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An epitope shared by cellular cytokeratin and Orientia tsutsugamushi
ARTICLE IN PRESS
MICROBIAL PATHOGENESIS Microbial Pathogenesis 41 (2006) 125–132 www.elsevier.com/locate/micpath
An epitope shared by cellular cytokeratin and Orientia tsutsugamushi Mi-Jeong Kim, Mee-Kyung Kim, Jae-Seung Kang Department of Microbiology and Research Institute for Medical Science, Inha University College of Medicine, Jungsuk B/D, 3rd Street, Shinheung-Dong, Choong-Gu, Incheon 400-712, Republic of Korea Received 14 February 2006; accepted 6 April 2006 Available online 9 August 2006
Abstract Many microbial pathogens have epitopes shared with host cell components and these epitopes may induce transient or longer-term tissue-damaging autoantibody responses. We observed that several mouse monoclonal antibodies (MAbs) raised against Orientia tsutsugamushi were also reactive with host cells. One such antibody, MAb Rb105, cross-reacted with the cytoskeleton, as shown by immunofluorescent staining. Biochemical studies identified the cross-reacting component as a cytokeratin protein. These results identify an epitope shared by O. tsutsugamushi and the cytokeratins of host cells. In addition, antibodies cross-reactive with the cytoskeleton were detected in the sera of scrub typhus patients, suggesting that an epitope similar to that detected by MAb Rb105 may be recognized by human antibodies. r 2006 Elsevier Ltd. All rights reserved. Keywords: Orientia tsutsugamushi; Epitope; Monoclonal antibody; Cytokeratin
1. Introduction Orientia tsutsugamushi (OT), a member of the Rickettsiaceae family, is the causative agent of scrub typhus. This disease is an acute febrile illness characterized by fever, rash, and eschar. The disease is endemic in many countries in the Asia-Pacific region, including Korea [1]. The mechanism of pathogenesis has not been extensively studied, but is believed to be similar to that shown by other Rickettsia. Pathogenesis includes endothelial damage and subsequent vasculitis [2]. Many microbial pathogens have epitopes shared with host cell components, and antibody responses to such epitopes may cause transient or long-term damage if the antibodies react with the cytoskeleton, the mitochondrion, the nucleus, lipid, or other components of the host cell [3]. Such autoantibodies are mostly subject to transient synthesis, but some are produced for a protracted time. The presence of epitopes shared by rickettsial organisms and humans, and the induction of autoantibodies, has been reported. Cytoplasmic, Corresponding author. Tel.: +82 32 890 0952; fax: +82 32 881 8559.
E-mail address: [email protected] (J.-S. Kang). 0882-4010/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2006.04.008
nuclear, and platelet autoantibodies are found in the sera of human granulocytic ehrlichiosis patients [4]. An antiphospholipid antibody appears in persons suffering from Mediterranean spotted fever [5] while patients with Q fever develop an anticardiolipin antibody [6]. However, such developments have not been reported following infection with OT. Mouse monoclonal antibodies (MAbs) have been used to measure the antigen specificity of cross-reacting antibodies produced after microbial infection. For example, strong immunofluorescence (IF) staining of heart tissue sections by anti-Streptococcus MAbs was reported, indicating the presence of an epitope shared by both heart tissue and Streptococcus [7]. During characterization of MAbs against OT we found that several MAbs were reactive against host cell components. Among these crossreacting MAbs, we characterized one MAb (Rb105) that reacts with both an epitope of OT and an epitope of the cytoskeleton. In addition, anticytoskeleton antibodies were detected in the sera of scrub typhus patients, suggesting that the OT epitope to which MAb Rb105 is directed may induce an antibody response during human infection.
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2. Results
2.2. Reaction of MAb Rb105 with host proteins
2.1. MAbs reacting with both OT and the cytoskeleton
To characterize the target antigen of MAb Rb105, we analyzed proteins of cells infected with OT by western blotting using MAb Rb105 as primary antibody. A 46 kDa band was detected by Rb105 and the expression level of this protein did not change over 11 days of infection. However, the expression of a 47 kDa OT protein detected by MAb M686-8 increased as infection proceeded (Fig. 3). Further, the 46 kDa band was detected in uninfected cells at the same level seen in infected cells. This suggested that MAb Rb105 detected only a host protein upon western blotting and that no bacterial proteins could be visualized, using western blotting, with MAb Rb105. To explain the fact that MAb Rb105 stained both bacteria and the cytoskeleton in the IF protocol but that no bacterial protein was detected upon western blotting, we hypothesized that a host cytoskeleton protein might bind to bacteria resulting in simultaneous staining of both bacteria and the cytoskeleton. To address this issue, we stained uninfected ECV304 cells with MAb Rb105 and
In several separate fusion experiments we have produced a panel of MAbs against OT and found that some MAbs were also reactive with uninfected cells. These crossreacting MAbs appeared to be directed against epitopes of the nucleus, the mitochondrion, the endoplasmic reticulum, and the cytoskeleton of the host cell. Control MAb FS15 did not stain any structures in uninfected cells (Fig. 1). Among the reactive MAbs, one (Rb105) detected epitopes of both OT and the cytoskeleton as judged by IF staining of acetone/methanol-fixed cells. As shown (Fig. 2), MAb Rb105 simultaneously stained both bacteria and cytoskeletal structures in a slide preparation. The morphology of the stained cytoskeleton suggested that the reactive epitope might be located on the intermediate filament. As the results suggested that OT and the cytoskeleton might share epitopes, we further explored the antigen specificity of MAb Rb105.
Fig. 1. Cross-reaction of anti-OT MAbs with host cells. Uninfected ECV304 cells were treated with anti-OT MAbs followed by IF staining. Inset: Each MAb reacted with OT in infected ECV304 cells.
