Human pathogenic Borrelia spielmanii sp. nov. resist
complement-mediated killing by direct binding of immune
regulators factor H and FHL-1
Pia Herzberger
1, Corinna Siegel
1, Christine Skerka
2, Volker Fingerle
3, Ulrike
Schulte-Spechtel3, Alje van Dam
4, Bettina Wilske
3, Volker Brade
1, Peter F.
Zipfel2,5
, Reinhard Wallich6, and Peter Kraiczy
1*
Running title: Complement resistance of Borrelia spielmanii
1Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Paul-
Ehrlich-Str. 40, D-60596 Frankfurt, Germany
2Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection
Biology, Beutenbergstr. 11a, D-07745 Jena, Germany
3Max von Pettenkofer-Institut für Medizinische Mikrobiologie und Hygiene der Ludwig-
Maximilians-Universität München, D-80336 Munich, Germany
4Department of Medical Microbiology, University Medical Center, P.O. Box 9600, 2300RC
Leiden, The Netherlands
5Friedrich Schiller University, D-07745 Jena, Germany
6Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120
Heidelberg, Germany
*address correspondence and reprints requests to:
Peter Kraiczy
Institute of Medical Microbiology and Infection Control
University Hospital of Frankfurt
Paul-Ehrlich-Str. 40
D-60596 Frankfurt, Germany,
E-mail address: [email protected]
Keywords: Borrelia spielmanii, Complement, Innate immunity, Immune evasion, factor H,
CRASP
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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00532-07 IAI Accepts, published online ahead of print on 16 July 2007
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Abstract 1
B. spielmanii sp. nov. has recently been shown to be a novel human pathogenic 2
genospecies that cause Lyme disease in Europe. In order to elucidate immune evasion 3
mechanisms of B. spielmanii as a means of evading the innate immune system we have 4
compared the ability of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to 5
escape from complement-mediated bacteriolysis. Applying a growth inhibition assay, we show 6
that four B. spielmanii isolates, including PC-Eq17, are serum-resistant whereas a single isolate, 7
PMew, was more sensitive to complement-mediated lysis. All isolates activate complement in 8
vitro as demonstrated by covalent attachment of C3 fragments, however, deposition of later 9
activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew 10
and serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed 11
that all B. spielmanii isolates acquire the host alternative pathway regulators factor H and FHL-1 12
from human serum. Both complement regulators retain their factor I-mediated C3b inactivation 13
activity when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding 14
proteins, BsCRASP-1 and BsCRASP-2, were identified that we estimated to be approximately 15
23 to 25 kDa in size. A further factor H-binding protein, BsCRASP-3, was exclusively found in 16
the tick isolate PC-Eq17. In conclusion, this is the first report describing an immune evasion 17
mechanism utilized by B. spielmanii sp. nov. and it demonstrates capture of human immune 18
regulators to resist complement-mediated killing. 19
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Introduction 20
Lyme disease is a multisystemic disorder caused by species of the Borrelia (B.) 21
burgdorferi sensu lato (s.l.) complex (44). It is the most prevalent vector-borne zoonosis in 22
Eurasia and North America with about 23,000 newly reported clinical cases in 2005 occuring in 23
the USA (8, 44). The B. burgdorferi s.l. complex comprises at least 13 distinct species or 24
genomic groups including B. burgdorferi s.s., B. afzelii, B. garinii, B. japonica, B. valaisiana, B. 25
lusitaniae, B. andersonii, B. bissettii, B. tanukii, B. turdi, B. sinica, B. californiensis and B. 26
spielmanii (39, 40). In Central Europe, B. burgdorferi sensu stricto (s.s.), B. afzelii, and B. 27
garinii are the most important causative agents of Lyme disease, while also B. bissettii, B. 28
lusitaniae, and B. valaisiana appear to be associated with Lyme disease (9, 11, 41, 46). More 29
recently, B. spielmanii (formerly designated as genospecies A14S) spirochetes have been 30
isolated from patients with skin manifestations in the Netherlands, Germany, Denmark, Hungary, 31
and Slovenia (12, 13, 31, 34, 39, 47, 48, 52, 54). 32
