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The involvement of cAMP in the growth inhibition of filamentous fungus Rhizopus nigricans by steroids

Nataša Jeraj, Helena Lenasi, Katja Breskvar
DOI: http://dx.doi.org/10.1016/j.femsle.2004.10.051 147-154 First published online: 1 January 2005

Abstract

Several steroids, in particular progesterone, are toxic for the filamentous fungus Rhizopus nigricans and, at high concentrations, inhibit its growth. Previous studies on this microorganism revealed progesterone specific receptors coupled to G proteins at the plasma membrane. In this study, the next step of steroid signalling in R. nigricans following G protein activation is investigated, together with the possible impact of this pathway on fungal growth inhibition. The intracellular level of cAMP decreased in the presence of steroids, demonstrating the probable involvement of cAMP signalling in the response of R. nigricans to steroids. Results of the growth analysis in the presence of cAMP increasing agents suggest that the role of cAMP in fungal growth inhibition by steroids cannot be ruled out, but it would appear to be minor and not make a major contribution to growth inhibition.

Keywords
  • Steroid
  • Rhizopus nigricans
  • Growth inhibition
  • G protein
  • cAMP

1 Introduction

The investigation of progesterone metabolism in filamentous fungus Rhizopus nigricans (class Zygomycetes) dates from the discovery of inducible enzymes in this microorganism that convert progesterone into the pharmaceutically interesting 11α-hydroxyprogesterone [1]. The progesterone-induced response of the fungus is twofold, one leading to inhibition of fungal growth, and the other, to fungal defence against toxic steroids, including the induction of steroid hydroxylases [2]. An investigation of progesterone signalling revealed progesterone receptors in the cytosol, pointing to the classical action of progesterone [3]. In addition, receptors coupled to trimeric guanine nucleotide binding proteins (G proteins) were found in the plasma membrane, indicating the non-classical action of progesterone [4]. Previous results indirectly suggest the involvement of cytosolic receptors in the induction of progesterone hydroxylase [5], although the biological role of progesterone binding to membrane receptors, as well as of the resulting signalling pathway, is not known.

Signalling cascades that regulate cell function exhibit a high degree of conservation between fungi and animals [6]. Signalling via membrane receptors coupled to G proteins is very important and is well established in animals. Binding of an inducing ligand, such as a neurotransmitter or hormone, to a G protein-coupled receptor triggers activation of the G protein. This involves GDP-to-GTP exchange of the guanine nucleotide bound to the Gα subunit, followed by release of the latter from the Gβγ dimer [7]. Effectors such as adenylyl cyclase can then be stimulated by either Gα or Gβ[8,9]. Although non-classical steroid signalling through G proteins has not been studied in fungi other than R. nigricans, there are numerous examples of this signalling in higher organisms, including progesterone-mediated maturation of amphibian oocytes [10]. Progesterone induces the resumption of meiosis (maturation) in Xenopus oocytes through a non-classical mechanism involving G protein-coupled receptors and heterotrimeric G proteins, as well as inhibition of an oocyte adenylyl cyclase and transient reduction of intracellular cAMP levels [11]. Progesterone action in Xenopus oocytes is not blocked by pertussis toxin (PTX), an inactivator of Gi/o type of G proteins, indicating that α subunits of Gi type of G proteins are not involved in the inhibition of the oocyte adenylyl cyclase. It was demonstrated that G protein βγ subunits, rather than α subunits, play a key role in regulating oocyte maturation [11].

Fungi employ cAMP/protein kinase A signalling in a variety of processes. cAMP is a critical mediator of growth and response to nutritional stress in Saccharomyces cerevisiae [12]. Proper pathway function is also required for mating and sporulation in the fission yeast Schizosaccharomyces pombe, and differentiation and virulence of the pathogenic fungi, Cryptococcus neoformans and Ustilago maydis [13]. So far, non-classical progesterone action via the cAMP signalling pathway has not been reported in fungi. In this study, we examined the cAMP content in R. nigricans in response to progesterone and other steroids, as well as the involvement of the G protein/cAMP pathway in steroid-induced inhibition of fungal growth.

2 Materials and methods

2.1 Materials

Cyclic AMP [3H] assay system was from Amersham Biosciences UK Limited (Little Chalfont, England). PTX, cholera toxin (CTX), forskolin, 8-bromo-cAMP (8-Br-cAMP) and all other chemicals were obtained from Sigma–Aldrich (St. Louis, MO).

