Isolation of carotenoid producing yeast strains
To isolate carotenoid-producing yeast strains, the isolation source selected was soil/ compost, peels, flower petals, etc. About 1g of the above material were added to different flasks containing isolation medium (10ml) and incubated for 24-48h. After growing for 48h, the medium was serially diluted from 10-1 to 10-10. 0.1 ml of the each dilution was spread on yeast extract peptone dextrose agar (YPD) plates. Best colony formation was observed in 10-6 dilution spread plate. Following 2-4 days of incubation at room temperature, morphologically different isolates were selected and transferred to fresh YPD plates and stored appropriately at 4°C and at -80°C as glycerol stocks.
Identification and characterization of selected yeast strains
Selective yeast strains Y1, ab1, WEP, CS1 and CS2 were characterized, first on the basis of morphology, followed by rDNA sequencing. Phenotypic characterization was done on the basis of colony and cell morphology after visualizing them under Nikon Eclipse Ni Microscope (NIS Elements).
Genomic DNA isolation and rDNA sequencing
Yeast genomic DNA isolation
Total yeast DNA was extracted by standardized method of Guillamon et al., 1994 . Yeast colonies were picked from YPD medium selective plates after two days of growth at 30°C. Alternatively, single colonies were picked from plates and inoculated in 5 ml Yeast Peptone Dextrose (YPD), and grown at 30°C for approximately 24h. About 2ml of the overnight cultures were centrifuged at 1,254× g for 5 min and the cell pellets obtained, were re-suspended in 1 ml 1X PBS buffer and again centrifuged at 2,822× g for 4 min. The pellet was re-suspended in 0.5 ml of TE buffer [50 mMTris-HCl (pH7.4), 20 mM EDTA]; the mixture was then incubated for 30 min at 65° C. Followed by addition of glass beads and P:C:I (Phenol:Chloroform:Isoamyl alcohol) solution to the mixture, which was placed on ice and centrifuged for 5 min. The supernatant was transferred to a fresh tube, mixed with an equal volume of 2-propanol, and incubated for 5 min at room temperature. The pellet was air-dried and dissolved in sterile distilled water.
The 5.8S-internally transcribed spacer (ITS) rDNA regions (ITS1 and ITS2) of all strains were PCR amplified from the genomic DNA using the ITS1 and ITS4 primers. PCR amplifications were performed in a 50 µL reaction volume supplemented with 50 ng of genomic DNA, 10pmol of each primer, 10 mM of each dNTP and 2.5 units of Taq DNA Polymerase (G-Biosciences, USA). All the amplifications were programmed in a T100 thermo cycler (BIO RAD, India) as follows: 95°C for 3 min, followed by 30 cycles of 95°C for 30 sec, 52°C for 30 sec and 72° C for 1 min, with a final extension step at 72° C for 10 min. The amplified PCR products were separated on a 0.8% (w/v) agarose gel in 1X TAE (40 mMTris-Acetate, 1 mM EDTA (pH 8.0) buffer and detected by staining with ethidium bromide. The amplified PCR products of the 5.8S rDNA regions were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) and sequenced using the ITS1 and ITS4 primers. The sequences were submitted to NCBI to get the accession number.
Carotenoid extraction and determination
About 5 ml of 48-h-old seed culture (log phase) was used to inoculate 100 ml of growth media and incubated in an orbital shaker at 120 rpm. Samples were collected at every 6-12 h interval for determination of OD (600 nm), dry and wet cell weight, total sugar and reducing sugar of the medium as well as carotenoid content . Carotenoids were extracted by a modified method of Kim et al., 2004 . After centrifugation, the cells were collected, washed and treated with DMSO at 50°C for 1h and finally transferred to the hexane phase by liquid-liquid extraction. The OD of the supernatant was determined at 450 nm against hexane using a Systronics UV-Vis spectrophotometer. Quantification was done using standard curve of β-carotene (prepared in hexane). Standard β-carotene was obtained from SRL PVT Ltd, India. Total carotenoid content was expressed as the content of β-carotene that was determined spectrophotometrically at 475nm. All statistical calculations (t-Test) were done using MEDCALC Software bvba statistical software (www.medcalc.org) for determination of p-value.
Thin Layer Chromatography (TLC) analysis for the separation of the pigment fractions
Thin Layer Chromatographic separation of the different fractions from the crude carotenoid pigment of all isolated yeast strains was carried out using silica gel 60 TLC plates (Merck, Germany) using petroleum ether and acetone (80:20, v/v) as a mobile phase and determined their Rf values. The samples were identified by comparing the distance travelled by the standard to the distance travelled by the test sample β-carotene. The Rf values is a mathematical representation of the ration of the distance travelled by the solvent . Perrier et al. (1995), reported that β-carotene was the major pigment (78.6%) present in the Rhodotorula strain . The isolated yeast strain, Rhodotorula glutinis ab1 produced good amount of carotenoids and shows different bands in TLC analysis. So we chose the carotenoid extract of Rhodotorula glutinis ab1 strain for further study.