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Fig. 4B). These results suggest that the cellular target antigen of MAb Rb105 does not bind bacteria. 2.3. Identification of the cellular antigen detected by MAb Rb105
Fig. 2. MAb Rb105 is reactive with both OT and the cytoskeleton. ECV304 cells infected with OT for 4 days were stained with MAb Rb105, which was reactive with both OT (arrow) and the cytoskeleton (arrowhead).
Fig. 3. Expression of the target antigen of MAb Rb105 during infection. ECV304 cells infected with OT for 11 days were solubilized. Total proteins were separated and analyzed by western blotting at the indicated times. MAb M686-8 was used to detect the 47 kDa OT protein. C, uninfected control cells; 11A, cells attached to the culture substrate at 11 days after infection; 11D, cells detached from the culture substrate at 11 days after infection.
anti-cytoskeleton antibodies. MAb Rb105 stained the filamentous structures of the uninfected ECV304 cells like other anti-cytoskeleton antibodies (Fig. 4A). Next, we stained the infected ECV304 cells with MAb Rb105 or anticytoskeleton antibodies and simultaneously stained OT with anti-OT MAb (FS15). As expected, both MAb Rb105 and the anticytoskeleton antibodies stained filamentous structures of the infected ECV304 cells (left column, Fig. 4B). However, anticytoskeleton antibodies alone did not stain any bacteria, while MAb Rb105 alone stained bacteria both inside and outside the cells left column. Further, images of bacteria stained with anti-OT MAb (middle column, Fig. 4B) did not merge with images of the cytoskeleton stained with anticytoskeleton antibodies (right column, Fig. 4B). This was in contrast to the merging of images seen when an image of infected cells stained with MAb Rb105 was merged with an image of infected cells stained with anti-OT MAb (lower row,
To identify the host target protein recognized by MAb Rb105 we performed IF staining and immunoprecipitation analysis. First, we stained uninfected ECV304 cells with both MAb Rb105 and antiactin/tubulin antibodies simultaneously. As shown (Fig. 5A), images stained with MAb Rb105 appeared distinct from those obtained with antiactin antibody and the images did not merge, indicating clearly that the MAb Rb105 target antigen is not actin. When we stained uninfected ECV304 cells with MAb Rb105 and antitubulin antibody, the images obtained with the two antibodies appeared very similar (upper row, Fig. 5B). As intermediate filament fibers are associated closely with microtubules, we treated cells with 33 mM nocodazole to selectively disrupt microtubules. As shown (lower row, Fig. 5B), images of nocodazole-treated cells stained with MAb Rb105 were distinct from images obtained using antitubulin antibody, indicating that the MAb Rb105 target antigen is not tubulin. We therefore reasoned that the MAb Rb105 target protein might be one of the intermediate filament proteins. Next, we performed immunoprecipitation analysis with various anticytoskeleton antibodies to confirm that the target antigen was an intermediate filament protein. MAb Rb105 did not immunoprecipitate a target protein from uninfected ECV304 cells, but was active in western blotting of proteins from uninfected cells, as shown (seventh lane, Fig. 6A). Nevertheless, MAb Rb105 reacted strongly with protein immunoprecipitated by pan-cytokeratin antibody (fourth lane, Fig. 6A) and was weakly reactive with protein precipitated with antibodies anticytokeratin 8 and anticytokeratin 18. Proteins immunoprecipitated by the other anticytokeratin antibodies, anticytokeratin 14 and anticytokeratin 19, were not recognized by MAb Rb105 (data not shown). Similar results were obtained by immunoprecipitation analyses of infected cells (Fig. 6B). We conclude that the MAb Rb105 target antigen is a cytokeratin protein. 2.4. Detection of anticytoskeleton antibody in the sera of scrub typhus patients To examine whether anticytoskeleton antibodies develop during human OT infections we tested sera from 41 scrub typhus patients for the presence of anticytoskeleton antibody. Uninfected and infected ECV304 cells were subjected to IF staining using 1:80 dilutions of sera. We confirmed that all patients’ sera reacted with OT in infected ECV304 cells (data not shown). As shown (Fig. 7), eight sera contained anticytoskeleton antibodies staining filamentous structures of uninfected cells. None of 30 sera from healthy
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Rb105
CK Pan
Vimentin
α-Tubulin
(A) Anti-cytoskeleton
Anti-OT
Merge
Rb105
CK Pan
Vimentin
α-Tubulin
(B) Fig. 4. IF staining of cells using anticytoskeleton antibodies: (A) Uninfected ECV304 cells were stained with MAb Rb105, anti-pan-cytokeratin antibody (CK Pan), anti-vimentin antibody, or anti-a-tubulin antibody. (B) ECV304 cells infected with OT were stained simultaneously with anticytoskeleton antibodies (left column, red) and MAb FS15 for the detection of the 56 kDa OT protein (middle column, green spots).
humans contained such antibodies. One patient had nuclear antibody (data not shown). 3. Discussion This work suggests that cellular cytokeratin and OT share an epitope. This epitope elicits antibody responses when OT infects mice. We first noticed the OT-mediated
induction of various cross-reacting antibodies during characterization of several MAbs reactive with some components of uninfected cells. Such cross-reacting MAbs may be directed against the nucleus, the mitochondrion, the endoplasmic reticulum or the cytoskeleton of the host cell, in addition to detecting bacterial epitopes. It seems likely, therefore, that many antigenic epitopes are shared between OT and human cells. We chose one MAb (Rb105)
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Rb105
Actin
Merge
Heochst
α-Tubulin
Rb105
Merge
Con
Heochst
Noco
Con
(A)
(B) Fig. 5. Comparison of images obtained with MAb Rb105 and antiactin or antitubulin antibodies: (A) Uninfected ECV304 cells were stained simultaneously with MAb Rb105 (green), antiactin (red), and Heochst (blue). (B) Uninfected ECV304 cells were stained simultaneously with antitubulin (green), MAb Rb105 (red), and Heochst (blue). Uninfected ECV304 Cells were treated with nocodazole (33 mM) and stained with the same antibodies (lower row).
