Borrelia burgdorferi possesses a collagenolytic activity

MICROBIOLOGY LETTERS

ELSEVIER

FEMS

Microbiology

Letters

144 (1996) 39-45

Borrelia burgdorferi possesses a collagenolytic Dennis J. Grab alb,*, Richard

activity

Kennedy a, Mario T. Philipp a

a Department of Parasitology, Tulane Regional Primate Research Center (TRPRC), Covington, LA, USA b Department of Tropical Medicine, Tulane University School of Public Health and Tropical Medicine, New Orleans, LA, USA Received 30 May 1996; revised 11 August

1996; accepted

17 August

1996

Abstract

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi, an invasive spirochete. B. burgdorferi has a predilection for collagenous tissue and one major clinical manifestation of the disease is arthritis. We have identified a collagenolytic activity in B. burgdorferi detergent lysates using iodinated gelatin as well as iodinated pepsinized human collagen types II and IV as protein substrates. In addition, we describe several proteolytic activities in B. burgdorferi with molecular masses greater than 200 kDa on sodium dodecyl sulfate polyacrylamide gels containing copolymerized gelatin. We propose that the collagenolytic activity of B. burgdorferi has a role in invasion, in the pathogenesis of Lyme arthritis, and perhaps also in other manifestations of Lyme borreliosis. Keywords: Borrelia burgdorferi; Lyme disease; Collagenolysis

1. Introduction Borrelia burgdorferi, the spirochete that causes Lyme disease, is an invasive bacterium that is transmitted to humans and other animals by the bite of infected Ixodes scapularis ticks. Following infection, spirochetes remain in the periphery of the site of the tick bite for a limited time, after which they disperse to multiple organs, including the nervous, musculoskeletal, reticula-endothelial and cardiovascular systems, as well as the eye [l]. The molecular mechanism for tissue invasion by B. burgdorferi has remained a mystery for some time. It had been assumed that B. burgdorferi does

* Corresponding author. E-mail: [email protected]

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not produce any hydrolytic activities needed to degrade the extracellular matrix. Recently, there have been several exciting reports describing the ability of B. burgdorferi to bind to plasminogen, the zymogen of the serine protease plasmin [2-51. The bound plasminogen could be activated to plasmin by host-derived plasminogen activators [2-51. Putative plasminogen receptors include a 70 kDa protein [5] as well as OspA [2,3,5]. There is little doubt that plasmin(ogen) plays a role in invasion of tissues by B. burgdorferi. However, it is especially pertinent that B. burgdorferi has a predilection for collagenous connective tissue [6]. Spirochetes have been detected between collagen fibers of bradytrophic dense connective tissue [7] as well as other collagen-rich sites [8]. This manifest predilection for collagenous tissues prompted us to search for a collagenase-like activity in extracts of Microbiological

Societies. Published

by Elsevier Science B.V.

D.J. Grrrh et al. I FEMS Microhiolqy

40

B. burgdorferi,

for plasmin does not hydrolyze collagen. We report here on the results of this search.

2. Materials and methods 2.1. Materials Young rabbit serum was obtained from Pel-Freeze Biologicals (Rogers, Arkansas). Sephadex G-25 M PD-10 columns were obtained from Pharmacia LKB Biotechnology AB (Uppsala, Sweden). Gelatin for zymography was obtained either from Difco laboratories (Detroit, MI) or J.T. Baker Chemical Co. (Phillipsburg, NJ). All other reagents used for sodium dodecyl sulfate polyacrylamide gels (SDSPAGE) were from Bio-Rad Laboratories (Hercules, CA). Protease-free gelatin, pepsinized human collagen types I, II, III, and IV, gelatin-free BSK-H medium, trypsin-TPCK (trypsin treated with ~-l-tosylamide-2-phenylethyl chloromethyl ketone), TLCK (N-a-p-tosyl-L-lysine chloromethyl ketone), EDTA (ethylenediaminetetraacetic acid) and all other biochemicals were from Sigma Chemical Company (St. Louis, MO). 2.2. In vitro cultivation of B. burgdorferi B. burgdorferi isolates were routinely cultured at 34°C (in 5% COP, 3% 02 and 92% Nz) in gelatin-free BSK-H medium [9] supplemented with 10% heat-inactivated young rabbit serum. Low-passage isolates (below 12) of several strains were used.

