IAI Accepts, published online ahead of print on 19 December 2011 Infect. Immun. doi:10.1128/IAI.05903-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved. 1 2 The antibacterial activity of LL-37 against Treponema denticola is 3 dentilisin-protease independent and facilitated by the major outer sheath 4 protein virulence factor 5 D o w 6 n lo a d 7 e d f 8 Graciela Rosen, Michael N. Sela and Gilad Bachrach* r o m 9 h t t p 10 Institute of Dental Sciences, the Hebrew University-Hadassah School of Dental Medicine, :/ / ia i. 11 Jerusalem, Israel. a s m 12 .o r g / 13 Running title: Antibacterial activity of LL-37 against T. denticola o n A 14 p r 15 il 4 , 2 16 *For correspondence. Gilad Bachrach, Institute of Dental Sciences, The Hebrew 0 1 9 17 University-Hadassah School of Dental Medicine, Jerusalem P.O.B 12272, Israel 91120 b y g 18 Tel: 972 2 675 7117; Fax: 972 2 675 8561; e-mail: [email protected] u e s t 19 20 21 22 Key words: Treponema denticola, periodontal disease, antimicrobial peptides, LL-37, 23 MSP, dentilisin. 1 24 Abstract 25 Host defense peptides are innate immunoeffectors that possess both bactericidal activities 26 and immunomodulatory functions. Deficiency in the human LL-37 host defense peptide has 27 been previously correlated with severe periodontal disease. Treponema denticola is an oral 28 anaerobic spirochete closely associated with the pathogenesis of periodontal disease. The T. D 29 denticola Major Surface Protein (MSP), involved in adhesion and in cytotoxicity, and the o w n 30 dentilisin serine protease are key virulence factors of this organism. In this study, we lo a d 31 examined the interactions between LL-37 and T. denticola. The three tested T. denticola e d f r 32 strains were susceptible to LL-37. Dentilisin was found to inactivate LL-37 by cleaving it at o m h 33 Lys, Phe, Gln and Val residues. However, dentilisin deletion did not increase susceptibility t t p : / 34 of T. denticola to LL-37. Furthermore, dentilisin activity was found to be inhibited by / ia i. a 35 human saliva. s m . 36 In contrast, deficiency of the T. denticola MSP increased resistance to LL-37. The MSP o r g / 37 deficient mutant bound less fluorescently-labelled LL-37 than the wild type strain. MSP o n A 38 demonstrated specific, dose dependent LL-37 binding. In conclusion, though capable of LL- p r il 39 37 inactivation, dentilisin does not protect T. denticola from LL-37. Rather, the rapid, MSP- 4 , 2 0 40 mediated binding of LL-37 to the treponemal outer sheath precedes cleavage by dentilisin. 1 9 b 41 Moreover, in vivo, saliva inhibits dentilisin thus preventing LL-37 restriction and ensuring y g u 42 its bactericidal and immunoregulatory activities. e s t 43 44 2 45 Introduction 46 Host defense peptides (HDPs) are cationic and amphipathic molecules produced mainly on 47 epithelial surfaces and by phagocytic cells (18). HDPsare inducible by injury or microbial 48 burden and protect the host by direct killing of pathogens and by acting as multifunctional 49 effectors that elicit cellular processes to promote anti-infective and repair responses (4). D 50 Direct killing of pathogens by host defense peptides is a major protective mechanism for o w n 51 generating an antimicrobial barrier that protects against systemic and skin pathogens (11, lo a d 52 12, 55), lung infections (3), and to balance the microflora in the oral cavity (32, 61, 79). e d f r 53 The oral HDPs include α- and β-defensins, histatins and the cathelicidin LL-37 (16, 29). o m h 54 Their importance has been shown in Kostman syndrome, in which patients deficient in LL- t t p : / 55 37 and α-defensin HNP-1 develop severe periodontal disease promoted by LL-37 sensitive /ia i. a 56 Aggregatibacter actinomycetemcomitans (61). Periodontal diseases are polybacterial- s m . o 57 induced (70), multifactorial (80) inflammatory processes of the tooth attachment apparatus r g / o 58 (67) and are the primary cause of tooth loss after the age of 35 (45). n A 59 p r il 4 60 Proteolysis, is a common microbial escape strategy from HDPs. Treponema denticola, , 2 0 61 Porphyromonas gingivalis and Tannerella forsythia are strongly associated with periodontal 1 9 b 62 diseases (70). These microorganisms, characterized by their high proteolytic and y g u 63 peptidolytic capacity (35, 42, 47), can hydrolyze the oral HDPs and inactivate their e s t 64 antimicrobial activity (17, 42, 60). Nevertheless, the above bacteria show different 65 susceptibility to oral HDPs. While T. forsythia is susceptible to β-defensins (38) and P. 66 gingivalis shows selective strain susceptibility to these peptides (38, 39), oral treponemes 3 67 seem to be relatively resistant to human β-defensins through a combination of decreased 68 defensin binding and effective efflux (6, 7). 