2.2 Microorganism and media

Filamentous fungus R. nigricans B154 (class Zygomycetes) obtained from Microbial Culture Collection of National Institute of Chemistry (Ljubljana, Slovenia) was grown in liquid nutrient medium in a rotary shaker at 28 °C (180 rpm) for 18 h [14].

The growth medium used for steroid cytotoxicity and germination kinetics assays was wort agar. Wort agar was prepared from filtered, autoclaved brewery lager wort and distilled water (1:1, v/v) to which 2% agar was added. The final pH of the medium was 6.5.

2.3 Hyphal growth inhibition assay

The inhibition of hyphal growth by progesterone at concentrations from 5 to 300 μM and inhibition by 300 μM deoxycorticosterone, testosterone, estradiol and 11α-hydroxyprogesterone were evaluated semi-quantitatively. Steroids were dissolved in dimethylformamide. Conidia of R. nigricans were harvested with sterile distilled water and inoculated on the centre of wort media plates containing various steroids and incubated at 28 °C for 21 h in the dark. Apical extension was determined by measuring colony diameters considering the size of a spot where conidia were inoculated onto agar medium. To check the involvement of cAMP signalling pathway in the growth inhibition by 15 and 100 μM progesterone, fungal growth was examined in the presence of cAMP increasing agents in the media: 0.1, 0.5 and 1.0 mM 8-Br-cAMP and 10 μM forskolin. The concentrations of 8-Br-cAMP and forskolin were chosen based on work in other filamentous fungi as described by Rosenberg and Pall [15] and Kinane and Oliver [16], respectively. Forskolin was dissolved in dimethyl sulfoxide. Vehicle controls were performed with the appropriate amount of the respective solvent. Each experiment was conducted at least three times.

2.4 Germination kinetics

Conidiospore germination was monitored by microscopic examination of slides coated with wort agar medium (prepared as described above) containing 300 μM progesterone, deoxycorticosterone, testosterone, estradiol or 11α-hydroxyprogesterone and spot inoculated with 1–5 × 104 conidiospores. Spores were incubated at 28 °C. The percentage of germinated spores was followed in time by examining 50–100 spores in at least two microscopic fields at 30 min intervals.

2.5 Measurement of intracellular cAMP concentrations

Eighteen-hour fungus grown in the liquid culture (as described above) was treated with 15 μM solutions of various steroids (progesterone, deoxycorticosterone, testosterone and estradiol) and, in the accompanying experiment, with 10 μM forskolin for 10 min. The mycelia were ground to a fine powder in liquid nitrogen and extracted in 10 mM Tris and 4 mM EDTA buffer, pH 7.5. Samples were centrifuged at 10,000g at 4 °C for 10 min. cAMP content in the supernatant of each sample was determined using a commercially available cAMP [3H] assay system following instructions of the manufacturer (Amersham Biosciences). Total protein concentration was determined in each sample using the Bradford protein assay. The data from at least duplicate experiments were analysed.

To assess the involvement of Gα proteins in the growth inhibition by progesterone, cAMP content was determined in PTX- or CTX-treated samples. After growing R. nigricans for 6 h, growth medium was supplemented with 40 ng ml−1 PTX or 200 ng ml−1 CTX [17]. Fungal growth proceeded for an additional 12 h. The fungus was then treated with 15 μM progesterone for 10 min. Intracellular cAMP concentration was determined as described above.

2.6 Statistical analysis

All statistics were done with Microsoft Excel program and statistical significance was calculated by one tailed, paired Student's t-test for means with 95% confidence interval.

3 Results and discussion

3.1 Effect of steroids on the growth of R. nigricans

Steroids are toxic for R. nigricans and, at 300 μM concentration, inhibit fungal growth in agar medium (Figs. 1 and 2). Progesterone almost completely inhibited growth after 21 h (99.0 ± 1.0%), whereas the inhibition by deoxycorticosterone, testosterone, estradiol and 11α-hydroxyprogesterone was partial, 78.8 ± 7.8, 64.5 ± 9.5, 26.0 ± 6.6 and 16.9 ± 4.4%, respectively. In addition to steroid induced inhibition of fungal growth, colonies exhibited major changes in morphology when cultivated in progesterone-containing media (Fig. 2). Growth in the presence of 300 μM progesterone (Fig. 2(b)) was more compact and not so fluffy as in the control plate (Fig. 2(a)), probably as a consequence of enhanced hyphal branching. The different branching pattern due to progesterone is clearly seen in lower panel of Fig. 2(b) since, after prolonged time of growth (25 h), slow apical extension was detected in the presence of progesterone. The morphology of colonies and the hyphal branching did not change much when 300 μM 11α-hydroxyprogesterone was added to the medium (Fig. 2(c)).

