Nanoscopic Localization of Surface-Exposed Antigensof 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
1Department of Infectious Diseases – Parasitology, Im Neuenheimer Feld 324, University of Heidelberg Medical School, 69120,Heidelberg, Germany2Laboratory of Microscopy for Life Sciences, Diretoria de Metrologia Aplicada às Ciências da Vida – Dimav, Instituto Nacionalde Metrologia, Qualidade e Tecnologia – Inmetro, 25250-020, Duque de Caxias, Rio de Janeiro, Brazil3Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, UFRJ, 21941-902, Rio de Janeiro, Brazil4Gene Expression and Biophysics Group, Synthetic Biology Emerging Research Area, Council for Scientific and IndustrialResearch, Box 395, Pretoria 0001S, South Africa5Institute 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 throughthe bite of infected Ixodes spp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means ofthe pathogen to escape the vertebrates’ immune system. One strategy employed by Lyme disease spirochetes toevade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins thatis vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterialsurface antigens using different microscopy techniques, from fluorescent wide field to super-resolution andimmunogold-scanning electron microscopy. A fluorescent strain of B. burgdorferi spirochetes was labeled withmonoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is moreevenly distributed over the surface than OspB and OspC that were present as punctate areas.
Lyme disease is a major vector-borne disease in the NorthernHemisphere and is caused by members of the Borrelia burg-dorferi complex (sensu lato) (Radolf et al., 2012). Borreliae areGram-negative spiroform shaped bacteria composed of twomembranes sandwiching a periplasmic flagellum, rangingfrom 10 to 30 µm in length and 0.25–0.5 µm in diameterbelonging to the order of Spirochaetales (Kudryashev et al.,2009, 2011; Radolf et al., 2012). Borrelia spirochetes aretransmitted during the blood meal of infected Ixodes spp. ticksto the skin of the hosts (Frischknecht, 2007; Hovius et al., 2007;Radolf et al., 2012).
Although our knowledge on the pathogenesis of Lymedisease has increased, there remains major interest in unra-veling the interaction of Borrelia with tick vectors and theirmammalian hosts during the course of infection. Thebacterial-host interface depends to some extent on compo-nents present on the cell envelope. The bacterial outermembrane contains lipid-raft microdomains and severalouter surface lipoproteins (OSPs), outer-membrane proteins(OMPs) and OMPs harboring transmembrane-spanningdomains (Anguita et al., 2003; Floden et al., 2011; Gesslbauer
et al., 2012; Önder et al., 2012; Radolf et al., 2012; Thein et al.,2012; LaRocca et al., 2013; Toledo et al., 2014). There havebeen several studies utilizing different microscopy techni-ques in the Borrelia field, such as confocal and two-photonmicroscopy, intravital and video microscopy, freeze-fracture,cryo-immunolabeling, and cryo-electron tomography;however, these studies did not specifically evaluate the dis-tribution 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; Flodenet al., 2011; Crowley et al., 2013; Bockenstedt et al., 2014;Toledo et al., 2014). Despite efforts in recent years to increaseour understanding of the biology of Borrelia spp., little isknown about the function and structure of outer surfaceproteins that mediate interaction with host cells.
To localize and precisely characterize cell outer surfaceantigens, different light microscopy techniques, mainly immu-nofluorescence assays (IFA) in wide-field (WFM) and confocalscanning laser microscopy (CSLM) were used. Moreover, dif-ferent high-resolution techniques, e.g., super-resolution micro-scopy (SRM) and immunogold-scanning electron microscopy(immunoSEM) have gained attention (Goldberg, 2008; Huanget al., 2010). In this study, we will review recent advances inlabeling surface antigens of Borrelia spirochetes employingmonoclonal antibodies and analyzing their expression patternsusing WFM, CSLM, SRM, and immunoSEM.*Corresponding author. [email protected]
Received September 16, 2014; accepted February 11, 2015
AntibodiesThe affinity and specificity of some of the monoclonalantibodies against Borrelia antigens used in this study werepreviously characterized (Kramer et al., 1990). The antibodiesused were LA2 (OspA), LA25 (OspB), LA97 (OspC), LA111(P39), and LA114 (P83/100). Appropriate control experimentsbased on antibody characterization by Kramer et al. (1990) andremoval of the primary antibody as a control for the secondaryantibody was performed. Results for all control experimentswere as expected and are not shown.
Borrelia CultureB. burgdorferi [strain GCB726, constitutively expressinggreen fluorescent protein (GFP)] was cultured essentially asdescribed previously (Wallich et al., 1995; Moriarty et al.,2008). Briefly, the spirochetes were cultivated in BSK-Hmedium (PAN Biotech, Aidenbach, Germany) supplementedwith 5% rabbit serum (Cell Concept, Umkirch, Germany) at30°C. Cells were harvested in the log phase during rapidgrowth. Cells were enriched by centrifugation followed byresuspension of the pellet in BSK medium (described below).
