Microsc. Microanal., page 1 of 9 doi:10.1017/S1431927615000318
© MICROSCOPY SOCIETY OF AMERICA 2015
Nanoscopic Localization of Surface-Exposed Antigens of Borrelia burgdorferi Leandro Lemgruber,1,2,3,* Celso Sant’Anna,2,3 Caron Griffths,4 Yuri Abud,2 Musa Mhlanga,4 Reinhard Wallich,5 and Friedrich Frischknecht1 1 Department of Infectious Diseases – Parasitology, Im Neuenheimer Feld 324, University of Heidelberg Medical School, 69120, Heidelberg, Germany 2 Laboratory of Microscopy for Life Sciences, Diretoria de Metrologia Aplicada às Ciências da Vida – Dimav, Instituto Nacional de Metrologia, Qualidade e Tecnologia – Inmetro, 25250-020, Duque de Caxias, Rio de Janeiro, Brazil 3 Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, UFRJ, 21941-902, Rio de Janeiro, Brazil 4 Gene Expression and Biophysics Group, Synthetic Biology Emerging Research Area, Council for Scientific and Industrial Research, Box 395, Pretoria 0001S, South Africa 5 Institute for Immunology, Im Neuenheimer Feld 305, University of Heidelberg Medical School, 69120, Heidelberg, Germany
Abstract: Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, is transmitted to humans through the bite of infected Ixodes spp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means of the pathogen to escape the vertebrates’ immune system. One strategy employed by Lyme disease spirochetes to evade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins that is vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterial surface antigens using different microscopy techniques, from fluorescent wide field to super-resolution and immunogold-scanning electron microscopy. A fluorescent strain of B. burgdorferi spirochetes was labeled with monoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is more evenly distributed over the surface than OspB and OspC that were present as punctate areas. Key words: spirochete, immunolabeling, immunoSEM, super-resolution microscopy
I NTRODUCTION Lyme disease is a major vector-borne disease in the Northern Hemisphere and is caused by members of the Borrelia burgdorferi complex (sensu lato) (Radolf et al., 2012). Borreliae are Gram-negative spiroform shaped bacteria composed of two membranes sandwiching a periplasmic flagellum, ranging from 10 to 30 µm in length and 0.25–0.5 µm in diameter belonging to the order of Spirochaetales (Kudryashev et al., 2009, 2011; Radolf et al., 2012). Borrelia spirochetes are transmitted during the blood meal of infected Ixodes spp. ticks to the skin of the hosts (Frischknecht, 2007; Hovius et al., 2007; Radolf et al., 2012). Although our knowledge on the pathogenesis of Lyme disease has increased, there remains major interest in unraveling the interaction of Borrelia with tick vectors and their mammalian hosts during the course of infection. The bacterial-host interface depends to some extent on components present on the cell envelope. The bacterial outer membrane contains lipid-raft microdomains and several outer surface lipoproteins (OSPs), outer-membrane proteins (OMPs) and OMPs harboring transmembrane-spanning domains (Anguita et al., 2003; Floden et al., 2011; Gesslbauer Received September 16, 2014; accepted February 11, 2015 *Corresponding author. [email protected]
et al., 2012; Önder et al., 2012; Radolf et al., 2012; Thein et al., 2012; LaRocca et al., 2013; Toledo et al., 2014). There have been several studies utilizing different microscopy techniques in the Borrelia field, such as confocal and two-photon microscopy, intravital and video microscopy, freeze-fracture, cryo-immunolabeling, and cryo-electron tomography; however, these studies did not specifically evaluate the distribution pattern of the surface proteins (Brusca et al., 1991; Radolf et al., 1995; Moriarty et al., 2008; Dunham-Ems et al., 2009; Kudryashev et al., 2009, 2011; Lee et al., 2010; Floden et al., 2011; Crowley et al., 2013; Bockenstedt et al., 2014; Toledo et al., 2014). Despite efforts in recent years to increase our understanding of the biology of Borrelia spp., little is known about the function and structure of outer surface proteins that mediate interaction with host cells. To localize and precisely characterize cell outer surface antigens, different light microscopy techniques, mainly immunofluorescence assays (IFA) in wide-field (WFM) and confocal scanning laser microscopy (CSLM) were used. Moreover, different high-resolution techniques, e.g., super-resolution microscopy (SRM) and immunogold-scanning electron microscopy (immunoSEM) have gained attention (Goldberg, 2008; Huang et al., 2010). In this study, we will review recent advances in labeling surface antigens of Borrelia spirochetes employing monoclonal antibodies and analyzing their expression patterns using WFM, CSLM, SRM, and immunoSEM.
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M ATERIALS AND M ETHODS
PALM/STORM
Antibodies
For SRM, GFP-expressing Borrelia were fixed in 4% paraformaldehyde in PBS prepared as described above for immunofluorescence microscopy and following the protocol previously described (Henriques et al., 2010). The secondary antibody was coupled to Cy3, a molecule with good photoswitching properties, allowing imaging of the bacteria in the super-resolution system. For observation, bacteria were incubated in a blinking-inducing buffer (0.5 mg/mL glucose oxidase, 40 μg/mL catalase, 10% w/v glucose, and 50 mM of β-mercaptoethylamine) in PBS (pH 7.4) to limit the production of reactive oxygen species, preventing fluorophore photobleaching and promoting better fluorophore blinking. The material was observed using a custom built Nikon Ti Eclipse widefield TIRF microscope (Nikon, Japan) using a 100× Apochromat TIRF oil immersion objective (NA 1.49). Particle detection, localization, and visualization were done using the QuickPalm plugin for ImageJ as previously described (Henriques et al., 2010).