Figure 1

Growth inhibition of R. nigricans by steroids. R. nigricans was spot inoculated on the centre of wort agar plates containing various steroids at 300 mM and allowed to grow for 21 h at 28 °C. Apical extensions were determined by measuring colony diameters. The values represent the inhibition of mycelial elongation in the presence of steroids as a percentage of the control value, and are the mean of three independent determinations; *p < 0.05 versus growth control.

Figure 2

Steroids inhibit growth and change the morphology of R. nigricans. Fungal growth after 21 h at 28 °C in wort agar medium: (a) without addition of steroids, (b) with 300 μM progesterone and (c) with 300 μM 11α-hydroxyprogesterone. Hatched circle in (a) indicates area where conidia from R. nigricans were inoculated onto agar medium. Lower panels represent a magnification of the colonies in upper panels after 25 h of growth. The scale bar shown in (a) is 100 μm.

Previous studies on R. nigricans demonstrated that some steroids are subject to 11α-hydroxylation [18]. The hydroxylated products are less toxic since they are more water-soluble and, in a liquid medium, easily removed from the cell, leading to a lower content of toxic steroids [2]. In the present study agar medium was used and, in this respect, results are not directly comparable. Our previous results showed a reduction in dry weight of the fungus grown in the presence of progesterone, testosterone, deoxycorticosterone and estrone, each in a slightly different manner. Deoxycorticosterone was a very efficient growth inhibitor while progesterone, estrone and testosterone had only slight inhibitory effects [2]. A similar example of differences in reduction of fungal growth in solid and liquid media was reported for the Gαi mutant of Neurospora crassa [19]. The inhibition of fungal growth in the presence of progesterone, testosterone and estradiol was also demonstrated for dermatophytes, using an agar dilution assay [20].

The influence of progesterone on the growth of R. nigricans was dose-dependent (Fig. 3). At 15 μM concentration of progesterone only minor effects on fungal growth were detected, at 100 μM the growth inhibition was approximately 50% and at 300 M growth was completely blocked. It is known that steroid hormones can intercalate into the bilayer of target cell plasma membranes producing a non-specific, dose-dependent effect on membrane fluidity and function of the membrane [21]. Moreover, intercalation of lipophilic steroids into membranes, resulting in perturbation of lipid–lipid interaction, may in turn alter the function of membrane proteins [22] such as, in our case, G protein coupled receptors and consequently, the following signalling pathways. We therefore selected 15 and 100 μM concentrations for further studies of the impact of progesterone on fungal growth. For studies of steroid interference with fungal signalling pathways we chose 15 μM concentration of steroids; this being the lowest concentration of progesterone with measurable effect on fungal growth and presumably also low enough to follow specific signalling triggered by steroids.

Figure 3

Dose-dependent growth inhibition of R. nigricans by progesterone. The growth of R. nigricans was assayed in the presence of various concentrations of progesterone in wort agar medium. After 21 h of fungal growth at 28 °C apical extensions were determined by measuring colony diameters. The values represent the mycelial elongation in the presence of progesterone as a percentage of the control value, and are the mean of three independent determinations.

To analyse the impact of possible conidiospore germination delay on fungal growth inhibition by steroids, germination was examined on solid medium. The germination kinetics of R. nigricans conidia were similar in the presence of 300 μM estradiol, 11α-hydroxyprogesterone and control (Fig. 4). Germination of conidia in the medium with addition of 300 μM progesterone was slightly delayed (30 min), but conidia in the presence of 300 μM deoxycorticosterone and testosterone germinated markedly more slowly than conidia in the control medium (1.0–1.5 h). Therefore, we can exclude the possibility that the dramatic growth inhibition on plates with addition of 300 μM progesterone could be due to slight germination delay, although slower germination in the presence of 300 μM deoxycorticosterone or testosterone could in part contribute to fungal growth inhibition.

Figure 4

Germination kinetics of R. nigricans in the presence of different steroids. Germination was monitored as described in Section 2 using wort agar medium at 28 °C. The effects of following 300 μM steroids were analysed: Embedded Image , control; Embedded Image, progesterone; ○, deoxycorticosterone; ▪, testosterone; ◻, estradiol; ●, 11α-hydroxyprogesterone. Each point is the average of three independent determinations. Standard errors of the mean never exceeded 15% and are omitted for clarity.

