Relationship between cell envelope ultrastructure and the antibacterial properties of a novel hydrophobic eumelanin-inspired derivative

Abstract

IntroductionUnique impermeability properties of the gram-negative outer cell envelope typically render these organisms intrinsically resistant to hydrophobic antibacterial compounds. Eumelanin-inspired indoylenephenyleneethynylene (EIPE) compounds possess scaffolding to which functional groups were attached to potentially provide antibacterial properties in the forms of hydrophilic (EIPE-HCl) and hydrophobic (EIPE-1) derivatives.MethodsStandardized disk agar diffusion and microbroth dilution bioassays were employed to assess the susceptibility of disparate gram-negative and gram-positive bacterial pathogens to the two compounds. EIPE-1 mechanisms of action and intrinsic resistance were further investigated turbidimetrically in batch cultures with the aid of the gram-negative outer membrane permeabilizer compound 48/80.ResultsHydrophobic derivative EIPE-1 exhibited a gram-positive antibacterial spectrum, while hydrophilic derivative EIPE-HCl possessed no antibacterial properties. EIPE-1 exhibited minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values below 2.0 µg/mL against all gram-positive bacteria, including two methicillin-resistant strains. In contrast, MIC/MBC values greater than 128 µg/mL were obtained for all gram-negative bacteria examined. Susceptibility of two strains of the strict anaerobe Clostridioides difficile indicated the EIPE-1 mechanism of action does not require molecular oxygen. Turbidimetric growth curves revealed EIPE-1 induced rapid bacteriolysis of Bacillus subtilis ATCC 6633, thereby suggesting a membrane-directed modality. Lastly, the outer membrane permeabilizer compound 48/80 failed to markedly sensitize any of three phylogenetically disparate gram-negative organisms to EIPE-1.ConclusionThese data suggest that the hydrophobic melanin-inspired derivative EIPE-1 inhibits gram-positive bacteria in a cytoplasmic membrane-directed manner independent of oxygen. Moreover, a secondary mechanism may function concomitantly with outer membrane exclusionary properties to underly the intrinsic resistance of gram-negative pathogens.

Antifungal activity of eumelanin-inspired indoylenepheyleneethynylene against Cryptococcus neoformans

“…While its original purpose was as a building block for organic semiconductors, it was later discovered that this core has the interesting capability of serving as a foundation for the integration of antibactericidal moieties and possesses intrinsic antimicrobial activity that causes cytoplasmic membrane disruption in gram positive bacteria. Additionally, it was revealed that thirteen different strains from eight gram-positive bacteria, including two methicillin resistant strains were found to be susceptible to EIPE-1 ( Adhikari et al, 2022 ; Reed et al, 2023 ). While not previously studied in fungal organisms, we found enough evidence to support studying this compound as an antifungal against Cryptococcus isolates.…”

“…This membrane maintains the viability of a cell and prevents the free exchange of molecules from the cytoplasm to the cell’s environment and vice versa ( Upadhya et al, 2023 ). Previously, microorganism membrane permeability to hydrophobic molecules was identified as being pertinent for susceptibility to the molecule ( Reed et al, 2023 ). Since C. neoformans has a high cellular surface hydrophobicity due to the presence of mannoproteins, lipids, glucan, and chitin molecules, it could allow the hydrophobic EIPE-1 molecule to passively diffuse across the membrane into the cell ( van der Rest et al, 1995 ; Danchik and Casadevall, 2020 ; Vij et al, 2020 ).…”

“…3,3′-(((5,6-dimethoxy-2-(methoxyxarbonyl)-1-methyl- 1H- indole-4,7-diyl)bis(ethyne-2,1-diyl))bis(4,1-phenylene)bis(oxy))bis( N,N,N -trimethylpropan-1-aminium) iodide (EIPE-1) ( Figure 1 ) was synthesized and provided by Dr. Nelson’s laboratory ( Adhikari et al, 2022 ; Reed et al, 2023 ). EIPE-1 powder was then reconstituted with dimethyl sulfoxide (DMSO) to a stock concentration of 5 mg/mL.…”

“…A recent study by Adhikari et al utilized vanillin for the synthesis of a eumelanin-inspired indolyenepheneethylene synthetic compound, EIPE-1 ( Adhikari et al, 2022 ). Through the application of synthetic approaches for derivatization of the methyl-4,7-dibromo-5,6-dimethoxy-1-methyl-1H-indole-2-carboxylate (DBI), a eumelanin-inspired indole core decorated at the 4- and 7- positions, EIPE-1 was prepared as a potential new antimicrobial agent ( Selvaraju et al, 2015 ; Adhikari et al, 2022 ; Reed et al, 2023 ). EIPE-1 has two bactericidal moieties ligated to the DBI core that were intended to exhibit similar antibiotic mechanisms to cationic cell-wall disrupting compounds ( Baker et al, 1941 ; Velkov et al, 2013 ; Adhikari et al, 2022 ).…”

“…EIPE-1 has two bactericidal moieties ligated to the DBI core that were intended to exhibit similar antibiotic mechanisms to cationic cell-wall disrupting compounds ( Baker et al, 1941 ; Velkov et al, 2013 ; Adhikari et al, 2022 ). This compound demonstrated antimicrobial effects against 13 strains of gram-positive bacteria, including two methicillin resistant strains ( Adhikari et al, 2022 ; Reed et al, 2023 ). Thus, we hypothesized that EIPE-1 may be effective as an antifungal treatment.…”