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Antimicrob Agents Chemother. 2009 October; 53(10): 4490–4494.
Published online 2009 August 3. doi:  10.1128/AAC.00558-09
PMCID: PMC2764146

Borrelia burgdorferi Resistance to a Major Skin Antimicrobial Peptide Is Independent of Outer Surface Lipoprotein Content [down-pointing small open triangle]


We hypothesize a potential role for Borrelia burgdorferi OspC in innate immune evasion at the initial stage of mammalian infection. We demonstrate that B. burgdorferi is resistant to high levels (>200 μg/ml) of cathelicidin and that this antimicrobial peptide exhibits limited binding to the spirochetal outer membrane, irrespective of OspC or other abundant surface lipoproteins. We conclude that the essential role of OspC is unrelated to resistance to this component of innate immunity.

Borrelia burgdorferi, a spirochete and the causal organism of Lyme disease, is naturally transmitted to mammals through the bite of infected Ixodes ticks (5, 8). A significant change in B. burgdorferi gene expression accompanies transmission between these diverse environments. This was first described for the inverse relationship between two abundant outer surface proteins of B. burgdorferi, in which synthesis of OspA declines and that of OspC increases during tick feeding (41). We and others have demonstrated the essential nature of OspC for colonization of the murine host (23, 35, 42, 45, 47, 49). These findings suggest a critical role for OspC in evasion of host innate immunity immediately after transmission (47). However, the essential contribution of OspC to early mammalian infection by B. burgdorferi remains undefined.

Microorganisms induce a variety of responses from the skin epithelial cells of their hosts, including the production of antimicrobial peptides, which are recognized as integral components of the innate immune system (20, 22). Defensins and cathelicidins comprise two major families of cationic antimicrobial peptides secreted by human and other mammalian skin neutrophils (20). Mouse neutrophils lack α defensins (14, 24), but about 30 cathelicidin members have been identified in various mammalian species, including mice (21, 50). These small, cationic, amphipathic molecules are primarily stored as inactive propeptides in the secretory granules of skin neutrophils. The mature bioactive peptides assume an α-helical structure in solution and preferentially interact with negatively charged cell surface components of a broad spectrum of bacteria and fungi, in which they disrupt cell membrane integrity (6, 9, 12, 20, 34). The importance of the sole murine cathelicidin, known as mCRAMP (mouse cathelin-related antimicrobial peptide) (19, 36), to innate host defense is well established, and mCRAMP has been shown to provide protection against bacterial skin infections in mice (33).

Resistance of B. burgdorferi to cathelicidin.

Treatment of Lyme borreliosis with antibiotics is generally successful, but there are rare instances of resistance (26), and several B. burgdorferi genes have been identified with potential roles in resistance to antibiotics (7, 10, 18, 40). However, potential mechanisms employed by the spirochete to evade the innate host response are not well understood yet. It has been demonstrated that unlike many other bacterial pathogens, B. burgdorferi is highly resistant to cathelicidin-derived peptides (27, 39), consistent with the spirochete's ability to persistently colonize the skin, where CRAMP is present. Sambri et al. (39) suggest that the resistance of B. burgdorferi to antimicrobial peptides may derive from the spirochete's lack of lipopolysaccharide, a negatively charged membrane component to which cationic peptides typically bind (25, 43). However, the B. burgdorferi outer membrane contains abundant lipoproteins with exposed charged residues that could mediate or repel cathelicidin binding, such as OspC (13, 30, 31), which is made by B. burgdorferi during the initial phase of mammalian infection, when the spirochete would encounter antimicrobial peptides in the skin. Although OspC is a basic protein with an isoelectric point of ~9.0 and a net positive charge, the three-dimensional structure of OspC indicates the presence of a surface region with a strong negative electrostatic potential that would project away from the positively charged, membrane-proximal region (13, 30). This negatively charged, exposed surface of OspC is postulated to be important for binding to unidentified positively charged host molecules or ligands (13, 30). We hypothesize that as an abundant surface lipoprotein with limited membrane contact, OspC could shield the spirochete from lytic components of innate defense like cathelicidin by binding and sequestering them, thus preventing access to the cell membrane. This potential role of OspC in resistance is consistent with the rapid clearance from skin of mutant spirochetes that lack OspC (45).