69 Proteases from both P. gingivalis and T. forsythia degrade LL-37 in vitro (1, 42). 70 Nevertheless, T. forsythia is susceptible to LL-37 (42) and resistance of P. gingivalis to 71 direct killing by LL-37 is protease independent and at least partially due to the low affinity D 72 of the peptide for this bacteria (1). o w n 73 LL-37 is poorly active against the systemic pathogenic spirochetes Leptospira interrogans, lo a d 74 Borrelia burgdorferi and Treponema pallidum with minimum bactericidal concentration e d f r 75 values ranging from 150-450 μg/ml (33-100 μM) (66). Thus, it seemed of interest to o m h 76 evaluate the antimicrobial activity of LL-37 against periodontopathogenic spirochetes. t t p : / 77 The periodontopathic T. denticola (70), possesses a number of virulence factors. These /ia i. a 78 include motility, the ability to attach to host tissues (21), coaggregation with other oral s m . o 79 bacteria (41, 62), complement evasion mechanisms (53) and the presence of several outer r g / o 80 sheath and periplasmic proteolytic and peptidolytic activities (47, 63, 64). The proteolytic n A 81 capacity of T. denticola sustains the nutritional requirements (69) and ATP production (65, p r il 4 82 68) of these spirochetes. Two components associated with the spirochetes’ outer sheaths , 2 0 83 and extracellular vesicles are the major surface protein [also known as the major outer 1 9 b 84 sheath protein (MSP)] and a serine protease, dentilisin, previously known as the y g u 85 chymotrypsin-like protease (74). Recent bioinformatics analysis reclassified dentilisin as a e s t 86 member of the subtilisin rather than the chymotrypsin family (13, 37). Dentilisin is involved 87 in the degradation of membrane basement proteins (laminin, fibronectin and collagen IV) 88 (64), serum proteins (fibrinogen, transferrin, IgG and IgA) including protease inhibitors 89 (α1-antitrypsin, antichymotrypsin, antithrombin and antiplasmin) (30, 74), and bioactive 4 90 peptides (50). Degradation of tight junctional proteins by dentilisin seems to enable 91 penetration of epithelial cell layers by this oral spirochete (10). 92 93 MSP is a major antigen (9, 28) with pore-forming activity (20). This abundant membrane 94 protein mediates binding of T. denticola to fibronectin, fibrinogen, laminin and collagen D 95 (19, 25), induces macrophage tolerance to further activation with LPS (56), and elicits o w n 96 cytotoxic effects in different cell types (24, 75). lo a d 97 e d f r 98 The objectives of this study were to examine the effectiveness of LL-37 against T. denticola o m h 99 and to study the interactions between the two oral components. We found that in contrast to t t p : / 100 systemic spirochetes, LL-37 possesses an effective bactericidal activity against T. denticola. / ia i. a 101 This activity was targeted and enhanced by the presence of the treponeme’s major outer s m . 102 sheath protein, and preceded degradation by the spirochete’s dentilisin protease. o r g / 103 Surprisingly, saliva was found to inhibit dentilisin, attenuating its virulence properties and o n A 104 conserving LL-37 activity despite the presence of the protease. p r il 105 4 , 2 0 106 Materials and Methods 1 9 b 107 LL-37 peptide. LL37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was y g u 108 synthesized as described previously by the solid-phase method on a fully automated, e s t 109 programmable peptide synthesizer (2). Peptide integrity and purity (greater than 95%) were 110 determined by analytical high-performance liquid chromatography and mass spectrometry. 