2.3. Preparation of ‘251-labeled gelatin or human collagen types II and IV

Pepsinized human collagen types II and IV were solubilized in 20 mM acetic acid, 100 mM NaCl, pH 3.3 The proteins were further purified by HPLC-gel permeation chromatography on a 10 X 300 mm BioSep 2000 (Phenomenex, Torrance, CA) column equilibrated in 2% glycerol in phosphate buffered saline (PBS), pH 7.4. The flow rate was 2 ml/min. The proteins were iodinated using the Iodo-Beads iodination reagent according to the manufacturer’s instructions (Pierce, Rockford, IL). Briefly, the proteins (160-200 ug in 1 ml PBS) were incubated at ambient

Letters 144 (1996) 39-45

temperature in the presence of 0.6 mCi Na1251 and Iodo-Beads (one bead per reaction). After 15 min, the protein samples were passed through a Sephadex G-25 M PD-10 column to remove free iodine and other small reaction products. The specific activities for 1251-labeled gelatin, collagen type II and collagen type IV were 812 000, 215 000 and 354 000 cpm/ug protein respectively. 2.4. Analysis of collagenolytic activity To assess proteolytic activity with iodinated gelatin or collagen substrates, B. burgdorferi was solubilized in PBS containing l-5% (w/v) CHAPS (3-[(3cholamidopropyl)dimethlyammonio]1-propanesulfonate), a zwitterionic detergent. Typically, extracts of solubilized B. burgdorferi of 0.3-2.5 X IO8 spirochetes in 100 1.11CHAPS buffer were incubated with l-5 ug iodinated protein in a final volume of 100 ul for 18 h at 37°C. Assay blanks not containing enzyme were also done. Sodium azide (0.05-0.25%) was added to prevent bacterial contamination during the long incubations. The reaction (in duplicate) was stopped by the addition of l/2 volume 0.04 N phosphotungstic acid in 2 N HCl [lo]. After 15 min at ambient temperature the samples were centrifuged at 17000Xg for 15 min in an MR1812 Jouan microfuge to precipitate any undegraded gelatin/collagen and the acid soluble phase counted for released radioactivity. The activity is expressed as the mean acid soluble cpm released in the presence of spirochete protein minus the blank controls. Analysis of collagenolytic activity by zymography was performed in gelatin-containing SDS-PAGE as described by Lacks and Springhorn [ll]. The B. burgdorferi proteins were solubilized in sample buffer [12] containing 2% SDS and 2.3 M urea without 2mercaptoethanol (2-ME), but were not boiled, to avoid irreversible denaturation of the putative proteases. SDS-PAGE was done in 16 X 20 cm gels using the Bio-Rad Protean II xi system (Bio-Rad). After electrophoresis, the gel was washed twice (10 min) in Hz0 and incubated overnight in 100 mM HEPES buffer, pH 7.5, to allow for protein renaturation while the SDS diffused out of the gel matrix. The gel was then stained with Coomassie brilliant blue, and destained in 7% acetic acid-30% methanol. Proteolytic activity was revealed by clear areas (bands).

41

D.J. Grab et al. IFEMS Microbiology Letters 144 (1996) 39-45

25 ul Bb + Collagen type II

25 ul Bb + Gelatin

5 ul Bb + Gelatin

Cpm released [x IO51 Fig. 1. Hydrolysis of iodinated gelatin and human soluble collagen types II and IV by 8. burgdorferi detergent lysates. Protease-free gelatin and HPLC-purified human soluble collagens types II and IV (from Sigma) were iodinated using Iodo-Beads. Approximately 2.5 X 1O’O E. burgdorferi JDl were solubilized in 2.5 ml 10% (v/v) glycerol in PBS containing 5% (w/v) CHAPS and 0.25% sodium azide. After 10 min on ice the sample was centrifuged at 17400 Xg for 20 min (4°C). The CHAPS-soluble fractions were assayed for collagenase activity as described in Section 2. 5 pg ‘251-labeled gelatin (protease free), soluble human collagens type II, or human collagen type IV were incubated with either 5 or 25 ~1 CHAPS soluble spirochete fraction representing 5 X 10’ or 2.5 X lOsspirochetes/ml respectively. The cpm initially incubated were 2.4 X 106, 8.9 X 105, and 1.6 X 10s cpm (5 Kg) gelatin, collagen type II and collagen type IV respectively.