that reacts with both OT and the cytoskeleton on IF staining for further characterization. Although MAb Rb105 stained both bacteria and the cytoskeleton, the MAb detected only a single protein band in western blotting and the expression level of this protein did not change during the course of an 11 days infection. This result was puzzling at first as we expected two protein bands, one from bacteria and the other from host cells. However, the bacterial antigen was not detected in western blotting. Two explanations are possible. First, the Rb105reactive antigen from the host cell might coat intracellular bacteria. The coating of intracellular bacteria with host protein has been demonstrated in other systems [8,9]. Second, OT and cytokeratin may indeed share an epitope, but the bacterial epitope may be heat-sensitive and thus undetectable on western blotting. To address this issue we first stained both uninfected and infected cells with an anticytokeratin antibody. The results showed that neither the anticytokeratin antibody nor control antibodies stained bacterial particles in infected cells. Thus, we could conclude that cytokeratin did not coat OT. Instead, it seemed likely that OT and cytokeratin did indeed share an epitope. The identity of the cell antigen targeted by MAb Rb105 was examined by IF staining and immunoprecipitation. IF staining using antiactin and antitubulin antibodies clearly indicated that the target antigen was neither actin nor
tubulin. When the MAb Rb105 antigen was characterized by immunoprecipitation it seemed clear that MAb Rb105 was reactive against cytokeratins of the host cell. However, it has yet to be explained why the MAb Rb105-reactive epitope of OT could not be detected in western blotting, in contrast to the facile detection of the cytokeratin epitope. One explanation is that a nonprotein antigen of OT was detectable in IF staining but was not detectable by western blotting. Thus, it is possible that MAb Rb105 is reactive against bacterial carbohydrate and carbohydrate-mimetic peptides [10] of cytokeratin. Another possibility is that MAb Rb105 detects carbohydrate moieties of both OT and cytokeratin. This is possible as cytokeratins are glycosylated at multiple sites with single O-linked N-acetylglucosamine residues [11]. Epitopes shared by microbial pathogens and host cells may activate autoreactive T cells and B cells during infection [12]. The most probable mechanism by which infection of mice with OT induces the synthesis of selfreactive antibody may be molecular mimicry. This is believed to involve the triggering, by many pathogens, of immune responses against host tissues. The pathogens present epitopes that are identical or nearly so to host components. Self-tolerance is broken, resulting in the appearance of autoantibodies. The best known example of this phenomenon is the appearance of autoantibodies
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Rickettsia conorii infection, may cause membrane damage in endothelial cells [5]. However, in OT infection, further studies are required to assess the role of antibodies to the shared epitope observed in this study. In our tests, eight of 41 patients (19.5%) had antibodies reactive with the cytoskeleton of ECV304 cells. This suggests that autoantibodies against the cytoskeleton may be induced, at least transiently, by scrub typhus infection. These antibodies will cause a strong nonspecific fluorescence that interferes with reading the result of serologic test with IFA staining, as pointed in a previous study [4]. In addition, it may be worth exploring the possible pathogenic role of anticytoskeleton antibodies during human OT infections. 4. Materials and methods 4.1. Cell culture and OT
Fig. 6. Reaction of MAb Rb105 with cytokeratin. Uninfected (A) and infected (B) cells were lysed and immunoprecipitated with normal mouse serum (N), antivimentin antibody (Vim), antitubulin antibody (a-tub), anti-pan-cytokeratin antibody (CK Pan), anticytokeratin 18 antibody (CK 18), anticytokeratin 8 antibody (CK 8), or MAb Rb105. The precipitated proteins were analyzed with MAb Rb105 or anti-pan-cytokeratin as primary antibodies.
Cells of the human umbilical vein-derived endothelial cell line ECV304 were maintained with medium 199 (Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco BRL) in a humidified atmosphere containing 5% (v/v) CO2 at 37 1C. The Boryong serotype of OT (strain MF) was cultivated in ECV304 cells as described previously [17]. When infected ECV304 cells showed maximum cytopathic effects the cells were disrupted with glass beads (diameter 1.0 mm) and centrifuged at 300g for 5 min. The resulting supernatant was immediately used to infect further ECV304 cells. 4.2. Antibodies
cross-reactive with streptococcal antigens after rheumatic fever [7]. These autoantibodies are reactive against various cytoskeletal proteins including actin, vimentin, and cytokeratin. Interestingly, autoantibodies triggered by microbial molecules are not necessarily reactive against host components with similar chemical structures. Multireactive antibodies reactive against host components of dissimilar chemical natures are also produced. For example, antiN-acetyl-b-D-glucosamine antibodies against Streptococcus also react also with cytoskeletal proteins of the animal cell [13]. Generally, autoantibodies have been considered to be of little importance in pathogenesis. They occur at low levels in the sera of normal individuals [14]. However, in some infectious diseases, cross-reactions between human antigens and microbial antigens have been implicated in the development of autoimmune disease. Infection with rickettsial organisms induces various autoantibodies. The contribution of these antibodies to rickettsial pathogenesis remains largely unknown, but some experimental data suggest they may indeed contribute to ill effects in humans. Serum platelet-binding antiplatelet antibodies, appearing in dogs after experimental infection with Ehrlichia canis, may contribute to the immune destruction of platelets [15,16]. Antiphospholipid antibodies, appearing after
The production of MAb FS15 was described previously [18] and other MAbs (M37-2, M716G, Rb105, M686-20, and M716B) were produced using the same method. The antibody MAb FS15 is reactive against a 56 kDa protein of OT [18]. MAbs against pan-cytokeratin, cytokeratin 18, cytokeratin 8, vimentin, and a-tubulin were purchased from Sigma–Aldrich (St. Louis, MO). 4.3. Patient sera Sera from 41 scrub typhus patients were obtained for this study. Diagnosis was performed using an OT-specific IF test or by isolation of OT. Sera from 30 healthy adults were used as control sera. All serum samples were dispensed into aliquots and stored at 20 1C until required. 4.4. IF staining ECV304 cells grown in eight-well chamber slides were infected with OT. After infection, cells were washed with PBS, permeabilized with 0.25% (w/v) Triton X-100 (Sigma–Aldrich) and 0.4% (v/v) paraformaldehyde for 2 min, and then fixed using 4% (v/v) paraformaldehyde at 4 1C for 30 min. After several washes with PBS the cells
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Fig. 7. Anticytoskeleton antibody in scrub typhus patients. Some 41 human sera from scrub typhus patients were examined for the presence of anticytoskeleton antibodies. Uninfected and infected ECV304 cells were stained with sera at a dilution of 1:80. The stained images of uninfected and infected ECV304 cells were presented for representative three sera (patients 1–3), and the stained images of uninfected ECV304 cells were presented for other sera. Arrowheads indicate filamentous cell structures stained by patients’ sera.
