Summary of Study ST002747

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001710. The data can be accessed directly via it's Project DOI: 10.21228/M8P43M This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002747
Study Title	Evolutionary genomics identifies host-directed therapeutics to treat intracellular bacterial infections
Study Summary	Obligate intracellular bacteria from the Rickettsiaceae family have shed essential biosynthetic pathways during their evolution towards host dependency. By systematically comparing this cytosolic family of bacteria to the related vacuolar Anaplasmataceae family using a novel computational pipeline called PoMeLo, we identified 20 metabolic pathways that may have been lost since the divergence of Anaplasmataceae and Rickettsiaceae, corresponding to the latter’s change to a cytosolic niche. We hypothesized that drug inhibition of these host metabolic pathways would reduce the levels of metabolic products available to the bacteria, thereby inhibiting bacterial growth. We tested 22 commercially available inhibitors for 14 of the identified pathways and found that 59% of the inhibitors reduced bacterial growth at concentrations that did not contribute to host cell cytotoxicity. Of these, 5 inhibitors with an IC50 under 5 µM were tested to determine whether their mode of inhibition was bactericidal or bacteriostatic. Both mycophenolate mofetil, an inhibitor of inosine-5'-monophosphate dehydrogenase in the purine biosynthesis pathway, and roseoflavin, an analog of riboflavin, displayed bactericidal activity. We then took an unbiased metabolomics approach to Rickettsia-infected cells to determine whether there was any overlap between our predicted host pathways and depletion of metabolite levels in infected cells, as measured by mass spectrometry. Our results show that 13 pathways were identified as metabolic gaps in both our computational predictions and our metabolomics analysis. These in vitro validation studies support the feasibility of a novel evolutionary genomics-guided approach for antibiotic drug development against obligate pathogens.
Institute	CZ Biohub
Last Name	DeFelice
First Name	Brian
Address	1291 Welch Rd., Rm. G0821 (SIM1), Stanford CA, California, 94305, USA
Email	bcdefelice@ucdavis.edu
Phone	5303564485
Submit Date	2023-06-23
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-07-07
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001710
Project DOI:	doi: 10.21228/M8P43M
Project Title:	Evolutionary genomics identifies host-directed therapeutics to treat intracellular bacterial infections
Project Summary:	Obligate intracellular bacteria from the Rickettsiaceae family have shed essential biosynthetic pathways during their evolution towards host dependency. By systematically comparing this cytosolic family of bacteria to the related vacuolar Anaplasmataceae family using a novel computational pipeline called PoMeLo, we identified 20 metabolic pathways that may have been lost since the divergence of Anaplasmataceae and Rickettsiaceae, corresponding to the latter’s change to a cytosolic niche. We hypothesized that drug inhibition of these host metabolic pathways would reduce the levels of metabolic products available to the bacteria, thereby inhibiting bacterial growth. We tested 22 commercially available inhibitors for 14 of the identified pathways and found that 59% of the inhibitors reduced bacterial growth at concentrations that did not contribute to host cell cytotoxicity. Of these, 5 inhibitors with an IC50 under 5 µM were tested to determine whether their mode of inhibition was bactericidal or bacteriostatic. Both mycophenolate mofetil, an inhibitor of inosine-5'-monophosphate dehydrogenase in the purine biosynthesis pathway, and roseoflavin, an analog of riboflavin, displayed bactericidal activity. We then took an unbiased metabolomics approach to Rickettsia-infected cells to determine whether there was any overlap between our predicted host pathways and depletion of metabolite levels in infected cells, as measured by mass spectrometry. Our results show that 13 pathways were identified as metabolic gaps in both our computational predictions and our metabolomics analysis. These in vitro validation studies support the feasibility of a novel evolutionary genomics-guided approach for antibiotic drug development against obligate pathogens.
Institute:	CZ Biohub
Last Name:	DeFelice
First Name:	Brian
Address:	1291 Welch Rd., Rm. G0821 (SIM1), Stanford CA, California, 94305, USA
Email:	bcdefelice@ucdavis.edu
Phone:	5303564485

Subject:

Subject ID:	SU002854
Subject Type:	Cultured cells
Subject Species:	Rickettsia parkeri; Homo sapiens
Taxonomy ID:	35792; 9606

Factors:

