SUPPLEMENTARY MATERIAL
Penicillium commune Metabolic Profile as a Promising Source of Antipathogenic Natural Products
Lorena Diblasiab, Federico Arrighic, Julio Silvab, Alicia Bardónacd & Elena Cartagenaa*
a Instituto de Química Orgánica, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471, Tucumán 4000, Argentina; b Cátedra de Micología, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471, Tucumán 4000, Argentina;c LISA-CONICET, d INQUINOA-CONICET. Ayacucho 471, Tucumán 4000, Argentina
Abstract
Penicillium is an important genus of ascomycetous fungi in the environment and in food and drug production. This paper aims to investigate statins and antipathogenic natural products from a P. commune environmental isolate. Fractions (F1, F2, F3 and F4) were obtained from an ethyl acetate extract. DIP/EI/ITD mass spectrometry (MS and MS/MS) identified lovastatin (1) in F1, while GC-MS showed that 3-isobutylhexahydropyrrolo [1,2-a]pyrazine-1,4-dione (2) was the main constituent of F2 (49.34%). F4 presented 3 (16.38%) as an analogue of 2 and their known structures were similar to that of an autoinducer-signal. F1 produced a significant decrease in the Pseudomonas aeruginosa biofilms, which is the main cause of bacterial pathogenicity. F2 and F4 were effective against Staphylococcus aureus biofilms, but when F2 was associated with oxacillin it showed an important activity against both bacteria. These novel results suggest that P commune INTA1 is a new source of promising antipathogenic products.
Keywords: Penicillium commune metabolic profile; DIP/EI/ITD mass spectrometry (MS and MS/MS); GC-MS analysis; lovastatin; pyrrolopyrazines; biofilms
Experimental
Fungal strain and media
Penicillium commune INTA1 mod was isolated from environment, and classified by morphological criteria and a microscope method (Sanidad Aviar laboratory of INTA, Entre Ríos, Argentina). The fungal strain was maintained in Petri dishes of PGA (potato, glucose, and agar). After incubation from the original slant, the dishes were incubated at 28 ºC for 5 days, and subsequently stored at 5 ºC.
A suspension of spores (harvested from plates) was obtained with sterile saline solution (NaCl, 7.8 g L−1) and concentrations adjusted to ~107 conidia mL-1 (λ 550 nm, DO = 0.3) (Chakravarti & Sahai, 2002; Casas López et al., 2003). The cultivation medium, contained per liter glucose: 20 g, glycerol: 24 mL, peptone: 8 g, NaNO3: 2 g, MgSO4: 1 g. All components were dissolved in soybean water (soybean: 40 g, sterile water: 1000 mL), and maintained for 6 days at 4 ºC, before fermentation. Then, the liquid medium was sterilized at 121 ºC and ¾ de atm (Chakravarti & Sahai, 2002). A second medium was prepared in the same way, but containing 10 g glucose per liter.
The culture medium contained glucose and glycerol as carbon source, and peptone and soybean meal as nitrogen source.
Fermentation and conditions
Fungal pellets were obtained by germination from spores suspended in shake flasks in a preliminary fermentation stage, and used for further inoculation of corresponding bioreactors. Seed cultures (1% , v/v) were carried out in a 500 mL flasks containing 150 mL medium in duplicate, held on a rotary shaker at 110 rpm, 28 ºC and pH = 6.3 (Szakács et al., 1998; Kumar et al., 2000; Casas López et al., 2004; Bizukojc et al., 2007). A medium control (without fungal strain) was also performed in duplicate.
Fermentations lasted 10 days (Chakravarti & Sahai, 2002), and thereafter the pH of each culture was adjusted to 6.3 again.
Extraction and isolation of fungal metabolites
The biomasses obtained were removed by filtering with cellulose filter and vacuum, the filtrate media were extracted with ethyl acetate twice a room temperature. The AcOEt extracts were dried over anhydrous Na2SO4 and were evaporated in rotavapor (Büchi R-3000) under vacuum at 40 ºC to give 0.4665 g of extract (E), and 0.0229 g of extract from the second medium containing 50% glucose (E2).
The isolation of the fungal metabolites from extracts was carried out by routine chromatographic techniques.
Chromatography techniques
Analytical thin layer chromatography (TLC) was performed on Merck precoated silica gel G 60 F254 plates to analyze each fungal extracts, and controls. Different mobile phases were employed for development of method. UV irradiation (λ: 254 nm and 366 nm), and the Godin reagent were employed as physical and chemical revealing, respectively. Lovastatin (Merck®), simvastatin (Merck®), and mevastatin (MP Biomedicals LLC®) were used as standards, and an extract from culture medium was employed as negative control.
