Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Bacterial lipolysis of immune-activating ligands promotes evasion of innate defenses

doi: 10.1073/pnas.1817248116

Figure Lengend Snippet: Geh inactivates bacterially derived PAMPs. (A) GelCode Blue-stained 4–20% gradient SDS/PAGE gel of 5 μg of purified Geh and pNp-palmitate hydrolysis (absorbance at 410 nm) in the presence of 1 nM, 4 nM, 10 nM, and 20 nM Geh. (B) GelCode Blue-stained 12% SDS/PAGE gel of TCA-precipitated exoproteins isolated from the indicated strains after 9 h of growth in RPMI (Δgeh + Geh is supernatant isolated from a Δgeh mutant that was supplemented with 10 nM Geh). The asterisk indicates the mature form of Geh. (C) IL-6 (ng/mL) production by BMMs after addition of cell-free supernatant from WT, Δgeh, and Δgeh + geh strains and from Δgeh supernatant supplemented with 1 nM, 10 nM, or 100 nM Geh or Geh alone (1 nM, 10 nM, and 100 nM). (-), medium alone. (D) IL-6 (pg/mL) production by BMMs after addition of Pam2CSK4/Pam3CSK4 (10 ng/mL) or Geh-treated Pam2CSK4/Pam3CSK4 (10 ng/mL). (E) GelCode Blue-stained 4–20% gradient SDS/PAGE gel of 1 μg of SitC/PrsA, 1 μg of SitC/PrsA + Geh, and 1 μg of FPLC-purified SitC*/PrsA* (Geh-treated). (F) IL-6 (ng/mL) production by BMMs after addition of Pam3CSK4 (10 ng/mL), SitC/PrsA (100 ng/mL), Geh-treated SitC/PrsA (SitC*/PrsA*; 100 ng/mL), and Geh (40 ng/mL). All experiments were repeated at least three times. Data shown B–D and F are mean ± SD from three representative independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: For Geh-6xHis or Geh(S412A)-6xHis lipopeptide reactions, 300 ng of LPS-free Geh-6xHis or Geh(S412A)-6xHis was incubated with 10 ng of Pam3CSK4 (Invivogen) or Pam2CSK4 (Invivogen) at 37 °C for 2 h. Geh- or GehS412A-treated Pam3CSK4 and Pam2CSK4 or untreated controls were incubated with Ni-NTA resin for 1 h at room temperature, followed by centrifugation at 21,130 × g for 5 min at 4 °C to remove Geh-6xHis– and Geh(S412A)-6xHis–containing resin.

Techniques: Derivative Assay, Staining, SDS Page, Purification, Isolation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Bacterial lipolysis of immune-activating ligands promotes evasion of innate defenses

doi: 10.1073/pnas.1817248116

Figure Lengend Snippet: Ester hydrolase activity of Geh is required for lipoprotein inactivation. (A) pNp-palmitate hydrolysis (absorbance at 410 nm) in the presence of 1 nM, 4 nM, 10 nM, and 20 nM Geh(S412A). (B) IL-6 (pg/mL) production by BMMs after addition of cell-free supernatant from WT, Δgeh, and Δgeh + geh strains and from Δgeh supernatant supplemented with 10 nM Geh, Δgeh supernatant supplemented with 10 nM Geh(S412A), and Geh(S412A) alone (10 nM). (C) IL-6 (ng/mL) production by BMMs after addition of Pam2CSK4/Pam3CSK4 (10 ng/mL), Geh-treated Pam2CSK4/Pam3CSK4 (10 ng/mL), and Geh(S412A)-treated Pam2CSK4/Pam3CSK4 (10 ng/mL). (-), medium alone. (D) IL-6 (ng/mL) production by BMMs after addition of Pam3CSK4 (10 ng/mL), SitC/PrsA (100 ng/mL), Geh-treated SitC/PrsA (100 ng/mL), Geh(S412A)-treated SitC/PrsA (100 ng/mL), Geh (40 ng/mL), and Geh(S412A) (40 ng/mL). All experiments were repeated at least three times. Data shown in B–D are mean ± SD from three representative independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: For Geh-6xHis or Geh(S412A)-6xHis lipopeptide reactions, 300 ng of LPS-free Geh-6xHis or Geh(S412A)-6xHis was incubated with 10 ng of Pam3CSK4 (Invivogen) or Pam2CSK4 (Invivogen) at 37 °C for 2 h. Geh- or GehS412A-treated Pam3CSK4 and Pam2CSK4 or untreated controls were incubated with Ni-NTA resin for 1 h at room temperature, followed by centrifugation at 21,130 × g for 5 min at 4 °C to remove Geh-6xHis– and Geh(S412A)-6xHis–containing resin.