Subject type: Cultured cells; Subject species: Rickettsia parkeri; Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype	Treatment
SA289292	VIAH002_BK_3_Pos_QE1_Lipids_030	blank	blank
SA289293	VIAH002_BK_1_Neg_QE1_Lipids_007	blank	blank
SA289294	VIAH002_BK_3_Neg_QE1_Lipids_031	blank	blank
SA289295	VIAH002_BK_2_Pos_QE1_Lipids_018	blank	blank
SA289296	VIAH002_BK_2_Neg_QE1_Lipids_019	blank	blank
SA289297	VIAH002H_BK_2_Pos_QE2_HILIC_018	blank	blank
SA289298	VIAH002H_BK_3_Pos_QE2_HILIC_030	blank	blank
SA289299	VIAH002H_BK_1_Pos_QE2_HILIC_006	blank	blank
SA289300	VIAH002H_BK_1_Neg_QE2_HILIC_007	blank	blank
SA289301	VIAH002H_BK_2_Neg_QE2_HILIC_019	blank	blank
SA289302	VIAH002H_BK_3_Neg_QE2_HILIC_031	blank	blank
SA289303	VIAH002_BK_1_Pos_QE1_Lipids_006	blank	blank
SA289252	M5_uninfected_NA_5__012	Human	control
SA289253	M5_uninfected_NA_5__013	Human	control
SA289254	M1_uninfected_NA_1__017	Human	control
SA289255	VIAH002H_Pos_M1_QE2_HILIC_008	Human	control
SA289256	M3_uninfected_NA_3__011	Human	control
SA289257	VIAH002H_Pos_M4_QE2_HILIC_028	Human	control
SA289258	M2_uninfected_NA_2__009	Human	control
SA289259	M2_uninfected_NA_2__008	Human	control
SA289260	VIAH002H_Neg_M4_QE2_HILIC_029	Human	control
SA289261	VIAH002H_Neg_M3_QE2_HILIC_021	Human	control
SA289262	VIAH002H_Pos_M3_QE2_HILIC_020	Human	control
SA289263	M4_uninfected_NA_4__029	Human	control
SA289264	VIAH002H_Neg_M2_QE2_HILIC_011	Human	control
SA289265	VIAH002H_Pos_M2_QE2_HILIC_010	Human	control
SA289266	VIAH002H_Neg_M1_QE2_HILIC_009	Human	control
SA289267	VIAH002H_Pos_M5_QE2_HILIC_014	Human	control
SA289268	VIAH002H_Neg_M5_QE2_HILIC_015	Human	control
SA289269	M3_uninfected_NA_3__010	Human	control
SA289270	M1_uninfected_NA_1__016	Human	control
SA289271	M4_uninfected_NA_4__028	Human	control
SA289272	Inf2_infected_NA_2__020	Rickettsia parkeri & human	PostInfection
SA289273	Inf1_infected_NA_1__024	Rickettsia parkeri & human	PostInfection
SA289274	Inf3_infected_NA_3__026	Rickettsia parkeri & human	PostInfection
SA289275	Inf3_infected_NA_3__027	Rickettsia parkeri & human	PostInfection
SA289276	Inf1_infected_NA_1__025	Rickettsia parkeri & human	PostInfection
SA289277	Inf4_infected_NA_4__023	Rickettsia parkeri & human	PostInfection
SA289278	Inf4_infected_NA_4__022	Rickettsia parkeri & human	PostInfection
SA289279	Inf2_infected_NA_2__021	Rickettsia parkeri & human	PostInfection
SA289280	Inf5_infected_NA_5__014	Rickettsia parkeri & human	PostInfection
SA289281	VIAH002H_Pos_Inf3_QE2_HILIC_016	Rickettsia parkeri & human	PostInfection
SA289282	VIAH002H_Neg_Inf5_QE2_HILIC_013	Rickettsia parkeri & human	PostInfection
SA289283	VIAH002H_Pos_Inf5_QE2_HILIC_012	Rickettsia parkeri & human	PostInfection
SA289284	Inf5_infected_NA_5__015	Rickettsia parkeri & human	PostInfection
SA289285	VIAH002H_Pos_Inf1_QE2_HILIC_022	Rickettsia parkeri & human	PostInfection
SA289286	VIAH002H_Neg_Inf3_QE2_HILIC_017	Rickettsia parkeri & human	PostInfection
SA289287	VIAH002H_Neg_Inf1_QE2_HILIC_023	Rickettsia parkeri & human	PostInfection
SA289288	VIAH002H_Neg_Inf2_QE2_HILIC_027	Rickettsia parkeri & human	PostInfection
SA289289	VIAH002H_Neg_Inf4_QE2_HILIC_025	Rickettsia parkeri & human	PostInfection
SA289290	VIAH002H_Pos_Inf2_QE2_HILIC_026	Rickettsia parkeri & human	PostInfection
SA289291	VIAH002H_Pos_Inf4_QE2_HILIC_024	Rickettsia parkeri & human	PostInfection