The ability of Borreliae to perpetuate in their natural cycle in different reservoir hosts 33
requires an array of strategies to survive in diverse environments and to overcome innate and 34
adaptive immune responses. Certain Lyme disease genospecies are resistant to complement-35
mediated killing in vitro. Most B. afzelii isolates are serum-resistant, B. burgdorferi isolates were 36
classified as moderate serum-resistant, and B. garinii isolates are sensitive to complement-37
mediated killing (21, 22, 29, 30, 49). The distinct pattern of complement susceptibility is 38
consistent with the finding that serum resistant B. afzelii isolates deposit low amounts of late 39
activation products C6 and C5b-9 membrane attack complex on their cell surface. In contrast, 40
serum-sensitive B. garinii isolates show considerably higher amounts of activation products 41
deposited on their surfaces (4, 5, 21). Recent studies have shown that resistance to complement-42
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mediated killing correlates with the ability of serum-resistant B. burgdorferi and B. afzelii 43
isolates to acquire host immune regulators factor H and FHL-1 (1, 17, 23, 51). Protection against 44
complement attack by binding of complement regulators factor H and FHL-1 has also been 45
demonstrated for a number of other important human pathogens such as relapsing fever 46
spirochetes Borrelia hermsii, B. recurrentis and B. duttonii (32, 33, 42), Leptospira interrogans 47
(50), Neisseria gonorrhoeae (37), N. meningitidis (38), Streptococcus pyogenes (3, 20), and S. 48
pneumoniae (14, 18, 19). 49
Factor H and FHL-1, the main immune regulators of the alternative pathway of 50
complement activation are structurally related proteins and composed of several protein domains 51
termed short consensus repeats (SCRs). Factor H is a 150 kDa glycoprotein composed of 20 SCR 52
domains. In contrast, FHL-1 is a 42 kDa glycoprotein and corresponds to a product of an 53
alternatively spliced transcript of the factor H gene and consists of seven SCRs. The N-terminal 54
seven SCRs of both complement regulators are identical with the exception of the C-terminal 55
four amino acids of FHL-1 (26, 55, 56). Both plasma glycoproteins act as co-factors for factor I-56
mediated inactivation of C3b, accelerate the decay of the C3bBb convertase and protect self 57
surfaces from harmful attacks (26, 28, 35, 53). 58
In the present study, we have investigated susceptibility of B. spielmanii isolates obtained 59
from Lyme disease patients as well as type strain PC-Eq17 (tick isolate) to resist complement-60
mediated killing. We demonstrate that serum resistance correlates with the ability to acquire 61
immune regulators factor H and FHL-1. Surface-bound both immune regulators retain their 62
complement regulatory activity for factor I-mediated C3b inactivation. Finally, we have 63
identified three surface-exposed proteins, designated BsCRASP-1 to -3 in B. spielmanii isolates. 64
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Materials and Methods 65
Bacterial isolates and culture conditions. B. spielmanii isolates PC-Eq17 (DSM No. 16813T = 66
CIP 108855T), A14S, PHap, PMai, and PMew, as well as B. burgdorferi isolate LW2, B. afzelii 67
clonal isolate FEM1-D15, and B. garinii isolate G1 were grown at 33ºC for 4 days up to cell 68
densities of 1 × 107
ml-1
in modified Barbour-Stoenner-Kelly (BSK) medium as described 69
previously (23). B. spielmanii strain PC-Eq17 was isolated from Ixodes ricinus (40), A14S, 70
PHap, PMai and PMew are skin isolates from erythema migrans patients (12, 52). The density of 71
spirochetes was determined using dark-field microscopy and a Kova counting chamber (Hycor 72
Biomedical, Garden Grove, CA). 73
74
Human sera, monoclonal and polyclonal antibodies 75
Non-immune human serum (NHS) obtained from 20 healthy human blood donors without known 76
history of spirochetal infections was used as source for factor H. Sera that proved negative for 77
anti-Borrelia antibodies were pooled, stored as aliquots at -80º C and thawed on ice before use. 78
Polyclonal rabbit αSCR1-4 antiserum, polyclonal goat anti-factor H antiserum (Calbiochem) or 79
mAb B22 was used for detection of FHL-1 and factor H (26) and the mAb VIG8 was applied to 80