ImmunocytochemistryFor wide-field immunofluorescence microscopy, GFP-expressing bacteria were fixed by adding 4% recently pre-pared paraformaldehyde to the bacteria culture medium.After washing with phosphate buffered saline (PBS), cellswere incubated for 30 min in the blocking buffer (albumin3% in PBS, pH 8) and then incubated with primary anti-bodies for 30 min. Cells were washed and incubated withAlexa Fluor® 546-labeled secondary antibody (MolecularProbes, USA) for 30 min. Afterwards, the bacteria wereallowed to adhere onto coverslips and were observed in aninverted Zeiss Axiovert 200M microscope (Zeiss, Germany)at room temperature. For confocal analysis, the cells wereprocessed as described above and observed in a ZeissLSM 710 (Zeiss, Germany).
For immunoSEM, the protocol established by Sant’Anna et al. (2005) was used. Cells were treated as describedpreviously for immunofluorescence. After incubating with theprimary antibody and the subsequent wash step, thecells were incubated with the secondary antibody conjugatedwith a 15-nm gold particle (BBInternational, Madison, USA)for 30min at room temperature. After incubation, the sampleswere washed and fixed in 2% glutaraldehyde in 0.1M caco-dylate buffer. The cells were washed again, dehydrated in anethanol series (50, 70, 90, and 100%), critical point-dried in aBaltec CPD 030 apparatus (Leica, Germany) and mounted onspecimen stubs. The samples were ion-sputtered with a 5-nmcarbon layer using a Leica electron microscope (EM) SCD500high-resolution ion beam coater (Leica, Germany) to avoidcharging effects and imaged in a FEI Quanta FEG 450 micro-scope (Netherlands) at an accelerating voltage of 10 kV. Themicroscope was operated in backscattered (BSE) mode toimage the gold particles at the spirochete surface.
PALM/STORMFor SRM, GFP-expressing Borrelia were fixed in 4% paraf-ormaldehyde in PBS prepared as described above forimmunofluorescence microscopy and following the protocolpreviously described (Henriques et al., 2010). The secondaryantibody was coupled to Cy3, a molecule with good photo-switching properties, allowing imaging of the bacteria in thesuper-resolution system. For observation, bacteria wereincubated in a blinking-inducing buffer (0.5 mg/mL glucoseoxidase, 40 μg/mL catalase, 10% w/v glucose, and 50 mM ofβ-mercaptoethylamine) in PBS (pH 7.4) to limit the pro-duction of reactive oxygen species, preventing fluorophorephotobleaching and promoting better fluorophore blinking.The material was observed using a custom built Nikon TiEclipse widefield TIRF microscope (Nikon, Japan) using a100× Apochromat TIRF oil immersion objective (NA 1.49).Particle detection, localization, and visualization were doneusing the QuickPalm plugin for ImageJ as previouslydescribed (Henriques et al., 2010).
Image AnalysisAll images were analyzed and processed in Adobe Photo-shop® and FIJI software. Figures were assembled usingAdobe Illustrator® software.
RESULTS AND DISCUSSION
During transmission between hosts, members of the Borreliagenus, like other pathogens, modify their surface antigenexpression with some lipoproteins being most expressed inthe tick and others in the mammalian host as driven bydifferent 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-terminallyanchored lipoproteins, with the few integral membraneproteins in the outer membrane most likely acting as pore-forming proteins, e.g. DipA (Thein et al., 2012). The lipo-proteins are formed in the cytoplasm, but the mechanismsthat control their retention in the periplasm or cause them tobe exposed on the surface are yet to be characterized. Themost apparent change in protein exposure is OspA-OspCbetween the tick and the mammalian host (Schwan et al.,1995; Schwan & Piesman, 2000), in which the bacteriatemporally down regulate the surface expression of OspAand upregulate that of OspC during transmission. OspA isimportant for attachment of the bacteria to the host cell andcolonization (Schmit et al., 2011), while OspC, a plasmino-gen receptor, is important during the tick-to-host transition(Önder et al., 2012).