were incubated with primary antibodies or human sera for 1 h. After several further washes with PBS containing 0.1% (w/v) Tween-20 cells were incubated with rhodamineconjugated antimouse IgG antibody (Jackson Laboratories, Bar Harbor, MA) or fluorescein-isothiocyanate conjugated antihuman IgG antibody (Jackson Laboratories) in the dark for 1 h. For double staining of OT, the cells were further fixed with 4% (v/v) paraformaldehyde followed by treatment with MAb FS15 conjugated with fluorescein-isothiocyanate. To stain nuclei, cells were incubated with Heochst 33258 (0.05 mg/mL, Sigma–Aldrich) in PBS for 10 min at room temperature in the dark. Cover glasses were mounted on glass slides with Antifade (Vector Laboratories, Burlingame, CA) and viewed with a confocal microscope (Bio-Rad, Hercules, CA). 4.5. Western blotting Cells were resuspended in lysis buffer [1% (w/v) SDS,5% (v/v) glycerol,0.01% (w/v) bromophenol blue, and 0.031 M Tris–HCl, pH 7.0] and incubated at room temperature for 1 h. b-Mercaptoethanol was added to 5% (v/v), the solutions heated at 100 1C for 5 min, and solubilized proteins electrophoresed through 11% (w/v) SDS-polyacrylamide (SDS-PAGE) gels. The separated proteins were subsequently transferred electrophoretically to polyvinylidene difluoride membrane (Immobilon-P, Millipore Co.,
Bedford, MA). Membranes were blocked with a suspension of 5% (w/v) nonfat dry milk powder in water and then incubated with primary antibodies for 1 h. After membranes were thrice washed with TBS with 0.1% (v/v) Tween-20 (TBST) they were incubated with horseradish peroxidase-conjugated goat antimouse IgG for 1 h at room temperature and developed using a protocol involving an enhancement of chemiluminescence (Amersham, UK).
4.6. Immunoprecipitation Cells were resuspended in RIPA buffer [50 mM Tris pH 7.4,1% (w/v) Empigen,0.5% (v/v) sodium deoxycholate, 150 mM NaCl, 5 mM EDTA, 0.1% (w/v) SDS,1 mM phenylmethylsulfonyl fluoride,1 mM leupeptin,1 mM aprotinin,1 mM NaF, 1 mM Na3VO4, and 1 mM iodoacetamide] and proteins were immunoprecipitated by overnight incubation at 4 1C with primary antibodies and protein G/A-Agarose beads (Calbiochem, La Jolla, CA). Beads were pelleted by centrifugation at 10,000g for 1 min and washed three times. The immunoprecipitated proteins were eluted with sample buffer [1% (w/v) SDS, 5% (v/v) glycerol, 0.01% (w/v) bromophenol blue, 0.031 M Tris–HCl (pH 7.0), and 5% (v/v) b-mercaptoethanol] by boiling for 5 min and the proteins were then separated on an SDS-PAGE gel with acrylamide concentration of 7.5% (w/v).
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Acknowledgment This work was supported by Korea Research Foundation Grant KRF-2003-015-E00097.
References [1] Seong SY, Choi MS, Kim IS. Orientia tsutsugamushi infection: overview and immune responses. Microbes Infect 2001;3:11–21. [2] Moron CG, Popov VL, Feng HM, Wear D, Walker DH. Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus. Mod Pathol 2001;14:752–9. [3] Oldstone MB. Molecular mimicry and immune-mediated diseases. FASEB J 1998;12:1255–65. [4] Wong SJ, Thomas JA. Cytoplasmic, nuclear, and platelet autoantibodies in human granulocytic ehrlichiosis patients. J Clin Microbiol 1998;36:1959–63. [5] Sotto A, Berard M, Bessis D, Porneuf M, Jourdan J, Boffa MC. Antiphospholipid antibody production during Mediterranean spotted fever. Autoimmunity 1995;21:123–6. [6] Ordi-Ros J, Selva-O’Callaghan A, Monegal-Ferran F, MonasterioAspiri Y, Juste-Sanchez C, Vilardell-Tarres M. Prevalence, significance, and specificity of antibodies to phospholipids in Q fever. Clin Infect Dis 1994;18:213–8. [7] Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470–511. [8] Finlay BB, Ruschkowski S, Dedhar S. Cytoskeletal rearrangements accompanying salmonella entry into epithelial cells. J Cell Sci 1991; 99:283–96.
