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1.
FIGURE 7.

FIGURE 7. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Factors interfere with bactericidal activity of AMPs. A and B, bactericidal activities of AMPs assayed by the diameter of inhibition zone on Luria-Bertani agar plates are shown. P. aeruginosa (panel A) and K. pneumoniae (panel B) were plated on Luria-Bertani agar plates in which AMPs were dotted (4 μg each) as indicated. C and D, effects of MgCl2 and NaCl on the bactericidal activity of AMPs are shown. The bactericidal activities of 10 μm SMAP-29, 10 μm LL-37, 10 μm Polymyxin B, and 10 μm (R3S1)3 against K. pneumoniae were determined in the presence of MgCl2 and NaCl as indicated. E and F, shown is the effect of MgCl2 on the binding of AMP to Lpp. SMAP-29 and LL-37 (4 μg each) were incubated with 1.5 μg Lpp-His in the presence of MgCl2 as indicated before pulling down by Ni-NTA Agarose and visualized by Coomassie blue staining.

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.
2.
FIGURE 5.

FIGURE 5. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Protection of bacteria from AMP-mediated toxicity. A and B, repression of antimicrobial activity of AMP by the addition of exogenous recombinant Lpp or anti-Lpp antibody is shown. The viability of K. pneumoniae or P. aeruginosa (5–10 × 104 cfu) treated with 0.1 μm SMAP-29, 0.2 μm CAP-18, 0.2 μm LL-37, 0.1 μm Protegrin-1, or 0.1 μm Polymyxin B was measured in the presence of increasing amounts of recombinant Lpp (A) or anti-Lpp antibody (B) as indicated. C, protection of various microbes from the action of SMAP-29 by the pretreatment with anti-Lpp antibody is shown. D, repression of AMP-induced increase of membrane permeability by anti-Lpp antibody is shown. Bacteria (107 cfu) containing 1 μm SYTOX® Green were incubated with or without anti-Lpp antibody in a dark 96-well plate for 15 min before the treatment of 5 μm SMAP-29, 5 μm CAP-18, and 40 μm Polymyxin B and assayed for fluorescence as described in Fig. 1C.

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.
3.
FIGURE 4.

FIGURE 4. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Specific recognition of native Lpp of K. pneumoniae by cationic α-helical AMPs. A, shown is antigenicity of native OprI and Lpp to anti-OprI and anti-Lpp antibodies. The total lysates of indicated bacteria (0.5 μg each) were separated by 14% SDS-PAGE and detected by Western blot using anti-OprI (top) and anti-Lpp (bottom) antibody, respectively. B, shown is binding of native Lpp by biotinylated SMAP-29. The membrane fraction of K. pneumoniae (2 μg each) was incubated with increasing amounts of biotinylated SMAP-29 in 10 mm sodium phosphate buffer, pH 7.5, containing 0.15 m NaCl and 0.15% Triton X-100, precipitated with streptavidin-conjugated beads, and analyzed by Western blot using anti-Lpp antibody (Ab). Anti-Lpp or anti-OprI antibodies (4 μg each) were added to the mixture as control. C, competition of the formation of Lpp·SMAP-29 complex by free cationic α-helical AMPs is shown. The membrane fraction of K. pneumoniae (2 μg each) was incubated with biotinylated SMAP-29 in the presence of free SMAP-29, CAP-18, LL-37, and Polymyxin B, precipitated, and analyzed as panel B.

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.
4.
FIGURE 8.

FIGURE 8. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Screening of bactericidal AMPs by His-tagged Lpp. The mixture of human RNase 7 (2 μg, lane 1), RC-RNase from bullfrog, Rana Catesbeiana (2 μg, lane 2), SMAP-29 (4 μg, lane 3), and Polymyxin B (4 μg, lane 4) were incubated with His-tagged Lpp (1.5 μg, lane 5) in 10 mm sodium phosphate, pH 7.5, containing 0.15 m NaCl and 40 mm imidazole in the presence of crude cell lysates of human lung adenocarcinoma cell CL1-0 (30 μg, lane 6), then pulled down by Ni-NTA Agarose and analyzed by SDS-PAGE and Coomassie Blue staining. The human RNase 7 and SMAP-29 were pulled down as shown on the right (lane 9). No peptide/protein was pulled down by Ni-NTA Agarose from the two peptide/two protein mixture (lane 7) and the crude cell lysate (lane 8) without the His-tagged Lpp ligand.

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.
5.
FIGURE 3.

FIGURE 3. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Specific binding of cationic α-helical AMPs to recombinant Lpp. A, binding of cationic AMPs to recombinant Lpp is shown. Cationic AMPs as indicated (4 μg each) were incubated with 1.5 μg of His-tagged Lpp in 10 mm sodium phosphate buffer, pH 7.5, containing 0.15 m NaCl and 40 mm imidazole, precipitated by Ni-NTA Agarose, and analyzed by SDS-PAGE and Coomassie Blue staining. B–D, specific interaction between Lpp and SMAP-29 is shown. Recombinant Lpp of K. pneumoniae (0.8 μg each) was incubated with increasing amounts of biotinylated SMAP-29 for 30 min, cross-linked with glutaraldehyde for 20 min, separated by 14% SDS-PAGE, and analyzed by silver staining (B), Western blot analysis with anti-Lpp antibody (C), or anti-biotin antibody(D). Numbers 1–6 represent the status of Lpp polymerization, and a–d indicate the positions of biotinylated SMAP-29·Lpp complexes. E, binding of recombinant Lpp/OprI by biotinylated SMAP-29 is shown. The recombinant OprI of P. aeruginosa and Lpps of K. pneumoniae, E. coli, and S. typhimurium (2 μg each) were incubated with 2 μg of biotinylated SMAP-29 and analyzed by Western blot with anti-Lpp antibody (Ab).