The affinity and specificity of some of the monoclonal antibodies against Borrelia antigens used in this study were previously characterized (Kramer et al., 1990). The antibodies used were LA2 (OspA), LA25 (OspB), LA97 (OspC), LA111 (P39), and LA114 (P83/100). Appropriate control experiments based on antibody characterization by Kramer et al. (1990) and removal of the primary antibody as a control for the secondary antibody was performed. Results for all control experiments were as expected and are not shown.
Borrelia Culture B. burgdorferi [strain GCB726, constitutively expressing green fluorescent protein (GFP)] was cultured essentially as described previously (Wallich et al., 1995; Moriarty et al., 2008). Briefly, the spirochetes were cultivated in BSK-H medium (PAN Biotech, Aidenbach, Germany) supplemented with 5% rabbit serum (Cell Concept, Umkirch, Germany) at 30°C. Cells were harvested in the log phase during rapid growth. Cells were enriched by centrifugation followed by resuspension of the pellet in BSK medium (described below).
Immunocytochemistry For wide-field immunofluorescence microscopy, GFPexpressing bacteria were fixed by adding 4% recently prepared paraformaldehyde to the bacteria culture medium. After washing with phosphate buffered saline (PBS), cells were incubated for 30 min in the blocking buffer (albumin 3% in PBS, pH 8) and then incubated with primary antibodies for 30 min. Cells were washed and incubated with Alexa Fluor 546-labeled secondary antibody (Molecular Probes, USA) for 30 min. Afterwards, the bacteria were allowed to adhere onto coverslips and were observed in an inverted Zeiss Axiovert 200 M microscope (Zeiss, Germany) at room temperature. For confocal analysis, the cells were processed as described above and observed in a Zeiss LSM 710 (Zeiss, Germany). For immunoSEM, the protocol established by Sant’ Anna et al. (2005) was used. Cells were treated as described previously for immunofluorescence. After incubating with the primary antibody and the subsequent wash step, the cells were incubated with the secondary antibody conjugated with a 15-nm gold particle (BBInternational, Madison, USA) for 30 min at room temperature. After incubation, the samples were washed and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer. The cells were washed again, dehydrated in an ethanol series (50, 70, 90, and 100%), critical point-dried in a Baltec CPD 030 apparatus (Leica, Germany) and mounted on specimen stubs. The samples were ion-sputtered with a 5-nm carbon layer using a Leica electron microscope (EM) SCD500 high-resolution ion beam coater (Leica, Germany) to avoid charging effects and imaged in a FEI Quanta FEG 450 microscope (Netherlands) at an accelerating voltage of 10 kV. The microscope was operated in backscattered (BSE) mode to image the gold particles at the spirochete surface.
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Image Analysis All images were analyzed and processed in Adobe Photoshop® and FIJI software. Figures were assembled using Adobe Illustrator® software.
RESULTS
AND
DISCUSSION
During transmission between hosts, members of the Borrelia genus, like other pathogens, modify their surface antigen expression with some lipoproteins being most expressed in the tick and others in the mammalian host as driven by different immune pressures (Templeton, 2004; Palmer et al., 2009; Kumru et al., 2011; Kenedy et al., 2012; Tilly et al., 2013; Wang et al., 2013; Yang et al., 2013; Raffel et al., 2014). The vast majority of the surface proteins are N-terminally anchored lipoproteins, with the few integral membrane proteins in the outer membrane most likely acting as poreforming proteins, e.g. DipA (Thein et al., 2012). The lipoproteins are formed in the cytoplasm, but the mechanisms that control their retention in the periplasm or cause them to be exposed on the surface are yet to be characterized. The most apparent change in protein exposure is OspA-OspC between the tick and the mammalian host (Schwan et al., 1995; Schwan & Piesman, 2000), in which the bacteria temporally down regulate the surface expression of OspA and upregulate that of OspC during transmission. OspA is important for attachment of the bacteria to the host cell and colonization (Schmit et al., 2011), while OspC, a plasminogen receptor, is important during the tick-to-host transition (Önder et al., 2012). To observe the distribution of antigens on the Borrelia surface we used an IFA in WFM. In this technique, fluorescent-conjugated antibodies detect specific target antigens. In indirect IFA, labeling of the antigen is done in a two-step procedure; with a primary, unlabeled antibody that
Localization of Borrelia Outer Surface Proteins
binds to the target antigen, followed by a secondary antibody coupled to a fluorophore that specifically recognizes the Fc portion of the primary antibody. In our study we used a Borrelia strain that constitutively expressed GFP (Moriarty et al., 2008), allowing easy identification of the bacteria. The overlay of the GFP signal from the bacterial cytoplasm and the antibodies on the surface were used to visualize the surface antigens. We used a set of antibodies previously characterized to recognize antigens exposed on the surface of Borrelia: OspA, OspB, OspC, P39, and P83/100 (Kramer et al., 1990). Using WFM, we observed different patterns of distribution of these antibodies. OspA was evenly distributed over the bacteria surface (Figs. 1a–1c), while OspB (Figs. 1d–1f) and OspC (Figs. 1g–1i) presented a punctate distribution of 12 ± 6 and 6.45 ± 4.1 punctae/cell, respectively. P39 (Figs. 1j–1l) and P83/100 (Figs. 1m–1o) also showed a punctuated pattern but much less frequent at 3.1 ± 1.7 and 1.9 ± 0.9 punctae/cell, respectively. This is unexpected considering the fact that both P39 and P83/100 are immunogenic antigens producing high amounts of antibodies during infection in the host (Simpson et al., 1991; Wallich et al., 1995; Roessler et al., 1997; Hauser et al., 1998; Bauer et al., 2001; Miller, 2003). However, one has to keep in mind that an immunological response is not directly proportional to the amount of antigen