specifically detect factor H (36). Monoclonal antibody L41 1C11 was used for the detection of 81
flagellin (16). The goat anti-human C3 (dilution 1/1000 for immunofluorescense microscopy and 82
1/2000 for Western blotting) and C6 antibodies (dilution 1/50) were purchased from 83
Calbiochem, and the monoclonal anti-human C5b-9 antibody (dilution 1/10) was from Quidel 84
(San Diego, CA, USA). 85
86
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Expression of recombinant FHL-1 87
Recombinant FHL-1 were expressed in insect cells infected with recombinant baculovirus (27). 88
Briefly, Spodoptera frugiperda cells (Sf9) were grown at 28°C in monolayer cultures in protein-89
free expression medium for insect cells (BioWhittaker, Verviers, Belgium). Adherent Sf9 cells 90
were infected with recombinant virus using a multiplicity of infection of five. The culture 91
supernatant was harvested after 9 days and subjected to affinity purification using Ni-NTA-92
Agarose (Qiagen, Hilden, Germany). 93
94
Serum susceptibility testing 95
Serum susceptibility of B. spielmanii isolates and B. garinii isolate G1 was assessed by applying 96
a growth inhibition assay (21). Briefly, cells grown to mid-logarithmic phase were harvested, 97
washed and resuspended in fresh modified BSK medium. Spirochetes (1.25 × 107) diluted in a 98
final volume of 100 µl in BSK medium containing 240 µg ml-1
phenol red were incubated with 99
50% normal human serum or 50% heat-inactivated human serum in microtiter plates for 10 days 100
at 33 °C (Costar, Cambridge, MA). Modified BSK medium instead of human serum was 101
included in all assays as growth control. Growth of spirochetes was monitored by measuring the 102
indicator color shift of the medium at 562/630nm using an ELISA reader (PowerWave 200, Bio-103
Tek Instruments, Winooski, VT). For calculation of the growth curves the Mikrowin Version 3.0 104
software (Mikrotek, Overath, Germany) was used. 105
106
Serum adsorption experiments 107
Spirochetes grown to mid-log phase, harvested by centrifugation (5000 × g; 30 min; 4°C) were 108
resuspended in 500 µl veronal-buffered saline (VBS, supplemented with 1 mM Mg2+
, 0.15 mM 109
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Ca2+
, 0.1% gelatin, pH 7.4) and after cell counting a portion of 2 × 109 organisms were 110
sedimented by centrifugation. The cell sediment was then resuspended in 750 µl NHS 111
supplemented with 34 mM EDTA and incubated for 1 h at room temperature with gentle 112
agitation. After three washes with PBSA (0.15 M NaCl, 0.03 M phosphate, 0.02% sodium azide, 113
pH 7.2) containing 0.05% Tween-20, the proteins bound to the Borreliae were eluted by 114
incubation with 0.1 M glycine-HCl, pH 2.0, for 15 min. The bacterial cells were sedimented by 115
centrifugation (14,000 × g; 20 min; 4°C), and the proteins in the supernatant were analyzed by 116
SDS-PAGE and Western blotting. 117
118
SDS-PAGE, ligand affinity blot and western blot analysis 119
Borrelial cell lysates (15 µg) were subjected either to 10% Tricine-SDS-PAGE under reducing 120
conditions or to 12.5% Laemmli SDS-PAGE under non-reducing condition and transferred to 121
nitrocellulose membranes (Protran BA83, Whatman, Dassel, Germany) as previously described 122
(24). Briefly, after transfer of proteins onto nitrocellulose, nonspecific binding sites were 123
blocked using 5% (w/v) dried milk in TBS (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 0.1% 124
Tween 20) for 1 h at room temperature. Subsequently, membranes were rinsed four times in 125
TBS and incubated at 4°C overnight with NHS or culture supernatants containing recombinant 126
FHL-1 protein. After four washings with 50 mM Tris-HCl pH 7.5, 150mM NaCl, 0.2% 127
Tween20 (TBST), membranes were incubated for 1 h with a 1/500 dilution of mAb B22 128
recognizing the N-terminal region SCR5 of factor H and FHL-1 or with mAb VIG8 (undiluted) 129
directed against the C-terminus of factor H. Following four washes with TBST, membranes were 130
incubated with a secondary peroxidase-conjugated anti-mouse IgG antibody at a final dilution of 131
1/1000 (DakoCytomation, Glostrup, Denmark) for 1 h at room temperature. Detection of bound 132