[9] Kocks C, Gouin E, Tabouret M, Berche P, Ohayon H, Cossart P. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 1992;68:521–31. [10] Johnson MA, Pinto BM. Molecular mimicry of carbohydrates by peptides. Aust J Chem 2002;55:13–25. [11] Chou CF, Smith AJ, Omary MB. Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18. J Biol Chem 1992;267:3901–6. [12] Wucherpfennig KW. Mechanisms for the induction of autoimmunity by infectious agents. J Clin Invest 2001;108:1097–104. [13] Shikhman AR, Greenspan NS, Cunningham MW. A subset of mouse monoclonal antibodies cross-reactive with cytoskeletal proteins and group A streptococcal M proteins recognizes N-acetyl-b-D-glucosamine. J Immunol 1993;151:3902–13. [14] Dighiero G, Guilbert B, Avrameas S. Naturally occurring antibodies against nine common antigens in human sera. II. High incidence of monoclonal Ig exhibiting antibody activity against actin and tubulin and sharing antibody specificities with natural antibodies. J Immunol 1982;128:2788–92. [15] Harrus S, Waner T, Bark H, Jongejan F, Cornelissen AW. Recent advances in determining the pathogenesis of canine monocytic ehrlichiosis. J Clin Microbiol 1999;37:2745–9. [16] Waner T, Harrus S, Weiss DJ, Bark H, Keysary A. Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis. Vet Immunol Immunopathol 1995;48:177–82. [17] Kim MK, Kee SH, Cho KA, Chung MH, Lim BU, Chang WH, et al. Apoptosis of endothelial cell line ECV304 persistently infected with Orientia tsutsugamushi. Microbiol Immunol 1999;43:751–7. [18] Seong SY, Kim MK, Lee SM, Odgerel Z, Choi MS, Han TH, et al. Neutralization epitopes on the antigenic domain II of the Orientia tsutsugamushi 56-kDa protein revealed by monoclonal antibodies. Vaccine 2000;19:2–9.
MICROBIAL PATHOGENESIS Microbial Pathogenesis 41 (2006) 125–132 www.elsevier.com/locate/micpath
An epitope shared by cellular cytokeratin and Orientia tsutsugamushi Mi-Jeong Kim, Mee-Kyung Kim, Jae-Seung Kang Department of Microbiology and Research Institute for Medical Science, Inha University College of Medicine, Jungsuk B/D, 3rd Street, Shinheung-Dong, Choong-Gu, Incheon 400-712, Republic of Korea Received 14 February 2006; accepted 6 April 2006 Available online 9 August 2006
Abstract Many microbial pathogens have epitopes shared with host cell components and these epitopes may induce transient or longer-term tissue-damaging autoantibody responses. We observed that several mouse monoclonal antibodies (MAbs) raised against Orientia tsutsugamushi were also reactive with host cells. One such antibody, MAb Rb105, cross-reacted with the cytoskeleton, as shown by immunofluorescent staining. Biochemical studies identified the cross-reacting component as a cytokeratin protein. These results identify an epitope shared by O. tsutsugamushi and the cytokeratins of host cells. In addition, antibodies cross-reactive with the cytoskeleton were detected in the sera of scrub typhus patients, suggesting that an epitope similar to that detected by MAb Rb105 may be recognized by human antibodies. r 2006 Elsevier Ltd. All rights reserved. Keywords: Orientia tsutsugamushi; Epitope; Monoclonal antibody; Cytokeratin
1. Introduction Orientia tsutsugamushi (OT), a member of the Rickettsiaceae family, is the causative agent of scrub typhus. This disease is an acute febrile illness characterized by fever, rash, and eschar. The disease is endemic in many countries in the Asia-Pacific region, including Korea [1]. The mechanism of pathogenesis has not been extensively studied, but is believed to be similar to that shown by other Rickettsia. Pathogenesis includes endothelial damage and subsequent vasculitis [2]. Many microbial pathogens have epitopes shared with host cell components, and antibody responses to such epitopes may cause transient or long-term damage if the antibodies react with the cytoskeleton, the mitochondrion, the nucleus, lipid, or other components of the host cell [3]. Such autoantibodies are mostly subject to transient synthesis, but some are produced for a protracted time. The presence of epitopes shared by rickettsial organisms and humans, and the induction of autoantibodies, has been reported. Cytoplasmic, Corresponding author. Tel.: +82 32 890 0952; fax: +82 32 881 8559.
E-mail address: [email protected] (J.-S. Kang). 0882-4010/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2006.04.008
nuclear, and platelet autoantibodies are found in the sera of human granulocytic ehrlichiosis patients [4]. An antiphospholipid antibody appears in persons suffering from Mediterranean spotted fever [5] while patients with Q fever develop an anticardiolipin antibody [6]. However, such developments have not been reported following infection with OT. Mouse monoclonal antibodies (MAbs) have been used to measure the antigen specificity of cross-reacting antibodies produced after microbial infection. For example, strong immunofluorescence (IF) staining of heart tissue sections by anti-Streptococcus MAbs was reported, indicating the presence of an epitope shared by both heart tissue and Streptococcus [7]. During characterization of MAbs against OT we found that several MAbs were reactive against host cell components. Among these crossreacting MAbs, we characterized one MAb (Rb105) that reacts with both an epitope of OT and an epitope of the cytoskeleton. In addition, anticytoskeleton antibodies were detected in the sera of scrub typhus patients, suggesting that the OT epitope to which MAb Rb105 is directed may induce an antibody response during human infection.
