Effect of Mn(II) and Co(II) on Anti- Candida Metabolite Production by Aspergillus sp. an Endophyte Isolated from Dizygostemon riparius (Plantaginaceae)

Abstract

Background/Objectives: This study evaluates the effect of Mn(II) and Co(II) ions on the production of anti-Candida metabolites by the endophytic fungus Aspergillus sp., isolated from Dizygostemon riparius. The objective was to identify metal-induced secondary metabolites with antifungal potential against drug-resistant Candida species. Methods: Aspergillus sp. was cultivated in Czapek agar supplemented with MnCl₂ (400 µM) or CoCl₂ (200 µM). Metabolite profiles were analyzed using UHPLC-DAD and LC-ESI-HRMS, followed by structural elucidation via NMR. Antifungal and biofilm inhibition activities were tested against Candida albicans and Candida parapsilosis. Toxicity was assessed using Tenebrio molitor larvae. Results: Key metabolites, including pyrophen, penicillquei B, and fonsecinone B, demonstrated antifungal activity with MIC values of 4.37-280.61 µg/mL. Fonsecinone B exhibited superior biofilm inhibition, surpassing fluconazole in reducing biofilm biomass and viability. In vivo assays showed low toxicity, with survival rates above 80% at 2× MIC/kg. Conclusions: Mn(II) and Co(II) significantly modulated the production of antifungal metabolites in Aspergillus sp. Fonsecinone B emerged as a promising candidate for antifungal therapy due to its potent activity and low toxicity. These findings support further investigation into the therapeutic potential of metal-induced fungal metabolites for combating drug-resistant Candida infections.

Keywords: anti-Candida metabolites; endophytic fungi; metal-induced secondary metabolism; natural products biotechnology.

Figure 1

Compounds annotated by LC-ESI-HRMS in positive mode.

Figure 2

Comparative chromatogram of total ions of crude extracts of the EtOAc phase (EtOAc Control in blue; Mn_EtOAc in red; Co_EtOAc in green) derived from the liquid culture medium of Aspergillus sp.

Figure 3

Comparative selected ion chromatograms for pyrophen with m/z 288.1229 (EtOAc Control in blue; Mn_EtOAc in red; Co_EtOAc in green) derived from the liquid culture medium of Aspergillus sp.

Figure 4

Comparative selected ion chromatograms for nigragillin with m/z 223.1783 (EtOAc Control in blue; Mn_EtOAc in red; Co_EtOAc in green) derived from the liquid culture medium of Aspergillus sp.

Figure 5

Comparative selected ion chromatograms for penicillquei B with m/z 246.1125 (EtOAc Control in blue; Mn_EtOAc in red; Co_EtOAc in green) derived from the liquid culture medium of Aspergillus sp.

Figure 6

Comparative selected ion chromatograms for aurasperone D with m/z 557.1451 (EtOAc Control in blue; Mn_EtOAc in red; Co_EtOAc in green) derived from the liquid culture medium of Aspergillus sp.

Figure 7

Comparative selected ion chromatograms for Fonsecinone B with m/z 589.1702 (EtOAc Control in blue; Mn_EtOAc in red; Co_EtOAc in green) derived from the liquid culture medium of Aspergillus sp.

Figure 8

Comparative selected ion chromatograms for fonsecinone A with m/z 571.1618 (EtOAc Control in blue; Mn_EtOAc in red; Co_EtOAc in green) derived from the liquid culture medium of Aspergillus sp.

Figure 9

Structural Elucidation of the Isolated Compounds.

Figure 10

Percentage of inhibition in young (A,C) and mature (B,D) biofilms treated with pyrophen, penicillquei B, and fonsecinone B in strains of C. albicans (ATCC 90028) (A,B) and C. parapsilosis (ATCC 22019) (C,D) assessed by the MTT assay. The control is represented by Candida strains treated with sterile phosphate-buffered saline (PBS), while 2% dimethyl sulfoxide (DMSO) serves as the solubility agent control for the tested chemical isolates and was designated as a treatment. Treatments were based on the values of 1/4 and 1/2 MIC for forming biofilms and 2× and 4× MIC for mature biofilms and compared with untreated controls or the reference antifungal Fluconazole. (*) p < 0.05 compared to control. (#) p < 0.005 compared to the other experimental groups. (@) p < 0.05 compared to the different concentrations.

Figure 11

Percentage reduction in biofilm biomass eradication stained with crystal violet for C. albicans (ATCC 90028) (A,C) and C. parapsilosis (ATCC 22019) (B,D). Treatments considered the MIC values (1/4 and 1/2 MIC for forming biofilms and 2× and 4× MIC for mature biofilms) and were compared with untreated controls for each Candida strain that received sterile phosphate-buffered saline (PBS) and with a dimethyl sulfoxide (DMSO) group used as solubility agent for the chemical isolates or the reference antifungal Fluconazole. (*) p < 0.05 compared to control. (#) p < 0.005 compared to other groups. (@) p < 0.05 compared different concentration.

Figure 12

Evaluation of acute toxicity and survival percentage following injection of test substances, pyrophen (A,B), penicillquei B (C,D), Fonsecinone B (E,F), and Fluconazole (G,H). Treatments were based on concentrations of 2×, 4×, and 8× MIC (µg/kg) for C. albicans (ATCC 90028) (A,C,E,G) and C. parapsilosis (ATCC 22019) (B,D,F,H). Controls correspond to Candida strains treated with sterile PBS or with dimethyl sulfoxide (DMSO) (2%) as this solubility agent was used for chemical isolates. Survival was assessed using the log-rank test. (*) p < 0.05 compared to the control group.

Figure 13

Survival percentage of T. molitor larvae after infection with C. albicans (ATCC 90028) and treatment at concentrations of 2× and 4× MIC (µg/kg) with pyrophen (A,B), penicillquei B (C,D), fonsecinone B (E,F). Negative controls were treated with sterile PBS or with DMSO (2%), and the positive control was Fluconazole. Survival was assessed using the log-rank test. (*) p < 0.05 compared to the control group.

Figure 14

Survival percentage of T. molitor larvae after infection with C. parapsilosis (ATCC 22019) and treatment at concentrations of 2× and 4× MIC (µg/kg) with pyrophen (A,B), penicillquei B (C,D), Fonsecinone B (E,F). Negative controls were treated with sterile PBS or DMSO (2%), and the positive control was Fluconazole. Survival was assessed using the log-rank test. (*) p < 0.05 compared to the control group.