A portion of the acetate ethyl extract from P. commune (87.7 mg) was separated on silica gel column chromatography (1:100, extract/silica gel relation) using CHCl3:AcOEt (0-100%) as mobile phase. The eluted fractions were monitored by TLC employing statins like standards.
The fractions with particular aroma (F2-4) were analyzed by GC-MS (EI) technique. The analysis was carried out using a Thermo electron TraceTM Ultra couple with split-split less injector and Polaris Q ion trap mass spectrometer equipped with a DB-5 capillary column (30 m x 0.25 mm, film thickness 0.25 µm). The initial temperature of the column was 60 °C (0 min). A temperature programming was applied from 60 °C to 246 °C at a rate flow of 3 °C min-1, and finally 246 °C for 3 min. Carrier gas was helium (flow 0.3 mL min−1). Injection mode split-less with surge (30 s, surge pressure 100 kPa). The main volatile constituents were determined by comparison of their mass spectra with standard data of NIST GC/MS library.
Fourier transformed infrared spectroscopy (FT-IR)
FT-IR spectra of the fungal culture extracts, medium control, and fraction F1 were recorded with a Perkin Elmer spectrophotometer 1600 FT-IR, in order to determine the δ-lactone ring absorption (~ 1735 cm−1), and absorption bands associated with C=O bond stretching (1180 cm−1) of δ or γ-lactones (Pretsch et al. 1998).
DIP/EI/ITD Mass spectrometry (MS and MS/MS)
The fungal extracts (10 µg) , F1 (10 µg) , and statins standards (1.7 µg) were dissolved in CH2Cl2 to determine their fragmentation patterns at 70 eV by direct introduction into a mass spectrometer (DIP: Direct Introduction Probe/EI: 70eV-Electron Ionization/ITD: Ion Trap Detector Polaris Q Thermo Electron 250 ms (max ion time); gas carrier He; ion trap detector; ion source temperature 200 °C; acquisition range from 40 to 450 Da/DIP: temperature program 40-320 °C, temperature gradient 120 °C min−1). MS/MS or MS2 experiments were performed by acquiring full MS spectra of both F1 and lovastatin standard. The identification of lovastatin was clearly achieved by comparison with MS and MS2 fragmentation patterns from precursor [M+H]+ ion (m/z 405) of an authentic standard of lovastatin Merck®.
Lovastatin was also analysed in amounts ranging from 76 to 2,052 ng for determining the presence of ion quasi-molecular and its relationship with sample size.
Optical rotation
Optical rotation of lovastatin standard and F1 were carried out on a HORIBA SEPA-300 High Sensitive Polarimeter.
Antibacterial and antibiofilm activities of volatiles from P. commune INTA1 strain
Fungal metabolites and antibiotics: Solutions of F1-4 from P. commune, lovastatin standard, and antibiotics: azithromycin and oxacillin were screened.
Bacterial strains and media: Staphylococcus aureus ATCC 6538 P, and Pseudomonas aeruginosa ATCC 27853 from American Type Culture Collection were grown in Müller Hinton (MH, Britania), and Luria-Bertani media (LB, Cabeo, Rockville, MD, USA), respectively, and employed for the bioassays.
Bacterial growth assay: Overnight cultures of each strain were diluted to reach 105 CFU mL−1 in LB or MH medium. The diluted culture (190 µL) was placed in each of the 96 wells of a microtitre polystyrene plate. Solutions containing 50, and 25 µg mL−1 of F1-4, and mixture of F2 (6 µg mL-1) with oxacillin (3 µg mL-1) in DMSO-distilled water (50:50) were prepared separately and 10 µL of each one was pipetted to the plastic microtitre plate wells individually (eight replicates). Control wells (eight replicates) contained the diluted culture (190 µL) and 10 µL of a solution of DMSO-H2O (50:50). Medium control was prepared using sterile LB or MH. Bacteria grew in liquid medium at 37 °C, and the growth was detected as turbidity (600 nm) using a microtitre plate reader (Power Wave XS2, Biotek, VT, USA). The maximum level of DMSO to which the cells were exposed was 2.5%. The negative control was oxacillin as cell wall synthesis inhibitor. Then, it was quantified the biofilm formed after 24 h incubation using the crystal violet dye (O'Toole & Kolter, 1998).
Biofilm assays: Biofilms developed after treatment was quantified by the technique described by O'Toole and Kolter (1998) modified. This assay is based on the ability of bacteria to form biofilms on polystyrene. The technique requires the addition of violet crystal solution which stains the cells but not the polystyrene.
