Epigenetic Induction of Secondary Metabolites Production in Endophytic Fungi Penicillium chrysogenum and GC-MS Analysis of Crude Metabolites with Anti-HIV-1 Activity

Abstract

The continuous burden of human immunodeficiency virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated four endophytic fungal isolates from a medicinal plant, Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate, and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Penicillium chrysogenum P03MB2 showed anti-HIV activity with an IC₅₀ of 0.6024 µg/mL compared to untreated fungal crude extract (IC₅₀ 5.053 µg/mL) when treated with sodium butyrate. The profile of secondary metabolite compounds from the bioactive, partially purified extracts were identified by gas chromatography-mass spectrometry (GC-MS), and more bioactive compounds were detected in treated P. chrysogenum P03MB2 fractions than in untreated fractions. Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro (13.64%), cyclotrisiloxane, hexamethyl (8.18%), cyclotetrasiloxane, octamethyl (7.23%), cyclopentasiloxane, decamethyl (6.36%), quinoline, 1,2-dihydro-2,24-trimethyl (5.45%), propanenitrile (4.55%), deca-6,9-diene (4.55%), dibutyl phthalate (4.55%), and silane[1,1-dimethyl-2-propenyl)oxy]dimethyl (2.73%) were the most abundant compounds. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with stronger anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites which can be developed into therapeutic compounds.

Keywords: biosynthetic gene clusters; endophytic fungi; epigenetic modifiers; human immunodeficiency virus; secondary metabolites.

Figure 1

The maximum-likelihood tree was constructed based on ITS gene sequences of P03MB2, with closely-related fungal ITS sequences accessed from GenBank using the BLASTN tool. Panel (A) shows the similarity search of strain P03MB2, while Panel (B) shows the maximum likelihood of endophytic fungi P. chrysogenum P03MB2. The sequences were aligned using ClustalW. Bootstrap values included 1000 replicates using MEGA software (version 11.06) and are displayed on the tree branches.

Figure 2

Percentage of cell viability of TZM-bl cell lines against methanol crude fungal extracts treated and untreated with sodium butyrate using the MTT viability assay. Results were obtained from two independent experiments; data represent the mean SEM. The x-axis shows the different treatments (treated or not treated crude extracts and a positive control, AZT (zidovudine), SB (sodium butyrate), PO3 (plant 3A, A. adianthifolia), M (malt extract agar), P (potato dextrose agar), B (bark), 2 (isolate number), T (treated extract), NT (not treated crude extract).

Figure 3

Dose-response curve showing anti-HIV-1 activity of sodium butyrate-treated crude extracts of P. chrysogenum P03MB2T (blue) and untreated crude extracts of P. chrysogenum P03MB2NT (pink) tested in TZM-bl cell lines. Fungi-free sodium butyrate (SB) crude extract (green) was included as a negative control, while AZT (red) was used as a positive control. The graph shows inhibitory concentration at 50% inhibition (µg/mL). The IC₅₀ of P03MB2T was 0.048 µg/mL, P03MB2NT was 0.0090 µg/mL, and AZT (positive) control IC₅₀ was 0.0330 µg/mL. The x-axis shows the serial dilution of crude extract concentrations in Log scale and the y-axis shows the percentage of HIV-1 inhibition.

Figure 4

Dose-response curves of three crude extract fractions eluted in pre-concentration cartridges ((A) HLB 45%, (B) MCX 45%, and (C) MAX 45%). The sodium butyrate-treated P. chrysogenum P03MB2T (blue), untreated P. chrysogenum P03MB2NT (pink), AZT positive control (red), and fungi-free sodium butyrate-treated crude extract fraction (SB) as negative control (green). The x-axis shows serial dilution of crude extract concentrations in the Log scale and the y-axis shows the percentage of HIV-1 inhibition. The anti-HIV activity of fractionated crude extracts was tested against the HIV-1 infected TZM-bl cells. In Panel A (HLB 45%: P. chrysogenum PO3MB2T, IC₅₀ 0.6024 µg/mL, P. chrysogenum PO3MB2NT, IC₅₀ 5.053 µg/mL); Panel B (MCX 45%: P. chrysogenum PO3MB2T, IC₅₀ 4.246 µg/mL; P. chrysogenum PO3MB2NT, IC₅₀ 2.707 µg/mL); Panel C (MAX 45%: P. chrysogenum PO3MB2T, IC₅₀ 1.456 µg/mL, P. chrysogenum PO3MB2NT, IC₅₀ 0.4809 µg/mL).