To test this hypothesis, we compared the resistance of B. burgdorferi variants that differ in outer surface lipoprotein composition to mouse cathelicidin-related antimicrobial peptide (mCRAMP). The bacterial strains and plasmids used in this study are described in Table Table1,1, and the relative amounts of OspC produced by the B. burgdorferi study strains are shown in Fig. 1A and C. We initially compared the survival, following incubation with mCRAMP, of three B. burgdorferi clones synthesizing or lacking OspC (A3, the ospCK1 strain, and the ospCK1+ospC strain). Briefly, mid-log phase B. burgdorferi cultures were washed and resuspended in 10 mM sodium phosphate buffer (pH 7.4) at a concentration of ~107 organisms/ml, and 10 μl of bacterial culture was added to duplicate wells of a 96-well polypropylene plate (Sigma-Aldrich, St. Louis, MO) containing mCRAMP (Axxora, San Diego, CA) (1 mg/ml in 0.01% acetic acid containing 0.2% bovine serum albumin) at various concentrations using Mueller-Hinton broth as the assay medium, in a total volume of 100 μl. All three strains were found to be highly resistant to killing at a wide range of antimicrobial peptide concentrations, irrespective of their OspC phenotype (Fig. 2A and B). This experiment was conducted at even higher concentrations of mCRAMP (300 to 500 μg/ml), but no killing was observed for any of these strains (data not shown). However, the cathelicidin-susceptible species Escherichia coli and Leptospira biflexa were found to be highly sensitive to mCRAMP when assayed under identical experimental conditions (Fig. 2C and D). A similar killing assay conducted with polymyxin B, another standard cationic antimicrobial agent, demonstrated resistance of B. burgdorferi to this compound as well (Fig. (Fig.2E).2E). These results indicate that the resistance of B. burgdorferi to these antimicrobial peptides is not OspC dependent.

FIG. 1.
Outer surface protein profiles of B. burgdorferi clones. (A) Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell lysates of B31 clones: A3, the ospCK1 strain, the ospCK1+ospC strain, AC2, B312, ...
FIG. 2.
Role of OspC in cathelicidin resistance of B. burgdorferi. The wild-type strain A3, the ospCK1 mutant, and the constitutively OspC-expressing ospCK1+ospC strain were incubated with various concentrations of cathelicidin (A and B) and polymyxin ...
Bacterial strains and plasmids used in this study

Surface lipoprotein contribution to cathelicidin resistance.

The in vitro-grown spirochetes tested in this initial assay continuously synthesized OspA, while OspC synthesis varied (Fig. (Fig.1).1). It was observed recently that when synthesis of OspA is manipulated to remain artificially high following inoculation into a mouse, OspA can restore mouse infectivity to an ospC mutant, albeit inefficiently (48). To determine whether the presence of OspA contributed to the observed resistance to cathelicidin and masked an otherwise critical role of OspC, we constructed and tested a mutant strain that lacks genes for ospAB and ospC and hence does not produce any of these lipoproteins (AC2) (Table (Table11 and Fig. Fig.1).1). However, this strain still maintained a high level of resistance to cathelicidin (Fig. (Fig.3).3). We also found that despite lacking many lipoproteins, two highly attenuated B. burgdorferi clones, B312 and B314, were highly resistant to cathelicidin-mediated lysis (Fig. (Fig.3).3). We therefore conclude that physical properties of the outer membrane of B. burgdorferi, not the presence of specific lipoproteins, likely confer the spirochete's high level of resistance to these cationic antimicrobial peptides.

FIG. 3.
Outer surface lipoprotein content of B. burgdorferi and cathelicidin resistance. B312, B314, and the double mutant AC2 (lacking OspAB and OspC) were incubated with the indicated concentrations of mouse cathelicidin for 3 h. Bacterial survival was determined ...

Cathelicidin binding.

The mechanism by which B. burgdorferi maintains resistance to cathelicidin and other antimicrobial peptides is largely unknown. To determine whether sensitivity to cathelicidin reflects peptide binding to the outer surface of the spirochete, we employed an indirect immunofluorescence assay to assess mCRAMP binding to B. burgdorferi and L. biflexa (which was used as a positive control). We observed limited mCRAMP binding to B. burgdorferi, in contrast to extensive mCRAMP binding to L. biflexa (Fig. (Fig.4).4). The highly attenuated clones B312 and B314, varying in OspAB and OspC synthesis, as well as strain AC2, lacking OspAB and OspC, similarly demonstrated very low levels of cathelicidin binding, consistent with their resistant phenotypes (Fig. (Fig.4C4C).