present. Previously, it was shown that OspA, OspB, and OspC were not exposed exclusively on the Borrelia outer surface, but were also located in the periplasm (Brusca et al., 1991; Cox et al., 1996; Hefty et al., 2002; Kenedy et al., 2012). Using methanol-fixed spirochetes (therefore with permeabilized membranes), a more intense and uniform label was observed by the authors (Cox et al., 1996). This finding was also obtained when cryo-immuno labeling was used, with the authors getting an intense signal for anti-OspA and OspB intra as well as extracellularly (Brusca et al., 1991). So far it is not clear how these lipoproteins can be directed to and from the outer surface. For the next experiments, we limited analysis to the antibodies that presented the best signals— OspA, OspB, and OspC. A key limitation of standard fluorescence microscopy is that when a sample is illuminated, some portion of the exiting light is scattered by the sample, exciting fluorescence in regions of the specimen other than just the focal plane. This generates a “cloud” of diffuse and out-of-focus fluorescence around the center of the beam (Lichtman & Conchello, 2005). Thus, this out-of-focus signal can mask fluorescence from the focal plane, reducing the contrast and the signal-to-noise ratio. A way to mitigate this problem in a WFM is to use computational sectioning. Alternatively, CSLM can be used for optical sectioning as it removes the out-of-focus light using a pinhole in front of the detector. In CSLM the sample is scanned during image acquisition and, using a laser as a source of illumination, a very small spot is focused on the specimen at a specific time, with the excitation and detection in focus at the same spot (Conchello & Lichtman, 2005; Schermelleh et al., 2010). CSLM allowed us to acquire a series of images of thin slices of a thick specimen. Later these images were combined in a stack allowing
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generation of a three-dimensional reconstructed model of the sample. We used a CSLM to image the GFP-expressing Borrelia labeled with anti-OspA (Fig. 2a), anti-OspB (Fig. 2c), and anti-OspC (Fig. 2e). As in the WFM images, OspA presented an even distribution, while OspB and OspC presented a dot-like pattern. Although the images obtained presented better visualization of the antigens’ punctae, the overall resolution was not improved. In conventional light microscopy, the resolution is constrained by the diffraction of light, which causes the signal from a point source to spread as it travels to the detector. The resulting signal is termed the “point spread function” and determines the resolution limit. This is approximately half the wavelength of detected light; around 250–300 nm (Fischer et al., 2011). In the past few years, new technologies, collectively called SRM have been developed to overcome the diffraction limit, bringing the optical resolution to a macromolecular level. Some of these techniques allow resolution down to tens of nanometers, approaching the resolution obtained in the electron microscope (Huang et al., 2010; Schermelleh et al., 2010; Henriques et al., 2011). SRM techniques use the physical properties of fluorescent probes to distinguish the emission from two nearby molecules. One such technique is termed photoactivated localization microscopy/stochastic optical reconstruction microscopy (PALM/STORM). Essentially, one stimulates photoswitching of the fluorophores such that individual fluorophores within the sample are activated in a stochastic manner. Only a small, random sub-population of the fluorophores in the sample will be activated and fluoresce (“on” state) at a point in time and can be imaged as individual particles before they return to the dark (“off” state)—as other fluorophores are activated in turn. This limits the number of fluorophores emitting light at one time and reduces the probability of adjacent fluorophores being imaged simultaneously. Thus, one can record the signals of individual fluorophores throughout a large series of images, as the fluorophores progress through thousands of light–dark cycles. From this, a computational algorithm is able to determine high-precision localization of each fluorophore compiling these localization points to form a superresolution single-image reconstruction of the original sample, where single molecules and cell structure can be highly resolved (Rust et al., 2006; Henriques et al., 2010). Researchers have been applying SRM techniques to resolve the architecture and composition of complex intracellular structures in bacteria (reviewed in Cattoni et al., 2012; Coltharp & Xiao, 2012), e.g., distribution of chemoreceptor clusters, and imaging of subcellular machineries involved in cell shape, growth, and division. We applied PALM/STORM to test if OspA is indeed uniformly localized around the bacterial membrane or rather in focal spots too small to distinguish from each other. The labeling obtained with anti-OspA (Fig. 2b), anti-OspB (Fig. 2d), and anti-OspC (Fig. 2f) was similar to the one obtained with CSLM. Comparing the intensity plot profile of OspC labeling obtained with WFM, CSLM, and SRM (Fig. 3), it is clear that the punctae were more precisely resolved
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Figure 1. Wide-field immunofluorescence microscopy reveals the distribution of five different antigens on GFPexpressing Borrelia burgdorferi: OspA (a,b,c), OspB (d,e,f), OspC (g,h,i), P39 (j,k,l), P83/100 (m,n,o). OspA was more evenly distributed over the bacteria surface, while the other antigens presented a punctate distribution. GFP, green fluorescent protein.
Localization of Borrelia Outer Surface Proteins
Figure 2. Confocal and super-resolution microscopy (PALM/STORM) were used to compare the distribution pattern of OspA (a,b), OspB (c,d), and OspC (e,f). For confocal, GFP-expressing spirochetes were used, and the antigens labeled in red. For super-resolution, the antigen distribution patterns are shown in white. PALM/STORM, photoactivated localization microscopy/stochastic optical reconstruction microscopy; GFP, green fluorescent protein.
