Glycogen synthase kinase 3 inhibition controls Mycobacterium tuberculosis Infection
Data files
Nov 22, 2024 version files 29.52 MB
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KinexusReport.pdf
1.48 MB
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R1_UP_Parental_Uninf_Untreated.txt
1.33 MB
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R2_IP_Parental_WT_Untreated.txt
1.33 MB
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R3_UPD1_Parental_Uninf_Treated.txt
1.32 MB
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R4_IPD1_Parental_WT_Treated.txt
1.33 MB
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R5_UO_GSK3BKO_Uninf_Untreated.txt
1.33 MB
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R6_IO_GSK3BKO_WT_Untreated.txt
1.34 MB
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R7_IPK01_Parental_ptpAKO_Untreated.txt
1.34 MB
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README.md
2.17 KB
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S1_UP_vs_UO__UPD1.xlsx
6.57 MB
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S2_IP_vs_IO__IPD1__IPK01.xlsx
9.28 MB
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S3_UPD1_vs_IPD1.xlsx
2.85 MB
Abstract
Compounds targeting host control of infectious diseases provide an attractive alternative to antimicrobials. A phenotypic screen of a kinase library identified compounds targeting glycogen synthase kinase 3 as potent inhibitors of Mycobacterium tuberculosis (Mtb) intracellular growth in the human THP-1 cell line and primary human monocytes-derived macrophages (hMDM). CRISPR knockouts and siRNA silencing showed that GSK3 isoforms are needed for the growth of Mtb and that a selected compound, P-4423632 targets GSK3β. GSK3 inhibition was associated with macrophage apoptosis governed by the Mtb secreted protein tyrosine phosphatase A (PtpA). Phospho-proteome analysis of macrophages response to infection revealed a wide array of host signaling and apoptosis pathways controlled by GSK3 and targeted by P-4423632. P-4423632 was additionally found to be active against other intracellular pathogens. Our findings strengthen the notion that targeting host signaling to promote the infected cell's innate antimicrobial capacity is a feasible and attractive host-directed therapy approach.
Data showing modulation of macrophage phosphorylation upon infection and treatment with P-4423632 using antibody microarray analysis.
Description of the data and file structure
The phosphorylation status of THP-1 cells were analysed from seven experimental conditions:
UP: Parental THP-1 cells, Uninfected, Untreated
IP: Parental THP-1 cells, Mtb-infected, Untreated
UPD1: Parental THP-1 cells, Uninfected, P-4423632-treated
IPD1: Parental THP-1 cells, Mtb-infected, P-4423632-treated
UO: GSK3B KO THP-1 cells, Uninfected, Untreated
IO: GSK3B KO THP-1 cells, Mtb-infected, Untreated
IPK01: Parental THP-1 cells, ptpA KO Mtb-infected, Untreated
Excel Data
Excel files include descriptions in the Read Me First tab
Excel File S1 compares UO and UPD1 to the UP control
Excel File S2 compares IO, IPD1 and IPK01 to the IP control
Excel File S3 compares IPD1 to UPD1
Calculations: Standard error and percent standard deviation of 2 separate measurements of globally normalized signal intensity values for each different antibody on the microarray were calculated. Data were determined as percent change from selected controls (% CFC). A positive value corresponds to an increase in signal intensity in response to the treatment, with a value of 100% corresponding to a 2-fold increment in signal intensity. A negative CFC value indicates the degree of reduction in signal intensity from that of the control.
Raw Txt Files
Text files R1 - R7 are the raw Kinexus data files for the seven conditions tested. These files can be directly uploaded into CAT-PETR for plotting Volcano Plots, Scatter Plots, and Heatmaps in Figure 4A-D.
CAT-PETR settings: Phospho-specific comparisions using Log Transform normalization, Cyber-T t-test, and Benjamini Hochberg correction were used to plot the data.
PDF File
KinexusReport.pdf gives a detailed description of the methods, results and analysis of the antibody microarray.
Lysates were prepared from parental or GSK3β knockout THP-1 cells (0.5 x 106 cells/well) that were differentiated overnight and then infected with WT Mtb or ΔptpA Mtb and treated with or without 10 µM P-4423632. Lysates were subjected to Kinexus Kinex™ KAM-2000 antibody microarray analyses as described (Yue, L., et al Clinical Proteomics & Bioinformatics. 2017;2(1):1-10). The KAM-2000 microarrays utilized 2059 commercial, pan-specific antibodies for 939 non-redundant human proteins targets that included protein kinases, phosphatases, transcription factors, stress proteins and many other signaling proteins. The KAM-2000 microarray featured 1165 pan-specific and 894 phosphosite-specific commercial antibodies, produced principally by Kinexus Bioinformatics (Vancouver, BC, Canada) as well as from other suppliers following their in-house validation, with each antibody printed in quadruplicate on each Nexterion P slide (Schott AG, Jena, Germany).
Two 16-bit images from each KAM-2000 microarray were then captured using a ScanArray Reader (Perkin-Elmer). Signal quantification was performed with ImaGene 9.0 from BioDiscovery (El Segundo, CA) with predetermined settings for spot segmentation and background correction. The output of the array consisted of the average normalized net signals (i.e., the average of 2 normalized net signal values of each antibody on the microarray).
See the README file for processing details.
Microsoft Excel
CAT-PETR (av-gay-ubc.shinyapps.io/CAT-PETR/): Flanagan K, Pelech S, Av-Gay Y, Dao Duc K. CAT PETR: A graphical user interface for differential analysis of phosphorylation data. Statistical Applications in Genetics and Molecular Biology. 2023 Aug 21;22(1). doi: 10.1515/sagmb-2023-0017.