Actin enables the antimicrobial action of LL-37 peptide in the presence of microbial proteases

Abstract

Host defense peptides play an important host-protective role by their microcidal action, immunomodulatory functions, and tissue repair activities. Proteolysis is a common strategy of pathogens used to neutralize host defense peptides. Here, we show that actin, the most abundant structural protein in eukaryotes, binds the LL-37 host defense peptide, protects it from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis, and enables its antimicrobial activity despite the presence of the proteases. Co-localization of LL-37 with extracellular actin was observed in necrotized regions of samples from oral lesions. Competition assays, cross-linking experiments, limited proteolysis, and mass spectrometry revealed that LL-37 binds by specific hydrophobic interactions to the His-40-Lys-50 segment of actin, located in the DNase I binding loop. The integrity of the binding site of both LL-37 and actin is a prerequisite to the binding. Our results demonstrate that actin, presumably released by dead cells and abundant in infected sites, might be utilized by the immune system to enhance spatio-temporal immunity in an attempt to arrest infection and control inflammation.

Keywords: Actin; Antimicrobial Peptide (AMP); Host Defense; Protease; Transglutaminase.

FIGURE 1.

Actin binds rhCAP18 and LL-37. Coomassie Blue-stained SDS-PAGE (A) and mass spectrometry analysis (B) of salivary factors (open arrow) that were pulled down (see “Experimental Procedures”) with Ni-NTA-agarose beads coated (+) or not (−) with rhCAP18 (closed arrow) used as bait. C, co-localization of LL-37 (red) with extracellular actin (green). Confocal microscopy imaging of immunofluorescence (see under “Experimental Procedures”) was performed on a sample of granular tissue collected from a human oral lesion. The merged channel contains nuclei staining with Hoechst (blue). Right panel shows the inset indicated in the merged image. Arrows point to co-localization of actin and LL-37.

FIGURE 2.

F-actin protects and enables the antibacterial activity of LL-37 in the presence of bacterial proteases. A and B, Western immunodetection and densitometry analysis (mean ± S.D. of two independent experiments) of 3 μm LL-37 incubated with 5 μl of culture supernatant (SN) of P. gingivalis (P.g) (A) or P. aeruginosa (P.a) (B), in the absence or presence of 9 μm F-actin. C and D, growth inhibition of B. subtilis by 1.1 μm LL-37 treated or not with P. gingivalis (P.g) (C) or P. aeruginosa (P.a) (D) supernatant (SN), in the presence or absence of 3 μm F-actin. E and F, killing of B. subtilis (displayed as colony-forming units, CFU) by 1.1 μm LL-37 treated or not with P. gingivalis (P.g) (E) or P. aeruginosa (P.a) (F) supernatant (SN) in the presence or absence of 3 μm F-actin. * represents p < 0.05 in Student's t test, ** represents p < 0.01 in one-way analysis of variance.

FIGURE 3.

Prevention of LL-37 proteolysis by microbial proteases is mediated by protection rather than protease inhibition. A, actin does not inhibit the proteolytic activity of R-gingipain of P. gingivalis (P.g). Protease activity (quantified using BAPNA as an R-gingipain substrate, see “Experimental Procedures”) of P. gingivalis conditioned medium co-incubated with or without 3 μm F-actin or with 1 μl of protease inhibitor mixture used as control. B, unlike actin, BSA (see Fig. 2C) does not protect the antimicrobial activity of LL-37 from the proteases of P. gingivalis. Presented data are mean ± S.D. of two independent experiments performed in triplicate. SN, supernatant.

FIGURE 4.

Binding of LL-37 to the bacterial membrane is maintained in the presence of F-actin. FACS histogram (representative experiment) (A) and analysis of three independent experiments (B) of B. subtilis PY79 labeling when incubated with 1.1 μm tetramethylrhodamine-labeled LL-37, in the presence (blue dashed in A) or absence (red in A) of 3 μm F-actin, are shown. Gray histogram in A represents the background of B. subtilis without LL-37.