FIG. 4.
Relative CRAMP binding of B. burgdorferi and L. biflexa. B. burgdorferi (A) and L. biflexa (B) were fixed with 3% paraformaldehyde at 37°C for 10 min, washed, resuspended in phosphate-buffered saline, and incubated with CRAMP (50 μg/ml) ...

Our data are in agreement with the previous observation that B. burgdorferi is highly resistant to the innate skin antimicrobial peptide cathelicidin (39), and the data presented here indicate that the critical early role of OspC during mammalian infection is unrelated to resistance to major antimicrobial peptides in the skin; hence, the role of OspC remains elusive. Our results support a model by which the observed resistance of B. burgdorferi to cathelicidin correlates with a lack of antimicrobial peptide binding to the spirochete membrane, and we suggest that this characteristic is consistent with the unique biophysical properties of the Borrelia outer membrane, independent of lipoprotein content.


We thank David A. Haake and Marija Pinne, School of Medicine, University of California, Los Angeles, CA, for providing the Leptospira strain; Tom Schwan, Rocky Mountain Laboratories, Hamilton, MT, for providing monoclonal antibodies; Jonathan Warawa, Kevin Rigby, and members of the Rosa laboratory for critical reading of the manuscript; and Gary Hettrick for assistance with illustrations.

This research work was supported by the Intramural Research Program of the NIAID, NIH.


[down-pointing small open triangle]Published ahead of print on 3 August 2009.


1. Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521-525. [PMC free article] [PubMed]
2. Barbour, A. G., S. F. Hayes, R. A. Heiland, M. E. Schrumpf, and S. L. Tessier. 1986. A Borrelia-specific monoclonal antibody binds to a flagellar epitope. Infect. Immun. 52:549-554. [PMC free article] [PubMed]
3. Barbour, A. G., S. L. Tessier, and W. J. Todd. 1983. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41:795-804. [PMC free article] [PubMed]
4. Battisti, J. M., J. L. Bono, P. A. Rosa, M. E. Schrumpf, T. G. Schwan, and P. F. Policastro. 2008. Outer surface protein A protects Lyme disease spirochetes from acquired host immunity in the tick vector. Infect. Immun. 76:5228-5237. [PMC free article] [PubMed]
5. Bosler, E. M., J. L. Coleman, J. L. Benach, D. A. Massey, J. P. Hanrahan, W. Burgdorfer, and A. G. Barbour. 1983. Natural distribution of the Ixodes dammini spirochete. Science 220:321-322. [PubMed]
6. Braff, M. H., M. Zaiou, J. Fierer, V. Nizet, and R. L. Gallo. 2005. Keratinocyte production of cathelicidin provides direct activity against bacterial skin pathogens. Infect. Immun. 73:6771-6781. [PMC free article] [PubMed]
7. Bunikis, I., K. Denker, Y. Ostberg, C. Andersen, R. Benz, and S. Bergstrom. 2008. An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds. PLoS Pathog. 4:e1000009. [PMC free article] [PubMed]
8. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis. 1982. Lyme disease—a tick-borne spirochetosis? Science 216:1317-1319. [PubMed]
9. Chromek, M., Z. Slamova, P. Bergman, L. Kovacs, L. Podracka, I. Ehren, T. Hokfelt, G. H. Gudmundsson, R. L. Gallo, B. Agerberth, and A. Brauner. 2006. The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection. Nat. Med. 12:636-641. [PubMed]
10. Criswell, D., V. L. Tobiason, J. S. Lodmell, and D. S. Samuels. 2006. Mutations conferring aminoglycoside and spectinomycin resistance in Borrelia burgdorferi. Antimicrob. Agents Chemother. 50:445-452. [PMC free article] [PubMed]
11. Cullen, P. A., D. A. Haake, D. M. Bulach, R. L. Zuerner, and B. Adler. 2003. LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species. Infect. Immun. 71:2414-2421. [PMC free article] [PubMed]
12. De Smet, K., and R. Contreras. 2005. Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol. Lett. 27:1337-1347. [PubMed]
13. Eicken, C., V. Sharma, T. Klabunde, R. T. Owens, D. S. Pikas, M. Höök, and J. C. Sacchettini. 2001. Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi. J. Biol. Chem. 276:10010-10015. [PubMed]
14. Eisenhauer, P. B., and R. I. Lehrer. 1992. Mouse neutrophils lack defensins. Infect. Immun. 60:3446-3447. [PMC free article] [PubMed]
15. Elias, A. F., P. E. Stewart, D. Grimm, M. J. Caimano, C. H. Eggers, K. Tilly, J. L. Bono, D. R. Akins, J. D. Radolf, T. G. Schwan, and P. Rosa. 2002. Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background. Infect. Immun. 70:2139-2150. [PMC free article] [PubMed]
16. Ellinghausen, H. C., Jr., and W. G. McCullough. 1965. Nutrition of Leptospira pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am. J. Vet. Res. 26:45-51. [PubMed]
17. Frank, K. L., S. F. Bundle, M. E. Kresge, C. H. Eggers, and D. S. Samuels. 2003. aadA confers streptomycin resistance in Borrelia burgdorferi. J. Bacteriol. 185:6723-6727. [PMC free article] [PubMed]
18. Galbraith, K. M., A. C. Ng, B. J. Eggers, C. R. Kuchel, C. H. Eggers, and D. S. Samuels. 2005. parC mutations in fluoroquinolone-resistant Borrelia burgdorferi. Antimicrob. Agents Chemother. 49:4354-4357. [PMC free article] [PubMed]
19. Gallo, R. L., K. J. Kim, M. Bernfield, C. A. Kozak, M. Zanetti, L. Merluzzi, and R. Gennaro. 1997. Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse. J. Biol. Chem. 272:13088-13093. [PubMed]
20. Ganz, T., and R. I. Lehrer. 2003. Antimicrobial peptides, p. 287-296. In R. A. B. Ezekowitz and J. A. Hoffmann (ed.), Infectious disease: innate immunity. Humana Press Inc., Totowa, NJ.
21. Ganz, T., and R. I. Lehrer. 1998. Antimicrobial peptides of vertebrates. Curr. Opin. Immunol. 10:41-44. [PubMed]
22. Ganz, T., M. E. Selsted, D. Szklarek, S. S. Harwig, K. Daher, D. F. Bainton, and R. I. Lehrer. 1985. Defensins. Natural peptide antibiotics of human neutrophils. J. Clin. Investig. 76:1427-1435. [PMC free article] [PubMed]
23. Grimm, D., K. Tilly, R. Byram, P. E. Stewart, J. G. Krum, D. M. Bueschel, T. G. Schwan, P. F. Policastro, A. F. Elias, and P. A. Rosa. 2004. Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals. Proc. Natl. Acad. Sci. USA 101:3142-3147. [PubMed]
24. Hancock, R. E., and H. G. Sahl. 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol. 24:1551-1557. [PubMed]
25. Hardy, P. H., Jr., and J. Levin. 1983. Lack of endotoxin in Borrelia hispanica and Treponema pallidum. Proc. Soc. Exp. Biol. Med. 174:47-52. [PubMed]
26. Hunfeld, K. P., and V. Brade. 2006. Antimicrobial susceptibility of Borrelia burgdorferi sensu lato: what we know, what we don't know, and what we need to know. Wien Klin. Wochenschr. 118:659-668. [PubMed]