111 5 112 Bacterial strains and growth conditions. T. denticola strains ATCC 35404 (TD4), ATCC 113 33520 and GM-1, and T. denticola mutants K1(lacking the outer sheath dentilisin protease) 114 (36), and MHE (lacking the major outer sheath protein, MSP) (26), were grown in GM-1 115 media (5) in a Bactron anaerobic (85% N , 10% H and 5% CO ) environmental chamber 2 2 2 116 (Sheldon Manufacturing Inc., Cornelius, OR) at 37°C. Late exponential phase cultures, D 117 corresponding to an optical density of 0.250-0.300 at 660 nm, were obtained following o w n 118 incubation for 4 days. Escherichia coli ATCC 25922 was grown in BHI broth under aerobic lo a d 119 conditions. Bacterial purity was determined by phase microscopy and Gram staining. e d f r 120 o m h 121 Growth inhibition by LL-37. Susceptibility of T. denticola to LL-37 was evaluated t t p : / 122 following four days of bacterial growth spectrophotometrically at 660 nm with or without / ia i. a 123 increasing concentrations of LL-37. Optical density at time 0 was considered 100% growth s m . 124 inhibition. Optical density of bacterial growth in the absence of LL-37 was considered as o r g / 125 0% growth inhibition. o n A 126 Growth inhibition of T. denticola TD4 following 1 hour of exposure to LL-37 was p r il 127 performed as follows: T. denticola cells (1.0x108 cells/ml) from 4 day-old cultures were 4 , 2 0 128 incubated in GM-1 culture media with or without LL-37 (50 μg/ml) for 1 hour. Bacteria 1 9 b 129 were diluted x 20 in GM-1 media and grown for 36 hours. Optical density at time 0 (after y g u 130 dilution) was considered 100% growth inhibition. Optical density of bacterial growth in the e s t 131 absence of LL-37 was considered as 0% growth inhibition. Presented results are mean and 132 standard deviation of two independent experiments performed in quadruplicates. 133 134 6 135 Bacterial viability. Bacterial killing by LL-37 was evaluated by measuring intracellular 136 ATP levels, an energetic parameter commonly used as an indicator of cell injury and 137 viability (71). Briefly, T. denticola cells (1.0x108 cells/ml) from 4 day-old cultures were 138 incubated in GM-1 culture media with or without LL-37 for the time stated. The cells were 139 then centrifuged (10,000 x g, 5 min), resuspended in 1 ml PBS and transferred into a 2 ml D 140 microfuge tube containing glass beads (diameter 160 µm; Sigma, St. Louis, MO). The cells o w n 141 were disrupted with the aid of a Fast Prep Cell Disrupter (Bio 101, Savant Instruments, Inc., lo a d 142 Holbrook, NY). ATP levels were determined using the ATP Bioluminescence assay kit e d f r 143 (Roche Applied Science Mannheim, Germany). ATP levels of bacteria not treated with LL- o m 144 37 was considered 0% killing and ATP levels of heat-killed bacteria (exposure to 100 0C for h t t p : / 145 5 min) was defined as 100% killing. / ia i. a 146 s m . 147 Purification of dentilisin. The protease was purified from isolated T. denticola outer o r g / 148 sheaths or extracellular vesicles followed by preparative SDS-PAGE and electroelution as o n A 149 described before (64). The specific activity of the purified dentilisin was of the order of 100 p r il 150 to 150 U/mg, a 100-fold purification from that of total treponemal cells (1.2 U/mg). 4 , 2 0 151 1 9 b 152 Protease assays. Dentilisin activity was determined by cleavage of the chromogenic y g u 153 substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) (64). Trypsin-like activity e s t 154 was determined with the chromogenic substrate Nα benzoyl-L-Arg-p-nitroanilide 155 (BAAPNA), as described before (48). 156 7 157 Enzymatic degradation of LL-37. Four day-old T. denticola cultures were centrifuged for 158 10 min at 10,000 x g in a microcentrifuge (Eppendorf, Germany) and the supernatant was 159 collected. LL-37 (10 μg) was incubated with 10 μl of T. denticola supernatant (0.15-0.2 160 units/ml of SAAPNA degrading activity) or with 10 ng purified dentilisin in 50 mM Tris- 161 HCl buffer pH 8.0 for 2 hr at 37°C. The reaction was terminated by addition of SDS-PAGE D 162 sample buffer and boiling for 3 min at 100°C. The samples were centrifuged at 10,000 x g o w n 163 in a microcentrifuge for 2 min and subjected to 15% SDS-PAGE. The gels were stained lo a d 164 with Coomassie blue. The effect of PMSF (1 mM), chymostatin (0.4 mM), and saliva (13 μl ed f r 165 of saliva cleared by centrifugation at 13,000 x g for 10 min/2x10-3 units SAAPNA o m h 166 degrading activity) on the enzymatic degradation of LL-37 was evaluated by incubating the t t p : / 167 supernatant with the inhibitors for 20 min at room temperature prior to addition of the /ia i. a 168 peptide. s m . o 169 r g / o 170 Mass Spectrometry (MS). LL-37 was incubated with purified dentilisin as described n A 171 above. The hydrolyzed peptide mixture was solid phase extracted with a C18 resin filled tip p r il 4 172 (ZipTip Milipore, Billerica, MA USA) and nanosprayed into the Orbi-trap MS system in , 2 0 173 50% CH3CN 1% CHOOH solution. 