#METABOLOMICS WORKBENCH danielpardo88_20240930_065556 DATATRACK_ID:5250 STUDY_ID:ST003511 ANALYSIS_ID:AN005765 PROJECT_ID:PR002156 VERSION 1 CREATED_ON October 3, 2024, 10:01 pm #PROJECT PR:PROJECT_TITLE Lipidomic analysis of Malassezia globosa at different growth stages and the PR:PROJECT_TITLE dynamics of uptake and secreted lipids with growth media PR:PROJECT_TYPE Lipidomics PR:PROJECT_SUMMARY Malassezia is one of the most abundant genera found on human skin; specifically, PR:PROJECT_SUMMARY M. globosa is one of the yeast species dominant in this organ as it has been PR:PROJECT_SUMMARY associated with several skin diseases. Malassezia cannot synthesize fatty acids. PR:PROJECT_SUMMARY In response, the yeast cell intakes external fatty acids from the host or the PR:PROJECT_SUMMARY growth media for survival. Several studies have focused on investigating the PR:PROJECT_SUMMARY identity of lipids and enzymes in M. globosa to understand its lipid metabolism PR:PROJECT_SUMMARY and the biology of the yeast cell-host interaction. In this work, we performed a PR:PROJECT_SUMMARY supernatant lipidomic analysis on the mDixon media and the supernatant and on PR:PROJECT_SUMMARY the M. globosa at early and late stationary phase (72h and 90h, respectively) to PR:PROJECT_SUMMARY determine the lipid dynamics (lipids consumed vs. lipids secreted) between the PR:PROJECT_SUMMARY growth media and the two stages of growth. We were able to identify 87 lipids PR:PROJECT_SUMMARY within 17 classes of lipids; during the analysis, the increment of several PR:PROJECT_SUMMARY lipids increased throughout time concerning the growth media, suggesting a PR:PROJECT_SUMMARY secretion pattern from the cell to the media; some lipids found in this group PR:PROJECT_SUMMARY were conjugated Sterols (ST) such as Glycochenodeoxycholic acid (GCDCA), PR:PROJECT_SUMMARY Glycerophospholipids (GP), specifically phosphocholine's (PCs), Cardiolipins PR:PROJECT_SUMMARY (CL), in particular those with chains of (47 to 54 carbons) and Sphingolipids PR:PROJECT_SUMMARY (SP) such as Cer-PI which might have some role in pathogenicity. Likewise, the PR:PROJECT_SUMMARY increment of some lipids decreased, but some only reduced at the late stationary PR:PROJECT_SUMMARY phase (90h) only when the nutrients available was minimal. Finally, we observed PR:PROJECT_SUMMARY a third pattern in which the amount of some lipids decreased throughout time PR:PROJECT_SUMMARY (starting in the early stationary phase and finishing in the late stationary PR:PROJECT_SUMMARY phase), hinting at a distinctive consumption pattern. The principal lipids PR:PROJECT_SUMMARY consumed wereSterols (ST) bile acids, cholic acid, and its derivates, some PR:PROJECT_SUMMARY phosphocholines (PCs), Fatty acyls (FA), and cardiolipins (CL). The consumption PR:PROJECT_SUMMARY of these lipids was associated with different metabolic roles of the lipids in PR:PROJECT_SUMMARY the cell as it lacks production of these lipids in M. globosa. PR:INSTITUTE Universidad de los Andes PR:LAST_NAME Cala PR:FIRST_NAME Monica PR:ADDRESS CALLE 46 N 3 35 Bogota-Colombia PR:EMAIL mp.cala10@uniandes.edu.co PR:PHONE +573164316037 #STUDY ST:STUDY_TITLE Lipidomic analysis of Malassezia globosa at different growth stages and the ST:STUDY_TITLE dynamics of uptake and secreted lipids with growth media ST:STUDY_SUMMARY Malassezia is one of the most abundant genera found on human skin; specifically, ST:STUDY_SUMMARY M. globosa is one of the yeast species dominant in this organ as it has been ST:STUDY_SUMMARY associated with several skin diseases. Malassezia cannot synthesize fatty acids. ST:STUDY_SUMMARY In response, the yeast cell intakes external fatty acids