FEMS Yeast Research Advance Access published May 7, 2015
Synergistic fungicidal activity of the lipopeptide Bacillomycin D with Amphotericin B against pathogenic Candida species Tabbene Olfa 1, Di Grazia Antonio2, Azaiez Sana 1, Ben Slimene Imen1, Elkahoui Salem1, Alfeddy Mohamed Najib3, Casciaro Bruno2, Luca Vincenzo2, Limam Ferid1 & Mangoni Maria Luisa2 1
Laboratoire des Substances Bioactives, Centre de Biotechnologie de Borj-Cedria, HammamLif, Tunisia; 2Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche, Universita` La Sapienza, Rome, Italy; 3Laboratoire de Phytobactériologie, UR Agrobiotechnologie, Institut National de Recherches Agronomiques, Marrakech, Morocco Correspondence: Tabbene Olfa, Laboratoire des Substances Bioactives, Centre de Biotechnologie de Borj-Cedria, BP-901, 2050 Hammam-Lif Cedex, Tunisia. Tel.: +216 79 325 728; fax: +216 79 325 728; e-mail:
[email protected] Abstract In the present study, the synergism of the lipopeptide bacillomycin D in combination with the polyene amphotericin B against pathogenic Candida species is described along with their potential cytotoxicity against mammalian cells. Bacillomycin D inhibited the growth of various Candida species at minimal concentrations from 12.5 µg mL-1 to 25 µg mL-1. Furthermore it showed a synergistic effect with the antifungal drug amphotericin B in inhibiting the growth of Candida strains, with fractional inhibitory concentration indices ranging from 0.28 to 0.5. Time killing studies revealed more than 2-log reduction in the viability of Candida albicans ATCC 10231 cells after 3h incubation with the combination amphotericin B plus bacillomycin D, at their sub-inhibitory concentration. Interestingly, when the two drugs were used together at those dosages displaying a synergism in the anti-Candida activity, no cytotoxic effect was observed against mammalian cells. Therefore, the combination bacillomycin D/amphotericin B may represent a valid alternative to conventional antifungals for topical treatment of Candida albicans infections. To the best of our knowledge, this is the first report describing the in vitro interaction between the antifungal drug amphotericin B and bacillomycin D against pathogenic Candida species Keywords: Synergy – Lipopeptides – Bacillomycin D – Amphotericin B – Candida – Cytotoxicity Running Title: Interaction of amphotericin B and bacillomycin D on Candida species
Introduction Members of Candida genus are commensally aerobic microorganisms commonly found in the oral cavity, digestive and vaginal tracts (Höfling et al., 2010). However, they may become pathogenic fungi causing a number of diseases in immunosuppressive hosts by the adherence of the hyphal form to the tissue surfaces (Höfling et al., 2010). About 20 species of Candida yeasts can cause infection in humans; the most common species is Candida albicans (Achkar & Fries, 2010). Among antifungal agents used to treat different types of candidiasis is the polyene amphotericin B (Kumar et al., 2013). The mechanism of action of amphotericin B is based on its binding to the fungal cell membrane ergosterol, followed by the production of a transmembrane channel allowing the leakage of cytoplasmic contents and therefore cell death (Laniado-Laborı´n & Cabrales-Vargas, 2009). However, amphotericin B has severe side effects and is highly toxic to kidneys, hematopoietic and central nervous system (Hong et al., 1999). Furthermore, beside its toxicity, the widespread use of such drug has led to the development of resistant Candida strains (Rex et al., 1995; Wanger et al., 1995; Nolte et al., 1997; Linares et al., 2013). The combination of two or more antimycotics could be an attempt to prevent the development of drug resistance as well as to enlarge their antimicrobial spectrum reducing toxic effects (Kumar et al., 2013). Lipopeptide compounds produced by microorganisms have gained attention because of their broad spectrum of activity including antibacterial, antifungal and antiviral activities (Arnusch et al., 2012). Lipopeptides have generally an acyl chain conjugated to a cyclic or a linear peptide sequence. The ability of Bacillus species to synthesize a wide variety of lipopeptide antimicrobials has been extensively exploited in medicine and agriculture (Moyne et al., 2001). Among them, members of the iturin family comprising bacillomycin D, iturin and mycosubtilin are the most potent antifungal agents but with a demonstrated haemolytic activity (Tenoux et al., 1991; Maget-Dana & Peypoux, 1994). The antifungal efficacy is related to the interaction of the iturin lipopeptides with the cytoplasmic membrane of the target cells, causing a significant increase in the permeability to K+ ions through the formation of ion-conducting pores, the characteristics of which depend on both the lipid composition of the membrane and the structure of the cyclic peptide (Maget-Dana & Peypoux, 1994). In a previous work (Tabbene et al., 2011) we described the production of three bacillomycin D isoforms with 14, 15 and 16 carbon atoms fatty acid chains, by Bacillus subtilis B38 strain. It was found that the compound bacillomycin D C16 had the strongest fungicidal activity against pathogenic C albicans strains compared to the other isoforms. However, it was toxic
to human erythrocytes. Therefore, in the present study we evaluated the potential synergistic effect of bacillomycin D C16 when combined with amphotericin B, with the aim to minimize their toxicity towards normal mammalian cells while preserving antifungal activity.