3. Results We found that B. burgdorferi collagenase is active and can be solubilized with detergents such as CHAPS. In addition, the hydrolysis of gelatin or collagens was found to be dependent on both B. burgdorferi protein and substrate concentration. 25 ul of a CHAPS soluble B. burgdorferi (strain JDl) fraction (from 2.5 X 10s spirochetes) was able to catalyze the hydrolysis of iodinated gelatin, collagen type II and collagen type IV, cleaving approximately 59.8% (1.20X lo6 cpm), 22% (1.96~10~ cpm) and 11% (1.76X lo5 cpm) respectively, of the added radioactivity (Fig. 1). In the presence of 5 ul CHAPS soluble B. burgdorferi (from 0.5 X lo8 spirochetes) fraction the release of radioactivity from gelatin was 19.1% (4.6 X lo5 cpm) of the added radioactivity (Fig. 1). The amount of hydrolysis is also dependent

on gelatin or collagen (type II) concentration (Fig. 2). Using whole (CHAPS solubilized) B. burgdorferi, the total amount of radioactivity released from gelatin and collagen type II increased 4.6- and 7.5-fold respectively, when the concentration of radiolabeled protein substrate was increased from 1 ug to 5 ug (Fig. 2). In addition, in the presence of 5 ug [1251]gelatin, increasing the concentration of spirochetes from 0.3X lo8 (total) to 2.5X lo8 (from a CHAPS lysate) also resulted in a lo-fold increase in the release of acid soluble 1251 cpm (compare Bb+Gelatin (5 ug) sample in Fig. 2 with 25 ~1 Bb+Gelatin sample in Fig. 1). Proteolytic activity does not appear to be inhibited by EDTA, but was abolished by heating the B. burgdorferi preparation at 95°C (10 min) before addition into the assay (Fig. 2). The collagenolytic

activity

of B. burgdorferi

also

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D.J. Grab et al. I FEMS Microbiology

Letters 144 (1996) 39-45

Heated Bb + CollagenII (5 ug)

Bb + Collagen II (5 ug)

Bb + Collagen II (1 ug)

Heated Bb + Gelatin (5 ug)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cpm released [x IO51 Fig. 2. The effect of gelatin or collagen concentration on f3. hurgdorfrrl collagenase activity. Whole B. burgdurj&i JDl (3X 10’) was incubated with increasing concentrations of [‘2sI]gelatin or [““Ilhuman collagen type II (1 bg and 5 kg) in PBS, pH 7.5 containing 0.5% CHAPS and 0.05% sodium azide. Spirochetes heated at 95°C for 10 min prior to the start of the assay were used as controls.

was analyzed by SDS-PAGE zymography. On 7% (7% T/0.6% C) SDS-PAGE gels containing gelatin, a very high molecular mass single band of proteolytic activity for B. burgdorferi strain B31 was detected (Fig. 3). However, by reducing the concentration of acrylamide to 6% and by increasing the bis-acrylamide concentration (6% T/1.1% C) it was possible to resolve at least three bands of proteolytic activity in B.burgdorferi strains B31, JDl and 25015 (Fig. 4). The molecular mass of the enzyme(s) appeared to be greater than the mass of myosin (203 kDa); the enzymatic activity was present in extracts of cells harvested in both stationary and exponential phase growth (Fig. 4). The same three proteolytic activities seen on gelatin gels for B. burgdorferi (JDI) were also able to hydrolyze soluble human collagen types I, II, and IV when these were incorporated into the gels (not shown). The three bands were present even when the spirochetes were solubilized in 2% SDS alone or in 2% SDS containing 6.9 M urea prior to SDS-PAGE zymography. No bands of activity were

seen when conditioned medium was tested. The enzyme was active at pH 7.5 and, in one experiment, was still active at pH 5. The enzyme(s) was (were) not easily removed from the spirochetes since it was present even after extensive washing in PBS. In addition, the activity of the enzyme does not appear to be significantly affected by EDTA, 2-ME, ATP or divalent cations when these were added into the renaturation buffer in this system (not shown). To activate any collagenase zymogens we preincubated B.burgdorferi with plasmin for 30 min at 37°C prior to the addition of sample buffer and SDSPAGE. Plasmin did not appear to activate any additional collagenase activity nor did it produce any additional bands. Plasmin itself had no activity. Similar results were found using [iz51]gelatin (not shown). In contrast to plasmin, under these experimental conditions (zymography) trypsin-TPCK totally destroyed the B. burgdorferi gelatinase (not shown). When B. burgdorferi was preincubated with both trypsin and the trypsin inhibitor TLCK