from the host or the ST:STUDY_SUMMARY growth media for survival. Several studies have focused on investigating the ST:STUDY_SUMMARY identity of lipids and enzymes in M. globosa to understand its lipid metabolism ST:STUDY_SUMMARY and the biology of the yeast cell-host interaction. In this work, we performed a ST:STUDY_SUMMARY supernatant lipidomic analysis on the mDixon media and the supernatant and on ST:STUDY_SUMMARY the M. globosa at early and late stationary phase (72h and 90h, respectively) to ST:STUDY_SUMMARY determine the lipid dynamics (lipids consumed vs. lipids secreted) between the ST:STUDY_SUMMARY growth media and the two stages of growth. We were able to identify 87 lipids ST:STUDY_SUMMARY within 17 classes of lipids; during the analysis, the increment of several ST:STUDY_SUMMARY lipids increased throughout time concerning the growth media, suggesting a ST:STUDY_SUMMARY secretion pattern from the cell to the media; some lipids found in this group ST:STUDY_SUMMARY were conjugated Sterols (ST) such as Glycochenodeoxycholic acid (GCDCA), ST:STUDY_SUMMARY Glycerophospholipids (GP), specifically phosphocholine's (PCs), Cardiolipins ST:STUDY_SUMMARY (CL), in particular those with chains of (47 to 54 carbons) and Sphingolipids ST:STUDY_SUMMARY (SP) such as Cer-PI which might have some role in pathogenicity. Likewise, the ST:STUDY_SUMMARY increment of some lipids decreased, but some only reduced at the late stationary ST:STUDY_SUMMARY phase (90h) only when the nutrients available was minimal. Finally, we observed ST:STUDY_SUMMARY a third pattern in which the amount of some lipids decreased throughout time ST:STUDY_SUMMARY (starting in the early stationary phase and finishing in the late stationary ST:STUDY_SUMMARY phase), hinting at a distinctive consumption pattern. The principal lipids ST:STUDY_SUMMARY consumed wereSterols (ST) bile acids, cholic acid, and its derivates, some ST:STUDY_SUMMARY phosphocholines (PCs), Fatty acyls (FA), and cardiolipins (CL). The consumption ST:STUDY_SUMMARY of these lipids was associated with different metabolic roles of the lipids in ST:STUDY_SUMMARY the cell as it lacks production of these lipids in M. globosa. ST:INSTITUTE Universidad de los Andes, Colombia ST:LAST_NAME Cala ST:FIRST_NAME Mónica ST:ADDRESS CALLE 46 N 3 35 ST:EMAIL mp.cala10@uniandes.edu.co ST:PHONE +573164316037 ST:NUM_GROUPS 6 #SUBJECT SU:SUBJECT_TYPE Fungi SU:SUBJECT_SPECIES Malassezia globosa SU:TAXONOMY_ID 76773 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Sg1_1 Sample source:mDixon(media) | Sample_type:mDixon | Time of collection:na RAW_FILE_NAME=GL(-)-G1_001.mzXML SUBJECT_SAMPLE_FACTORS - Sg1_2 Sample source:mDixon(media) | Sample_type:mDixon | Time of collection:na RAW_FILE_NAME=GL(-)-G1_002.mzXML SUBJECT_SAMPLE_FACTORS - Sg1_3 Sample source:mDixon(media) | Sample_type:mDixon | Time of collection:na RAW_FILE_NAME=GL(-)-G1_003.mzXML SUBJECT_SAMPLE_FACTORS - Sg2_4 Sample source:Yeast | Sample_type:Mglobosa | Time of collection:72h RAW_FILE_NAME=GL(-)-G2_004.mzXML SUBJECT_SAMPLE_FACTORS - Sg2_5 Sample source:Yeast | Sample_type:Mglobosa | Time of collection:72h RAW_FILE_NAME=GL(-)-G2_005.mzXML SUBJECT_SAMPLE_FACTORS - Sg2_6 Sample source:Yeast | Sample_type:Mglobosa | Time of collection:72h RAW_FILE_NAME=GL(-)-G2_006.mzXML SUBJECT_SAMPLE_FACTORS - Sg3_7 Sample source:Yeast | Sample_type:Mglobosa | Time of collection:90h RAW_FILE_NAME=GL(-)-G3_007.mzXML SUBJECT_SAMPLE_FACTORS - Sg3_8 Sample source:Yeast | Sample_type:Mglobosa | Time of collection:90h RAW_FILE_NAME=GL(-)-G3_008.mzXML SUBJECT_SAMPLE_FACTORS - Sg3_9 Sample source:Yeast | Sample_type:Mglobosa | Time of collection:90h RAW_FILE_NAME=GL(-)-G3_009.mzXML