Summary of Study ST003483

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 PR002139. The data can be accessed directly via it's Project DOI: 10.21228/M84F91 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST003483
Study TitleTissue niche influences immune and metabolic profiles to Staphylococcus aureus biofilm infection (Extracellular data)
Study SummaryInfection is a devastating post-surgical complication, often requiring additional procedures and prolonged antibiotic therapy. This is especially relevant for craniotomy and prosthetic joint infections (PJI), both of which are characterized by biofilm formation on the bone or implant surface, respectively, with S. aureus representing a primary cause. The local tissue microenvironment likely has profound effects on immune attributes that can influence treatment efficacy, which becomes critical to consider when developing therapeutics for biofilm infections. However, the extent to which distinct tissue niches influence immune function during biofilm development remains relatively unknown. To address this, we compare the metabolomic, transcriptomic, and functional attributes of leukocytes in mouse models of S. aureus craniotomy and PJI complemented with patient samples from both infection modalities, which reveals profound tissue niche-dependent differences in nucleic acid, amino acid, and lipid metabolism with links to immune modulation. These signatures are both spatially and temporally distinct, differing not only between infection sites but evolving over time within a single model. Collectively, this demonstrates that biofilms elicit unique immune and metabolic responses that are heavily influenced by the local tissue microenvironment, which will likely have important implications when designing therapeutic approaches targeting these infections. This submission contains the extracellular metabolomic data for the larger project.
Institute
University of Nebraska Medical Center
DepartmentDepartment of pathology, microbiology and immunology
Last NameShinde
First NameDhananjay
AddressDRC 2, 7066, UNMC, Emily st
Emaildhananjay.shinde@unmc.edu
Phone4025597623
Submit Date2024-09-14
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2024-09-24
Release Version1
Dhananjay Shinde Dhananjay Shinde
https://dx.doi.org/10.21228/M84F91
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002139
Project DOI:doi: 10.21228/M84F91
Project Title:Tissue niche influences immune and metabolic profiles to Staphylococcus aureus biofilm infection
Project Type:Intracellular data
Project Summary:Infection is a devastating post-surgical complication, often requiring additional procedures and prolonged antibiotic therapy. This is especially relevant for craniotomy and prosthetic joint infections (PJI), both of which are characterized by biofilm formation on the bone or implant surface, respectively, with S. aureus representing a primary cause. The local tissue microenvironment likely has profound effects on immune attributes that can influence treatment efficacy, which becomes critical to consider when developing therapeutics for biofilm infections. However, the extent to which distinct tissue niches influence immune function during biofilm development remains relatively unknown. To address this, we compare the metabolomic, transcriptomic, and functional attributes of leukocytes in mouse models of S. aureus craniotomy and PJI complemented with patient samples from both infection modalities, which reveals profound tissue niche-dependent differences in nucleic acid, amino acid, and lipid metabolism with links to immune modulation. These signatures are both spatially and temporally distinct, differing not only between infection sites but evolving over time within a single model. Collectively, this demonstrates that biofilms elicit unique immune and metabolic responses that are heavily influenced by the local tissue microenvironment, which will likely have important implications when designing therapeutic approaches targeting these infections.
Institute:UNMC
Department:Department of pathology, microbiology and immunology
Laboratory:Pathology, Microbiology and Immunology
Last Name:Dhananjay
First Name:Shinde
Address:DRC 2, 7066, UNMC, Emily st
Email:dhananjay.shinde@unmc.edu
Phone:4025597623

Subject:

Subject ID:SU003611
Subject Type:Mammal
Subject Species:Mus musculus
Gender:Male and female
Species Group:Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Experimental Variable
SA383829d14-B5Brain d14
SA383830d14-B4Brain d14
SA383831d14-B3Brain d14
SA383832d14-B2Brain d14
SA383833d14-B1Brain d14
SA383834d3-B2Brain d3
SA383835d3-B1Brain d3
SA383836d3-B3Brain d3
SA383837d3-B5Brain d3
SA383838d3-B4Brain d3
SA383839d7-B3Brain d7
SA383840d7-B1Brain d7
SA383841d7-B2Brain d7
SA383842d7-B4Brain d7
SA383843d7-B5Brain d7
SA383844d14-G5Galea d14
SA383845d14-G4Galea d14
SA383846d14-G3Galea d14
SA383847d14-G2Galea d14
SA383848d14-G1Galea d14
SA383849d3-G1Galea d3
SA383850d3-G4Galea d3
SA383851d3-G3Galea d3
SA383852d3-G5Galea d3
SA383853d3-G2Galea d3
SA383854d7-G4Galea d7
SA383855d7-G5Galea d7
SA383856d7-G3Galea d7
SA383857d7-G2Galea d7
SA383858d7-G1Galea d7
SA383859d14-T2Tissue (PJI) d14
SA383860d14-T5Tissue (PJI) d14
SA383861d14-T4Tissue (PJI) d14
SA383862d14-T3Tissue (PJI) d14
SA383863d14-T1Tissue (PJI) d14
SA383864d3-T4Tissue (PJI) d3
SA383865d3-T5Tissue (PJI) d3
SA383866d3-T3Tissue (PJI) d3
SA383867d3-T2Tissue (PJI) d3
SA383868d3-T1Tissue (PJI) d3
SA383869d7-T2Tissue (PJI) d7
SA383870d7-T4Tissue (PJI) d7
SA383871d7-T1Tissue (PJI) d7
SA383872d7-T5Tissue (PJI) d7
SA383873d7-T3Tissue (PJI) d7
Showing results 1 to 45 of 45

Collection:

Collection ID:CO003604
Collection Summary:The intracellular metabolome of granulocytes isolated from the galea and PJI tissue of mouse craniotomy and PJI models, respectively, were profiled by LC-HRMS. Fifteen mice were infected for each biofilm model (craniotomy and PJI) with single-cell suspensions prepared by pooling tissue homogenates from 3 mice into one sample, resulting in 5 biological replicates. Samples were then enriched for granulocytes using Ly6G magnetic beads (Miltenyi Biotec, Bergishch Gladbach, Germany). Magnetic beads were utilized for purification instead of FACS to limit alterations in cellular metabolism that may occur with extended processing times. Samples were washed twice in 1X PBS, resuspended in 80% MeOH, and frozen at -80°C for 15 min. Metabolomics was also performed on supernatants prepared from brain, galea, and PJI tissue homogenates at days 3, 7, and 14 post-infection. Similar to granulocyte intracellular metabolite collection, tissue supernatants were centrifuged at high speed to remove residual cellular material, combined with 100% MeOH to yield an 80% MeOH solution, and frozen at -80°C.
Sample Type:Extracellular infection sites homogenate

Treatment:

Treatment ID:TR003620
Treatment Summary:All mice were group-housed at 21-23°C (22°C average) and 30-70% humidity (55% average) under 12 h light/dark cycle with free access to food (2019S Teklad Global 19% Protein Extruded Rodent Diet; Inotiv, West Lafayette, IN) and water. S. aureus craniotomy infection was established in male and female 8- to 10-week-old C57BL/6J mice (RRID:IMSR_JAX:000664). Briefly, ketamine and xylazine were administered to achieve a surgical plane of anesthesia before skin disinfection with betadine. A midline incision of the scalp was made, whereupon a bone flap was created using a high-speed pneumatic drill (Stryker Corporation, Kalamazoo, MI). The bone flap was incubated with 0.5 mL of S. aureus USA300 LAC13C diluted to 10^6/mL in brain-heart infusion broth at 37°C for 5 min and subsequently rinsed in PBS, blotted dry on a sterile field, and reimplanted. This procedure reliably produces an infectious inoculum of 10^3 CFUs per bone flap, which more accurately models surgical site infection that progresses to biofilm formation. The scalp incision was closed using 6-0 nylon suture and animals were continuously monitored until they regained ambulation, and daily thereafter. S. aureus PJI was established in male and female 8- to 10-week-old C57BL/6J mice. Mice were anesthetized with ketamine and xylazine prior to skin disinfection with betadine and initial incision of the quadriceps. A secondary incision was made to laterally displace the patellar tendon and a burr hole was created in the distal end of the femur with a 26-gauge needle. An orthopedic-grade nickel-titanium Kirschner wire (0.6 mm diameter; Custom Wire Technologies, Port Washington, WI) was inserted through the burr hole into the medullary canal and 10^3 CFUs of S. aureus USA300 LAC13C were inoculated at the tip of the wire and the incision was closed with nylon suture. Mice received buprenorphine SR to alleviate any post-surgical pain and were continuously monitored until ambulatory and daily thereafter.