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Figure 3. Intensity plot profile of the anti-OspC labeling in wide-field microscopy (a), confocal microscopy (b), and super-resolution microscopy (c). The peaks were more distinct in confocal and in super-resolution microscopy, reaching higher gray values. X-axis correspond to a full bacterial long axis.
in CSLM and SRM, with greater sensitivity and signal contrast, demonstrated by the higher gray values obtained over the background with these techniques. Despite the variable level of Osp expression on the Borrelia bacteria, their presentation as surface proteins facilitates their labeling without extensive sample permeabilization. This high-efficiency labeling contributes to the superior resolution obtained with PALM/STORM. Thus, these SRM techniques would be particularly suitable for co-localization assays of different surface proteins at the single-molecule level. Electron microscopy surpasses the resolution limit of optical microscopy and is the routine approach to resolve cellular architecture at the ultra-structural scale. Immunocytochemistry allows the identification and localization of molecules using EM. Immunogold labeling is frequently used in transmission electron microscopy, especially in resin embedded material and cryo-sections (Griffihs, 1993; Slot &
Geuze, 2007). However, only the antigens exposed in the sections are accessible for the antibodies. Therefore, antigens with few copies on the surface are easily missed. ImmunoSEM can overcome this problem, allowing a “tri-dimensional” analysis of the cell surface, facilitating the identification of cell structure and function. The secondary electron detectors provide high-resolution topographic surface images, while a BSE detector provides visualization of the gold marker, based on the detected mass difference between the gold particles and the biological material (Hermann et al., 1996; Sant’Anna et al., 2005). When we used immunoSEM (Fig. 4), the labeling patterns obtained were largely similar to those obtained in fluorescence microscopy. However, intensity of the labeling was much weaker than those observed in the previous techniques. The punctae normally observed in WFM or CSLM were observed at a lower frequency in immunoSEM, with the gold more
Localization of Borrelia Outer Surface Proteins
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immunoSEM offers the best resolution, the sample preparation and labeling is more challenging than that required for light microscopy-based techniques. All these techniques showed that the antigens located on the surface of Borrelia presented different distribution patterns. Although further analysis are still required, this could be an indication of a different role for each protein in parasite transmission, establishment of the infection, and interaction with host immune cells.
ACKNOWLEDGMENTS We thank Tara Moriarty and George Chaconas for the GFPexpressing Borrelia strain. The work was funded by grants from the German Research Foundation, the Chica and Heinz Schaller Foundation, and the Germany-South Africa initiative from the South African National Science Foundation and the Federal German Ministry for Science and Education to M.M. and F.F. The authors gratefully acknowledge Carl Zeiss for providing the LSM 710 confocal scanning laser microscope during the EMBO practical course Imaging Infection and Immunity (2011); and to Luis Sergio Cordeiro and Mirko Singer for assistance and discussion during the project. L.L. was a recipient of a postdoctoral fellowship from the Cluster of Excellence CellNetworks at the University of Heidelberg and is now supported by a PRONAMETRO fellowship from INMETRO.
REFERENCES
Figure 4. Immuno-gold SEM showing the location of the antigens (arrows) OspA (a), OspB (b), and OspC (c). SEM, scanning electron microscopy.
sparsely distributed over the bacteria surface. Although immunoSEM did not add much information about the antigen distribution in Borrelia spirochetes, one cannot discard this technique to characterize a distribution pattern in another system; in addition this technique may be used to study the presence of antigens in secreted vesicles.
CONCLUSION Several imaging techniques for localization of antigens on the surface of cells are available, ranging from light to electron microscopy, including SRM. Each technique has its advantages and disadvantages, and presents challenges when deciding which technique to use in a particular study. Here we compared different techniques for the localization of antigens on the surface of the spirochete Borrelia. Although
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LAROCCA, T.J., PATHAK, P., CHIANTIA, S., TOLEDO, A., SILVIUS, J.R., BENACH, J.L. & LONDON, E. (2013). Proving lipid rafts exist: Membrane domains in the prokaryote Borrelia burgdorferi have the same properties as eukaryotic lipid rafts. PLoS Pathog 9, e1003353. LEE, W.-Y., MORIARTY, T.J., WONG, C.H.Y., ZHOU, H., STRIETER, R.M., VAN ROOIJEN, N., CHACONAS, G. & KUBES, P. (2010). An intravascular immune response to Borrelia burgdorferi involves kupffer cells and inkt cells. Nat Immunol 11, 295–302. LICHTMAN, J.W. & CONCHELLO, J.-A. (2005). Fluorescence microscopy. Nat Met 2, 910–919. MILLER, J.C. (2003). Immunological and genetic characterization of Borrelia burgdorferi BapA and EppA proteins. Microbiology 149, 1113–1125. MORIARTY, T.J., NORMAN, M.U., COLARUSSO, P., BANKHEAD, T., KUBES, P. & CHACONAS, G. (2008). Real-time high resolution 3D imaging of the Lyme disease spirochete adhering to and escaping from the vasculature of a living host. PLoS Pathog 4, e1000090. ÖNDER, Ö., HUMPHREY, P. & MCOMBER, B. (2012). OspC is potent plasminogen receptor on surface of Borrelia burgdorferi. J Biol Chem 287, 16860–16868. PALMER, G.H., BANKHEAD, T. & LUKEHART, S.A. (2009). ‘Nothing is permanent but change’–antigenic variation in persistent bacterial pathogens. Cell Microbiol 11, 1697–1705. RADOLF, J.D., CAIMANO, M.J., STEVENSON, B. & HU, L.T. (2012). Of ticks, mice and men: Understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 10, 87–99. RADOLF, J.D., GOLDBERG, M.S., BOURELL, K., BAKER, S.I., JONES, J.D. & NORGARD, M.V. (1995). Characterization of outer membranes isolated from Borrelia burgdorferi, the Lyme disease spirochete. Infect Immun 63, 2154–2163. RAFFEL, S.J., BATTISTI, J.M., FISCHER, R.J. & SCHWAN, T.G. (2014). Inactivation of genes for antigenic variation in the relapsing fever spirochete Borrelia hermsii reduces infectivity in mice and transmission by ticks. PLoS Pathog 10, e1004056. ROESSLER, D., HAUSER, U. & WILSKE, B. (1997). Heterogeneity of BmpA (P39) among European isolates of Borrelia burgdorferi sensu lato and influence of interspecies variability on serodiagnosis. J Clin Microbiol 35, 2752–2758. RUST, M.J., BATES, M. & ZHUANG, X. (2006). Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Met 3, 793–796. SANT’ANNA, C., CAMPANATI, L., GADELHA, C., LOURENÇO, D., LABATITERRA, L., BITTENCOURT-SILVESTRE, J., BENCHIMOL, M., CUNHA-ESILVA, N.L. & DE SOUZA, W. (2005). Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM). Histochem Cell Biol 124, 87–95. SCHERMELLEH, L., HEINTZMANN, R. & LEONHARDT, H. (2010). A guide to super-resolution fluorescence microscopy. J Cell Biol 190, 165–175. SCHMIT, V.L., PATTON, T.G. & GILMORE, R.D. Jr (2011). Analysis of Borrelia burgdorferi surface proteins as determinants in establishing host cell interactions. Front Microbiol 2, 141. SCHWAN, T.G. & PIESMAN, J. (2000). Temporal changes in outer surface proteins A and C of the Lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. J Clin Microbiol 38, 382–388. SCHWAN, T.G., PIESMAN, J., GOLDE, W.T., DOLAN, M.C. & ROSA, P.A. (1995). Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad Sci U S A 92, 2909–2913.