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.
6.
FIGURE 2.

FIGURE 2. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Production of oligomeric Lpps. A, shown is analysis of recombinant Lpp of K. pneumoniae at different purification steps by 14% SDS-PAGE and Coomassie Blue staining. Lane 1, crude lysate from E. coli BL21(DE3) transformed with thioredoxin/Lpp-fused gene; lane 2, eluate of nickel column; lane 3, protease Factor Xa-digested product; lane 4, flow-through of second nickel column; lane 5, eluate of SuperoseTM 12 column chromatography. B, purity of recombinant OprI/Lpp is shown. Equal amounts of recombinant OprI of P. aeruginosa and Lpps of K. pneumoniae, E. coli, and S. typhimurium (3 μg each) were taken for 14% SDS-PAGE separation and Coomassie Blue staining. C, a Western blot analysis of recombinant OprI and Lpp is shown. The recombinant proteins as mentioned in panel B (0.2 μg each) were taken for Western blot analysis using anti-OprI (left panel) and anti-Lpp (right panel) antibody (Ab), respectively. D and E, cross-linking of Lpp by EDC is shown. The recombinant Lpp of K. pneumoniae (0.8 μg each) was incubated with increasing amounts of EDC as indicated at pH 5.5 for 30 min and subjected to 14% SDS-PAGE and silver staining (panel D) and Western blot analysis (panel E).

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.
7.
FIGURE 6.

FIGURE 6. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Morphological changes and entry of surface Lpp caused by SMAP-29. A, internalization of Lpp and SMAP-29 is shown. Bacteria (1 × 107) were incubated with 60 μg of biotinyl-SMAP-29 at 37 °C for 5 and 20 min, respectively. Thin sections of bacteria were incubated with both anti-Lpp antibody from rabbit and anti-biotin antibody from mouse then with secondary antibody conjugated with large gold particles (18 nm, for Lpp (L)), for rabbit antibody and small gold particles (12 nm, SMAP-29 (S)), and for mouse antibody and examined under transmission electron microscopy. OM and IM represent outer membrane and inner membrane, respectively. B, cross-linking of bacterial surface Lpp by EDC is shown. Small aliquots of K. pneumoniae were incubated with EDC as indicated in 10 mm sodium phosphate, pH 5.5, and analyzed by Western blot using anti-Lpp antibodies. C, shown is protection of surface Lpp from EDC cross-linking by SMAP-29 treatment. The bacteria were treated with SMAP-29 for 10 min before cross-linking with 5 mm EDC and analyzed as described above. D, masking of surface Lpp by SMAP-29 treatment is shown. The binding capacities of SMAP-29-treated bacteria to anti-Lpp antibody (4 μg) were assayed by SDS-PAGE and Coomassie Blue staining (left panel) and Western blot ( of protein loaded to left panel) using anti-rabbit immunoglobulin antibody (right top panel) and anti-Lpp antibody (right bottom panel), respectively. Anti-Lpp antibody at left lane acts as a control, and H represents the heavy chain of immunoglobulin.

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.
8.
FIGURE 1.

FIGURE 1. From: Outer Membrane Lipoprotein Lpp Is Gram-negative Bacterial Cell Surface Receptor for Cationic Antimicrobial Peptides.

Antimicrobial spectra of cationic AMPs and their target protein on Gram-negative bacteria. A, bactericidal activities of AMPs on K. pneumoniae are shown. B, antimicrobial activity of SMAP-29 on various microbes is shown. Microbes (5–10 × 104 cfu) were treated with AMPs at 37 °C in 10 mm sodium phosphate, pH 7.5, for 3 h and then plated on agar plates for the determination of remaining cfu. C, increase of membrane permeability of K. pneumoniae after AMP treatment is shown. Bacteria (107 cfu) were incubated with 1 μm SYTOX® Green in a dark 96-well plate for 15 min before the addition of AMP. The increase of fluorescence was measured using 485- and 520-nm filters for excitation and emission wavelengths, respectively. RFU, relative fluorescence units. D, binding of AMPs to K. pneumoniae is shown. Overnight cultures of bacteria (107 cfu) were incubated with SAMP-29, LL-37, Polymyxin B, or (R3S1)3 (3 μg each) in 50 μl at 37 °C for 30 min, then spun at 10,000 × g for 10 min followed by SDS-PAGE and Coomassie Blue staining. NaCl or MgCl2 was added as indicated. T represents total AMPs employed, S is for supernatant, and P is for pellet. E, amino acid sequence alignment of Lpp-homologous proteins by the software ClustalX is shown. The Lpp sequences of S. typhimurium (NP_460342), Enterobacter cloacae (YP_003612855), Citrobacter youngae (ZP_06353081), E. coli (NP_416192), Shigella boydii (YP_001880438), S. marcescens (AAA26566), Y. enterocolitica (YP_001006405), K. pneumoniae (YP_001335792), V. cholerae (ZP_01972724), and OprI sequence of P. aeruginosa (NP_251543) were obtained from National Center for Biotechnology Information.

Ting-Wei Chang, et al. J Biol Chem. 2012 January 2;287(1):418-428.

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