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antibodies was performed using 3,3',5,5'-Tetramethylbenzidine as substrate. 133
134
Immunofluorescence assay for detection of complement proteins. 135
For indirect immunofluorescence assays, spirochetes were grown to mid-log phase, harvested by 136
centrifugation at 5000 × g for 30 min, washed and resuspended in 300 µl PBS. Spirochetes (6 × 137
106) were incubated with either 25% NHS or 25% heat-inactivated NHS (hiNHS) for 30 min at 138
37°C with gentle agitation, washed three times with PBS containing 1% BSA (PBS-BSA) and 139
resuspended in 100 µl of the same buffer. Aliquots of 10 µl were then spotted on microscope 140
slides and allowed to air dry overnight. After fixation with 100% acetone, slides were dried for 141
1h at room temperature and incubated for 1h in a humidified chamber with antibodies against 142
complement components C3 (dilution of 1/1000), C6 (dilution of 1/50), C5b-9 (dilution of 1/10), 143
factor H and FHL-1 (dilution of 1/20). Following three washes with PBS-BSA, the slides were 144
incubated for 1h at room temperature with 1:500 dilutions of appropriate Alexa 488-conjugated 145
secondary antibodies (Molecular Probes, Leiden, The Netherlands). Slides were then washes 146
three times with PBS-BSA and mounted in ProLong Gold antifade reagent containing the 147
DNA-binding dye DAPI (Molecular Probes) before being sealed with cover slips. Slides were 148
visualized at a magnification of × 1000 using an Olympus CX40 fluorescence microscope. 149
150
Functional assay for cofactor assay of cell-bound factor H and FHL-1 151
Cofactor activity of factor H and FHL-1 bound to borrelial cells was analyzed by measuring 152
factor I-mediated conversion of C3b to iC3b. Spirochetes (5 x 107) were incubated with either 153
factor H (Calbiochem, Darmstadt, Germany) or recombinant FHL-1 protein (3 µg/ml each) for 154
1h at room temperature with gently agitation. After extensive washing with PBS, C3b 155
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(Calbiochem) (10 µg/ml) and factor I (Calbiochem) (50 µg/ml) were added to the cells and the 156
mixture was incubated for 30 min at 37°C. Cells were sedimented by centrifugation at 14.000 × 157
g for 10 min and the supernatants were mixed with sample buffer. The samples were then 158
subjected to SDS-PAGE under reducing conditions and transferred onto a nitrocellulose 159
membrane. C3b degradation products were evaluated by detection of α´-chain cleavage 160
fragments of 68, 46 and 43 kDa by using polyclonal goat anti-C3 IgG at a final dilution of 161
1/2000) (Calbiochem) and a secondary peroxidase-conjugated anti-goat IgG antibody 162
(DakoCytomation, Glostrup, Denmark). For detection, 3,3',5,5'-Tetramethylbenzidine was used 163
as substrate. 164
165
In situ protease treatment of native spirochetes 166
Whole cells of B. spielmanii isolate A41S were treated with proteases using a modification of a 167
method described previously (7). Briefly, freshly harvested cells were washed twice with PBS-168
MgCl and, after centrifugation at 5000 rpm for 10 min, the sedimented spirochetes were 169
resuspended in 100 µl of this buffer. To 2 × 108 intact borrelial cells (final volume of 0.5 ml) 170
proteinase K in distilled water (Sigma-Aldrich, Deisenhofen, Germany) or trypsin in 0.001 N 171
HCl (Sigma-Aldrich) was added to a final concentration of 12.5 to 100 µg/ml. Following 172
incubation for 2h at room temperature, proteinase K was terminated by adding 5 µl 173
phenylmethylsulfonyl fluoride (Sigma-Aldrich) (50 mg/ml in isopropanol) and trypsin was 174
inhibited by adding 5 µl phenylmethylsulfonyl fluoride (Sigma-Aldrich) and 5 µl pefabloc SC 175
(Roche Diagnostic, Germany). The cells were then washed twice with PBS-Mg, resuspended in 176
20 µl of the same buffer and lysed by sonication 5 times using a Branson B-12 sonifier 177
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(Heinemann, Schwäbisch Gmünd, Germany). Aliquots (10 µl) were separated using 10 % 178
Tricine-SDS-PAGE. 179
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Results 180
Serum resistance of B. spielmanii isolates. 181
To assess serum sensitivity of B. spielmanii, human isolates A14S, PHap, PMai, and PMew, as 182