Sample Preparation:

Sampleprep ID:SP003618
Sampleprep Summary:A 13C15N-labeled canonical amino acid (13C15N-CAA) mix (Cambridge Isotope Laboratories, Andover, MA) was added to the extraction buffer comprised of 80% MeOH as an internal standard. Samples were then pelleted, and supernatants were dried by vacuum centrifuge for storage at -80°C. Cell pellets were stored for protein quantification to normalize metabolite peak areas. Prior to LC-HRMS, samples were reconstituted in 100 µL of 50% MeOH, pelleted, and the resulting supernatant was analyzed.

Combined analysis:

Analysis ID AN005719 AN005720
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system 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)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Orbitrap Exploris 480 Thermo Orbitrap Exploris 480
Ion Mode NEGATIVE POSITIVE
Units Relative intensities Relative intensities

Chromatography:

Chromatography ID:CH004339
Chromatography Summary:Metabolite separation was performed by liquid chromatography using a XBridge Amide analytical column (150 × 2.1mm ID; 1.7µm particle size; Waters Corporation, Milford, MA) and a binary solvent system infused at a flow rate of 0.3 mL/min. A guard XBridge Amide column (20 × 2.1mm ID; 3.5µm particle size; Waters Corporation) was connected in front of the analytical column. Mobile phase A was composed of ammonium acetate and ammonium hydroxide (10 mM each) containing 5% acetonitrile in LC-MS grade water; mobile phase B was 100% LC-MS grade acetonitrile. The pH of mobile phase A was adjusted to 8.0 using glacial acetic acid. The UHPLC pumps were operated in gradient mode. The amide column was maintained at 40°C, and the autosampler temperature was held at 5°C throughout data acquisition. The injection volume for all samples was 5 µl with the 13C15N-CAA mix as the internal standard.
Instrument Name:Thermo Vanquish
Column Name:Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:40°C
Flow Gradient:Started at 85% of B at 0-0.1 min, 84% B at 0.1-3.0 min; 65% B at 3.0-7.3 min, 60% B at 7.3-12.0 min, 55% B at 12.0-15.0 min, 50% B at 15.0-17.0 min, 50% B at 17.0-20.5 min, 70% B at 20.5-22.0 min. The initial conditions were recovered by 22.0 min to equilibrate the column. Total run time was 30 min.
Flow Rate:0.3 mL/min
Solvent A:95% Water/5% Acetonitrile; 10mM Ammonium acetate and 10 mM Ammonium hydroxide
Solvent B:100% Acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS005442
Analysis ID:AN005719
Instrument Name:Thermo Orbitrap Exploris 480
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:A HRMS Orbitrap (Exploris 480; Thermo Fisher Scientific) was operated in polarity switching mode and was used for untargeted metabolomics in a data-dependent MS/MS acquisition mode (DDA). Electrospray ionization (ESI) parameters were optimized as follows: electrospray ion voltage of -2700V and 3500V in negative and positive mode respectively, ion transfer tube temperature was maintained at 400°C, and m/z scan range was 70-1050 Da. Orbitrap resolution for precursor ion scans were maintained at 240,000 for intracellular and 120,000 for extracellular whereas fragment ion scans were maintained at 120,000 and 60,000, for intracellular and extracellular respectively. Normalized collision energies at 30, 50, and 150% were used for fragmentation and data was acquired in profile mode.
Ion Mode:NEGATIVE
  
MS ID:MS005443
Analysis ID:AN005720
Instrument Name:Thermo Orbitrap Exploris 480
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:A HRMS Orbitrap (Exploris 480; Thermo Fisher Scientific) was operated in polarity switching mode and was used for untargeted metabolomics in a data-dependent MS/MS acquisition mode (DDA). Electrospray ionization (ESI) parameters were optimized as follows: electrospray ion voltage of -2700V and 3500V in negative and positive mode respectively, ion transfer tube temperature was maintained at 400°C, and m/z scan range was 70-1050 Da. Orbitrap resolution for precursor ion scans were maintained at 240,000 for intracellular and 120,000 for extracellular whereas fragment ion scans were maintained at 120,000 and 60,000, for intracellular and extracellular respectively. Normalized collision energies at 30, 50, and 150% were used for fragmentation and data was acquired in profile mode.
Ion Mode:POSITIVE
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