Journal: Journal of Bacteriology

Article Title: Copper-Induced Expression of a Transmissible Lipoprotein Intramolecular Transacylase Alters Lipoprotein Acylation and the Toll-Like Receptor 2 Response to Listeria monocytogenes

doi: 10.1128/JB.00195-19

Figure Lengend Snippet: TLR2 response to synthetic lipopeptides. (A) HEK-Blue-TLR2/1/6 cells were exposed to 10-fold dilutions of the synthetic lipopeptides Pam3CSK4 (Pam3) and PamC(Pam)SK4 (Pam-Lyso), representing the TA-LP and lyso-LP, respectively, as well as Pam2CSK4 (Pam2) and FSL-1, both DA-LP ligands. (B and C) The same lipopeptides were exposed to HEK-Blue cells expressing only TLR2/TLR1 (B) or TLR2/TLR6 (C). (D) HEK-Blue-TLR2/1/6 cells pretreated with 10 μg/ml TLR-neutralizing antibodies were then exposed to two optimized concentrations of Pam3CSK4, PamC(Pam)SK4, and FSL-1. The percent activity when normalized to the water control wells is indicated. The data shown are the means ± standard deviations of the results of three biological replicates.

Article Snippet: HEK293 NF-κB/SEAP-reporter cells (HEK-Blue) expressing human TLR2/TLR1/TLR6, TLR2/TLR1, and TLR2/TLR6 (InvivoGen, San Diego, CA) were cultivated in 75-cm 2 culture flasks in 15 ml Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l -glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin (InvivoGen), and 4 μl/ml HEK-Blue selection antibiotics at 37°C in 5% CO 2 .

Techniques: Expressing, Activity Assay

Journal: Journal of Bacteriology

Article Title: Copper-Induced Expression of a Transmissible Lipoprotein Intramolecular Transacylase Alters Lipoprotein Acylation and the Toll-Like Receptor 2 Response to Listeria monocytogenes

doi: 10.1128/JB.00195-19

Figure Lengend Snippet: TLR2 response to whole bacteria. (A) HEK-Blue-TLR2/1/6 cells were exposed to 5-fold dilutions of heat-inactivated whole bacterial cells of L. monocytogenes CFSAN023459 (wild type [WT]) grown with or without 1 mM copper, as well as the derivative Δlit2 cells grown with or without 1 mM CuCl2. (B) HEK-Blue-TLR2/1/6 cells were pretreated with 10 μg/ml of TLR-neutralizing antibodies and then exposed to the same bacterial cell preparations as in panel A. The “WT + 1 mM Cu” sample was added to 8.0 × 105 CFU/ml, while the others were added to 3.2 × 104 CFU/ml. The percent activity normalized to the water control is indicated. (C and D) HEK-Blue-TLR2/1/6 cells were exposed to 5-fold dilutions of heat-inactivated, whole bacterial cells of strains L. monocytogenes CFSAN023459 (C) and E. faecalis ATCC 19433 (D). The data are shown as the means ± standard deviations of the results of three biological replicates.

Article Snippet: HEK293 NF-κB/SEAP-reporter cells (HEK-Blue) expressing human TLR2/TLR1/TLR6, TLR2/TLR1, and TLR2/TLR6 (InvivoGen, San Diego, CA) were cultivated in 75-cm 2 culture flasks in 15 ml Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l -glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin (InvivoGen), and 4 μl/ml HEK-Blue selection antibiotics at 37°C in 5% CO 2 .

Techniques: Bacteria, Activity Assay

Journal: Journal of Bacteriology

Article Title: Copper-Induced Expression of a Transmissible Lipoprotein Intramolecular Transacylase Alters Lipoprotein Acylation and the Toll-Like Receptor 2 Response to Listeria monocytogenes

doi: 10.1128/JB.00195-19

Figure Lengend Snippet: Model of transposon-mediated response to copper. Copper enters the cell nonspecifically or via transporters. The proposed high-affinity association of copper at the N terminus of DA-LP is shown. Elevated copper levels induce transcription through an undetermined copper-dependent regulator. Expression of genes downstream of lit2 contributes to copper resistance by various mechanisms, including copper efflux, copper oxidation, and regulation of additional genes. Coinduction of Lit2 simultaneously converts lipoproteins from the DA-LP form to the lyso-LP, which is proposed to reduce copper coordination at the lipoprotein N terminus. While both DA-LP and lyso-LP are sensed by the TLR2/TLR6 heterodimer, the lyso form is overall a less potent ligand at TLR2/TLR6 than DA-LP and a poor TLR2/TLR1 ligand.

Article Snippet: HEK293 NF-κB/SEAP-reporter cells (HEK-Blue) expressing human TLR2/TLR1/TLR6, TLR2/TLR1, and TLR2/TLR6 (InvivoGen, San Diego, CA) were cultivated in 75-cm 2 culture flasks in 15 ml Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l -glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin (InvivoGen), and 4 μl/ml HEK-Blue selection antibiotics at 37°C in 5% CO 2 .

Techniques: Expressing

Journal: PLoS ONE

Article Title: Murine Toll-Like Receptor 2 Activation Induces Type I Interferon Responses from Endolysosomal Compartments

doi: 10.1371/journal.pone.0010250

Figure Lengend Snippet: RAW 264.7 macrophages transfected with hTLR2-GFP were labelled with lysotracker then left untreated ( A ) or stimulated with 1 µg/ml of MALP-2 and imaged by confocal microscopy after 3 h ( B ). Panel C shows cells first labelled with lysotracker then incubated for 1 hour with 50 µM Bafilomycin A before stimulation with MALP-2. The co-localization fluorographs displayed in the right panels show the intensities and scatter pattern of all pixels within the merged images. Pixels with mostly one fluorescent component are placed along the axes 1 and 2 while the pixels with equal fluorescence intensity from both components (due to co-localization) are placed along the diagonal. Axis 1 and 2 show the green (TLR2-GFP) and red (Lysotracker) fluorescence intensities on an arbitrary scale of 0 to 250.

Article Snippet: hTLR2-GFP was bought from Invivogen.

Techniques: Transfection, Confocal Microscopy, Incubation, Fluorescence