well as tick isolate PC-Eq17 were incubated in 50% NHS or in 50% heat-inactivated NHS 183
(hiNHS) for up to 10 days. Applying a growth inhibition assay (21) different levels of serum 184
susceptibility were observed among the five B. spielmanii isolates. Isolates A14S, PC-Eq17, 185
PHap, and PMai are more resistant to complement-mediated lysis than isolate PMew as 186
demonstrated by a delay in growth in the presence of complement (Figure 1). In contrast, growth 187
of the serum-sensitive B. garinii isolate G1 which was used as control was strongly inhibited 188
under the same conditions as compared to the five B. spielmanii isolates. Applying heat-189
inactivated NHS instead of NHS, growth of borrelial isolates was not affected and led to a 190
continuous decrease of adsorbance. 191
192
Detection of deposited complement component C3, C6 and C5b-9 on the surface of B. 193
spielmanii. 194
Since the B. spielmanii isolates exhibit differential serum susceptibility, we analyzed deposition 195
of complement component C3 and late activated complement components C6 and C5b-9 196
(terminal complement complex, TCC) on the surface of isolates A14S, PC-Eq17, and PMew. 197
After incubation of spirochetes in NHS or heat-inactivated serum, binding of complement 198
components was analyzed by immunofluorescence microscopy. C3 bound strongly to all isolates 199
tested (Figure 2) while the intensity of C6 and C5b-9 binding varies markedly between the 200
resistant isolates A14S and PC-Eq17 and the moderate serum-resistant isolate PMew. A mixed 201
population containing few strongly labeled cells and many weakly stained cells was observed for 202
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isolates A14S and PC-Eq17 (Figure 2). In contrast, a higher number of cells of isolate PMew was 203
positive for both C6 and C5b-9. Analysing serum-sensitive B. garinii isolate G1, a strong 204
fluorescent staining for C3, C6, and C5b-9 was observed for the majority of the cells. We noticed 205
that spirochetes covered complement components exhibited blebs of various sizes, showed signs 206
of lysis and alterations in cell morphology (Figure 2). To identify all spirochetes in a given field 207
couterstaining with DAPI was performed. Interestingly, blebs exhibited a very strong 208
fluorescence signal whereas a number of complement-positive cells stained negative with DAPI 209
indicating that the borrelial DNA was highly concentrated in blebs and that DAPI-negative 210
spirochetes might represent cell ghosts. As a control, spirochetes incubated with heat-inactivatd 211
NHS (hiNHS) showed no fluorescent staining. Taken together, B. spielmanii isolates differ in 212
their ability to activate complement as previously demonstrated for B. burgdorferi s.s., B. afzelii, 213
and B. garinii (5, 21, 49). 214
215
Binding of complement regulators to B. spielmanii. 216
To assess the mechanism of complement resistance in B. spielmanii, we determined binding of 217
human complement regulators factor H and FHL-1 to the surface of borrelial cells. To this end B. 218
spielmanii isolates A14S, PC-Eq17, PHap, PMai, and PMew were incubated with NHS as a 219
natural source for factor H and FHL-1 that was supplemented with EDTA to prevent 220
complement activation. After serum incubation the wash and elute fractions were separated by 221
SDS-PAGE and subjected to Western blotting with anti-FHL-1 and anti-factor H antibodies. All 222
tested strains of B. spielmanii bound factor H and FHL-1 although with distinct capacities 223
(Figure 3). 224
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Binding of complement regulators to isolates A14S and PC-Eq17 was further analyzed by 225
immunofluorescence microscopy. Following incubation with NHS-EDTA, factor H was evenly 226
distributed on the surface of isolate A14S and PC-Eq17, suggesting that the factor H interacting 227
proteins were homogeneously expressed and distributed on the borrelial surface (Figure 4). As 228
negative control, serum-sensitive B. garinii isolate G1 was incubated with NHS-EDTA under 229
identical condition and stained for factor H detection. As expected, no fluorescent cells could be 230
detected. For detection of the spirochetes in a given microscopic field, the same slides were 231
incubated with mounting medium containing DAPI (right panels). 232