To observe the distribution of antigens on the Borreliasurface we used an IFA in WFM. In this technique,fluorescent-conjugated antibodies detect specific targetantigens. In indirect IFA, labeling of the antigen is done in atwo-step procedure; with a primary, unlabeled antibody that
2 Leandro Lemgruber et al.
binds to the target antigen, followed by a secondary antibodycoupled to a fluorophore that specifically recognizes the Fcportion of the primary antibody. In our study we used aBorrelia strain that constitutively expressed GFP (Moriartyet al., 2008), allowing easy identification of the bacteria. Theoverlay of the GFP signal from the bacterial cytoplasm andthe antibodies on the surface were used to visualize the sur-face antigens. We used a set of antibodies previously char-acterized to recognize antigens exposed on the surface ofBorrelia: OspA, OspB, OspC, P39, and P83/100 (Krameret al., 1990). Using WFM, we observed different patterns ofdistribution of these antibodies. OspA was evenly distributedover the bacteria surface (Figs. 1a–1c), while OspB (Figs. 1d–1f)and OspC (Figs. 1g–1i) presented a punctate distribution of12± 6 and 6.45±4.1 punctae/cell, respectively. P39 (Figs. 1j–1l)and P83/100 (Figs. 1m–1o) also showed a punctuated pat-tern but much less frequent at 3.1± 1.7 and 1.9± 0.9 punc-tae/cell, respectively. This is unexpected considering the factthat both P39 and P83/100 are immunogenic antigens pro-ducing high amounts of antibodies during infection in thehost (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 immunologicalresponse is not directly proportional to the amount of anti-gen present. Previously, it was shown that OspA, OspB, andOspC were not exposed exclusively on the Borrelia outersurface, 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 permea-bilized membranes), a more intense and uniform label wasobserved by the authors (Cox et al., 1996). This finding wasalso obtained when cryo-immuno labeling was used, with theauthors getting an intense signal for anti-OspA and OspBintra as well as extracellularly (Brusca et al., 1991). So far it isnot clear how these lipoproteins can be directed to and fromthe outer surface. For the next experiments, we limitedanalysis to the antibodies that presented the best signals—OspA, OspB, and OspC.
A key limitation of standard fluorescence microscopy isthat when a sample is illuminated, some portion of theexiting light is scattered by the sample, exciting fluorescencein regions of the specimen other than just the focal plane.This generates a “cloud” of diffuse and out-of-focus fluor-escence around the center of the beam (Lichtman &Conchello, 2005). Thus, this out-of-focus signal can maskfluorescence from the focal plane, reducing the contrast andthe signal-to-noise ratio. A way to mitigate this problem in aWFM is to use computational sectioning. Alternatively,CSLM can be used for optical sectioning as it removes theout-of-focus light using a pinhole in front of the detector. InCSLM the sample is scanned during image acquisition and,using a laser as a source of illumination, a very small spot isfocused on the specimen at a specific time, with the excita-tion and detection in focus at the same spot (Conchello &Lichtman, 2005; Schermelleh et al., 2010). CSLM allowed usto acquire a series of images of thin slices of a thick specimen.Later these images were combined in a stack allowing
generation of a three-dimensional reconstructed model ofthe sample. We used a CSLM to image the GFP-expressingBorrelia 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 OspCpresented a dot-like pattern. Although the images obtainedpresented better visualization of the antigens’ punctae, theoverall resolution was not improved.
In conventional light microscopy, the resolution isconstrained by the diffraction of light, which causes the sig-nal 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 approximatelyhalf 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 thediffraction limit, bringing the optical resolution to a macro-molecular level. Some of these techniques allow resolutiondown to tens of nanometers, approaching the resolutionobtained in the electron microscope (Huang et al., 2010;Schermelleh et al., 2010; Henriques et al., 2011). SRM tech-niques use the physical properties of fluorescent probes todistinguish the emission from two nearby molecules. Onesuch technique is termed photoactivated localizationmicroscopy/stochastic optical reconstruction microscopy(PALM/STORM). Essentially, one stimulates photo-switching of the fluorophores such that individual fluor-ophores within the sample are activated in a stochasticmanner. Only a small, random sub-population of the fluor-ophores in the sample will be activated and fluoresce (“on”state) at a point in time and can be imaged as individualparticles before they return to the dark (“off” state)—as otherfluorophores are activated in turn. This limits the number offluorophores emitting light at one time and reduces theprobability of adjacent fluorophores being imaged simulta-neously. Thus, one can record the signals of individualfluorophores throughout a large series of images, as thefluorophores progress through thousands of light–darkcycles. From this, a computational algorithm is able todetermine high-precision localization of each fluorophorecompiling these localization points to form a super-resolution single-image reconstruction of the original sam-ple, where single molecules and cell structure can be highlyresolved (Rust et al., 2006; Henriques et al., 2010).
Researchers have been applying SRM techniques toresolve the architecture and composition of complex intracel-lular structures in bacteria (reviewed in Cattoni et al., 2012;Coltharp & Xiao, 2012), e.g., distribution of chemoreceptorclusters, and imaging of subcellular machineries involved incell shape, growth, and division.We applied PALM/STORM totest if OspA is indeed uniformly localized around the bacterialmembrane or rather in focal spots too small to distinguishfrom each other. The labeling obtained with anti-OspA (Fig.2b), anti-OspB (Fig. 2d), and anti-OspC (Fig. 2f) was similar tothe one obtained with CSLM. Comparing the intensity plotprofile of OspC labeling obtained withWFM, CSLM, and SRM(Fig. 3), it is clear that the punctae were more precisely resolved
Localization of Borrelia Outer Surface Proteins 3
Figure 1. Wide-field immunofluorescence microscopy reveals the distribution of five different antigens on GFP-expressing 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 moreevenly distributed over the bacteria surface, while the other antigens presented a punctate distribution. GFP, greenfluorescent protein.