3.2 Involvement of cAMP signalling in fungal response to steroids

It is well established that hormonal stimulation of cAMP and the cAMP-dependent protein kinase A regulates cell growth by multiple mechanisms [23]. Both positive and negative effects of cAMP on growth have been demonstrated in fungi; for example, cAMP activates filamentous growth in S. cerevisiae, but plays a negative role and inhibits filamentous growth in U. maydis [6]. In Aspergillus niger, growth is reduced when glucose containing medium is supplemented with cAMP [24], while activation of adenylyl cyclase and the production of cAMP are necessary for normal vegetative growth and asexual development in A. nidulans [25]. N. crassa adenylyl cyclase mutants produce abundant conidia and have very low apical extension rates [26].

To determine the involvement of steroid signalling via cAMP, R. nigricans was exposed to progesterone, deoxycorticosterone, testosterone and estradiol at 15 μM concentration for 10 min, and the intracellular concentration of cAMP was assayed. In the accompanying experiment for testing the reliability of the method the intracellular concentration of cAMP was elevated after 10 min treatment with 10 μM forskolin, a direct activator of adenylyl cyclase. As shown in Fig. 5, all these steroids decreased cAMP levels in R. nigricans but significantly so only in the case of testosterone and progesterone. This reduction could be due to inhibition of adenylyl cyclase activity or stimulation of the cAMP inactivating enzyme cyclic nucleotide phosphodiesterases.

Figure 5

Effect of steroids and forskolin on the intracellular cAMP concentration. R. nigricans grown in nutrient medium for 18 h was exposed to various 15 μM steroids and to 10 μM forskolin for 10 min. cAMP content was determined using cAMP [3H] assay system and is expressed in pmol cAMP (mg protein)−1. Black bars, control; hatched bars, treatment with steroids. The data shown are means ± SD of duplicate determinations of three separate experiments; *p < 0.05 compared to corresponding control.

Although all the steroids had a similar effect on cAMP levels in R. nigricans, the inhibition of growth differed. Fifteen-micromolar concentration of steroids used in the analysis of intracellular cAMP level might be sufficiently high for all steroids to saturate steroid binding sites in the plasma membrane and consequently to activate the cAMP signalling pathway. Binding sites for different steroids have been detected in the plasma membrane of R. nigricans [4]. On the other hand, 15 μM concentration was still too low for non-specific effects on the membrane to be manifested [21]. Growth inhibition was studied at rather high steroid concentrations (100 μM) when non-specific effects at the membrane level occur. The difference in growth inhibition of the steroids tested at this concentration is best explained if the growth inhibition is partly due to the different non-specific interactions of steroids with membranes. Shivaji and Jagannadham [27] reported that progesterone decreases the fluidity of hamster spermatozoal membranes, whereas testosterone and estradiol at the same concentration had very little effect.

Our previous results showed that progesterone binds specifically to R. nigricans plasma membrane sites [28] and that further steps in the non-classical action of progesterone on R. nigricans include heterotrimeric G protein activation [4]. Since progesterone caused a decrease of intracellular cAMP in our system (Fig. 5), we anticipated that α subunits of Gi type of the heterotrimeric G proteins, rather than the Gs type, might be involved in progesterone signalling. To test this hypothesis, we added 40 ng ml−1 PTX or 200 ng ml−1 CTX to the growth medium. PTX and CTX ADP-ribosylate Gαi and Gαs, thus causing permanent inhibition of Gαi [29] and activation of Gαs [30], respectively, and, in both cases, activation of adenylyl cyclase. R. nigricans grown under these modified culture conditions retained the ability to respond to 15 μM progesterone by decreasing intracellular cAMP concentration (Table 1). In fact, PTX and CTX increased the basal concentration of cAMP, resulting in an even larger decrease of cAMP content after addition of progesterone in PTX- (50.0% of basal) and CTX- (61.4% of basal) treated cells compared with untreated cells (69.1% of basal). In the absence of progesterone, PTX induced a large increase in cAMP content, whereas CTX induced only a slight increase. This could mean that the Gi type of G proteins is more abundant than Gs. Similar results were obtained for mouse membranes by Bégin-Heick [31]. Since treatment with PTX or CTX did not affect the progesterone-induced decrease in cAMP level, we suggest that neither Gαi nor Gαs are involved in the R. nigricans response to progesterone.