233
Cell bound complement regulators displays cofactor activity. 234
We next determined if factor H and FHL-1 bound to the surface of B. spielmanii are functionally 235
acting as cofactor for the serum protease factor I in cleaving C3b. Spirochetes were first 236
incubated with factor H or FHL-1, and after washing of the spirochetes, factor I and C3b were 237
added. After incubation the cleavage products were detected by SDS-PAGE and Western 238
blotting. As shown in Figure 5, surface-bound factor H and FHL-1 retained cofactor activity as 239
indicated by the presence of representative C3b inactivation products (68, 46 and 43 kDa α´-240
chain). Borrelial cells preincubated in buffer alone with factor I did not promote cleavage of C3b 241
indicating that the studied B. spielmanii isolates lack endogenous C3b degradation activity or 242
cofactor activity for cleavage. Thus, binding of factor H and FHL-1 to the surface of B. 243
spielmanii enhances their complement control capacity. 244
245
Identification of the borrelial protein(s) interacting with factor H and FHL-1. 246
To identify the bacterial protein(s) involved in factor H and FHL-1 binding, cell extracts from 247
isolates A14S, PC-Eq17, PHap, PMai, and PMew were separated by a 10% Tris/Tricine gel, 248
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transferred to nitrocellulose and incubated with either NHS as source for factor H or recombinant 249
FHL-1. Following incubation with factor H or FHL-1 specific antibodies, a dominant factor H 250
and FHL-1 binding protein of approximately 24.9 kDa, termed BsCRASP-1, and a second 251
borrelial protein of 22.1 kDa, BsCRASP-2 was identified (Figure 6). BsCRASP-1 was present in 252
all B. spielmanii isolates studied while expression of BsCRASP-2 was restricted to isolates 253
A14S, PMai, and PMew. A stronger binding of BsCRASP-2 to factor H and FHL-1 as compared 254
to BsCRASP-1 was detected in isolates A14S and PMai. Tick isolate PC-Eq17 expressed an 255
additional factor H binding protein of approximately 15 kDa, termed BsCRASP-3 (Figure 6A). 256
Cell extracts from either serum-resistant isolates B. burgdorferi LW2 and B. afzelii FEM1-D15, 257
expressing up to five CRASP proteins, and a serum-sensitive, CRASP negative isolate B. garinii 258
G1 served as controls. 259
260
Surface exposure and protease sensitivity of BsCRASP-1 261
To assess surface exposition of BsCRASP-1 and BsCRASP-2 in situ, spirochetes were treated 262
with proteinase K and trypsin to analyse accessibility of proteins to proteolytic degradation. 263
Treatment with proteinase K at concentrations up to 50 µg/ml resulted in the complete 264
elimination of factor H binding by isolate A14S indicating that BsCRASP-1 and, in particular 265
BsCRASP-2 were highly susceptible of to proteolytic cleavage (Figure 7). Lower concentrations 266
of proteinase K led to partial inhibition of factor H binding. Similarly, treatment with trypsin 267
resulted in decreased binding of factor H and FHL-1 indicating that BsCRASP-1 and BsCRASP-268
2 are more resistant to trypsin digestion (Figure 7). The limited accessibility of OspA to 269
proteinase K is reminiscent of previous reports using various B. burgdorferi strains (7). In 270
contrast, OspB was highly sensitive even at low concentrations ≤12.5 µg/ml to both proteases. 271
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As a negative control, membranes was also screened with anti-FlaB antiserum. As expected, 272
according to the periplasmic localization to the FlaB protein in the Borrelia, FlaB was not 273
degraded by either of the two proteases. These analyses demonstrate that BsCRASP-1 and 274
BsCRASP-2 are exposed at the outer surface and thus is potentially available in vivo to interact 275
with factor H and FHL-1. 276
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Discussion 277
Lyme disease spirochetes employ a broad range of strategies to survive and persist in the 278
human host. It is far from being completely understood by which sophisticated means Borreliae 279
overcome the host´s destructive immune defence, however, immune escape has recently attracted 280
particular attention. Several studies demonstrated that serum-resistant B. burgdorferi s.s. and B. 281