Techniques: Activity Assay

Journal: Gastric Cancer

Article Title: Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6

doi: 10.1007/s10120-023-01461-7

Figure Lengend Snippet: Rac1, pPI3K, tPI3K, pGSK-3β, tGSK-3β, NF-κB p50 and NF-κB p65, pJNK, tJNK, pJUN and tJUN expression in control cells, Hp- 1 cells and Hp- 30 cells ( A ). TLR6 and pJNK expression in control cells, Hp- 1 cells, GES-1 cells treated with 1 μM C29 for 24 h and GES-1 cells treated with H. pylori and 1 μM C29 for 24 h ( B ). TLR6 and pJNK expression in control cells, GES-1 cells treated with 0.25 μg/mL, 0. 5 μg/mL, 1 μg/mL and 2 μg/mL Pam2CSK4 for 24 h ( C ). TLR6 and pJNK expression in GES-1 cells, Hp- 1 cells, GES-1 cells treated with 1 μg/mL Pam2CSK4 for 24 h, GES-1 cells treated with 2 μg/mL SP600125 for 24 h, GES-1 cells treated with H. pylori and 2 μg/mL SP600125 for 24 h and GES-1 cells treated with 1 μg/mL Pam2CSK4 and 2 μg/mL SP600125 for 24 h ( D ). All experiment were performed in triplicate and all data represents means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The TLR2/6 heterodimer agonist Pam2CSK4 was purchased from Apexbio (B5665).

Techniques: Expressing, Control

Journal: Gastric Cancer

Article Title: Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6

doi: 10.1007/s10120-023-01461-7

Figure Lengend Snippet: TLR6 mRNA expression ( A ) and IL-1β mRNA expression ( B ) in Hp- 30 cells, Hp- 30 cells co-culture with H. pylori for 24 h and Hp -30 cells co-culture with H. pylori and 10 μg/ml Pam2CSK4 for 24 h. Supernatant IL-1β content ( C ) in control cells, Hp- 1 cells, Hp -30 cells, Hp -30 cells infected with H. pylori for 24 h and Hp -30 cells co-culture with H. pylori and 1 μg/ml Pam2CSK4 for 24 h. pJNK expression ( D ) in Hp- 30 cells, Hp- 30 cells co-culture with H. pylori for 24 h, Hp -30 cells co-culture with H. pylori and 1 μg/ml Pam2CSK4 for 24 h. All experiment were performed in triplicate and all data represents means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The TLR2/6 heterodimer agonist Pam2CSK4 was purchased from Apexbio (B5665).

Techniques: Expressing, Co-Culture Assay, Control, Infection

Journal: Gastric Cancer

Article Title: Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6

doi: 10.1007/s10120-023-01461-7

Figure Lengend Snippet: Schematic ( A ) of the in vivo treatment experimental plan. Serum TLR6 and IL-8 expression ( B, C ) and gastric mucosa TLR6 and IL-8 IHC staining ( D , E ) of the H. pylori- negative Mongolian gerbils or in the H. pylori- infected gerbils after 24 h, 48 h and 72 h intraperitoneal administration of Pam2CSK4 or DMSO. pJNK expression ( F ) in the gastric mucosa of H. pylori- negative Mongolian gerbils or in the H. pylori- infected gerbils after 72 h intraperitoneal administration of Pam2CSK4 or DMSO. The Immunofluorescence staining of CD11b + /Ly6G + cells ( G ), monocyte/macrophage ( I ), the number of CD11b + /Ly6G + cells ( H ) and the number of monocyte/macrophage ( J ) in gastric mucosa of H. pylori -negative gerbils, H. pylori -positive gerbils treated with DMSO or Pam2CSK4. The level of H. pylori 23srRNA ( K ) in gastric mucosa of H. pylori -positive Mongolian gerbils treated with DMSO or Pam2CSK4. IHC staining of TLR6 expression ( L ) in the gastric mucosa of patients. × 100. The average optical density (AOD) of TLR6 expression ( M ) in patients’ gastric mucosa. All experiment were performed in triplicate and all data represents means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The TLR2/6 heterodimer agonist Pam2CSK4 was purchased from Apexbio (B5665).

Techniques: In Vivo, Expressing, Immunohistochemistry, Infection, Immunofluorescence, Staining