Antifungal activity of cercosporamide produced by Phaeosphaeriaceae GV-1 against pathogenic fungi

Abstract

Fungal infections affect millions of people worldwide, and the several cases are related to invasive infections, which is a problem mainly for immunocompromised people, such as transplant and cancer patients with high mortality and morbidity rates. In addition, the number of emerging and multidrug-resistant fungal species has increased in the last decade. The search for new antifungal compounds is necessary, due to the increase in cases of resistance and the toxicity of drugs used in fungal infection treatment. This work aimed to study the antifungal activity of cercosporamide produced by Phaeosphaeriaceae GV-1. Cercosporamide was tested against pathogenic fungi by determining the minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations, using the broth microdilution method. Cercosporamide showed antifungal activity in vitro against 13 of 16 strains of medical importance tested, with the most susceptible species being Candida tropicalis, with MIC and MFC of 15.6 μg/mL. Thus, cercosporamide might be considered a promising therapeutic antifungal agent.

Introduction

Superficial fungal infections affect about 25% of the world population, being mainly caused by Candida species [1]. Invasive fungal infections are associated with high mortality rates in patients with compromised immune systems [1]. According to the World Health Organization, in 2016, deaths caused by fungal infections were the fifth leading cause of mortality in the world, and invasive infections are the most severe, causing about 1.6 million deaths per year [2].

Candida species are the most frequent causes of fungal infections, with Candida albicans being the most frequently isolated species, followed by Candida tropicalis, Candida parapsilosis, and Candida glabrata, with some clinical isolates resistant to the reported classes of antifungal agents. In addition, Candida auris and closed species have emerged, and they are considered multidrug-resistant strains [3]. There is also an increasing threat from pathogenic molds that are intrinsically resistant to a wide range of antifungals, such as Aspergillus terreus, Scedosporium spp., Fusarium spp., and members of the order Mucorales [4].

Currently, the therapeutic choices for the treatment of invasive fungal infections are limited to three main classes of antifungal agents: polyenes, azoles, and echinocandins. However, these drugs have several disadvantages associated to toxicity, spectrum of action, emergence of resistant strains, and unfavorable pharmacokinetic properties [5].

In previous studies by our research group, a fungus strain of the family Phaeosphaeriaceae was isolated from guava and identified as a producer of a metabolite compound with inhibitory activity against fungi of agricultural relevance [6]. The active principle related to this antifungal activity was identified as cercosporamide by chromatographic techniques coupled with mass spectrometry MS/MS and nuclear magnetic resonance (NMR) [7]. There are few studies in the literature on cercosporamide (Fig. 1), but the existing ones report biological properties of great interest, such as anticancer activity, blood glucose reduction, herbicidal activity, and antifungal activity [9–12].

Fig. 1.

Cercosporamide chemical structure [8]

Cercosporamide is a highly selective inhibitor of Pkc1 (Protein kinase C-like 1), a protein kinase that mediates the maintenance of cell wall integrity, being essential for the biosynthesis of this structure. The signaling pathway mediated by Pkc1 is highly conserved in fungi, and the loss of the function of this protein leads to rapid cell autolysis, presenting great potential as a molecular target for the development of antifungal agents [8]. Cercosporamide is the only compound exhibiting antifungal activity associated with Pkc1 inhibition described in the literature, presenting a new strategy for the development of antifungal agents [13].

Therefore, this work aims to expand the knowledge about the properties of cercosporamide by evaluating the antifungal activity of cercosporamide produced by Phaeosphaeriaceae GV-1 against pathogenic fungi.

Material and methods

Microorganisms

A fungal isolate of the family Phaeosphaeriaceae (strain GV-1) was used for the production of cercosporamide, as described in previous reports [6, 7, 14]. The antifungal activity of cercosporamide was evaluated against 15 human pathogen species of yeasts and molds: Candida albicans (SC5314), Candida glabrata (ATCC 2001), Candida krusei (ATCC 6258), Candida parapsilosis (ATCC 22019), Candida tropicalis (ATCC 200956), Candida auris (CBS 10913 and CBS 12766), Candida duobushaemulonii (CBS 7799), Candida pseudohaemulonii (CBS 10004), Cryptococcus gattii (ATCC 56990), Cryptococcus neoformans (H99), Aspergillus fumigatus (ATCC 16913), Aspergillus flavus (998), Fusarium solani (CBS 224.34), Cunninghamella sp. (strain 196), and Lomentospora prolificans. The strains used are part of the culture collection from Laboratory of Antifungal Chemotherapy (LQA), Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, and stored in brain and hearth infusion broth with 20% glycerol at −80 °C.