D.J. Grab et al. I FEMS Microbiology Letters 144 (1996) 39-45

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Fig. 4. Gelatinase activity in B. burgdorferi B31, JDl and 20515 in exponential and stationary growth phase. B. burgdorferi B31, JDl and 20515 were run on an SDS-PAGE gel (6% T/1 .l% C) containing 680 pg gelatin/ml. 191 pg B. burgdorferi protein was loaded per lane (2-7). Lane 1: myosin (203 kDa); lane 2: stationary growth phase I?. burgdorferi B31; lane 3: exponential growth phase B. burgdorferi B31; lane 4: stationary growth phase B. burgdorferi JDI; lane 5: exponential growth phase B. burgdorferi JDl ; lane 6: stationary growth phase B, burgdorferi 20515 ; lane 7: exponential growth phase B. burgdorferi 20515.

prior to zymography, full collagenase activity was found. This suggests that the collagenase activity is both trypsin sensitive and is not inhibited by TLCK.

a

b

C d

Fig. 3. 7% SDS-PAGE gelatin gel of B. burgdorferi B31. B. burgdorferi B31 were solubilized in sample buffer and run on an SDS-PAGE gel (7% T/0.6% C) containing 100-112 Kg gelatin/ml as described in Section 2. Lane 1: 20 pl Bio-Rad prestained SDS-PAGE protein standards: 106 kDa (a), 80 kDa (b), 49.5 kDa (c), and 32.5 kDa (d). Lane 2: The equivalent of approximately 10s spirochetes. The arrow indicates the area of proteolytic activity.

4. Discussion We have shown that B. burgdorferi possesses a collagenolytic activity which can hydrolyze gelatin and several different collagen types. Most importantly, this enzymatic activity is a specific collagenase/gelatinase activity since the telopeptide ends of the collagen substrates had been removed by pepsinization, thereby eliminating detection of non-specific proteases [13]. The collagenase(s) of B. burgdorferi that we have identified is unusual because of its size. Most bacterial collagenases have molecular masses less than 120 kDa [10,11,14,15]. The collagenolytic/ gelatinolytic activity of B. burgdorferi is of high molecular mass (greater than 200 kDa). The enzyme is unlike the collagenase of Treponema denticola, which prefers small peptides [16]. In addition, the collagenase is active in the presence of plasmin, indicating that both proteolytic activities can functionally coexist within the spirochete. As far as we can ascertain from the literature, this is the first example of a collagenolytic activity described in B. burgdorferi. Recent findings by Moser et al. [17] have provided evidence that together, plasminogen/plasmin (after tissue-type plasminogen activation) and metalloproteinases (gelatinase/collagen-

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ase) provide the biochemical mechanism for tumor invasiveness by ovarian epithelial carcinoma. Young et al. [18] found that plasma membrane fractions from ovarian adenocarcinoma cells not only promote zymogen activation of MMP-2 (matrix metalloproteinase-2) but also increase the efficiency of gelatinolysis. They hypothesized that even cells that do not express endogenous MMP-2 may be able to utilize exogenous MMP-2 to mediate proteolysis associated with invasion and metastasis. It is also possible that there exist tissue inhibitors of B. burgdorferz collagenase in a manner analogous to the inhibition of progelatinase A and B by tissue inhibitors of metalloproteinases-2 (TIMP-2) and TIMP-1, respectively [10,19]. How B. burgdorferi targets to specific tissues may depend, in part, on whether the tissue can activate or inhibit the bacterial collagenase. Based on the above findings, and findings in other systems, our working hypothesis is that the B. burgdorferi gelatinase/collagenase(s) activity, together with plasmin(ogen) plays an important role in the process of bacterial dissemination through the skin and endothelium, and probably plays a major role in Lyme arthritis. We hypothesize that B. burgdorferi collagenase(s), when released into the extracellular matrix of cartilage has the potential to dissociate collagen type II fibers. Release of collagen fragments could elicit an autoimmune type of arthritis. It is also possible that B. burgdorjhri collagenase may act to degrade not only collagens but the monomeric form of high density cartilage proteoglycan (aggrecan), as has recently been shown by Singer et al. [20] for matrix metalloproteinases, MMP (gelatinase A or stromelysin). Generation of a cleavage product between Asn-341 and Phe-342 (VDIPEN) demonstrated for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis. In an exciting preliminary finding, it has been shown that B. burgdorferi is able to degrade proteoglycans from human articular cartilage (H.D. Adkisson (Washington University, St. Louis, MO) and D.J. Grab, unpublished results). Whether the B. burgdorferi proteoglycanase and collagenase activities are the same remains to be tested. In conclusion, many mechanisms have been reported in the scientific literature to explain the ability of B. burgdorferi to bind and penetrate tissues and

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cells. Proteases such as plasmin [2-51 and collagenase (this paper) may both play a role.