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2. Results
2.2. Reaction of MAb Rb105 with host proteins
2.1. MAbs reacting with both OT and the cytoskeleton
To characterize the target antigen of MAb Rb105, we analyzed proteins of cells infected with OT by western blotting using MAb Rb105 as primary antibody. A 46 kDa band was detected by Rb105 and the expression level of this protein did not change over 11 days of infection. However, the expression of a 47 kDa OT protein detected by MAb M686-8 increased as infection proceeded (Fig. 3). Further, the 46 kDa band was detected in uninfected cells at the same level seen in infected cells. This suggested that MAb Rb105 detected only a host protein upon western blotting and that no bacterial proteins could be visualized, using western blotting, with MAb Rb105. To explain the fact that MAb Rb105 stained both bacteria and the cytoskeleton in the IF protocol but that no bacterial protein was detected upon western blotting, we hypothesized that a host cytoskeleton protein might bind to bacteria resulting in simultaneous staining of both bacteria and the cytoskeleton. To address this issue, we stained uninfected ECV304 cells with MAb Rb105 and
In several separate fusion experiments we have produced a panel of MAbs against OT and found that some MAbs were also reactive with uninfected cells. These crossreacting MAbs appeared to be directed against epitopes of the nucleus, the mitochondrion, the endoplasmic reticulum, and the cytoskeleton of the host cell. Control MAb FS15 did not stain any structures in uninfected cells (Fig. 1). Among the reactive MAbs, one (Rb105) detected epitopes of both OT and the cytoskeleton as judged by IF staining of acetone/methanol-fixed cells. As shown (Fig. 2), MAb Rb105 simultaneously stained both bacteria and cytoskeletal structures in a slide preparation. The morphology of the stained cytoskeleton suggested that the reactive epitope might be located on the intermediate filament. As the results suggested that OT and the cytoskeleton might share epitopes, we further explored the antigen specificity of MAb Rb105.
Fig. 1. Cross-reaction of anti-OT MAbs with host cells. Uninfected ECV304 cells were treated with anti-OT MAbs followed by IF staining. Inset: Each MAb reacted with OT in infected ECV304 cells.
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Fig. 4B). These results suggest that the cellular target antigen of MAb Rb105 does not bind bacteria. 2.3. Identification of the cellular antigen detected by MAb Rb105
Fig. 2. MAb Rb105 is reactive with both OT and the cytoskeleton. ECV304 cells infected with OT for 4 days were stained with MAb Rb105, which was reactive with both OT (arrow) and the cytoskeleton (arrowhead).
Fig. 3. Expression of the target antigen of MAb Rb105 during infection. ECV304 cells infected with OT for 11 days were solubilized. Total proteins were separated and analyzed by western blotting at the indicated times. MAb M686-8 was used to detect the 47 kDa OT protein. C, uninfected control cells; 11A, cells attached to the culture substrate at 11 days after infection; 11D, cells detached from the culture substrate at 11 days after infection.
anti-cytoskeleton antibodies. MAb Rb105 stained the filamentous structures of the uninfected ECV304 cells like other anti-cytoskeleton antibodies (Fig. 4A). Next, we stained the infected ECV304 cells with MAb Rb105 or anticytoskeleton antibodies and simultaneously stained OT with anti-OT MAb (FS15). As expected, both MAb Rb105 and the anticytoskeleton antibodies stained filamentous structures of the infected ECV304 cells (left column, Fig. 4B). However, anticytoskeleton antibodies alone did not stain any bacteria, while MAb Rb105 alone stained bacteria both inside and outside the cells left column. Further, images of bacteria stained with anti-OT MAb (middle column, Fig. 4B) did not merge with images of the cytoskeleton stained with anticytoskeleton antibodies (right column, Fig. 4B). This was in contrast to the merging of images seen when an image of infected cells stained with MAb Rb105 was merged with an image of infected cells stained with anti-OT MAb (lower row,
To identify the host target protein recognized by MAb Rb105 we performed IF staining and immunoprecipitation analysis. First, we stained uninfected ECV304 cells with both MAb Rb105 and antiactin/tubulin antibodies simultaneously. As shown (Fig. 5A), images stained with MAb Rb105 appeared distinct from those obtained with antiactin antibody and the images did not merge, indicating clearly that the MAb Rb105 target antigen is not actin. When we stained uninfected ECV304 cells with MAb Rb105 and antitubulin antibody, the images obtained with the two antibodies appeared very similar (upper row, Fig. 5B). As intermediate filament fibers are associated closely with microtubules, we treated cells with 33 mM nocodazole to selectively disrupt microtubules. As shown (lower row, Fig. 5B), images of nocodazole-treated cells stained with MAb Rb105 were distinct from images obtained using antitubulin antibody, indicating that the MAb Rb105 target antigen is not tubulin. We therefore reasoned that the MAb Rb105 target protein might be one of the intermediate filament proteins. Next, we performed immunoprecipitation analysis with various anticytoskeleton antibodies to confirm that the target antigen was an intermediate filament protein. MAb Rb105 did not immunoprecipitate a target protein from uninfected ECV304 cells, but was active in western blotting of proteins from uninfected cells, as shown (seventh lane, Fig. 6A). Nevertheless, MAb Rb105 reacted strongly with protein immunoprecipitated by pan-cytokeratin antibody (fourth lane, Fig. 6A) and was weakly reactive with protein precipitated with antibodies anticytokeratin 8 and anticytokeratin 18. Proteins immunoprecipitated by the other anticytokeratin antibodies, anticytokeratin 14 and anticytokeratin 19, were not recognized by MAb Rb105 (data not shown). Similar results were obtained by immunoprecipitation analyses of infected cells (Fig. 6B). We conclude that the MAb Rb105 target antigen is a cytokeratin protein. 2.4. Detection of anticytoskeleton antibody in the sera of scrub typhus patients To examine whether anticytoskeleton antibodies develop during human OT infections we tested sera from 41 scrub typhus patients for the presence of anticytoskeleton antibody. Uninfected and infected ECV304 cells were subjected to IF staining using 1:80 dilutions of sera. We confirmed that all patients’ sera reacted with OT in infected ECV304 cells (data not shown). As shown (Fig. 7), eight sera contained anticytoskeleton antibodies staining filamentous structures of uninfected cells. None of 30 sera from healthy
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(B) Fig. 4. IF staining of cells using anticytoskeleton antibodies: (A) Uninfected ECV304 cells were stained with MAb Rb105, anti-pan-cytokeratin antibody (CK Pan), anti-vimentin antibody, or anti-a-tubulin antibody. (B) ECV304 cells infected with OT were stained simultaneously with anticytoskeleton antibodies (left column, red) and MAb FS15 for the detection of the 56 kDa OT protein (middle column, green spots).