Antifungals

Amphotericin B (AMB), voriconazole (VCZ), fluconazole (FLC), and caspofungin (CAS) were dissolved in dimethyl sulfoxide (DMSO) (all from Sigma-Aldrich, St. Louis, USA) and the stock solutions were stored at −20 °C. For antifungal activity evaluation, the drugs were diluted to the final concentration in culture media.

Cercosporamide production

Fermentation was carried out in a bioreactor (New Brunswick Bioflo 310; 5 L; pitched blade impeller), using YM liquid medium (10 g/L glucose, 5 g/L peptone, 3 g/L malt extract, and 3 g/L yeast extract) at 25 °C/100 rpm for 96 h. Inoculation was performed with the fungus GV-1 previously activated in a Petri dish containing solid YM medium (20 g/L of agar), in the proportion of 1 cm² of fungal culture per 100 mL of YM medium, crushed with Ultra Turrax IKA T25 [6].

The recovery and purification of cercosporamide (CCP) were carried out in accordance with the methodology described in a patent application filed by our research group [7]. For extraction, 200 mL of ethyl acetate was added to 200 mL of the fermented medium previously filtered in a 0.22-μm filter. After homogenization and phase separation, the acetoethylic phase was alkalinized with 40 mL of 0.1 M NaOH solution. Subsequently, the aqueous phase (alkaline CCP solution) was recovered and neutralized with 8 mL of 2 M HCl solution. The resulting solution was filtered through a 0.45-μm filter and dried at 50 °C overnight. According to the patent application, the powder obtained contained c.a. 94% CCP (% area at 254 nm), which is similar to the purity of the cercosporamide commercial standard (95%). This powder was stored in microtubes at −18 °C until use. For the antifungal tests, CCP-containing powder was solubilized in a 0.1 M NaOH solution, followed by pH correction to 7 with a 1 M HCl solution and adjustment to a concentration of 4000 μg/mL with sterile distilled water.

Antifungal activity evaluation

Minimum inhibitory concentration (MIC) was determined by using the broth microdilution technique, following protocol M27-A3 for yeast and M38 for filamentous fungi [15, 16]. The concentrations tested for each compound ranged from 0.03 to 16 μg/mL (AMB, VCZ and CAS), 0.12 to 64 μg/mL (FLC), and 1.95 to 1000 μg/mL (CCP), diluted in RPMI 1640 medium buffered with 0.16 M MOPS into wells of a 96-well flat-bottom plate. The MIC values were determined by visual reading and were defined as the lowest concentration inhibiting 90% of fungal growth for AMB and 50% for other drugs and CCP.

To assess the minimum fungicidal concentration (MFC), 10 μL aliquots from each well from the MIC assay that did not show visible growth were inoculated into Potato Dextrose Agar medium, and incubated at 35 °C for 48 h. MFC is defined as the lowest concentration that kills 99.9% of cells compared to the initial inoculum by colony forming unit count [17]. All determinations were performed in independent triplicates and two biological replicates. The final MIC and MFC values were considered modal mean.

Results

The growth of C. albicans, C. glabrata, and C. krusei was not inhibited by CCP within the tested concentration range. All other yeasts were susceptible to the compound, with MIC ranging from 15.6 to 500 μg/mL and C. tropicalis being the most susceptible to CCP (MIC = 15.6 μg/mL) (Table 1). It is noteworthy that this strain presents resistance to AMB and azoles (VCZ and FLC) (Table 1). CCP had a fungicidal effect against C. auris CBS 12766, C. parapsilosis, C. duobushaemulonii, C. tropicalis, Cryptococcus gatti, and Cryptococcus neoformans, with concentrations from 15.6 to 1000 μg/mL (Table 2).

Table 1.