Preparation of stock and working concentrations of carotenoids
Standard β-carotene (SRL Pvt Ltd, India) and carotenoid extract were resuspended in dimethyl sulphoxide (DMSO) for preparation of 1 mg/ml and 2.5 mg/ml stock solution respectively. Further 0.1 mg/ml working stocks were prepared and eventually used to set up the various assays. Negative controls contained parasites in solvent without treatment.
In-vitro P. falciparum culture
Parasites were cultured at 2.5% hematocrit in RPMI-HEPES medium at pH 7.4 supplemented with hypoxanthine at 50 μg/ml, NaHCO3 at 25 mM, gentamicin at 2.5 μg/ml and Albumax II (Gibco) at 0.5% (w/v). To obtain enriched ring stage parasites, a standard sorbitol synchronization method was used. P. falciparum strain 3D7 was used for all experiments.
Microscopic examination of P. falciparum
Thin smears of P. falciparum cultures were prepared on glass slides, fixed with 100% methanol, and stained with fresh Giemsa (Sigma) solution made in filtered distilled water. Smears were examined with a 100X oil immersion objective and a standard phase-contrast microscope (Leica). Images from the smears were captured with Nikon camera and processed with Adobe Photoshop.
Growth inhibition assay by FACS
In vitro growth inhibitory activities of carotenoid extract and standard β-carotene against strain 3D7 were evaluated at concentrations ranging between 10-3µg/µl–10-5µg/µl for standard β-carotene and between 10-3µg/µl–10-6µg/µl for carotenoid extract respectively. Around 34–38h of sorbitol synchronized cells of strain 3D7 at a late trophozoite-stage with an initial parasitaemia of 0.8%, at 2% haematocrit, were incubated with the compounds at above-mentioned concentration over one intra erythrocytic cycle of parasite growth; untreated parasites served as control. Following a 48h incubation, the whole sample was collected and washed twice with 0.01M PBS and stained with ethidium bromide (10μM) (Sigma Aldrich, USA) for 15 min at room temperature in dark. The cells were washed with 0.01M PBS, and analysed by flow cytometry on ARIAIII (Becton Dickinson) using Flow-Jo software. Fluorescence signal (PE) was detected with the 590 nm band pass filter using an excitation laser of 488 nm collecting 100,000 cells per sample. Following acquisition, parasitaemia was estimated by quantifying number of infected erythrocytes which were determined by the proportion of PE-positive cells using Flow-Jo software.
Tightly synchronized, ring stage culture (10-12 hours) was diluted to 1% parasitemia & 2% hematocrit to determine any delay in progression upon treatment. Briefly, each well with 1% parasitemia and 2% hematocrit was treated with 1 µg/ml and 0.1µg/ml of standard β- carotene as well as carotenoid extract for period of 48 hours. Untreated cells served as control. To evaluate any morphological and progression variation, Giemsa stained smears were prepared at respective time points 24, 36, 48 and 56 hours. Nearly 2000 cells were scored by light microscopy.
Detection of parasitaemia using SYBR Green1 fluorescence
P. falciparum3D7 synchronized culture at late trophozoite stage with initial parasitaemia of 5% was diluted to 0.8% and packed erythrocytes were added to maintain 2% haematocrit in each well of 96-well microtitre plate keeping total volume of 100μl per well. The wells were treated with above mentioned concentration of carotenoids and incubated for 48h. Further, after completion of one intraerythrocytic cycle 100 μl of lysis buffer [20 mMTris (pH 7.5), 5 mM EDTA, 0.008 % (W/V) saponin, and 0.08 % (V/V) Triton X-100] containing Syber Green (1× final concentration) was added in each well. The plate was then incubated in the dark for 2h, fluorescence was measured at 485 nm (excitation) and 528 nm (emission). Parasite level was then plotted against fluorescence intensity.
Monitoring of the P. falciparum egress phenotype
To evaluate the egress of parasite from erythrocytes Egress assay was performed as mentioned by Garg et al., 2013 . Tightly synchronized schizont stage parasites (∼44-46 hpi) were enriched using 65% percoll with 85-90 % purity. Parasite culture was diluted at a final parasitaemia of 10% schizonts in 2% hematocrit. These were then seeded in a 96 well flat bottom plate (100 µl /well) and were exposed to respective treatments of carotenoids for 7-8h. BAPTA-AM was taken as positive control of egress inhibition in the study. After 8h the thin blood smears were made on glass slides and were stained with Giemsa stain for microscopic analysis.
Parasite egress % was calculated as the percent of schizonts ruptured in treatment and control during the incubation time as compared with the initial number of parasites at 0h, using the formula described below.