#METABOLOMICS WORKBENCH zouzhen1_20240827_013708 DATATRACK_ID:5150 STUDY_ID:ST003440 ANALYSIS_ID:AN005656 PROJECT_ID:PR002123 VERSION 1 CREATED_ON August 28, 2024, 11:02 pm #PROJECT PR:PROJECT_TITLE Gut symbiont-derived sphingosine modulates vector competence in Aedes mosquitoes PR:PROJECT_SUMMARY The main vectors of Zika virus (ZIKV) and dengue virus (DENV) are Aedes aegypti PR:PROJECT_SUMMARY and Ae. albopictus, with Ae. aegypti being more competent. However, the PR:PROJECT_SUMMARY underlying mechanisms remain unclear. Here, we find Ae. albopictus shows PR:PROJECT_SUMMARY comparable vector competence to ZIKV/DENV with Ae. aegypti by blood-feeding PR:PROJECT_SUMMARY after antibiotic treatment or intrathoracic injection. This suggests that midgut PR:PROJECT_SUMMARY microbiota can influence vector competence. Enterobacter hormaechei_B17 (Eh_B17) PR:PROJECT_SUMMARY is isolated from field-collected Ae. albopictus and conferred resistance to PR:PROJECT_SUMMARY ZIKV/DENV infection in Ae. aegypti after gut-transplantation. Sphingosine, a PR:PROJECT_SUMMARY metabolite secreted by Eh_B17, effectively suppresses ZIKV infection in both Ae. PR:PROJECT_SUMMARY aegypti and cell cultures by blocking viral entry during the fusion step, with PR:PROJECT_SUMMARY an IC50 of approximately 10 μM. A field survey reveals that Eh_B17 PR:PROJECT_SUMMARY preferentially colonizes Ae. albopictus compared to Ae. aegypti. And field Ae. PR:PROJECT_SUMMARY aegypti positive for Eh_B17 are more resistant to ZIKV infection. These findings PR:PROJECT_SUMMARY underscore the potential of gut symbiotic bacteria, such as Eh_B17, to modulate PR:PROJECT_SUMMARY the arbovirus vector competence of Aedes mosquitoes. As a natural antiviral PR:PROJECT_SUMMARY agent, Eh_B17 holds promise as a potential candidate for blocking ZIKV/DENV PR:PROJECT_SUMMARY transmission. PR:INSTITUTE Institute of Zoology, Chinese Academy of Sciences PR:LAST_NAME Zou PR:FIRST_NAME Zhen PR:ADDRESS 1 Beichen West Road, Chaoyang District, Beijing, Beijing, 100101, China PR:EMAIL zouzhen@ioz.ac.cn PR:PHONE +86 15010747660 #STUDY ST:STUDY_TITLE Non-targeted metabolomic analysis of Enterobacter hormaechei_B17 ST:STUDY_TITLE (Eh_B17),Enterobacter sichuanensis_B36(Es_B36), and Enterobacter hormaechei_B56 ST:STUDY_TITLE (Eh_B56) ST:STUDY_SUMMARY All samples were acquired by the LC-MS system according to the machine ST:STUDY_SUMMARY instructions. Firstly, all chromatographic separations were performed using an ST:STUDY_SUMMARY UPLC system (SCIEX). An XBridge BEH C18 column (3.5um,2.1mm × 100mm, Waters) ST:STUDY_SUMMARY was used for the reversed-phase separation. The column oven was maintained at ST:STUDY_SUMMARY 50°C. The flow rate was 0.3 mL/min and the mobile phase consisted of solvent A ST:STUDY_SUMMARY (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% ST:STUDY_SUMMARY formic acid). The gradient conditions were set as follows: 0-0.5 min, 5% phase ST:STUDY_SUMMARY B; 0.5-5 min, 5% to 80% phase B; 5-7 min, 80%-100% phase B; 7-8 min,100% phase ST:STUDY_SUMMARY B; 8-8.1 min, 100%-5% phase B; 8.1-10 min, 5% phase B. The injection volume for ST:STUDY_SUMMARY each sample was 10 μL.A high-resolution tandem mass spectrometer TripleTOF5600 ST:STUDY_SUMMARY (SCIEX) was used to detect metabolites eluted from the column. The Q-TOF was ST:STUDY_SUMMARY operated in both positive ion and negative ion modes. For positive ion mode, the ST:STUDY_SUMMARY capillary voltages were set to 5 kV. For negative ion mode, the capillary ST:STUDY_SUMMARY voltages were set to -4.5 kV. Mass spectrometry data were acquired in IDA mode. ST:STUDY_SUMMARY The TOF mass range was 50 to 1200 Da. The top 8 precursors were fragmented for ST:STUDY_SUMMARY MS/MS detection. In addition, a quality control sample (pool of all samples) was ST:STUDY_SUMMARY acquired after every 10 samples to evaluate the stability of the LC-MS ST:STUDY_SUMMARY throughout the acquisition. ST:INSTITUTE Institute of Zoology, Chinese Academy of Sciences ST:LAST_NAME Zou ST:FIRST_NAME Zhen ST:ADDRESS 1 Beichen West Road, Chaoyang District, Beijing, Beijing, 100101, China ST:EMAIL zouzhen@ioz.ac.cn ST:PHONE +86 15010747660 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Enterobacter sp. #FACTORS #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 - B17-F6_N-1 Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B17-F6_N-1.mzML SUBJECT_SAMPLE_FACTORS - B17-F6_N-2 Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B17-F6_N-2.mzML SUBJECT_SAMPLE_FACTORS - B17-F6_N-3 Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B17-F6_N-3.mzML SUBJECT_SAMPLE_FACTORS - B17-F6_P-1 Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B17-F6_P-1.mzML SUBJECT_SAMPLE_FACTORS - B17-F6_P-2 Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B17-F6_P-2.mzML SUBJECT_SAMPLE_FACTORS - B17-F6_P-3 Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B17-F6_P-3.mzML SUBJECT_SAMPLE_FACTORS - B36_N Bacterium:Enterobacter sichuanensis | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B36_N.mzML SUBJECT_SAMPLE_FACTORS - B36_P Bacterium:Enterobacter sichuanensis | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B36_P.mzML SUBJECT_SAMPLE_FACTORS - B56_N Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B56_N.mzML SUBJECT_SAMPLE_FACTORS - B56_P Bacterium:Enterobacter hormaechei | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=B56_P.mzML SUBJECT_SAMPLE_FACTORS - QC1_N Bacterium:mixture of B17-F6, B36, and B56 | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=QC1_N.mzML SUBJECT_SAMPLE_FACTORS - QC1_P Bacterium:mixture of B17-F6, B36, and B56 | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=QC1_P.mzML SUBJECT_SAMPLE_FACTORS - QC2_N Bacterium:mixture of B17-F6, B36, and B56 | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=QC2_N.mzML SUBJECT_SAMPLE_FACTORS - QC2_P Bacterium:mixture of B17-F6, B36, and B56 | Sample source:bacterial supernatant RAW_FILE_NAME(raw data file names)=QC2_P.mzML #COLLECTION CO:COLLECTION_SUMMARY Enterobacter hormaechei_B17 (B17) and Enterobacter sichuanensis_B36 (B36) were CO:COLLECTION_SUMMARY isolated from the midgut of wild Aedes albopictus, while Enterobacter CO:COLLECTION_SUMMARY hormaechei_B56 (B56) was isolated from the midgut of lab-adapted Aedes aegypti. CO:COLLECTION_SUMMARY The flow-through of Eh_B17 was then separated into six fractions by CO:COLLECTION_SUMMARY semi-preparative high performance liquid chromatography (SPHPLC) according to CO:COLLECTION_SUMMARY the peak time. The parts were named F1 (0–6.5 min), F2 (6.5–10 min), F3 CO:COLLECTION_SUMMARY (10–15.5 min), F4 (15.5–20 min), F5 (20–25.5 min), and F6 (25.5–55 min). CO:SAMPLE_TYPE bacteria #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY B36 and B56 were grown overnight in LB medium at 37°C with an OD600 of 0.6. SP:SAMPLEPREP_SUMMARY Then, a 200-μL bacterial solution was applied to LB plates and maintained for 3 SP:SAMPLEPREP_SUMMARY d. The bacterial colonies were collected in 1 mL methanol and then put in a SP:SAMPLEPREP_SUMMARY water bath at 80°C until the methanol evaporated completely. Samples were SP:SAMPLEPREP_SUMMARY reconstituted in 0.5 mL of methanol and centrifuged at 12,000 × g for 3 min to SP:SAMPLEPREP_SUMMARY collect the supernatant. The liquid was evaporated using a rotary evaporator, SP:SAMPLEPREP_SUMMARY and the dry powder was dissolved in ddH2O to at a concentration of 50 mg/mL. SP:SAMPLEPREP_SUMMARY Liquid was filtrated with a membrane filter (0.45 µm) and stored at −20°C SP:SAMPLEPREP_SUMMARY for further testing. At least three independent experiments were performed. B17 SP:SAMPLEPREP_SUMMARY was grown in LB medium at 37°C until OD600 reached 0.6, and then transferred to SP:SAMPLEPREP_SUMMARY 16°C and incubated for 48 h. The bacterial supernatant was collected and SP:SAMPLEPREP_SUMMARY concentrated using vacuum freeze-drying (ALPHA LDplus, Germany). The dry powder SP:SAMPLEPREP_SUMMARY was resuspended in ddH2O in a 1/20 volume of the original supernatant. The SP:SAMPLEPREP_SUMMARY resuspended supernatant was separated into retention (mainly protein) and SP:SAMPLEPREP_SUMMARY flow-through (mainly metabolites) by a 3 kDa centrifugal filter. The fluids were SP:SAMPLEPREP_SUMMARY stored at −80°C for further experiments. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME ExionUPLC（SCIEX） CH:COLUMN_NAME Waters XBridge BEH C18 (100 x 2.1mm,3.5um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0–0.5 min, 5% phase B; 0.5–5 min, 5%–80% phase B; 5–7 min, 80%–100% CH:FLOW_GRADIENT phase B; 7–8 min,100% phase B; 8–8.1 min, 100%–5% phase B; 8.1–10 min, CH:FLOW_GRADIENT 5% phase B CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 50°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME TripleTOF5600（SCIEX） MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS A high-resolution tandem mass spectrometer TripleTOF5600（SCIEX）was used to MS:MS_COMMENTS detect metabolites eluted form the column. The Q-TOF was operated in both MS:MS_COMMENTS positive and negative ion modes. For positive ion mode, the capillary voltages MS:MS_COMMENTS were set at 5 kV. For negative ion mode, the capillary voltages were set at -4.5 MS:MS_COMMENTS kV. The mass spectrometry data were acquired in IDA mode. The TOF mass range was MS:MS_COMMENTS from 50 to 1200 Da. For the MS/MS detection, top 8 precursors were fragmented. MS:MS_COMMENTS Furthermore, in order to evaluate the stability of the LC-MS during the whole MS:MS_COMMENTS acquisition, a quality control sample (Pool of all samples) was acquired after MS:MS_COMMENTS every 10 samples. MS:MS_RESULTS_FILE ST003440_AN005656_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END