Inhibitory activity of amphotericin B (AMB), voriconazole (VCZ), fluconazole (FLC), caspofungin (CAS), and cercosporamide (CCP) against pathogenic yeasts

Strains	MIC (μg/mL)
	AMB	VCZ	FLC	CAS	CCP
Candida albicans	≤0.03	≤0.03	≤0.12	0.06	>1000
Candida glabrata	≤0.03	0.50	4SDD	0.12	>1000
Candida krusei	0.12	0.5	>64R	0.25	>1000
Candida parapsilosis	≤0.03	0.06	1	0.50	250
Candida tropicalis	2R	>16R	>64R	0.12	15.6
Candida auris CBS 12766	0.25	0.50	>64	0.25	500
Candida auris CBS 10913	0.06	≤0.03	2	<0.03	125
Candida pseudohaemulonii	2	0.12	4	0.12	125
Candida duobushaemulonii	0.50	2	8	0.06	62.5
Cryptococcus neoformans	≤0.03	0.06	2	>16	62.5
Cryptococcus gatti	≤0.03	0.12	2	8	125

^RResistant, ^SDDSusceptible-dose-dependent

Table 2.

Fungicidal effect of fluconazole (FLC) and cercosporamide (CCP) against pathogenic yeasts

Strains	MFC (μg/mL)
	FLC	CCP
Candida albicans	>64	>1000
Candida glabrata	32	>1000
Candida krusei	>64	>1000
Candida parapsilosis	2	500
Candida tropicalis	>64	15.6
Candida auris CBS 12766	>64	1000
Candida auris CBS 10913	>64	>1000
Candida pseudohaemulonii	>64	>1000
Candida duobushaemulonii	>64	500
Cryptococcus neoformans	4	500
Cryptococcus gatti	8	125

The filamentous fungi showed less susceptibility to CCP, with MIC values ranging from 62.5 to 500 μg/mL (Table 3). Indeed, CCP had a fungicidal effect on all filamentous fungi tested, except for F. solani (Table 4). Cunninghamella sp. was the most susceptible to CCP, with MIC of 62.5 and MFC of 500 μg/mL, while this compound was the only one with fungicidal action against L. prolificans (MFC = 250 μg/mL), considering the ranges tested. All filamentous fungi were resistant to FLC, and only L. prolificans showed no resistance to CAS at the concentrations tested (Tables 3 and 4).

Table 3.

Inhibitory effect of amphotericin B (AMB), voriconazole (VCZ), fluconazole (FLC), caspofungin (CAS), and cercosporamide (CCP) against pathogenic filamentous fungi

Strains	MIC (μg/mL)
	AMB	VCZ	FLC	CAS	CCP
Aspergillus flavus	0.12	0.06	>64	>16	500
Aspergillus fumigatus	0.12	0.50	>64	>16	500
Fusarium solani	0.50	>16	>64	>16	250
Lomentospora prolificans	4	1	>64	8	250
Cunninghamella sp.	0.5	1	>64	>16	62.5

Table 4.

Fungicidal effect of amphotericin B (AMB), voriconazole (VCZ), fluconazole (FLC), caspofungin (CAS), and cercosporamide (CCP) against pathogenic filamentous fungi

Strains	MFC (μg/mL)
	AMB	VCZ	FLC	CAS	CCP
Aspergillus flavus	1	>16	>64	>16	500
Aspergillus fumigatus	4	4	>64	>16	1000
Fusarium solani	0.50	>16	>64	>16	>1000
Lomentospora prolificans	>16	>16	>64	>16	250
Cunninghamella sp.	4	>16	>64	>16	500

Discussion

Many of the clinical isolates of C. auris are resistant to standard antifungals, with some being reported to be multidrug-resistant, i.e., resistant to the three main classes of antifungal agents used: echinocandins, azoles, and polyenes. Its effective treatment is still a challenge, hence the great interest in the discovery of new compounds effective against C. auris [18]. The C. auris strains tested herein showed an intermediate susceptibility to CCP when compared with other fungal species tested in this study. The growth of C. auris CBS 12766 strain was inhibited by CCP with a MIC of 500 μg/mL and MFC of 1000 μg/mL, but this strain was resistant to FLC within the concentration range tested. In contrast, C. auris CBS 10913 was susceptible to all the compounds tested, being inhibited by CCP (MIC = 125 μg/mL), but with no fungicidal effect. These results are similar to the inhibition range obtained by other natural compounds, such as crotamine, which presented a 50% inhibition of C. auris growth at a concentration range of 400 to 800 μg/mL [19].