SUBJECT_SAMPLE_FACTORS - Sg4_10 Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h RAW_FILE_NAME=GL(-)-G4_010.mzXML SUBJECT_SAMPLE_FACTORS - Sg4_11 Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h RAW_FILE_NAME=GL(-)-G4_011.mzXML SUBJECT_SAMPLE_FACTORS - Sg4_12 Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h RAW_FILE_NAME=GL(-)-G4_012.mzXML SUBJECT_SAMPLE_FACTORS - Sg4_13 Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h RAW_FILE_NAME=GL(-)-G4_013.mzXML SUBJECT_SAMPLE_FACTORS - Sg4_14 Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h RAW_FILE_NAME=GL(-)-G4_014.mzXML SUBJECT_SAMPLE_FACTORS - Sg4_15 Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h RAW_FILE_NAME=GL(-)-G4_015.mzXML SUBJECT_SAMPLE_FACTORS - Sg5_16 Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h RAW_FILE_NAME=GL(-)-G5_016.mzXML SUBJECT_SAMPLE_FACTORS - Sg5_17 Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h RAW_FILE_NAME=GL(-)-G5_017.mzXML SUBJECT_SAMPLE_FACTORS - Sg5_18 Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h RAW_FILE_NAME=GL(-)-G5_018.mzXML SUBJECT_SAMPLE_FACTORS - Sg5_19 Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h RAW_FILE_NAME=GL(-)-G5_019.mzXML SUBJECT_SAMPLE_FACTORS - Sg5_20 Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h RAW_FILE_NAME=GL(-)-G5_020.mzXML SUBJECT_SAMPLE_FACTORS - Sg5_21 Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h RAW_FILE_NAME=GL(-)-G5_021.mzXML SUBJECT_SAMPLE_FACTORS - QC_10 Sample source:Yeast | Sample_type:Quality control | Time of collection:na RAW_FILE_NAME=GL(-)-QC_010.mzXML SUBJECT_SAMPLE_FACTORS - QC_11 Sample source:Yeast | Sample_type:Quality control | Time of collection:na RAW_FILE_NAME=GL(-)-QC_011.mzXML SUBJECT_SAMPLE_FACTORS - QC_12 Sample source:Yeast | Sample_type:Quality control | Time of collection:na RAW_FILE_NAME=GL(-)-QC_012.mzXML SUBJECT_SAMPLE_FACTORS - QC_13 Sample source:Yeast | Sample_type:Quality control | Time of collection:na RAW_FILE_NAME=GL(-)-QC_013.mzXML SUBJECT_SAMPLE_FACTORS - QC_14 Sample source:Yeast | Sample_type:Quality control | Time of collection:na RAW_FILE_NAME=GL(-)-QC_014.mzXML #COLLECTION CO:COLLECTION_SUMMARY The reference strain Malassezia globosa CBS 7966 (Westerdijk Institute, Utrecht, CO:COLLECTION_SUMMARY The Netherlands) was used for the whole study. A frozen stock was reactivated CO:COLLECTION_SUMMARY and precultured at 33°C for seven days in modified Dixon (mDixon) agar [36 g CO:COLLECTION_SUMMARY L-1 mycosel agar [BD, USA], 20 g L-1 Ox Bile [Sigma Aldrich, USA], 36 g L-1 malt CO:COLLECTION_SUMMARY extract [Oxoid, UK], 0.02% glycerol [Sigma Aldrich, USA], 0.02% oleic acid CO:COLLECTION_SUMMARY [Sigma Aldrich, USA], and 0.1% Tween 40 [Sigma Aldrich, USA]]. Then, one colony CO:COLLECTION_SUMMARY was transferred to a new mDixon agar plate and was incubated for five days at CO:COLLECTION_SUMMARY 33°C. From this plate, yeasts were suspended in 3 mL inoculum in water plus CO:COLLECTION_SUMMARY 0.1% Tween 80 [Sigma Aldrich, USA] to a top standard of 2 on the McFarland scale CO:COLLECTION_SUMMARY and were used to inoculate 27 mL of mDixon broth [36 g L-1 malt extract [Oxoid, CO:COLLECTION_SUMMARY UK], 6 g L-1 peptone [BD, USA], 20 g L-1 Ox bile [Sigma Aldrich, USA, 0.02% CO:COLLECTION_SUMMARY glycerol [Sigma Aldrich, USA], 0.02% oleic acid [Sigma Aldrich, USA], and 0.1% CO:COLLECTION_SUMMARY Tween 40 [Sigma Aldrich, USA] for 96 hours at 33°C and 180 rpm. An aliquot of CO:COLLECTION_SUMMARY 300 mL was used to inoculate 29.7 mL of fresh mDixon broth and incubated at CO:COLLECTION_SUMMARY 33°C and 180 rpm for 72 h and 90 h to reach the early stationary and stationary CO:COLLECTION_SUMMARY phase. (doi:10.1007/978-3-642-03616-3_2; doi:10.3389/fcimb.2020.00338). CO:SAMPLE_TYPE Yeast cells #TREATMENT TR:TREATMENT_SUMMARY No treatments were applied to the samples. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples of M. globosa were collected at different growth stages, then SP:SAMPLEPREP_SUMMARY centrifuged at 4,500 rpm for 10 minutes, and the supernatants were collected. SP:SAMPLEPREP_SUMMARY Subsequently, 5 mL of isopropanol was added to the supernatant, followed by SP:SAMPLEPREP_SUMMARY centrifugation at 4,500 rpm for another 10 minutes. Lipid extraction was SP:SAMPLEPREP_SUMMARY performed according to Bligh and Dyer lipid extraction with some modifications SP:SAMPLEPREP_SUMMARY (doi:10.1038/nprot.2016.040; doi:10.1016/j.jchromb.2017.06.045; SP:SAMPLEPREP_SUMMARY doi:10.1021/acs.analchem.8b02839). 2mL of a citric acid buffer [0.1 M sodium SP:SAMPLEPREP_SUMMARY citrate tribasic dihydrate, 1 M sodium chloride, pH 3.6], 2 mL of MeOH, and 4 mL SP:SAMPLEPREP_SUMMARY of chloroform were added to 8 mL of supernatant collected previously. The SP:SAMPLEPREP_SUMMARY mixture was homogenized with vortex for 15 min and sonicated for 30 min. The SP:SAMPLEPREP_SUMMARY extracted lipids' organic phase was collected and dried on a Speed Vac. Then, SP:SAMPLEPREP_SUMMARY the dry extract was re-dissolved in 1 mL of ACN containing 0.1% NH3·H2O (v/v), SP:SAMPLEPREP_SUMMARY followed by strong anion-exchange solid-phase extraction using Strata SAX SP:SAMPLEPREP_SUMMARY SPE-cartridge (55 uM, 70 A, 100 mg, 1 mL Phenomenex) which was pre-conditioned SP:SAMPLEPREP_SUMMARY with 3 mL ACN. After sampling 1 mL of the lipid extract, the cartridge was SP:SAMPLEPREP_SUMMARY washed with 3 mL acetone/H2O (1/9, v/v), 3mL acetone, and eluted with 3 mL SP:SAMPLEPREP_SUMMARY formic acid/acetone (1/99, v/v) followed by evaporation using a Speed Vac. SP:SAMPLEPREP_SUMMARY Samples were stored at -80°C for one week and dissolved in 1 mL of MeOH for SP:SAMPLEPREP_SUMMARY further analysis (doi:10.1038/nprot.2016.040; doi:10.1016/j.jchromb.2017.06.045; SP:SAMPLEPREP_SUMMARY doi:10.1021/acs.analchem.8b02839). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic analysis was carried out at 65°C and constant flow 0.6 mL/min CH:CHROMATOGRAPHY_SUMMARY using gradient elution with phase A (60:40 ACN: type I water with 10 mM of CH:CHROMATOGRAPHY_SUMMARY ammonium formate and 0.1% v/v of formic acid) and mobile phase B (90:10 CH:CHROMATOGRAPHY_SUMMARY Isopropanol: Acetonitrile with 10 mM of ammonium formate and 0.1% v/v of formic CH:CHROMATOGRAPHY_SUMMARY acid). The elution gradient was 0 min 15% (B), 0–4 min 30% (B), 4–5 min 48% CH:CHROMATOGRAPHY_SUMMARY (B), 5–22 min 82% (B), 22–23 min 95% (B), 23–25 min 95% (B), 25–26 min CH:CHROMATOGRAPHY_SUMMARY 15% (B), and 26–31 min 15% (B). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1260 Infinity LC System CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) CH:SOLVENT_A 60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid CH:FLOW_GRADIENT The elution gradient was 0 min 15% (B), 0–4 min 30% (B), 4–5 min 48% (B), CH:FLOW_GRADIENT 5–22 min 82% (B), 22–23 min 95% (B), 23–25 min 95% (B), 25–26 min 15% CH:FLOW_GRADIENT (B), and 26–31 min 15% (B) CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 65 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Mass spectrometry detection was performed in negative ionization mode in a full MS:MS_COMMENTS scan from 100 m/z to 1200 m/z. The mass correction was performed during the MS:MS_COMMENTS analysis with reference masses: m/z 121.0509 (C5H4N4) y m/z 922.0098 MS:MS_COMMENTS (C18H18O6N3P3F24). MS:MS_RESULTS_FILE ST003511_AN005765_Results.txt UNITS: Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END