A volume of 180 µL of MH (S. aureus) or LB (P. aeruginosa) broth and 10 µL of each fractions and controls were placed in each well of a 96-well polystyrene microtiter plate. Then, each well was inoculated with 10 µL of an overnight culture with biofilm phenotype (108 CFU mL−1). The microplates were incubated at 37 °C for 1 h in a moist chamber. Control experiments were performed in octuplicate using DMSO-H2O (without fractions). The negative controls were azithromycin as a known quorum-sensing inhibitor, and oxacillin.
After this time period, 25 μL of violet crystal solution (0.1%) were added to the wells, incubated 15 min at room temperature and then rinsed thoroughly and repeatedly with water to remove planktonic cells and unattached dye. Biomass-attached dye was solubilized with ethanol; and the absorbance was then measured at 540 nm in a microplate spectrophotometer (Biotek- Power Wave XS2 with GEN5 data analysis software).
Statistical analysis: Differences in the mean values were evaluated by analysis of variance (ANOVA). The Tukey test was used for all pair wise multiple comparisons of groups. In all analyses, values of P < 0.05 were considered statistically different (Statistix 7.1, 2002).
References
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Chakravarti, R., & Sahai, V. (2002). Optimization of compactin production in chemically defined production medium by Penicillium citrinum using statistical methods. Process Biochemistry, 38, 481–486.
O'Toole, G.A., &Kolter, R. (1998). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Molecular Microbioly, 28, 449–461.
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Figure S1. P. commune INTA1 culture (A) and TLC profiles of extracts, statins, and control of broth culture.*
* E: Extract from a P. commune INTA1 culture (A), E2: extract from P. commune INTA1 culture containing 50% glucose, standards: Mev: mevastatin, Lov: lovastatin, Sim: simvastatin (actives under UV light at 254 nm, ), and control: BC: broth culture extract.
Figure S2. EI-MS spectra of the complex Penicillium commune extract (E) and standards of lovastatin (L) and simvastatin (S), respectively.
Figure S3a. Full MS scan spectra of lovastatin standard and F1, respectively, and their main assignments.
F1 [m/z]: 405 [M+H] +, 404 [M] +• or [C24H36O5] +•, 387 [MH-H2O] + or [C24H35O4]+, 303 [C19H27O3]+, 287 [C18H23O3] +, 285 [C18H21O3] +, 224 [C14H24O2] +•, 209 [C12H17O3] +, 200 [C14H16O] +•, 199 [C13H11O2]+, 198 [C13H10O2] +•, 159 [C12H15] +, 157 [C12H13] + (100%), 143 [C7H11O3] + .
Figure S3b. MS/MS (MS2) spectra of lovastatin standard and F1, respectively, and their main assignments.
F1 [m/z]: 248 [C15H20O3] +•, 247 [C15H19O3]+, 232 [C15H20O2] +•, 205 [C13H17O2]+, 199 [C13H11O2]+ , 198 [C13H10O2] +•, 191 [C13H19O] +, 179 [C12H19O]+ and [C11H15O2] +, 178 [C11H14O2] +•, 169 [C9H13O3] +, 166 [C13H10] +• , 165 [C13H9] + , 159 [C12H15] +, 158 [C12H14] +•, 157 [C12H13] + , 155 [C9H15O2] +, 153 [C9H13O2] +, 152 [C9H12O2] +• , 143 [C7H11O3] +, 142 [C7H10O3] +•, 141 [C7H9O3] + and [C11H9]+, 129 [C6H9O3] +, 128 [C7H12O2] +• and [C10H8] +•, 127 [C7H11O2] + and [C10H7]+. .
Table S1. GC-MS of odorant fractions F2-4 from P. commune INTA1 extract.
Molecular formula / Molecularweight / CAS number / Retention time / Area
Compounds of F2
Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- / C11H18N2O2 / 210 / 5654-86-4 / 44 min / 49.34%
Betulin / C30H50O2 / 442 / 473-98-3 / 56 min / 3.31%
Hexanedioic acid, mono (2-ethylhexyl) ester
/ C14H26O4 / 258 / 4337-65-9 / 57 min / 39.66%
Compounds of F3
Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- / C11H18N2O2 / 210 / 5654-86-4 / 44 min / 21.81%
Hexanedioic acid, mono (2-ethylhexyl) ester / C14H26O4 / 442 / 4337-65-9 / 57 min / 33.43%
Dihydroergotamine / C33H37N5O5 / 583 / 511-12-6 / 57.6 min / 4.80%
Compounds of F4
Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- / C7H10N2O2 / 154 / 19179-12-5 / 38 min / 16.38%
Hexanedioic acid, mono (2-ethylhexyl) ester / C14H26O4 / 258 / 4337-65-9 / 57 min / 83.62%
Figure S4|. Biofilms of S. aureus and P. aeruginosa strains after 1 h treatment.1