Materials and methods Antifungal compounds The lipopeptide compound bacillomycin D (NH2-STNYNPE-OH) was isolated from Bacillus subtilis B38 strain as previously described (Tabbene et al., 2011) and was dissolved in 50mM Tris-HCl buffer pH 8. Chemical composition of the compound was determined by mass spectrometry analysis (Tabbene et al., 2011). Fungizone intravenous (amphotericin B deoxycholate), dissolved in 1%DMSO, was used as anti-yeast agent.
Microorganisms and culture conditions American type culture collection Candida strains (Candida albicans ATCC 10231, Candida krusei ATCC 6258 and Candida parapsilopsis ATCC 22019) and human clinical isolates of Candida species (Candida albicans, Candida tropicalis, Candida riferii, Candida glabrata) were cultured in Sabouraud dextrose agar plates at 30°C.
Mammalian cells and culture condition The well-established human immortalized keratinocytes (HaCaT cell line, ATCC, USA) and human type II alveolar epithelial cell line (A549 cells line from the American Type Culture Collection, Manassas, Va) were used throughout the study. HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS), L-glutamine (4 mM) and 0.05 mg/ml gentamicin, at 37 °C and 5% CO2, in 25-cm2 flasks. A549 cells were cultured in DMEM supplemented with 10% FBS, L-glutamine (2 mM), and antibiotics (0.1 mg/ml of penicillin and streptomycin) at 37 °C and 5% CO2 in 25-cm2 flasks. Human erythrocytes were prepared as previously described (Ponti et al., 1999). Human erythrocytes were separated from plasma by centrifugation at 3500rpm for 10min and washed twice with 0.9% NaCl. Afterwards, they were suspended in 0.9% NaCl at a final concentration of about 10 8cells/ml and used for the haemolytic activity. Anti-Candida activity assay The antifungal activity was evaluated using a standard microbroth dilution method in sterile 96 well plates as previously described (Grieco et al., 2013). Candida strains were grown in Sabouraud medium at 30 °C to mid-log phase. Cells were harvested by centrifugation and washed twice with phosphate-buffered saline (PBS). Cells were then re-suspended in RPMI
1640 (Sigma) supplemented with 2 mM L-glutamine and buffered to pH 7 with 0,165 M morpholinepropanesulfonic acid (MOPS). Aliquots of cell suspension were added into each well of the microtiter plate at a final concentration of 3.5 x104 cells/ml and incubated for 24 h at 30°C. Minimal inhibitory concentration (MIC) was determined as the lowest concentration of the antimicrobial compound inhibiting yeast growth.
Killing assay The effect of the antifungal compounds bacillomycin D, amphotericin B and their combination on the viability of C. albicans was determined by counting the number of viable cells according to what was previously described (Uccelletti et al., 2010). The standard C. albicans ATCC 10231 was selected for these experiments, following the MIC data. About 3.5x104 cells mL-1 of C. albicans were incubated in RPMI 1640 medium with various concentrations of bacillomycin D or amphotericin B alone or in combination one with each other, at their sub-inhibitory concentrations. Aliquots of 10 μl were withdrawn at each time interval and spread on agar plates. Number of colony-forming units (CFUs) was determined after overnight incubation of plates at 30 °C. Controls were tested with fungal compound solvents Tris-HCl and DMSO at 2.5mM and 0.05% respectively.
Synergistic effect of bacillomycin D with the conventional antifungal product amphotericin B The synergism between amphotericin B and bacillomycin D was investigated by adding the combination of the compounds in a serial 2-fold dilution to a 96-well plate as previously described (Mangoni et al., 2008). Candida strains, listed in table 2, were added at a concentration of 3 x 10 4 CFU/ml. The fractional inhibitory concentration (FIC) index for the combination of the two compounds was calculated according to the following equation: Σ(FICA + FICB) = Σ(A/MICA + B/MICB) where A and B are the MICs of amphotericin B and bacillomycin D in the combination, MICA and MICB are the MICs of amphotericin B and bacillomycin D alone, FICA and FICB are the FICs of amphotericin B and Bacillomycin D. The FIC indices were interpreted as previously described (Mangoni et al., 2008): FIC ≤ 0.5, synergy; 0.5
