefully acknowledged. We also thank Dr Paul Vaughan, Dr Anna Johnson and Dr Connie Darmanin for their helpful comments in the preparation of this manuscript.