afzelii isolates acquire host immune regulators factor H and FHL-1 (1, 23, 32, 45). The primary 282
objective of the present study was to analyze the molecular mechanism(s) by which B. spielmanii 283
sp. nov. evades the innate immune system of the human host. Here, we demonstrate to our 284
knowledge for the first time that B. spielmanii strains isolated from Lyme disease patients resist 285
complement-mediated killing. The complement resistant phenotype appears to be accomplished 286
by acquiring immune regulators factor H and FHL-1. 287
B. spielmanii formerly designated as A14S-like spirochetes was recently delineated as 288
novel human pathogenic genospecies of the B. burgdorferi sensu lato-complex by multilocus 289
sequence analysis (40, 43). In Central Europe, B. spielmanii is closely associated with garden 290
and hazel dormice as the main reservoir hosts but not with mice or voles. Furthermore, sequence 291
analysis and polymorphic DNA fingerprinting distinguish these isolates from other Lyme disease 292
genospecies (39). First reports on the prevalence of B. spielmanii in ticks and mammals 293
contribute to a focal distribution of this genospecies at distinct areas in Central Europe, i.e. The 294
Netherlands, France, Germany, Denmark, Czech Republic, Slovenia, and Hungary (10, 13, 31, 295
39, 47, 52). Although B. spielmanii has frequently been detected in infected nymphal and adult 296
ticks, a limited number of isolates were isolated from Lyme disease patients with EM (12, 13, 31, 297
52). Here we present data on the serum susceptibility of the largest collection of human B. 298
spielmanii isolates. Previous studies on complement resistance of B. burgdorferi s.l. 299
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demonstrated that borrelial isolates differ substantially with regard to their sensitivity to human 300
serum as B. afzelii are mainly serum-resistant, whereas the majority of B. burgdorferi s.s. isolates 301
were classified as moderate serum-resistant, and isolates of the genospecies B. garinii were 302
frequently classified as serum-sensitive (5, 21, 49). Growth inhibition assays revealed that the 303
majority of B. spielmanii strains displayed a serum-resistant phenotype similar to B. afzelii 304
isolates. An earlier study of Lyme disease spirochetes provided evidence that differences in 305
serum susceptibility correlate with differential deposition of late complement components C6 306
and C5b-9 or the terminal complement complex (TCC) (21). Isolates A14S, PC-Eq17, and 307
PMew show deposition of various amounts of late complement activation products on their 308
surfaces and represent a mixed population of positively and negatively stained cells. In contrast, 309
higher amounts of surface-bound complement activation products were identified on isolate 310
PMew, suggesting that complement deposition contributes to limited growth. It is important to 311
note, however, that deposition of late activated products is regulated at the level of C3 312
implicating that factor H, the main immune regulator of the alternative pathway, plays an 313
important role. 314
Recent studies have shown that the potential of B. burgdorferi s.s. and B. afzelii isolates 315
to bind factor H and FHL-1 strictly correlates with the serum resistance (1, 17, 23, 32, 51). All 316
B. spielmanii isolates were able to acquire immune regulators factor H and FHL-1 from human 317
serum and both complement regulators were uniformly distributed on the borrelial cell surface. 318
This distribution suggests that factor H/FHL-1 interacting proteins on the spirochetal surface 319
bind to the host complement regulators and thereby efficiently inhibit the formation of the C3 320
convertase. It is of interest that both immune regulators when bound to the borrelial surface 321
maintain their cofactor activity for factor I-mediated C3b inactivation. Degradation of C3b is 322
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observed upon incubation with factor H and/or FHL-1 but not without complement regulators, 323
indicating that B. spielmanii isolates lack endogenous C3b cleaving activities. 324
Previous studies showed that B. burgdorferi s.s. and B. afzelii isolates express surface 325