Localization of Borrelia Outer Surface Proteins SIMPSON, W.J., BURGDORFER, W., SCHRUMPF, M.E., KARSTENS, R.H. & SCHWAN, T.G. (1991). Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals. J Clin Microbiol 29, 236–243. SLOT, J.W. & GEUZE, H.J. (2007). Cryosectioning and immunolabeling. Nat Protoc 2, 2480–2491. TEMPLETON, T.J. (2004). Borrelia outer membrane surface proteins and transmission through the tick. J Exp Med 199, 603–606. THEIN, M., BONDE, M., BUNIKIS, I., DENKER, K., SICKMANN, A., BERGSTRÖM, S. & BENZ, R. (2012). DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates. PLoS One 7, e36523. TILLY, K., BESTOR, A. & ROSA, P.A. (2013). Lipoprotein succession in Borrelia burgdorferi: Similar but distinct roles for OspC and VlsE at different stages of mammalian infection. Mol Microbiol 89, 216–227.
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TOLEDO, A., CROWLEY, J.T., COLEMAN, J.L., LAROCCA, T.J., CHIANTIA, S., LONDON, E. & BENACH, J.L. (2014). Selective association of outer surface lipoproteins with the lipid rafts of Borrelia burgdorferi. mBio 5, e00899–14. WALLICH, R., BRENNER, C., KRAMER, M. & SIMON, M. (1995). Molecular-cloning and immunological characterization of a novel linear-plasmid-encoded gene, Pg, of Borrelia burgdorferi expressed only in vivo. Infect Immun 63, 3327–3335. WANG, P., DADHWAL, P., CHENG, Z., ZIANNI, M.R., RIKIHISA, Y., LIANG, F.T. & LI, X. (2013). Borrelia burgdorferi oxidative stress regulator BosR directly represses lipoproteins primarily expressed in the tick during mammalian infection. Mol Microbiol 89, 1140–1153. YANG, X., HEGDE, S., SHRODER, D.Y., SMITH, A.A., PROMNARES, K., NEELAKANTA, G., ANDERSON, J.F., FIKRIG, E. & PAL, U. (2013). The lipoprotein La7 contributes to Borrelia burgdorferi persistence in ticks and their transmission to naïve hosts. Microbes Infect 15, 729–737.
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Nanoscopic Localization of Surface-Exposed Antigens of Borrelia burgdorferi Leandro Lemgruber,1,2,3,* Celso Sant’Anna,2,3 Caron Griffths,4 Yuri Abud,2 Musa Mhlanga,4 Reinhard Wallich,5 and Friedrich Frischknecht1 1 Department of Infectious Diseases – Parasitology, Im Neuenheimer Feld 324, University of Heidelberg Medical School, 69120, Heidelberg, Germany 2 Laboratory of Microscopy for Life Sciences, Diretoria de Metrologia Aplicada às Ciências da Vida – Dimav, Instituto Nacional de Metrologia, Qualidade e Tecnologia – Inmetro, 25250-020, Duque de Caxias, Rio de Janeiro, Brazil 3 Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, UFRJ, 21941-902, Rio de Janeiro, Brazil 4 Gene Expression and Biophysics Group, Synthetic Biology Emerging Research Area, Council for Scientific and Industrial Research, Box 395, Pretoria 0001S, South Africa 5 Institute for Immunology, Im Neuenheimer Feld 305, University of Heidelberg Medical School, 69120, Heidelberg, Germany
Abstract: Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, is transmitted to humans through the bite of infected Ixodes spp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means of the pathogen to escape the vertebrates’ immune system. One strategy employed by Lyme disease spirochetes to evade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins that is vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterial surface antigens using different microscopy techniques, from fluorescent wide field to super-resolution and immunogold-scanning electron microscopy. A fluorescent strain of B. burgdorferi spirochetes was labeled with monoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is more evenly distributed over the surface than OspB and OspC that were present as punctate areas. Key words: spirochete, immunolabeling, immunoSEM, super-resolution microscopy
I NTRODUCTION Lyme disease is a major vector-borne disease in the Northern Hemisphere and is caused by members of the Borrelia burgdorferi complex (sensu lato) (Radolf et al., 2012). Borreliae are Gram-negative spiroform shaped bacteria composed of two membranes sandwiching a periplasmic flagellum, ranging from 10 to 30 µm in length and 0.25–0.5 µm in diameter belonging to the order of Spirochaetales (Kudryashev et al., 2009, 2011; Radolf et al., 2012). Borrelia spirochetes are transmitted during the blood meal of infected Ixodes spp. ticks to the skin of the hosts (Frischknecht, 2007; Hovius et al., 2007; Radolf et al., 2012). Although our knowledge on the pathogenesis of Lyme disease has increased, there remains major interest in unraveling the interaction of Borrelia with tick vectors and their mammalian hosts during the course of infection. The bacterial-host interface depends to some extent on components present on the cell envelope. The bacterial outer membrane contains lipid-raft microdomains and several outer surface lipoproteins (OSPs), outer-membrane proteins (OMPs) and OMPs harboring transmembrane-spanning domains (Anguita et al., 2003; Floden et al., 2011; Gesslbauer Received September 16, 2014; accepted February 11, 2015 *Corresponding author. [email protected]
et al., 2012; Önder et al., 2012; Radolf et al., 2012; Thein et al., 2012; LaRocca et al., 2013; Toledo et al., 2014). There have been several studies utilizing different microscopy techniques in the Borrelia field, such as confocal and two-photon microscopy, intravital and video microscopy, freeze-fracture, cryo-immunolabeling, and cryo-electron tomography; however, these studies did not specifically evaluate the distribution pattern of the surface proteins (Brusca et al., 1991; Radolf et al., 1995; Moriarty et al., 2008; Dunham-Ems et al., 2009; Kudryashev et al., 2009, 2011; Lee et al., 2010; Floden et al., 2011; Crowley et al., 2013; Bockenstedt et al., 2014; Toledo et al., 2014). Despite efforts in recent years to increase our understanding of the biology of Borrelia spp., little is known about the function and structure of outer surface proteins that mediate interaction with host cells. To localize and precisely characterize cell outer surface antigens, different light microscopy techniques, mainly immunofluorescence assays (IFA) in wide-field (WFM) and confocal scanning laser microscopy (CSLM) were used. Moreover, different high-resolution techniques, e.g., super-resolution microscopy (SRM) and immunogold-scanning electron microscopy (immunoSEM) have gained attention (Goldberg, 2008; Huang et al., 2010). In this study, we will review recent advances in labeling surface antigens of Borrelia spirochetes employing monoclonal antibodies and analyzing their expression patterns using WFM, CSLM, SRM, and immunoSEM.