FIGURE 5.

Cross-linking of LL-37 to actin by TGase. A, SDS-PAGE of cross-linking of 10 μm LL-37 to 8 μm CaATP-G-actin by 0.2 mg/ml TGase. Lane a, actin only; lane b, actin cross-linked to LL-37 (see “Experimental Procedures”) visualized by Coomassie Blue (left) and by Western immunodetection (right, lane b*). B, effect of NaCl concentration on 0.2 mg/ml TGase-induced cross-link formation between 4 μm CaATP-G or Mg-F-actin and 9 μm LL-37 (mean ± S.D. of three independent experiments). C, actin-LL-37 cross-link formation is preferred over LL-37 dimerization. 66 μm LL-37 were incubated with (lanes a and c) and without (lane b) 0.4 mg/ml TGase in the absence (lane b) and in the presence (lane c) of 60 μm CaATP-G-actin at room temperature for 2 h (lanes a and b), Coomassie blue stain; lane a*, lane b*, and lane c*, Western immunodetection. D, antimicrobial activity of LL-37 is lost upon formation of LL-37-LL-37 or LL-37-actin covalent cross-links. 1.1 μm LL-37 was incubated with or without 1.1 μm F-actin and with 0.4 mg/ml TGase at room temperature for 2 h. Following incubation samples were added to B. subtilis cells in a 96-well plate, and antimicrobial activity was measured as described under “Experimental Procedures.”

FIGURE 6.

Pretreatment of LL-37 (but not of actin) with TGase inhibits actin-LL-37 cross-linking. A, 8 μm LL-37 was reacted with 0.2 mg/ml TGase at room temperature for 2 h and then cross-linked with 4 μm CaATP-G-actin, MgATP-G-actin, or Mg-F-actin for an additional 2 h. Lane a, actin alone; lanes b, e, and h, actin and TGase; lanes c, f, and j, LL-37 and TGase added simultaneously to actin and incubated for 2 h; lanes d, g, and i, LL-37 was preincubated for 2 h at room temperature with TGase before adding it to actin. B, effect of LL-37 pretreatment with TGase on the LL-37-actin cross-link formation. 82 μm LL-37 were treated with 1.8 mg/ml TGase at room temperature for 2 h and then 9 μm treated or untreated LL-37 and 0.2 mg/ml TGase was added to 4 μm actin and incubated for an additional 2 h (mean ± S.D. of three independent experiments). C, effect of actin pretreatment with TGase on the LL-37-actin cross-link formation. 8 μm actin was incubated with 0.2 mg/ml TGase on ice for 24 h and then 10 μm LL-37 was added, and the incubation was continued for an additional 24 h. Lanes a–d, CaATP-G-actin; lanes e and f, MgF-actin. Lane a, untreated actin; lane b, TGase-pretreated actin, no LL-37; lanes c and e, TGase pretreated actin with LL-37; lanes d and f, untreated actin with LL-37 and TGase added simultaneously; M, molecular weight marker.

FIGURE 7.