View this table:
Table 1

Effect of PTX and CTX on the decrease of intracellular cAMP concentration in response to progesterone

PTX/CTX additionIntracellular cAMP level (pmol mg protein−1)
No pretreatment (basal)Progesterone (15 μM) (% of basal)
No addition24.7 ± 14.317.1 ± 11.0a (69.1)
PTX41.3 ± 0.7b20.6 ± 7.1a (50.0)
CTX27.9 ± 15.3b17.2 ± 12.8a (61.4)
  • Note. After growing R. nigricans for 6 h, we added PTX to 40 ng ml−1 or CTX to 200 ng ml−1 in the growth medium. Fungal growth was allowed to continue for an additional 12 h. The fungus was then treated with 15 μM progesterone for 10 min, followed by determination of cAMP content using the cAMP [3H] assay system. The results are expressed in pmol cAMP (mg protein)−1 and as a percent of the basal cAMP concentration. The data shown are means SD of duplicate determinations of two separate experiments.

  • aIndicates a significant difference (p < 0.05) of samples treated with progesterone compared to corresponding samples not treated with progesterone (basal cAMP level).

  • bIndicates a significant difference (p < 0.05) of samples grown in the presence of PTX or CTX compared with samples grown without PTX/CTX (indicated only for samples not treated with progesterone).

Given that progesterone inhibited fungal growth in solid medium and simultaneously lowered the content of cAMP, we tried to reduce this inhibition by adding a cell-permeable cAMP analogue (0.1, 0.5 and 1.0 mM 8-Br-cAMP), or an agent that stimulates the production of cAMP (10 μM forskolin). In the presence of these agents the growth inhibition by 15 and 100 μM progesterone was seen to be less marked (Fig. 6), but they had no influence on growth by themselves (results not shown). These results indicate that the impairment of fungal growth by progesterone is connected to lowered cAMP content. In spite of the presence of cAMP elevating agents, 100 μM progesterone was still able to inhibit fungal growth remarkably, while growth inhibition by 15 μM progesterone was almost completely blocked. 0.5 mM concentration of 8-Br-cAMP was sufficient to reduce growth inhibition since, in the presence of higher concentration of 8-Br-cAMP (1.0 mM), the reduction of growth inhibition remained the same. This supports the hypothesis that, in addition to the cAMP pathway, progesterone interacts non-specifically with various membranes and in this way interferes directly with fungal growth. Accordingly, the non-specific interactions with membranes were more profound at higher concentration of progesterone.

Figure 6

Effect of 8-Br-cAMP and forskolin on the progesterone-induced growth inhibition of R. nigricans. R. nigricans was spot inoculated on the centre of wort agar plates containing 15 μM (open bars) or 100 μM (hatched bars) progesterone and/or cAMP increasing agents: 0.1, 0.5 and 1.0 mM 8-Br-cAMP and 10 μM forskolin. After 21 h of growth, apical extensions at 28 °C were determined by measuring colony diameters. The values represent the mycelial elongation after various treatments as a percentage of the control value, and are the mean of three independent determinations; *, indicates a significant difference (p < 0.05) of samples grown in the presence of 15 μM progesterone and cAMP increasing agents compared with samples grown in the presence of 15 μM progesterone alone; #, indicates a significant difference (p < 0.05) of samples grown in the presence of 100 μM progesterone and cAMP increasing agents compared with samples grown in the presence of 100 μM progesterone alone; all the samples were significantly different (p < 0.05) from the control.

4 Concluding remarks

In our previous studies on the isolated plasma membrane fraction of R. nigricans we identified specific progesterone receptors coupled to G proteins, which were activated by progesterone in concentrations up to 100 nM [4]. In the present study we were concerned with the subsequent steps of progesterone signalling and, in addition, aimed to find a possible biological role for this signalling pathway. To this end, we studied the effects of several steroids at μM levels in vivo. Although all the steroids reduced cAMP content in R. nigricans similarly, the inhibition of growth at higher steroid concentrations differed. The difference in growth inhibition under these experimental conditions is best explained if the growth inhibition is due partially to non-specific steroid interaction with membranes. These studies suggest that the role of cAMP in fungal growth inhibition by steroids cannot be ruled out, but it would appear to be minor and not make a major contribution to growth inhibition.

Acknowledgements

We thank Dr. Rok Romih for photomicrographs of hyphal development and Marjan Kužnik for excellent technical assistance. This work was supported by the Ministry of Education, Science and Sport of Slovenia.

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