Summary of Study ST002187

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 PR001394. The data can be accessed directly via it's Project DOI: 10.21228/M8J12Z 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 IDST002187
Study TitleInterplay of CodY and CcpA in regulating central metabolism and biofilm formation in S. aureus
Study TypeResearch
Study SummaryStaphylococcus aureus is a medically important pathogen that exhibit high metabolic versatility allowing it to infect various niches within a host. S. aureus utilizes two major transcriptional regulators, CodY and CcpA, to remodel metabolic and virulence gene expression in response to changing environmental conditions. Previous studies revealed that inactivation of either codY or ccpA has a pronounced impact on different aspects of staphylococcal physiology and pathogenesis. To determine the contribution and interplay of these two regulators in modulating central metabolism, virulence, and biofilm development we constructed and characterized codY ccpA double mutant in S. aureus UAMS-1. In line with previous studies, we found that CcpA and CodY control cellular metabolic status by altering carbon flow through the central and overflow metabolic pathways. Our results demonstrate that ccpA inactivation impairs biofilm formation and decreases incorporation of eDNA into the biofilm matrix while disrupting codY resulted in a robust structured biofilm tethered together with eDNA and PIA. Interestingly, inactivation of both codY and ccpA decreases biofilm biomass and neglects eDNA release in the double mutant. Compared to inactivation of codY, the codY ccpA mutant did not overexpress toxins but maintained overexpression of amino acid metabolism pathways. Furthermore, codY ccpA mutant produced higher amounts of PIA, in contrast to the wild-type strain and ccpA mutant. Overall, results of this study suggest that interplay between CodY and CcpA modulates central metabolism to optimize growth on preferred carbon sources while repressing virulence gene expression until nutrient limitation requires scavenging nutrients from the host.
Institute
University of Nebraska Medical Center
DepartmentPathology and Microbiology
Last NameSadykov
First NameMarat
AddressUNMC Department of Pathology and Microbiology 985900 Nebraska Medical Center Omaha, NE 68198-5900
Emailmsadykov@unmc.edu
Phone4025594186
Submit Date2022-05-25
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2022-06-14
Release Version1
Marat Sadykov Marat Sadykov
https://dx.doi.org/10.21228/M8J12Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001394
Project DOI:doi: 10.21228/M8J12Z
Project Title:Interplay of CodY and CcpA in regulating central metabolism and biofilm formation in S. aureus
Project Type:Staphylococcus aureus
Project Summary:Staphylococcus aureus is a medically important pathogen that exhibit high metabolic versatility allowing it to infect various niches within a host. S. aureus utilizes two major transcriptional regulators, CodY and CcpA, to remodel metabolic and virulence gene expression in response to changing environmental conditions. Previous studies revealed that inactivation of either codY or ccpA has a pronounced impact on different aspects of staphylococcal physiology and pathogenesis. To determine the contribution and interplay of these two regulators in modulating central metabolism, virulence, and biofilm development we constructed and characterized codY ccpA double mutant in S. aureus UAMS-1. In line with previous studies, we found that CcpA and CodY control cellular metabolic status by altering carbon flow through the central and overflow metabolic pathways. Our results demonstrate that ccpA inactivation impairs biofilm formation and decreases incorporation of eDNA into the biofilm matrix while disrupting codY resulted in a robust structured biofilm tethered together with eDNA and PIA. Interestingly, inactivation of both codY and ccpA decreases biofilm biomass and neglects eDNA release in the double mutant. Compared to inactivation of codY, the codY ccpA mutant did not overexpress toxins but maintained overexpression of amino acid metabolism pathways. Furthermore, codY ccpA mutant produced higher amounts of PIA, in contrast to the wild-type strain and ccpA mutant. Overall, results of this study suggest that interplay between CodY and CcpA modulates central metabolism to optimize growth on preferred carbon sources while repressing virulence gene expression until nutrient limitation requires scavenging nutrients from the host
Institute:University of Nebraska Medical Center
Department:Pathology and Microbiology
Last Name:Sadykov
First Name:Marat
Address:UNMC Department of Pathology and Microbiology 985900 Nebraska Medical Center Omaha, NE 68198-5900
Email:msadykov@unmc.edu
Phone:402-559-4186
Contributors:Dhananjay Shinde, Ahn, Jongsom

Subject:

Subject ID:SU002273
Subject Type:Bacteria
Subject Species:Staphylococcus aureus
Taxonomy ID:1280
Gender:Not applicable

Factors:

Subject type: Bacteria; Subject species: Staphylococcus aureus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA210074codY 3h_3ccpA
SA210075ccpA 6h_1ccpA
SA210076codY 3h_2ccpA
SA210077codY 3h_1ccpA
SA210078ccpA 3h_1ccpA
SA210079ccpA 6h_3ccpA
SA210080ccpA 6h_2ccpA
SA210081ccpA 3h_2ccpA
SA210082ccpA 3h_3ccpA
SA210083codY 6h_2ccpA
SA210084codY 6h_1ccpA
SA210085codY 6h_3ccpA
SA210086codY ccpA 6h_1codY ccpA
SA210087codY ccpA 6h_2codY ccpA
SA210088codY ccpA 6h_3codY ccpA
SA210089codY ccpA 3h_1codY ccpA
SA210090codY ccpA 3h_2codY ccpA
SA210091codY ccpA 3h_3codY ccpA
SA210068WT 6h_2WT
SA210069WT 3h_3WT
SA210070WT 3h_2WT
SA210071WT 6h_1WT
SA210072WT 6h_3WT
SA210073WT 3h_1WT
Showing results 1 to 24 of 24

Collection:

Collection ID:CO002266
Collection Summary:Strains were inoculated in 3 ml of TSB containing 14 mM glucose in glass tubes (Fisher Scientific, cat# 14-961-29) for 16 h and adjusted to an OD600 of 0.05 in 50 ml TSB containing 14 mM glucose in 500 ml flasks and grown at 250 rpm, 37oC for 3 h or 6 h. Culture volumes corresponding to OD600 of 10 optical units were harvested and rapidly filtered through a membrane (0.45 μm, Millipore Cat# MIHAWG100) using Microfil® Filtration Funnels (Millipore, Cat# HAWG047S6) and EZ-FITTM Manifold Base, 6 Place Stainless Steel (Millipore, Cat# EZFITBASE6). The cells on the membrane were washed twice with 5 ml of 0.6% saline and then immediately quenched in 5 ml of ice-cold 60% ethanol containing Br-ATP (final concentration 2 μM) as internal control. The cells were mechanically disrupted using 0.1 mm diameter Glass beads (biospec, Cat# 11079101) and bead homogenizer for 30 s at 6,800 rpm, 3 cycles, 10 sec pause, 4oC(Precellys Evolution, bertin technologies) and centrifuged at 13,000 rpm, 10 min at 4oC to remove cell debris. The supernatant containing intracellular metabolites were separated, and 3 ml was added to 17 ml DW, frozen, lyophilized using FreeZone 4.5 Liter (labconco, Catalog #: 720401050), , and stored in a ultra-low temperature freezer set at -80°C until further analysis.
Sample Type:Bacterial cells
Storage Conditions:-20℃

Treatment:

Treatment ID:TR002285
Treatment Summary:Staphylococcus aureus UAMS-1 wild-type strain (WT) Staphylococcus aureus UAMS-1 codY::ermC mutant (codY) Staphylococcus aureus UAMS-1 ccpA::tetM mutant (ccpA) Staphylococcus aureus UAMS-1 codY::ermC ccpA::tetM mutant (codY ccpA)

Sample Preparation:

Sampleprep ID:SP002279
Sampleprep Summary:Strains were inoculated in 3 ml of TSB containing 14 mM glucose in glass tubes (Fisher Scientific, cat# 14-961-29) for 16 h and adjusted to an OD600 of 0.05 in 50 ml TSB containing 14 mM glucose in 500 ml flasks and grown at 250 rpm, 37oC for 3 h or 6 h. Culture volumes corresponding to OD600 of 10 optical units were harvested and rapidly filtered through a membrane (0.45 μm, Millipore Cat# MIHAWG100) using Microfil® Filtration Funnels (Millipore, Cat# HAWG047S6) and EZ-FITTM Manifold Base, 6 Place Stainless Steel (Millipore, Cat# EZFITBASE6). The cells on the membrane were washed twice with 5 ml of 0.6% saline and then immediately quenched in 5 ml of ice-cold 60% ethanol containing Br-ATP (final concentration 2 μM) as internal control. The cells were mechanically disrupted using 0.1 mm diameter Glass beads (biospec, Cat# 11079101) and bead homogenizer for 30 s at 6,800 rpm, 3 cycles, 10 sec pause, 4oC(Precellys Evolution, bertin technologies) and centrifuged at 13,000 rpm, 10 min at 4oC to remove cell debris. The supernatant containing intracellular metabolites were separated, and 3 ml was added to 17 ml DW, frozen, lyophilized using FreeZone 4.5 Liter (labconco, Catalog #: 720401050), , and stored in a ultra-low temperature freezer set at -80°C until further analysis.
Processing Storage Conditions:On ice
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003581
Analysis type MS
Chromatography type HILIC
Chromatography system Waters Acquity I-Class
Column Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
MS Type ESI
MS instrument type QTRAP
MS instrument name ABI Sciex 6500+
Ion Mode UNSPECIFIED
Units CPS

Chromatography:

Chromatography ID:CH002648
Chromatography Summary:Triple-quadrupole-ion trap hybrid mass spectrometer viz., QTRAP6500+ (Sciex, USA) connected with ultra-performance liquid chromatography I-class (UPLC) system procured from Waters, USA was used for the metabolite analysis. The chromatographic separation was performed by liquid chromatography using XBridge Amide (150 × 2.1mm ID; 3.5µm particle size, Waters, USA) analytical column and a binary solvent system with a flow rate of 0.4 ml/min. A guard XBridge Amide column (20 × 2.1mm ID; 3.5µm particle size, Waters, USA) was connected in front of analytical column. Mobile phase A was composed of 10 mM ammonium acetate, 10mM ammonium hydroxide containing 5% acetonitrile in LC-MS grade water; mobile phase B was 100% LC-MS grade acetonitrile.
Instrument Name:Waters Acquity I-Class
Column Name:Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Chromatography Type:HILIC

MS:

MS ID:MS003338
Analysis ID:AN003581
Instrument Name:ABI Sciex 6500+
Instrument Type:QTRAP
MS Type:ESI
MS Comments:QTRAP6500+ operated in polarity switching mode was used for targeted quantitation of amino acids through Multiple Reaction Monitoring (MRM) process. Electrospray ionization (ESI) parameters were optimized are as follows: electrospray ion voltage of -4200V and 5500V in negative and positive mode respectively, source temperature of 400°C, curtain gas of 35, and gas 1 and 2 of 40 and 40 psi, respectively. Compound specific parameters were optimized for each compound using manual tuning. These parameters are declustering potential (DP) were 65V and -60V in positive and negative mode respectively, entrance potential (EP) was set at 10V and -10V in positive and negative mode respectively and collision cell exit potential (CXP) was maintained at 10V and -10V in positive and negative mode respectively. MRM data was analysed using Multiquant software.
Ion Mode:UNSPECIFIED
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