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M ATERIALS AND M ETHODS
PALM/STORM
Antibodies
For SRM, GFP-expressing Borrelia were fixed in 4% paraformaldehyde in PBS prepared as described above for immunofluorescence microscopy and following the protocol previously described (Henriques et al., 2010). The secondary antibody was coupled to Cy3, a molecule with good photoswitching properties, allowing imaging of the bacteria in the super-resolution system. For observation, bacteria were incubated in a blinking-inducing buffer (0.5 mg/mL glucose oxidase, 40 μg/mL catalase, 10% w/v glucose, and 50 mM of β-mercaptoethylamine) in PBS (pH 7.4) to limit the production of reactive oxygen species, preventing fluorophore photobleaching and promoting better fluorophore blinking. The material was observed using a custom built Nikon Ti Eclipse widefield TIRF microscope (Nikon, Japan) using a 100× Apochromat TIRF oil immersion objective (NA 1.49). Particle detection, localization, and visualization were done using the QuickPalm plugin for ImageJ as previously described (Henriques et al., 2010).
The affinity and specificity of some of the monoclonal antibodies against Borrelia antigens used in this study were previously characterized (Kramer et al., 1990). The antibodies used were LA2 (OspA), LA25 (OspB), LA97 (OspC), LA111 (P39), and LA114 (P83/100). Appropriate control experiments based on antibody characterization by Kramer et al. (1990) and removal of the primary antibody as a control for the secondary antibody was performed. Results for all control experiments were as expected and are not shown.
Borrelia Culture B. burgdorferi [strain GCB726, constitutively expressing green fluorescent protein (GFP)] was cultured essentially as described previously (Wallich et al., 1995; Moriarty et al., 2008). Briefly, the spirochetes were cultivated in BSK-H medium (PAN Biotech, Aidenbach, Germany) supplemented with 5% rabbit serum (Cell Concept, Umkirch, Germany) at 30°C. Cells were harvested in the log phase during rapid growth. Cells were enriched by centrifugation followed by resuspension of the pellet in BSK medium (described below).
Immunocytochemistry For wide-field immunofluorescence microscopy, GFPexpressing bacteria were fixed by adding 4% recently prepared paraformaldehyde to the bacteria culture medium. After washing with phosphate buffered saline (PBS), cells were incubated for 30 min in the blocking buffer (albumin 3% in PBS, pH 8) and then incubated with primary antibodies for 30 min. Cells were washed and incubated with Alexa Fluor 546-labeled secondary antibody (Molecular Probes, USA) for 30 min. Afterwards, the bacteria were allowed to adhere onto coverslips and were observed in an inverted Zeiss Axiovert 200 M microscope (Zeiss, Germany) at room temperature. For confocal analysis, the cells were processed as described above and observed in a Zeiss LSM 710 (Zeiss, Germany). For immunoSEM, the protocol established by Sant’ Anna et al. (2005) was used. Cells were treated as described previously for immunofluorescence. After incubating with the primary antibody and the subsequent wash step, the cells were incubated with the secondary antibody conjugated with a 15-nm gold particle (BBInternational, Madison, USA) for 30 min at room temperature. After incubation, the samples were washed and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer. The cells were washed again, dehydrated in an ethanol series (50, 70, 90, and 100%), critical point-dried in a Baltec CPD 030 apparatus (Leica, Germany) and mounted on specimen stubs. The samples were ion-sputtered with a 5-nm carbon layer using a Leica electron microscope (EM) SCD500 high-resolution ion beam coater (Leica, Germany) to avoid charging effects and imaged in a FEI Quanta FEG 450 microscope (Netherlands) at an accelerating voltage of 10 kV. The microscope was operated in backscattered (BSE) mode to image the gold particles at the spirochete surface.
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Image Analysis All images were analyzed and processed in Adobe Photoshop® and FIJI software. Figures were assembled using Adobe Illustrator® software.
