Antimicrobial and anti-inflammatory activities of endophytic fungi Talaromyces wortmannii extracts against acne-inducing bacteria

Abstract

Acne vulgaris is the most common skin disease, causing significant psychosocial problems such as anxiety and depression similar to a chronic illness for those afflicted. Currently, obtainable agents for acne treatment have limited use. Thus, development of novel agents to treat this disease is a high medical need. The anaerobic bacterium Propionibacterium acnes has been implicated in the inflammatory phase of acne vulgaris by activating pro-inflammatory mediators such as the interleukin-8 (IL-8) via the NF-κB and MAPK pathways. Talaromyces wortmannii is an endophytic fungus, which is known to produce high bioactive natural compounds. We hypothesize that compound C but also the crude extract from T. wortmannii may possess both antibacterial activity especially against P. acnes and also anti-inflammatory properties by inhibiting TNF-α-induced ICAM-1 expression and P. acnes-induced IL-8 release. Treatment of keratinocytes (HaCaT) with P. acnes significantly increased NF-κB and activator protein-1 (AP-1) activation, as well as IL-8 release. Compound C inhibited P. acnes-mediated activation of NF-κB and AP-1 by inhibiting IκB degradation and the phosphorylation of ERK and JNK MAP kinases, and IL-8 release in a dose-dependent manner. Based on these results, compound C has effective antimicrobial activity against P. acnes and anti-inflammatory activity, and we suggest that this substance or the crude extract are alternative treatments for antibiotic/anti-inflammatory therapy for acne vulgaris.

Figure 1. Analytical HPLC/MS profiles.

(A) HPLC/MS profile of the crude extract of T. wortmannii isolated with EtOAc. (B) Chromatogram after straight phase and sephadex-G50 separation.

Figure 2. Cytotoxicity of T. wormannii extract and isolates.

(A) HUVECs were plated at 10⁶ cells/ml on goldfilm electrods in 96 well plates and allowed to attach and form a monolayer for 24 h before cells were treated with 1, 10 or 100 µg/ml crude extract or 20% DMSO (control) or left untreated and cell toxicity measured every 5 min for 24 h. (B) HeLa, CaCo-2, HaCaT, HUVEC and HKER cells were plated at 2×10⁵ cells/ml in 96 well plates for 6 h and stimulated with 500 µg/ml–0.002 µg/ml crude extract for 24 h before Alamar blue was added (10%) and fluorescence intensity measured and the IC₅₀ determined. (C) HUVECs were treated with 20 µg/ml or 200 µg/ml crude extract, with staurosporine (positive control) or left untreated (neg. control) for 24 h and apoptotic and necrotic cells measured using AnnexinV and 7-AAD. HaCaT (D), HUVEC (E) and HKER (F) were plated as described in B and cells stimulated with AA, BB, C and D.

Figure 3. Screening for anti-inflammatory T. wortmannii components.

(A) Flow cytometric analysis of ICAM-1 expression on the cell surface of HUVECs. Cells were stimulated with 10 ng/ml hTNF-α with or without 25 µg/ml crude extract or left untreated as negative control and stained with mouse anti-human ICAM-1 mAB. (B) Screening for anti-inflammatory substances, using a cell-based ELISA. Cells were treated for 1h in the absence or presence of 25 µg/ml substances AA, BB, C, D and crude extract, prior to being stimulated for 24 h with 10 ng/ml hTNF-α and stained with human ICAM-1 mAB (C) HEK293 cells were transiently transfected with NF-κB-Luciferase construct and EGFP construct. 24 h later, transfected cells were treated with 0, 40, 20 or 10 µg/ml substances or crude extract, prior to being stimulated with 10 ng/ml hTNF-α. Luciferase activities was determined 8 h later, normalized to the EFGP activities and expressed as fold increase over the control. (*, p<0.05; **, p<0.01, Students t test).

Figure 4. Effect of T. wortmannii substance C on P. acnes-induced activation of NF-

κB and AP1. HaCaT cells were transiently transfected with NF-κB- (A) or AP-1- Luciferase (B) as described in Fig. 3C and 24 h later treated with 0, 10, 20 or 40 µg/ml component C. After 2 h cells were then stimulated by incubation with P. acnes suspension and luciferase activities measured 8 h later. The data are the mean of three experiments +− standard deviation. (C) HaCaT cells were pre-treated with component C (Sub. C) for 2 h, and then stimulated with P. acnes suspension for 20 min. Phosphorylation and total protein expression of ERK, JNK, and IkB-α were detected by western blot analysis using specific antibodies. Tubulin was used as a loading control.

Figure 5. Effect of T. wortmannii components on P. acnes-induced IL-8 expression.

(A) HaCaT cells were pre-treated with 20 µg/ml components AA, BB, C, D, Crude extract or left untreated for 2 h and IL-8 production stimulated with P. acnes suspension for 24 h. Control experiments were run with cells alone (n.c.) or cells stimulated with P. acnes suspension (pos. contr.). (B) Pre-, co-, and post-treatment effects of several concentrations of component C on P. acnes–induced IL-8 expression in HaCaT cells. (C) HaCaT cells were stimulated with different P. acnes strains (P. acnes, P. acnes (ERY^R), P. acnes_5-7) and post-treated with 20 µg/ml component C 3 h later. Data are presented as the mean+- standard deviation of three independent experiments. Statistical significance is indicated by *, p<0.05; **, p<0.01, Students t test.