Showing results 1 to 52 of 52

Showing results 1 to 52 of 52

Collection:

Collection ID:	CO002847
Collection Summary:	A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).
Sample Type:	A549 cells

Treatment:

Treatment ID:	TR002863
Treatment Summary:	A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).

Treatment ID:

TR002863

Treatment Summary:

A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).

Sample Preparation:

Sampleprep ID:	SP002860
Sampleprep Summary:	A549 cells were seeded into 10 x 6-well tissue culture treated plates the day before infection. On the day of infection, cells were either infected with an MOI of 0.05 in 1 mL of DMEM (5 replicates) or mock-infected with DMEM only (5 replicates). Plates were centrifuged at 300 × g for 5 minutes and placed in a 33°C in 5% CO2 for 4 days. After 4 days, cells from each well of a 6-well plate were washed with 2 mL of 1x PBS, scraped, and collected into an 1.5 mL centrifuge tube. Samples were washed twice with 500 µl of 1x PBS before resuspending in 225 µl ice-cold methanol with 1.5% iSTD-SPLASH and freezing on dry ice. Samples were processed within 48 h. Sample preparation and analysis have been detailed previously (DOI: dx.doi.org/10.17504/protocols.io.e6nvwj86dlmk/v1 (Private link for reviewers: https://www.protocols.io/private/8962C900975C11ED8E800A58A9FEAC02 to be removed before publication.) Analysis of metabolomics data was performed on MetaboAnalyst 5.0 and GraphPad Prism (Version 9.5.1 (528); Chong et al., 2018).

Sampleprep ID:

SP002860

Sampleprep Summary:

Combined analysis:

Analysis ID	AN004454	AN004455	AN004456	AN004457
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish
Column	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	counts, height	counts, height	counts, height	counts, height

Chromatography:

Chromatography ID:	CH003345
Chromatography Summary:	HILIC
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	45C
Flow Gradient:	Gradient elution was performed from 100% (B) at 0–2 min to 70% (B) at 7.7 min, 40% (B) at 9.5 min, 30% (B) at 10.25 min, 100% (B) at 12.75 min, isocratic until 16.75 min with a column flow of
Flow Rate:	0.4 mL/min.
Solvent A:	100% water; 10mM ammonium formate; 0.125% formic acid
Solvent B:	95% acetonitrile; 10mM ammonium formate; 0.125% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH003346
Chromatography Summary:	Lipids (ammonium acetate replaces ammonium formate in neg mode analysis
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	65C
Flow Gradient:	Gradient elution was performed from 15% (B) at 0 min to 30% (B) at 2 min, 48% (B) at 2.5 min, 82% (B) at 11 min, 99% (B) at 11.5 min, isocratic until 12 and resetting to initial conditions at 12.1 min through the end of the run at 15 min.
Flow Rate:	is 0.6 mL/min.
Solvent A:	60% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Solvent B:	10% acetonitrile/90% isopropanol; 10 mM ammonium formate; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004201
Analysis ID:	AN004454
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from the in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 50, 100 eV.
Ion Mode:	POSITIVE

MS ID:	MS004202
Analysis ID:	AN004455
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from the in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 50, 100 eV.
Ion Mode:	NEGATIVE

MS ID:	MS004203
Analysis ID:	AN004456
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected. Mass range was 220-1600 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 40, 60 eV.
Ion Mode:	POSITIVE

MS ID:	MS004204
Analysis ID:	AN004457
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Spectra were collected using a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer in both positive and negative mode ionization (separate injections). Full MS-ddMS2 data was collected. Mass range was 220-1600 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.0 Da. Collision energy was NCE 20, 40, 60 eV.
Ion Mode:	NEGATIVE