Replacing LL-37's Gln-22 with alanine and Ile-20 and Ile-24 with serine, and scrambling reduces binding of LL-37 to actin. A, cross-linking of 4 μm actin with 9 μm Q22A or LL-37 at low ionic strength and in the presence of 100 and 200 mm NaCl. Upper panel, SDS-PAGE. Lower panel, densitometry analysis. Lanes a–d, CaATP-G-actin; lanes e–j, MgF-actin. Lane a, G-actin alone; lane b, G-actin and TGase, no LL-37; lane c, G-actin, LL-37, and TGase; lane d, G-actin, Q22A, and TGase; lane e, F-actin, LL-37, and TGase; lane f, F-actin, Q22A, and TGase; lane g, F-actin, LL-37, TGase, and 100 mm NaCl; lane h, F-actin, Q22A, TGase, and 100 mm NaCl; lane i, F-actin, LL-37, TGase, and 200 mm NaCl; lane j, F-actin, Q22A, TGase, and 200 mm NaCl. B, bundling of 4 μm MgF-actin by 2–12 μm LL-37 and Q22A at low ionic strength and in the presence of 100 mm NaCl. C, bundling of 4 μm MgF-actin by 3–20 μm I20S/I24S and LL-37 at low ionic strength and in the presence of 100 mm NaCl. For bundling procedure see “Experimental Procedures.” D, F-actin protects the antimicrobial activity of Q22A from proteolysis. Antimicrobial activity of LL-37 and of Q22A and their protection assay was performed as described under “Experimental Procedures.” SN, supernatant. E, cross-linking of 4 μm CaATP-G-actin and MgF-actin with 9 μm LL-37 or s-LL37 in the presence of increasing concentrations of NaCl. s-LL-37 or LL-37 was incubated simultaneously with 4 μm actin and 0.2 mg/ml TGase. Upper panel, SDS-PAGE, Lane a, actin only; lane b, actin, LL-37, TGase; lane c, actin, LL-37, TGase, 100 mm NaCl; lane d, actin, LL-37, TGase, 200 mm NaCl; actin, s-LL-37, TGase; lane e, actin, s-LL-37, TGase; lane f, actin, s-LL-37, TGase, 100 mm NaCl; lane g, actin, s-LL-37, TGase, 200 mm NaCl. Lower panel, quantitative assessment of LL-37-actin cross-link obtained from densitometry of SDS-PAGE.

FIGURE 8.

DNase I and cofilin inhibit cross-linking of LL-37 to actin. A, representative gel (upper panel) and quantitative assessment (lower panel) of CaATP-G-actin (4 μm) cross-linked (“Experimental Procedures”) with 9 μm r-LL-37 or LL-37 by 0.2 mg/ml TGase in the presence and absence of 6 μm DNase I or 12 μm cofilin. All the constituents were incubated simultaneously at room temperature for 90 min. Lane a, actin alone; lane b, actin, TGase; lane c, actin, LL-37, TGase; lane d, actin, r-LL-37, TGase; lane e, actin, r-LL-37, cofilin, TGase; lane f, actin, r-LL-37, DNase I, TGase; *, rhodamine fluorescence. Samples were treated as in Fig. 6B. Evaluation is based on densitometry of SDS-PAGE. B, competition between 8 μm cofilin and 0–10 μm LL-37 for cross-linking to 4 μm CaATP-G-actin by TGase. LL-37 and cofilin were added simultaneously to CaATP-G-actin. For cross-linking procedure, see “Experimental Procedures.” Upper panel, SDS-PAGE. Lane a, actin only; lane b, actin, cofilin, TGase; lane c, actin, cofilin, TGase, 2 μm LL-37; lane d, actin, cofilin, TGase, 4 μm LL-37; lane e, actin, cofilin, TGase, 7 μm LL-37; lane f, actin, cofilin, TGase, 10 μm LL-37. Lower panel, quantitative evaluation of cross-linking of cofilin with actin from densitometry of SDS-PAGE.

FIGURE 9.

