Antifungal activity of eumelanin-inspired indoylenepheyleneethynylene against Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in >152,000 immunocompromised individuals annually, leading to 112,000 yearly deaths. The four classes of existing antifungal agents target plasma membrane sterols (ergosterol), nucleic acid synthesis, and cell wall synthesis. Existing drugs are not highly effective against Cryptococcus, and antifungal drug resistance is an increasing problem. A novel antimicrobial compound, a eumelanin-inspired indoylenepheyleneethynylene, EIPE-1, was synthesized and has antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MSRA), but not towards Gram-negative organisms. Based on EIPE-1's antibacterial activity, we hypothesized that EIPE-1 could have antifungal activity. For these studies, we tested EIPE-1 against C. neoformans strain H99 and 6 additional cryptococcal clinical isolates. We examined antifungal activity, cytotoxicity, effects on fungal gene expression, and mechanism of action of EIPE-1. Results showed that EIPE-1 has fungicidal effects on seven cryptococcal strains with MICs ranging from 1.56 to 3.125 μg/mL depending on the strain, and it is non-toxic to mammalian cells. We conducted scanning and transmission electron microscopy on the exposed cells to examine structural changes to the organism following EIPE-1 treatment. Cells exposed displayed structural changes to their cell wall and membranes, with internal contents leaking out of the cells. To understand the effect of EIPE-1 on fungal gene expression, RNA sequencing was conducted. Results showed that EIPE-1 affects several processes involved stress response, ergosterol biosynthesis, capsule biosynthesis, and cell wall attachment and remodeling. Therefore, our studies demonstrate that EIPE-1 has antifungal activity against C. neoformans, which affects both cellular structure and gene expression of multiple fungal pathways involved in cell membrane stability and viability.

Keywords: Cryptococcus neoformans; EIPE-1; antifungal; eumelanin; novel antifungal compound.

Figure 1

Molecular structure of eumelanin-inspired indolyenepheneethylene (EIPE-1).

Figure 2

Minimum Inhibition Concentration of EIPE-1 and Amphotericin B against Cryptococcal Strains. Cryptococcal yeast cells (strains H99, Cn145a, R265, R272, R4247, WSA87, or 52D) were incubated in RMPI-MOPs alone, in RPMI-MOPS with either EIPE-1, or Amphotericin B in a two-fold dilution for 48 h at 35°C, with humidity. Optical Densities were determined using a multi-mode plate reader. Data shown are from two independent experiments with each cryptococcal strain and means ± SEM are shown. Statistical significance (p < 0.05) is shown with an asterisk *. Some strains had no variation between experiments and do not have an error bar.

Figure 3

EIPE-1 is not cytotoxic to mammalian cells. (A) Human cervical epithelial cell line HeLa, (B) Murine fibroblast cell line McCoy, and (C) human lung epithelial cell line A549 were incubated in RPMI-MOPS, in RPMI-MOPS with either 6.25 µg/ml EIPE-1, 12.5 µg/ml EIPE-1, or 62.5 µg/ml EIPE-1 for 24 h at 37°C, 5% CO₂. Cytotoxicity was defined as greater than 30%. Fluorescence was measured on a Synergy HTX multi-mode plate reader. Data displayed are the mean ± SEM of results of 2 independent experiments.

Figure 4

Electron Microscopy of C. neoformans with EIPE-1 shows structural changes. C. neoformans H99 were grown in the presence of EIPE-1 at the calculated MIC of 1.749 μg/mL, for 4, 6, 8, and 12 h, fixed with 2.0% glutaraldehyde in 0.1 M cacodylate buffer, prepared for electron microscopy, and examined for TEM or SEM. (A) SEM of C. neoformans displayed structural changes (as indicated by the arrows) to the cells incubated with EIPE-1 for 4, 8, and 12 h, but not in the untreated cells. (B) TEM of C. neoformans displayed structural changes (arrows) to cells treated with EIPE-1 for 4, 8 and 12 h, but not in the untreated cells. (C) TEM of C. neoformans cells treated with EIPE-1 for 12 h cells displayed four variations in structure – rounded cell (undamaged), degraded membrane, black smudge present over the cell, and c-shape. Magnification is 10,000X for SEM and 8,000X for TEM. Images are representative of at least 8 fields per condition per time point examined.

Figure 5

EIPE-1 does not provide antifungal protection during in-vivo infection of Galleria mellonella. G. mellonella larvae were inoculated with either PBS alone or EIPE-1 at the concentration of 15 μg/mL, 20 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 150 μg/mL, or 200 μg/mL diluted in PBS. Infected G. mellonella were incubated at 37°C and were examined every 12 h for mortality for 10 days. Larvae were considered dead following full-body melanism and immobility. Data are representative of three independent experiments.

Figure 6

A putative model for the mechanism of antifungal activity of EIPE-1. C. neoformans H99 have a complex cell wall comprised of chitin, chitosan, a-1,3 glucan, B-1,3 glucan, B-1,6 glucan, mannoproteins and GPI-anchored proteins. Additionally, they possess a capsule that is consistently maintained on the outer cell wall and composed of GXM and GalXM. EIPE-1, a hydrophobic eumelanin inspired molecule, may enter the cell through a passive method and alters components of the cell by a downregulation in genes involved with capsule biosynthesis, cell wall attachment and remodeling, and ergosterol biosynthesis. (A) Glucuronoxylomannan (GXM) and Galactoxylomannan (GalXM). (B) Chitin and Chitosan. (C) GPI-anchored proteins. (D) Ergosterol.