RESULTS
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DISCUSSION
During transmission between hosts, members of the Borrelia genus, like other pathogens, modify their surface antigen expression with some lipoproteins being most expressed in the tick and others in the mammalian host as driven by different immune pressures (Templeton, 2004; Palmer et al., 2009; Kumru et al., 2011; Kenedy et al., 2012; Tilly et al., 2013; Wang et al., 2013; Yang et al., 2013; Raffel et al., 2014). The vast majority of the surface proteins are N-terminally anchored lipoproteins, with the few integral membrane proteins in the outer membrane most likely acting as poreforming proteins, e.g. DipA (Thein et al., 2012). The lipoproteins are formed in the cytoplasm, but the mechanisms that control their retention in the periplasm or cause them to be exposed on the surface are yet to be characterized. The most apparent change in protein exposure is OspA-OspC between the tick and the mammalian host (Schwan et al., 1995; Schwan & Piesman, 2000), in which the bacteria temporally down regulate the surface expression of OspA and upregulate that of OspC during transmission. OspA is important for attachment of the bacteria to the host cell and colonization (Schmit et al., 2011), while OspC, a plasminogen receptor, is important during the tick-to-host transition (Önder et al., 2012). To observe the distribution of antigens on the Borrelia surface we used an IFA in WFM. In this technique, fluorescent-conjugated antibodies detect specific target antigens. In indirect IFA, labeling of the antigen is done in a two-step procedure; with a primary, unlabeled antibody that
Localization of Borrelia Outer Surface Proteins
binds to the target antigen, followed by a secondary antibody coupled to a fluorophore that specifically recognizes the Fc portion of the primary antibody. In our study we used a Borrelia strain that constitutively expressed GFP (Moriarty et al., 2008), allowing easy identification of the bacteria. The overlay of the GFP signal from the bacterial cytoplasm and the antibodies on the surface were used to visualize the surface antigens. We used a set of antibodies previously characterized to recognize antigens exposed on the surface of Borrelia: OspA, OspB, OspC, P39, and P83/100 (Kramer et al., 1990). Using WFM, we observed different patterns of distribution of these antibodies. OspA was evenly distributed over the bacteria surface (Figs. 1a–1c), while OspB (Figs. 1d–1f) and OspC (Figs. 1g–1i) presented a punctate distribution of 12 ± 6 and 6.45 ± 4.1 punctae/cell, respectively. P39 (Figs. 1j–1l) and P83/100 (Figs. 1m–1o) also showed a punctuated pattern but much less frequent at 3.1 ± 1.7 and 1.9 ± 0.9 punctae/cell, respectively. This is unexpected considering the fact that both P39 and P83/100 are immunogenic antigens producing high amounts of antibodies during infection in the host (Simpson et al., 1991; Wallich et al., 1995; Roessler et al., 1997; Hauser et al., 1998; Bauer et al., 2001; Miller, 2003). However, one has to keep in mind that an immunological response is not directly proportional to the amount of antigen present. Previously, it was shown that OspA, OspB, and OspC were not exposed exclusively on the Borrelia outer surface, but were also located in the periplasm (Brusca et al., 1991; Cox et al., 1996; Hefty et al., 2002; Kenedy et al., 2012). Using methanol-fixed spirochetes (therefore with permeabilized membranes), a more intense and uniform label was observed by the authors (Cox et al., 1996). This finding was also obtained when cryo-immuno labeling was used, with the authors getting an intense signal for anti-OspA and OspB intra as well as extracellularly (Brusca et al., 1991). So far it is not clear how these lipoproteins can be directed to and from the outer surface. For the next experiments, we limited analysis to the antibodies that presented the best signals— OspA, OspB, and OspC. A key limitation of standard fluorescence microscopy is that when a sample is illuminated, some portion of the exiting light is scattered by the sample, exciting fluorescence in regions of the specimen other than just the focal plane. This generates a “cloud” of diffuse and out-of-focus fluorescence around the center of the beam (Lichtman & Conchello, 2005). Thus, this out-of-focus signal can mask fluorescence from the focal plane, reducing the contrast and the signal-to-noise ratio. A way to mitigate this problem in a WFM is to use computational sectioning. Alternatively, CSLM can be used for optical sectioning as it removes the out-of-focus light using a pinhole in front of the detector. In CSLM the sample is scanned during image acquisition and, using a laser as a source of illumination, a very small spot is focused on the specimen at a specific time, with the excitation and detection in focus at the same spot (Conchello & Lichtman, 2005; Schermelleh et al., 2010). CSLM allowed us to acquire a series of images of thin slices of a thick specimen. Later these images were combined in a stack allowing
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generation of a three-dimensional reconstructed model of the sample. We used a CSLM to image the GFP-expressing Borrelia labeled with anti-OspA (Fig. 2a), anti-OspB (Fig. 2c), and anti-OspC (Fig. 2e). As in the WFM images, OspA presented an even distribution, while OspB and OspC presented a dot-like pattern. Although the images obtained presented better visualization of the antigens’ punctae, the overall resolution was not improved. In conventional light microscopy, the resolution is constrained by the diffraction of light, which causes the signal from a point source to spread as it travels to the detector. The resulting signal is termed the “point spread function” and determines the resolution limit. This is approximately half the wavelength of detected light; around 250–300 nm (Fischer et al., 2011). In the past few years, new technologies, collectively called SRM have been developed to overcome the diffraction limit, bringing the optical resolution to a macromolecular level. Some of these techniques allow resolution down to tens of nanometers, approaching the resolution obtained in the electron microscope (Huang et al., 2010; Schermelleh et al., 2010; Henriques et al., 2011). SRM techniques use the physical properties of fluorescent probes to distinguish the emission from two nearby molecules. One such technique is termed photoactivated localization microscopy/stochastic optical reconstruction microscopy (PALM/STORM). Essentially, one stimulates photoswitching of the fluorophores such that individual fluorophores within the sample are activated in a stochastic manner. Only a small, random sub-population of the fluorophores in the sample will be activated and fluoresce (“on” state) at a point in time and can be imaged as individual particles before they return to the dark (“off” state)—as other fluorophores are activated in turn. This limits the number of fluorophores emitting light at one time and reduces the probability of adjacent fluorophores being imaged simultaneously. Thus, one can record the signals of individual fluorophores throughout a large series of images, as the fluorophores progress through thousands of light–dark cycles. From this, a computational algorithm is able to determine high-precision localization of each fluorophore compiling these localization points to form a superresolution single-image reconstruction of the original sample, where single molecules and cell structure can be highly resolved (Rust et al., 2006; Henriques et al., 2010). Researchers have been applying SRM techniques to resolve the architecture and composition of complex intracellular structures in bacteria (reviewed in Cattoni et al., 2012; Coltharp & Xiao, 2012), e.g., distribution of chemoreceptor clusters, and imaging of subcellular machineries involved in cell shape, growth, and division. We applied PALM/STORM to test if OspA is indeed uniformly localized around the bacterial membrane or rather in focal spots too small to distinguish from each other. The labeling obtained with anti-OspA (Fig. 2b), anti-OspB (Fig. 2d), and anti-OspC (Fig. 2f) was similar to the one obtained with CSLM. Comparing the intensity plot profile of OspC labeling obtained with WFM, CSLM, and SRM (Fig. 3), it is clear that the punctae were more precisely resolved
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Figure 1. Wide-field immunofluorescence microscopy reveals the distribution of five different antigens on GFPexpressing Borrelia burgdorferi: OspA (a,b,c), OspB (d,e,f), OspC (g,h,i), P39 (j,k,l), P83/100 (m,n,o). OspA was more evenly distributed over the bacteria surface, while the other antigens presented a punctate distribution. GFP, green fluorescent protein.