Acknowledgments We would like to thank Ms. Christina Givens for expert technical assistance, and Dr. H.D. Adkisson (Washington University, St. Louis, MO) and Drs. F. Cogswell, N. Lanners and M. Wiser (Tulane University) for their many helpful suggestions during the course of this work. We also thank Mr. Murphy Dowouis for his photographic work. This work was presented in part at the 35th Annual Meeting of The American Society for Cell Biology, December 9-13, 1995, Washington, D.C. [Grab et al. (1995) Mol. Cell Biol. 6, 43a]. Financial support for this work was provided in part by grants from the CDC (No. U5O/CCU606604-05) and from the NIH (No. PRO0164).

References 111Steere, A.C. (1989) Lyme disease. New Engl. J. Med. 321. 586--596.

[21Klempner,

M.S., Noring, R., Epstein, M.P., McCloud. B.. Hu, R., Limentani, S.A. and Rogers, R.A. (1995) Binding of human plasminogen and urokinase-type plasminogen activator to the Lyme disease spirochete, Borrelia burgdor/kri. J. Infect. Dis. 171, 1258-1265. [31Fuchs, H., Wallich, R., Simon, M.M. and Kramer, M.D. (1994) The outer surface protein A of the spirochete Borrelia burgdorferi is a plasmin(ogen) receptor. Proc. Nat]. Acad. Sci. USA 91, 1259412598. [41Coleman, J.L., Sellati, T.J., Testa, J.E., Kew, R.R., Furie, M.B. and Benach, J.L. (1995) Borrelia burgdorferi binds plasminogen, resulting in enhanced penetration of endothelial monolayers. Infect. Immun. 63, 2478-2484. [51Hu, L.T., Perides, G., Noring, R. and Klempner, M.S. (1995) Binding of human plasminogen to Borrelia burgdorferi. Infect. Immun. 63, 3491-3496. PI Barthold, S.W., Persing, D.H., Armstrong, A.L. and Peeples, R.A. (1991) Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. 139, 273-273. [71 Haupl, T., Hahn, G., Rittig, M., Krause, A., Schoerner, C., Schonherr, U., Kalden, J. R. and Burmester, G.R. (1993) Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme borreliosis. Arthritis Rheum. 36, 1621-1626. PI Pachner, A.R., Basta, J., Delaney, E. and Hulinska, D. (1995)

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Localization of Borrelia burgdorferi in murine Lyme borreliosis by electron microscopy. Am. J. Trop. Med. Hyg. 52, 128133. Barbour, A.G. (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57, 521-525. Seifter, S. and Harper, E. (1970) Collagenases. Methods Enzymol. 19, 613635. Lacks, S.A. and Springhom, S.S. (1980) Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J. Biol. Chem. 155, 7467773. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68&5. Dean, D.D. and Woessner, F.W. (1985) A sensitive specific assay for tissue collagenase using teleopeptide-free [3H]acetylated collagen. Anal. B&hem. 148, 174-181. Laskin, AI. and Lechevalier, H.A. (Eds.) (1973) In: Handbook of Microbiology, Vol. III, pp. 607611. Yoshihara, K., Matsushita, O., Minami, J. and Okabe, A. (1994) Cloning and nucleotide sequence analysis of the colH gene from Clostridim histolyticum encoding a collagenase and a gelatinase. J. Bacterial. 176, 64894i496. Makinen, K.K., Makinen, P.L. and Syed, S.A. (1992) Purifi-

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cation and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-leu-gly-pro-ala and bradykinin. J. Biol. Chem. 267, 14285-14293. Moser, T.L., Young, T.N., Rodriguez, G.C., Pizzo, S.V., Bast, R.C. Jr. and Stack, M.S. (1994) Secretion of extracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma. Int. J. Cancer 56, 552-559. Young, T.N., Pizzo, S.V. and Stack, M.S. (1995) A plasma membrane-associated component of ovarian adenocarcinoma cells enhances the catalytic efficiency of matrix metalloproteinase-2. J. Biol. Chem. 270, 999-1002. Coucke, P., Baramova, E., Leprince, P., Depauwgillet, M.C., Foidart, J.M., Bassieer, R. (1994) Metalloproteinases and serine proteases activities in mixed spheroids of mouse B16 melanoma cells and fibroblasts. Int. J. Oncol. 5, 1125-l 130. Singer II., Kawka, D.W., Bayne, E.K., Donatelli, S.A., Weidner, J.R., Williams, H.R., Ayala, J.M., Mumford, R.A., Lark, M.W., Glant, T.T. et al. (1995) VDIPEN, a metalloproteinax-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis. J. Clin. Invest. 95, 217882186.