4 Leandro Lemgruber et al.
Figure 2. Confocal and super-resolution microscopy (PALM/STORM) were used to compare the distribution patternof OspA (a,b), OspB (c,d), and OspC (e,f). For confocal, GFP-expressing spirochetes were used, and the antigenslabeled in red. For super-resolution, the antigen distribution patterns are shown in white. PALM/STORM, photo-activated localization microscopy/stochastic optical reconstruction microscopy; GFP, green fluorescent protein.
Localization of Borrelia Outer Surface Proteins 5
in CSLM and SRM, with greater sensitivity and signal contrast,demonstrated by the higher gray values obtained over thebackground with these techniques.
Despite the variable level of Osp expression on theBorrelia bacteria, their presentation as surface proteins facil-itates their labeling without extensive sample permeabilization.This high-efficiency labeling contributes to the superior reso-lution obtained with PALM/STORM. Thus, these SRM tech-niques would be particularly suitable for co-localization assaysof different surface proteins at the single-molecule level.
Electron microscopy surpasses the resolution limit ofoptical microscopy and is the routine approach to resolvecellular architecture at the ultra-structural scale. Immuno-cytochemistry allows the identification and localization ofmolecules using EM. Immunogold labeling is frequentlyused in transmission electron microscopy, especially in resinembedded material and cryo-sections (Griffihs, 1993; Slot &
Geuze, 2007). However, only the antigens exposed in thesections are accessible for the antibodies. Therefore, antigenswith few copies on the surface are easily missed. Immuno-SEM can overcome this problem, allowing a “tri-dimen-sional” analysis of the cell surface, facilitating theidentification of cell structure and function. The secondaryelectron detectors provide high-resolution topographic sur-face images, while a BSE detector provides visualization ofthe gold marker, based on the detected mass differencebetween the gold particles and the biological material (Her-mann et al., 1996; Sant’Anna et al., 2005). When we usedimmunoSEM (Fig. 4), the labeling patterns obtained werelargely similar to those obtained in fluorescence microscopy.However, intensity of the labeling was much weaker thanthose observed in the previous techniques. The punctaenormally observed in WFM or CSLM were observed at alower frequency in immunoSEM, with the gold more
Figure 3. Intensity plot profile of the anti-OspC labeling in wide-field microscopy (a), confocal microscopy (b), andsuper-resolution microscopy (c). The peaks were more distinct in confocal and in super-resolution microscopy, reach-ing higher gray values. X-axis correspond to a full bacterial long axis.
6 Leandro Lemgruber et al.
sparsely distributed over the bacteria surface. AlthoughimmunoSEM did not add much information about theantigen distribution in Borrelia spirochetes, one cannot dis-card this technique to characterize a distribution pattern inanother system; in addition this technique may be used tostudy the presence of antigens in secreted vesicles.
CONCLUSION
Several imaging techniques for localization of antigens onthe surface of cells are available, ranging from light to elec-tron microscopy, including SRM. Each technique has itsadvantages and disadvantages, and presents challenges whendeciding which technique to use in a particular study. Herewe compared different techniques for the localization ofantigens on the surface of the spirochete Borrelia. Although
immunoSEM offers the best resolution, the sample pre-paration and labeling is more challenging than that requiredfor light microscopy-based techniques.
All these techniques showed that the antigens located onthe surface of Borrelia presented different distributionpatterns. Although further analysis are still required, thiscould be an indication of a different role for each protein inparasite transmission, establishment of the infection, andinteraction with host immune cells.
ACKNOWLEDGMENTS
We thank Tara Moriarty and George Chaconas for the GFP-expressing Borrelia strain. The work was funded by grantsfrom the German Research Foundation, the Chica and HeinzSchaller Foundation, and the Germany-South Africa initia-tive from the South African National Science Foundationand the Federal German Ministry for Science and Educationto M.M. and F.F. The authors gratefully acknowledge CarlZeiss for providing the LSM 710 confocal scanning lasermicroscope during the EMBO practical course ImagingInfection and Immunity (2011); and to Luis Sergio Cordeiroand Mirko Singer for assistance and discussion during theproject. L.L. was a recipient of a postdoctoral fellowship fromthe Cluster of Excellence CellNetworks at the University ofHeidelberg and is now supported by a PRONAMETRO fel-lowship from INMETRO.
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