Localization of Borrelia Outer Surface Proteins
Figure 2. Confocal and super-resolution microscopy (PALM/STORM) were used to compare the distribution pattern of OspA (a,b), OspB (c,d), and OspC (e,f). For confocal, GFP-expressing spirochetes were used, and the antigens labeled in red. For super-resolution, the antigen distribution patterns are shown in white. PALM/STORM, photoactivated localization microscopy/stochastic optical reconstruction microscopy; GFP, green fluorescent protein.
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Figure 3. Intensity plot profile of the anti-OspC labeling in wide-field microscopy (a), confocal microscopy (b), and super-resolution microscopy (c). The peaks were more distinct in confocal and in super-resolution microscopy, reaching higher gray values. X-axis correspond to a full bacterial long axis.
in CSLM and SRM, with greater sensitivity and signal contrast, demonstrated by the higher gray values obtained over the background with these techniques. Despite the variable level of Osp expression on the Borrelia bacteria, their presentation as surface proteins facilitates their labeling without extensive sample permeabilization. This high-efficiency labeling contributes to the superior resolution obtained with PALM/STORM. Thus, these SRM techniques would be particularly suitable for co-localization assays of different surface proteins at the single-molecule level. Electron microscopy surpasses the resolution limit of optical microscopy and is the routine approach to resolve cellular architecture at the ultra-structural scale. Immunocytochemistry allows the identification and localization of molecules using EM. Immunogold labeling is frequently used in transmission electron microscopy, especially in resin embedded material and cryo-sections (Griffihs, 1993; Slot &
Geuze, 2007). However, only the antigens exposed in the sections are accessible for the antibodies. Therefore, antigens with few copies on the surface are easily missed. ImmunoSEM can overcome this problem, allowing a “tri-dimensional” analysis of the cell surface, facilitating the identification of cell structure and function. The secondary electron detectors provide high-resolution topographic surface images, while a BSE detector provides visualization of the gold marker, based on the detected mass difference between the gold particles and the biological material (Hermann et al., 1996; Sant’Anna et al., 2005). When we used immunoSEM (Fig. 4), the labeling patterns obtained were largely similar to those obtained in fluorescence microscopy. However, intensity of the labeling was much weaker than those observed in the previous techniques. The punctae normally observed in WFM or CSLM were observed at a lower frequency in immunoSEM, with the gold more
Localization of Borrelia Outer Surface Proteins
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immunoSEM offers the best resolution, the sample preparation and labeling is more challenging than that required for light microscopy-based techniques. All these techniques showed that the antigens located on the surface of Borrelia presented different distribution patterns. Although further analysis are still required, this could be an indication of a different role for each protein in parasite transmission, establishment of the infection, and interaction with host immune cells.
ACKNOWLEDGMENTS We thank Tara Moriarty and George Chaconas for the GFPexpressing Borrelia strain. The work was funded by grants from the German Research Foundation, the Chica and Heinz Schaller Foundation, and the Germany-South Africa initiative from the South African National Science Foundation and the Federal German Ministry for Science and Education to M.M. and F.F. The authors gratefully acknowledge Carl Zeiss for providing the LSM 710 confocal scanning laser microscope during the EMBO practical course Imaging Infection and Immunity (2011); and to Luis Sergio Cordeiro and Mirko Singer for assistance and discussion during the project. L.L. was a recipient of a postdoctoral fellowship from the Cluster of Excellence CellNetworks at the University of Heidelberg and is now supported by a PRONAMETRO fellowship from INMETRO.
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Figure 4. Immuno-gold SEM showing the location of the antigens (arrows) OspA (a), OspB (b), and OspC (c). SEM, scanning electron microscopy.
sparsely distributed over the bacteria surface. Although immunoSEM did not add much information about the antigen distribution in Borrelia spirochetes, one cannot discard this technique to characterize a distribution pattern in another system; in addition this technique may be used to study the presence of antigens in secreted vesicles.
CONCLUSION Several imaging techniques for localization of antigens on the surface of cells are available, ranging from light to electron microscopy, including SRM. Each technique has its advantages and disadvantages, and presents challenges when deciding which technique to use in a particular study. Here we compared different techniques for the localization of antigens on the surface of the spirochete Borrelia. Although
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