humans contained such antibodies. One patient had nuclear antibody (data not shown). 3. Discussion This work suggests that cellular cytokeratin and OT share an epitope. This epitope elicits antibody responses when OT infects mice. We first noticed the OT-mediated
induction of various cross-reacting antibodies during characterization of several MAbs reactive with some components of uninfected cells. Such cross-reacting MAbs may be directed against the nucleus, the mitochondrion, the endoplasmic reticulum or the cytoskeleton of the host cell, in addition to detecting bacterial epitopes. It seems likely, therefore, that many antigenic epitopes are shared between OT and human cells. We chose one MAb (Rb105)
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(B) Fig. 5. Comparison of images obtained with MAb Rb105 and antiactin or antitubulin antibodies: (A) Uninfected ECV304 cells were stained simultaneously with MAb Rb105 (green), antiactin (red), and Heochst (blue). (B) Uninfected ECV304 cells were stained simultaneously with antitubulin (green), MAb Rb105 (red), and Heochst (blue). Uninfected ECV304 Cells were treated with nocodazole (33 mM) and stained with the same antibodies (lower row).
that reacts with both OT and the cytoskeleton on IF staining for further characterization. Although MAb Rb105 stained both bacteria and the cytoskeleton, the MAb detected only a single protein band in western blotting and the expression level of this protein did not change during the course of an 11 days infection. This result was puzzling at first as we expected two protein bands, one from bacteria and the other from host cells. However, the bacterial antigen was not detected in western blotting. Two explanations are possible. First, the Rb105reactive antigen from the host cell might coat intracellular bacteria. The coating of intracellular bacteria with host protein has been demonstrated in other systems [8,9]. Second, OT and cytokeratin may indeed share an epitope, but the bacterial epitope may be heat-sensitive and thus undetectable on western blotting. To address this issue we first stained both uninfected and infected cells with an anticytokeratin antibody. The results showed that neither the anticytokeratin antibody nor control antibodies stained bacterial particles in infected cells. Thus, we could conclude that cytokeratin did not coat OT. Instead, it seemed likely that OT and cytokeratin did indeed share an epitope. The identity of the cell antigen targeted by MAb Rb105 was examined by IF staining and immunoprecipitation. IF staining using antiactin and antitubulin antibodies clearly indicated that the target antigen was neither actin nor
tubulin. When the MAb Rb105 antigen was characterized by immunoprecipitation it seemed clear that MAb Rb105 was reactive against cytokeratins of the host cell. However, it has yet to be explained why the MAb Rb105-reactive epitope of OT could not be detected in western blotting, in contrast to the facile detection of the cytokeratin epitope. One explanation is that a nonprotein antigen of OT was detectable in IF staining but was not detectable by western blotting. Thus, it is possible that MAb Rb105 is reactive against bacterial carbohydrate and carbohydrate-mimetic peptides [10] of cytokeratin. Another possibility is that MAb Rb105 detects carbohydrate moieties of both OT and cytokeratin. This is possible as cytokeratins are glycosylated at multiple sites with single O-linked N-acetylglucosamine residues [11]. Epitopes shared by microbial pathogens and host cells may activate autoreactive T cells and B cells during infection [12]. The most probable mechanism by which infection of mice with OT induces the synthesis of selfreactive antibody may be molecular mimicry. This is believed to involve the triggering, by many pathogens, of immune responses against host tissues. The pathogens present epitopes that are identical or nearly so to host components. Self-tolerance is broken, resulting in the appearance of autoantibodies. The best known example of this phenomenon is the appearance of autoantibodies
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Rickettsia conorii infection, may cause membrane damage in endothelial cells [5]. However, in OT infection, further studies are required to assess the role of antibodies to the shared epitope observed in this study. In our tests, eight of 41 patients (19.5%) had antibodies reactive with the cytoskeleton of ECV304 cells. This suggests that autoantibodies against the cytoskeleton may be induced, at least transiently, by scrub typhus infection. These antibodies will cause a strong nonspecific fluorescence that interferes with reading the result of serologic test with IFA staining, as pointed in a previous study [4]. In addition, it may be worth exploring the possible pathogenic role of anticytoskeleton antibodies during human OT infections. 4. Materials and methods 4.1. Cell culture and OT
Fig. 6. Reaction of MAb Rb105 with cytokeratin. Uninfected (A) and infected (B) cells were lysed and immunoprecipitated with normal mouse serum (N), antivimentin antibody (Vim), antitubulin antibody (a-tub), anti-pan-cytokeratin antibody (CK Pan), anticytokeratin 18 antibody (CK 18), anticytokeratin 8 antibody (CK 8), or MAb Rb105. The precipitated proteins were analyzed with MAb Rb105 or anti-pan-cytokeratin as primary antibodies.