Analysis of actin's LL-37-binding site by subtilisin digestion. A, cross-linking 6 μm r-LL-37 to 4 μm CaATP-G-actin protects actin's D-loop from subtilisin digestion. TGase reaction was stopped by 1 mm cysteamine; 30 min digestion with 1 μg/ml subtilisin was stopped by 1 mm PMSF; incubations were performed at room temperature. Samples were separated on SDS-PAGE and visualized by Coomassie Blue staining (lanes a–d) or by tetramethylrhodamine fluorescence (lanes c* and d*). Lane a, actin alone; lane b, actin and TGase; lanes c and c*, actin, r-LL-37, and TGase (added simultaneously); lanes d and d*, actin, r-LL-37, and TGase added simultaneously and followed by subtilisin digestion. Illustration on the left shows cleavage of CaATP-G-actin between Met-47 and Gly-48 by subtilisin (small black arrow) and covalent binding between Gln-41 and Lys-50 by TGase (two- headed arrow). B, subtilisin cleavage of the D-loop prevents LL-37-actin cross-linking. Other than using 9 μm LL-37, the same conditions as in A were applied. Lane a, actin; lane b, subtilisin-digested actin; lane c, subtilisin-digested actin treated by TGase; lane d, subtilisin-digested actin treated by TGase and then incubated with LL-37 for an additional 90 min; lane e, actin incubated with LL-37 and TGase (added simultaneously) for 90 min. C, LL-37 bundling of 4 μm MgF-actin digested or undigested by subtilisin in the presence or absence of 100 mm NaCl. 50 μm CaATP-G-actin were digested by subtilisin (at a 168:1 w/w/ratio) for 30 min, stopped by 1 mm PMSF and polymerized to MgF-actin by 2 mm MgCl₂ (subtilisin digested F-actin). To 4 μm subtilisin, digested F-actin LL-37 was added in increasing concentrations, and bundling was carried out as described under “Experimental Procedures.”

FIGURE 10.

LL-37 binds the actin His-40–Lys-113 10-kDa segment. A, illustration of thrombin digestion of EDTA-treated CaATP-G-actin. B, thrombin digestion of EDTA-treated r-LL-37-CaATP-G-actin cross-link. Lane a, 8 μm CaATP-G-actin was incubated with 1.5 mm EDTA for 21 h; lane b, same as lane a, but 30 μg/ml thrombin was also added together with EDTA, and proteolysis was stopped by 1 mm PMSF; lane c, 8 μm CaATP-G-actin cross-linked with 16 μm r-LL-37 by 0.3 mg of TGase for 2 h; the reaction was stopped by 1 mm cysteamine, and 1.5 mm EDTA was added followed by incubation for 21 h; lane d, same as lane c, but 30 μg/ml thrombin was also added together with EDTA, and proteolysis was stopped as in lane b; M, molecular weight marker. All incubations were performed at room temperature. Samples were separated on SDS-PAGE and visualized by Coomassie Blue stain (left panel) or by fluorescence (right panel, lanes c* and d*).

FIGURE 11.

Identification of the residues involved in cross-linking between actin and LL-37 by mass spectrometry. A, model of the F-actin molecule (green) and the D-loop (gray sticks) using the PyMOL software. Enlarged illustration shows the location of Gln-49 in the D-loop. B and C, relative abundance of the D-loop sequences in actin and in LL-37-actin cross-linked bands. B, His-40–Lys-50 peptide is practically missing from the digest of the actin-LL-37 cross-linked heterodimer, which indicates its involvement in the cross-link formation. C, quantity of VMVGMGQK peptide in the His-40–Lys-50 sequence, containing Gln-49, is greatly reduced in the digest of actin-LL-37 cross-linked heterodimer relative to its quantity in the actin digest. D and E, mass spectrometry analysis and relative abundance of selected peptides in the digest of LL-37-actin or Q22A-actin cross-linked heterodimers. D, mass spectrometry relative abundance analysis show that Gln-22 and five lysines (Lys-8, Lys-10, Lys-12, and to a lesser extent Lys-15 and Lys-18) in LL-37 can participate in the formation of LL-37-actin cross-link. E, similar analysis of the Q22A-actin cross-linked digest indicates the participation of the same lysine residues in the cross-linking reaction. The residues marked in red could participate in the cross-link reaction. A change in font size indicates the significance of the residues in the cross-linking according to the MS calculation of peak area ratio between LL-37 or Q22A and actin-LL-37 or Q22A cross-link.

FIGURE 12.

Schematic model of the significant residues that participate in cross-linking of LL-37 and actin by TGase. Arrow thickness signifies the residue's involvement in the cross-linking.