1 9 b 174 Mass spectrometry was carried out with Orbi-trap (Thermo Finnigen) using nanospray y g u 175 attachment (76). Data analysis was carried out using the Bioworks 3.3 package. e s t 176 177 FACS analysis. LL-37 was labeled with Alexa Fluor 350 (Molecular Probes, Oregon) 178 as described before (1). T. denticola were grown to late logarithmic phase, sedimented for 3 179 min at 10,000 x g, and brought to an OD of 1 in PBS. 200 μl of cells were incubated with 600 8 180 20 μl of labeled peptide for 10 min at room temperature. Cells were washed in 0.3 ml PBS, 181 and resuspended in 0.3 ml PBS. Samples were subjected to flow cytometry using the LSR II 182 instrument (BD Biosciences) and analyzed using FlowJo software (Tree Star). Runs were 183 repeated in three independent experiments with similar results. 184 D 185 LL-37 binding assay. The MSP complex was isolated and highly enriched by sequential o w n 186 detergent extraction and autoproteolysis of T. denticola extracts as previously described lo a d 187 (52). MSP (1 μl; 1, 0.1 or 0.01 μg/ml), fibrinogen or bovine serum albumin were attached to e d f r o 188 nitrocellulose at room temperature, blocked with 5% skim milk in PBS for 1 hr, and m h 189 incubated with LL-37 (1 μg/ml in PBS) for 1 hr. After three washes with PBS containing tt p : / / 190 0.05% Tween 20, LL-37 binding was detected with anti-LL37 rabbit serum as described ia i. a 191 before (33). s m . o 192 r g / o 193 Effect of proteolysis by T. denticola supernatant on the antimicrobial activity of LL-37. n A p 194 LL-37 was treated for 2 hours with T. denticola supernatant or with heat inactivated r il 4 195 supernatant (negative control) as described above. The remaining antimicrobial activity of , 2 0 1 196 the truncated LL-37 was tested on T. denticola and on E. coli as follows. Treated LL-37 was 9 b 197 added to a growing culture of T. denticola TD4 (50 μg/ml) and the percentage of y g u e 198 treponemal killing was determined by measuring intracellular ATP levels as described s t 199 above. ATP levels of bacteria not treated with LL-37 was considered 0% killing and ATP 200 levels of heat-killed bacteria (exposure to 100 0C for 5 min) was defined as 100% killing. 201 For growth inhibition of E. coli, treated or untreated LL-37 (4 µg) was brought to a total 202 volume of 50 µl with PBS and added to 150 µl of E. coli ATCC 25922 (overnight culture, 9 203 diluted 1:5,000 in BHI). Growth of E. coli was measured continually in 96-well plates 204 (NUNC, Denmark) at 37°C and absorbance was followed at OD (GENios reader 595 205 TECAN, Austria). 206 207 Statistical analysis. Data are reported as means ± standard deviations (SD). Statistical tests D 208 were performed using one-way ANOVA and Student t test. A p value <0.05 was considered o w n 209 significant. lo a d 210 e d f r 211 Results o m h 212 Antibacterial activity of LL-37 against T. denticola. Growth inhibition of T. denticola by t t p : 213 LL-37 was tested at LL-37 concentrations reaching 200 μg/ml. As shown in Fig. 1, LL-37 //ia i. a 214 concentration of 50 μg/ml (11 μM) or higher, inhibited growth of T. denticola ATCC 35404 s m . o 215 (TD4) by 85%. Higher peptide concentrations did not improve growth inhibition (Fig. 1). r g / o 216 To determine whether susceptibility to LL-37 is common among T. denticola strains, n A 217 growth inhibition by LL-37 (50 μg/ml) was also tested with T. denticola ATCC 33520 and p r il 4 218 GM-1. No significant differences in susceptibility to LL-37 were observed among the three , 2 0 1 219 tested T. denticola strains (data not shown). 9 b y 220 g u e 221 Next, the kinetics of T. denticola killing by the antimicrobial peptide was evaluated by s t 222 measuring intracellular ATP levels of T. denticola cells exposed to LL-37. Time dependent 223 killing of T. denticola TD4 is shown in Fig. 2. Incubation of T. denticola with LL-37 (50 224 μg/ml) consistently produced a sharp decay (89.7±7 %) in intracellular ATP after 1 hr. This 225 ATP decay corresponded with a 92.8±2% reduction in bacterial growth 36 hours after 10