Cells of the human umbilical vein-derived endothelial cell line ECV304 were maintained with medium 199 (Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco BRL) in a humidified atmosphere containing 5% (v/v) CO2 at 37 1C. The Boryong serotype of OT (strain MF) was cultivated in ECV304 cells as described previously [17]. When infected ECV304 cells showed maximum cytopathic effects the cells were disrupted with glass beads (diameter 1.0 mm) and centrifuged at 300g for 5 min. The resulting supernatant was immediately used to infect further ECV304 cells. 4.2. Antibodies
cross-reactive with streptococcal antigens after rheumatic fever [7]. These autoantibodies are reactive against various cytoskeletal proteins including actin, vimentin, and cytokeratin. Interestingly, autoantibodies triggered by microbial molecules are not necessarily reactive against host components with similar chemical structures. Multireactive antibodies reactive against host components of dissimilar chemical natures are also produced. For example, antiN-acetyl-b-D-glucosamine antibodies against Streptococcus also react also with cytoskeletal proteins of the animal cell [13]. Generally, autoantibodies have been considered to be of little importance in pathogenesis. They occur at low levels in the sera of normal individuals [14]. However, in some infectious diseases, cross-reactions between human antigens and microbial antigens have been implicated in the development of autoimmune disease. Infection with rickettsial organisms induces various autoantibodies. The contribution of these antibodies to rickettsial pathogenesis remains largely unknown, but some experimental data suggest they may indeed contribute to ill effects in humans. Serum platelet-binding antiplatelet antibodies, appearing in dogs after experimental infection with Ehrlichia canis, may contribute to the immune destruction of platelets [15,16]. Antiphospholipid antibodies, appearing after
The production of MAb FS15 was described previously [18] and other MAbs (M37-2, M716G, Rb105, M686-20, and M716B) were produced using the same method. The antibody MAb FS15 is reactive against a 56 kDa protein of OT [18]. MAbs against pan-cytokeratin, cytokeratin 18, cytokeratin 8, vimentin, and a-tubulin were purchased from Sigma–Aldrich (St. Louis, MO). 4.3. Patient sera Sera from 41 scrub typhus patients were obtained for this study. Diagnosis was performed using an OT-specific IF test or by isolation of OT. Sera from 30 healthy adults were used as control sera. All serum samples were dispensed into aliquots and stored at 20 1C until required. 4.4. IF staining ECV304 cells grown in eight-well chamber slides were infected with OT. After infection, cells were washed with PBS, permeabilized with 0.25% (w/v) Triton X-100 (Sigma–Aldrich) and 0.4% (v/v) paraformaldehyde for 2 min, and then fixed using 4% (v/v) paraformaldehyde at 4 1C for 30 min. After several washes with PBS the cells
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Fig. 7. Anticytoskeleton antibody in scrub typhus patients. Some 41 human sera from scrub typhus patients were examined for the presence of anticytoskeleton antibodies. Uninfected and infected ECV304 cells were stained with sera at a dilution of 1:80. The stained images of uninfected and infected ECV304 cells were presented for representative three sera (patients 1–3), and the stained images of uninfected ECV304 cells were presented for other sera. Arrowheads indicate filamentous cell structures stained by patients’ sera.
were incubated with primary antibodies or human sera for 1 h. After several further washes with PBS containing 0.1% (w/v) Tween-20 cells were incubated with rhodamineconjugated antimouse IgG antibody (Jackson Laboratories, Bar Harbor, MA) or fluorescein-isothiocyanate conjugated antihuman IgG antibody (Jackson Laboratories) in the dark for 1 h. For double staining of OT, the cells were further fixed with 4% (v/v) paraformaldehyde followed by treatment with MAb FS15 conjugated with fluorescein-isothiocyanate. To stain nuclei, cells were incubated with Heochst 33258 (0.05 mg/mL, Sigma–Aldrich) in PBS for 10 min at room temperature in the dark. Cover glasses were mounted on glass slides with Antifade (Vector Laboratories, Burlingame, CA) and viewed with a confocal microscope (Bio-Rad, Hercules, CA). 4.5. Western blotting Cells were resuspended in lysis buffer [1% (w/v) SDS,5% (v/v) glycerol,0.01% (w/v) bromophenol blue, and 0.031 M Tris–HCl, pH 7.0] and incubated at room temperature for 1 h. b-Mercaptoethanol was added to 5% (v/v), the solutions heated at 100 1C for 5 min, and solubilized proteins electrophoresed through 11% (w/v) SDS-polyacrylamide (SDS-PAGE) gels. The separated proteins were subsequently transferred electrophoretically to polyvinylidene difluoride membrane (Immobilon-P, Millipore Co.,
Bedford, MA). Membranes were blocked with a suspension of 5% (w/v) nonfat dry milk powder in water and then incubated with primary antibodies for 1 h. After membranes were thrice washed with TBS with 0.1% (v/v) Tween-20 (TBST) they were incubated with horseradish peroxidase-conjugated goat antimouse IgG for 1 h at room temperature and developed using a protocol involving an enhancement of chemiluminescence (Amersham, UK).
4.6. Immunoprecipitation Cells were resuspended in RIPA buffer [50 mM Tris pH 7.4,1% (w/v) Empigen,0.5% (v/v) sodium deoxycholate, 150 mM NaCl, 5 mM EDTA, 0.1% (w/v) SDS,1 mM phenylmethylsulfonyl fluoride,1 mM leupeptin,1 mM aprotinin,1 mM NaF, 1 mM Na3VO4, and 1 mM iodoacetamide] and proteins were immunoprecipitated by overnight incubation at 4 1C with primary antibodies and protein G/A-Agarose beads (Calbiochem, La Jolla, CA). Beads were pelleted by centrifugation at 10,000g for 1 min and washed three times. The immunoprecipitated proteins were eluted with sample buffer [1% (w/v) SDS, 5% (v/v) glycerol, 0.01% (w/v) bromophenol blue, 0.031 M Tris–HCl (pH 7.0), and 5% (v/v) b-mercaptoethanol] by boiling for 5 min and the proteins were then separated on an SDS-PAGE gel with acrylamide concentration of 7.5% (w/v).
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Acknowledgment This work was supported by Korea Research Foundation Grant KRF-2003-015-E00097.
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