C. parapsilosis is one of the most common Candida species from the non-albicans group causing invasive candidiasis [20]. Synthetic arylquinuclidine derivatives, which are inhibitors of squalene synthase, have been tested against C. parapsilosis, and the most effective compound presented a MIC₅₀ of 2 μg/mL, but no fungicidal effect [21]. In terms of CCP, a MIC value of 64 μg/mL was reported for C. parapsilosis [10], a lower value compared to the MIC obtained for this yeast in this study (250 μg/mL). Such variations are normally observed between different studies. For instance, MIC of CCP against C. albicans varied from 81 [10] to 10 μg/mL [8], depending on the study.

In this study, two species of the C. haemulonii complex were tested: C. pseudohaemulonii, for which CCP inhibited growth (MIC of 125 μg/mL) with no fungicidal effect; and C. duobushaemulonii, for which CCP inhibited growth (MIC of 62.5 μg/mL) and also presented a fungicidal effect (MFC of 500 μg/mL). These species belong to the family Metschnikowiaceae, which usually includes antifungal-resistant strains. Even though they are still not a frequent cause of candidemia, their genetic similarities with C. auris increase the awareness that species from this group may also emerge as drug-resistant pathogens in human populations [22]. In Korea, clinical isolates of C. pseudohaemulonii showed a MIC of 32 μg/mL for AMB, which is considered a high-level resistance for this drug [23], while clinical isolates of C. duobushaemulonii in Argentina had a MIC of 8, 128, 6.34, and 5.03 μg/mL for AMB, FLC, VCZ, and CAS, respectively [24].

The most susceptible yeast to CCP was C. tropicalis, which was inhibited at a concentration of 15.6 μg/mL, a concentration that also had a fungicidal effect. This strain of C. tropicalis was resistant to AMB and azoles as previously described [25]. C. tropicalis is the second most prevalent pathogenic Candida species (the first being C. albicans) and the most prevalent in the non-albicans Candida species group. The number of infections by C. tropicalis has increased dramatically worldwide, characterizing it as an emerging pathogenic yeast, and an aggravating factor in this scenario is the emergence of antifungal resistant isolates of C. tropicalis [26]. Clinical and experimental data suggest that morbidity and mortality rates are higher in infections by C. tropicalis than by C. albicans [27]. C. parapsilosis has been another threat in nosocomial environments with the recent emergence of multidrug-resistant and echinocandin-tolerant isolates [28]. Thus, the search for new antifungal compounds capable of inhibiting the growth of C. tropicalis, such as CCP, is of great interest. In former studies, the MIC₅₀ values reported for C. tropicalis were 2 μg/mL (no fungicidal effect) for synthetic derivatives of arylquinuclidines [21], 8 μg/mL for the antimicrobial peptide Lycosine-I [29], and 125 μg/ mL for the essential oil of Pelargonium graveolens [30]. In terms of CCP, the MIC value was 128 μg/mL [10], considerably higher than the MIC obtained in this study.

Cryptococcus neoformans is an opportunistic yeast responsible for cryptococcosis, a systemic infection clinically manifested as meningoencephalitis, from subacute to chronic, in immunocompromised patients. This condition is usually treated with AMB and flucytosine, but the high incidence of the disease, antifungal resistance, and the side effects caused by the drugs used have encouraged the search for alternatives to this treatment. The natural sources of antifungal agents against this yeast include thymol (MIC of 20 to 51 μg/mL and MFC in the range of 40 to 102 μg/mL depending on the strain) [31] and two new tetrahydrofuran derivatives isolated from the metabolites of Aspergillus sp. LS78 (MIC values of 50 and 128 μg/mL) [32]. The values for CCP obtained in this study for this yeast were in the same range as those examples.

Another Cryptococcus species evaluated in this study, C. gattii, can cause infections in immunocompromised and immunocompetent patients, and may produce severe neurological sequelae. The natural compounds previously tested against this yeast were the hexane-soluble fraction of the leaves of Lippia sidoides (a plant used in Brazilian folk medicine as an antiseptic), showing MIC of 62.5 μg/mL [33], and the hexane fraction of Eugenia calycina (another plant used in Brazilian folk medicine), with MIC of 31.2 μg/mL [34]. These values were significantly lower than the results obtained for CCP in this study.