27. Isogai, E., H. Isogai, K. Takahashi, M. Kobayashi-Sakamoto, and K. Okumura. 20 February 2009, posting date. Antimicrobial activity of three tick defensins and four mammalian cathelicidin-derived synthetic peptides against Lyme disease spirochetes and bacteria isolated from the midgut. Exp. Appl. Acarol., in press (doi ).10.1007/s10493-009-9251-5 [PubMed] [Cross Ref]
28. Johnson, R. C., and V. G. Harris. 1967. Differentiation of pathogenic and saprophytic letospires. I. Growth at low temperatures. J. Bacteriol. 94:27-31. [PMC free article] [PubMed]
29. Kelly, R. 1971. Cultivation of Borrelia hermsii. Science 173:443-444. [PubMed]
30. Kumaran, D., S. Eswaramoorthy, B. J. Luft, S. Koide, J. J. Dunn, C. L. Lawson, and S. Swaminathan. 2001. Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi. EMBO J. 20:971-978. [PubMed]
31. Li, H., J. J. Dunn, B. J. Luft, and C. L. Lawson. 1997. Crystal structure of Lyme disease antigen outer surface protein A complexed with an Fab. Proc. Natl. Acad. Sci. USA 94:3584-3589. [PubMed]
32. Louvel, H., and M. Picardeau. 2007. Genetic manipulation of Leptospira biflexa (Chapter 12, Unit 12E 4), p. 4.1-4.12. In R. Coico, T. Kowalik, J. Quarles, B. Stevenson, and R. Taylor (ed.), Current protocols in microbiology. Wiley Interscience, New York, NY.
33. Nizet, V., T. Ohtake, X. Lauth, J. Trowbridge, J. Rudisill, R. A. Dorschner, V. Pestonjamasp, J. Piraino, K. Huttner, and R. L. Gallo. 2001. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414:454-457. [PubMed]
34. Oren, Z., J. C. Lerman, G. H. Gudmundsson, B. Agerberth, and Y. Shai. 1999. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem. J. 341(Pt. 3):501-513. [PubMed]
35. Pal, U., X. Yang, M. Chen, L. K. Bockenstedt, J. F. Anderson, R. A. Flavell, M. V. Norgard, and E. Fikrig. 2004. OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J. Clin. Investig. 113:220-230. [PMC free article] [PubMed]
36. Pestonjamasp, V. K., K. H. Huttner, and R. L. Gallo. 2001. Processing site and gene structure for the murine antimicrobial peptide CRAMP. Peptides 22:1643-1650. [PubMed]
37. Rosa, P., D. S. Samuels, D. Hogan, B. Stevenson, S. Casjens, and K. Tilly. 1996. Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi. J. Bacteriol. 178:5946-5953. [PMC free article] [PubMed]
38. Sadziene, A., B. Wilske, M. S. Ferdows, and A. G. Barbour. 1993. The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid. Infect. Immun. 61:2192-2195. [PMC free article] [PubMed]
39. Sambri, V., A. Marangoni, L. Giacani, R. Gennaro, R. Murgia, R. Cevenini, and M. Cinco. 2002. Comparative in vitro activity of five cathelicidin-derived synthetic peptides against Leptospira, Borrelia and Treponema pallidum. J. Antimicrob. Chemother. 50:895-902. [PubMed]
40. Samuels, D. S., R. T. Marconi, W. M. Huang, and C. F. Garon. 1994. gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi. J. Bacteriol. 176:3072-3075. [PMC free article] [PubMed]
41. Schwan, T. G., J. Piesman, W. T. Golde, M. C. Dolan, and P. A. Rosa. 1995. Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc. Natl. Acad. Sci. USA 92:2909-2913. [PubMed]
42. Stewart, P. E., X. Wang, D. M. Bueschel, D. R. Clifton, D. Grimm, K. Tilly, J. A. Carroll, J. J. Weis, and P. A. Rosa. 2006. Delineating the requirement for the Borrelia burgdorferi virulence factor OspC in the mammalian host. Infect. Immun. 74:3547-3553. [PMC free article] [PubMed]
43. Takayama, K., R. J. Rothenberg, and A. G. Barbour. 1987. Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi. Infect. Immun. 55:2311-2313. [PMC free article] [PubMed]
44. Tilly, K., A. Bestor, D. P. Dulebohn, and P. A. Rosa. 2009. OspC-independent infection and dissemination by host-adapted Borrelia burgdorferi. Infect. Immun. 77:2672-2682. [PMC free article] [PubMed]
45. Tilly, K., A. Bestor, M. W. Jewett, and P. Rosa. 2007. Rapid clearance of Lyme disease spirochetes lacking OspC from skin. Infect. Immun. 75:1517-1519. [PMC free article] [PubMed]
46. Tilly, K., S. Casjens, B. Stevenson, J. L. Bono, D. S. Samuels, D. Hogan, and P. Rosa. 1997. The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene. Mol. Microbiol. 25:361-373. [PubMed]
47. Tilly, K., J. G. Krum, A. Bestor, M. W. Jewett, D. Grimm, D. Bueschel, R. Byram, D. Dorward, P. Stewart, and P. Rosa. 2006. Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect. Immun. 74:3554-3564. [PMC free article] [PubMed]
48. Xu, Q., K. McShan, and F. T. Liang. 2008. Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defenses. Mol. Microbiol. 69:15-29. [PMC free article] [PubMed]
49. Xu, Q., K. McShan, and F. T. Liang. 2007. Identification of an ospC operator critical for immune evasion of Borrelia burgdorferi. Mol. Microbiol. 64:220-231. [PubMed]
50. Zanetti, M., R. Gennaro, and D. Romeo. 1995. Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain. FEBS Lett. 374:1-5. [PubMed]

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