In terms of filamentous fungi, the two species of Aspergillus tested in this study may cause aspergillosis, which relates to a wide variety of diseases caused by Aspergillus species, mainly affecting the respiratory tract of immunocompromised people [35]. In this study, CCP inhibited the growth of these two species, but the values obtained (MIC of 500 μg/mL for both and MFC of 500 μg/mL and 1000 mg/mL for A. flavus and A. fumigatus, respectively) are much higher than the MIC previously reported for CCP against Aspergillus sp., i.e., 32 μg/mL [10].

The F. solani species complex is associated with fusariosis, usually presenting high MIC values for most of the therapeutic antifungal agents [36]. This was also observed in this study, since this fungus was not inhibited by most of the agents tested (except for AMB and CCP, but in this case at a relatively high concentration). The literature reports some active compounds against this species, such as polymyxin B (MIC ranging from 32 to 64 μg/ml and MFC from 32 and 128 μg/ml, depending on the clinical isolate) [37].

L. prolificans is commonly found in soil and polluted water and is increasingly recognized as a causal agent of severe invasive fungal infections, especially in stem cell and organ transplant patients, with a mortality rate reaching 90%. Treatment of these infections is challenging, since L. prolificans isolates are often drug resistant, with high MIC values for all commercially available antifungal agents. Thus, the combination of drugs (VCZ and terbinafine) is usually an option [38]. In this context, there is an interest in searching for new compounds with antifungal action against this fungus. An example is F901318, a dihydroorotate dehydrogenase inhibitor with antifungal potential, which showed antifungal activity against L. prolificans isolates, with MIC values ranging from 0.12 to 0.25 μg/mL [39]. In this study, the L. prolificans strain was susceptible to CCP, but the MIC and MFC were relatively high (250 μg/mL). However, it is noteworthy that CCP was the only compound tested that showed a fungicidal effect against this fungus.

Cunninghamella species include ubiquitous filamentous fungi, and despite being less frequent, infections caused by Cunninghamella species have high mortality rates [40]. In the few cases reported in the literature, treatment is often performed with combinations of drugs, such as AMB, CAS, and isavuconazole, or single drugs, such as AMB, which compromised the patient’s renal function [41]. In this study, Cunninghamella sp. was the most susceptible filamentous fungi to CPP (MIC of 62.5 μg/mL and MFC of 500 μg/mL).

The concentrations of CCP that were tested are still higher than the concentrations of standard drugs, which are compounds that are already used in the treatment of fungal infections. However, the results for CCP are in a same concentration range as those previously obtained for other natural compounds. There is still no consensus on the acceptable level of inhibition for natural products, but some authors consider a MIC of up to 500 μg/mL as strong inhibition, values that were used as reference in studies of essential oils and plant extracts [42]. Thus, CCP can be considered in further investigations, not only for its unique mechanism of action, which can be useful to overcome drug resistance, but also considering that the activity of natural products can be enhanced by chemical modifications or improvements in drug delivering systems. This compound was also active against fungi with a long history of resistant strains, and it also presented a fungicidal effect against several strains.

Conclusion

The search for new compounds with antifungal activity is highly desirable given the limitations found for the drugs currently available, such as toxicological concerns, lack of efficacy, and, above all, increase in cases of resistance to antifungals. In this study, cercosporamide showed antifungal activity against important strains of pathogenic fungi, which are responsible for causing severe invasive infections in immunocompromised patients. Cercosporamide is still the only compound described so far that acts in the inhibition of Pkc1, with highly selective inhibition, which may encourage the detailed study of a new class of antifungals through its use and also the development of derivatives based on its structure, aiming at possible applications in the health area. Despite the fact that the results between the different groups of fungi tested in this study were highly variable to some extent, which might be an obstacle for considering CCP a promising therapeutic antifungal agent, this compound was particularly active against C. tropicalis, encouraging further evaluations of this natural compound in this regard.

Funding

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Process numbers 143158/2018-0 and 303033/2021-5); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES - process number 23038.000795/2018-61, finance code 001); and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – 2020/04229-6 and 2021/01279-5).

Declarations

Competing interests

JLB and MYSK have two patent applications on the production and purification of cercosporamide resulting from their research on this subject.