exposed lipoproteins, collectively termed complement regulator-acquiring surface proteins 326
(CRASPs), which specifically interact with serum factor H and/or FHL-1 (24). Expression of 327
distinct CRASP proteins on the microbial surface has been implicated in persistence and survival 328
of spirochetes in the human host. Furthermore, complementation of serum-sensitive borrelial 329
strains with either BbCRASP-1, BbCRASP-2 or the factor H-binding OspE protein increases or 330
completely restore resistance to human serum (2, 6, 15) emphasizing a role of these lipoproteins 331
in evading the innate immune system of the human host. In this study B. spielmanii was shown 332
to express several, most likely two surface-exposed factor H and FHL-1 binding proteins, 333
designated BsCRASP-1 and BsCRASP-2. Applying ligand affinity blotting, BsCRASP-1 334
displayed a stronger binding intensity to FHL-1 as compared to factor H, which is reminiscent of 335
BbCRASP-1, BaCRASP-1, and BbCRASP-2 (Figure 6). Interestingly, BsCRASP-2 of A14S and 336
PMai showed a stronger binding capacity to both immune regulators than the dominant 337
BsCRASP-1 protein. Thus, it it tempting to speculate that differential expression levels of 338
BsCRASP-1 and BsCRASP-2 or sequence difference potentially account for their relative 339
binding properties to factor H and FHL-1 are involved in complement susceptibility of individual 340
B. spielmanii isolate. Moreover, tick isolate PC-Eq17 expressed an additional factor H-binding 341
protein, termed BbCRASP-3 comparable to that of the factor H binding BbCRASP-3 to 342
BbCRASP-5 proteins of B. burgdorferi and BaCRASP-4 and -5 of B. afzelii (23). Therefore, we 343
hypothesize that BsCRASP-3 belongs to the factor H-binding Erp protein family (25, 45). 344
Current investigations are underway to isolate and functional characterize BsCRASP-1 from 345
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distinct B. spielmanii isolates to provide further insights into the molecular interaction of factor 346
H and FHL-1 with BsCRASP-1 as well as their role in the virulence and pathogenesis of B. 347
spielmanii in humans. 348
Due to the limited number of isolated B. spielmanii strains and the fragmentary information 349
available yet, one could only speculate on their prevalence in humans (12, 34). It has been shown 350
by Richter et al. (39) that the garden and hazel dormice appear to be the main reservoir host for 351
B. spielmanii. Therefore, the geographical distribution of this genospecies is more restricted than 352
that of the other human pathogenic Lyme disease spirochetes. As the garden dormice have 353
adapted to distinct ecotonal habitats their distribution is somewhat restricted to particular 354
landscapes. Due to the exclusive host-pathogen relationship of the dormice-associated B. 355
spielmanii spirochetes together with the specific adaptation of their reservoir host(s) it is to be 356
expected that this genospecies should be rarely detected in human biospies. 357
Association of B. spielmanii with garden dormice might reflect an adaptation to the individual 358
hosts´complement system as previously shown for certain Lyme disease spirochetes, especially 359
to avian-associated B. garinii spirochetes (30). The fact that most B. spielmanii isolates exhibit 360
resistance to human complement might argue for their competence to infect and survive in the 361
human host. However, it has also been shown that B. spielmanii is transmitted more efficiently to 362
dormice as compared to B. afzelii spirochetes indicating that humans are not the preferred host 363
for B. spielmanii (39). Studies on the prevalence of B. spielmanii in patients with Lyme disease 364
who reside at the same geographical area where infected dormice are abundant will help to 365
elucidate the potential of this genospecies to cause clinical manifestations other than EM. 366
In summary, this study demonstrates that B. spielmanii acquire immune regulators, factor H and 367
FHL-1, to the borrelial surface, thereby contribute to resistance against complement-mediated 368
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lysis. The characterization of BsCRASP-1 represents an important step forward and will expand 369
our understanding of the molecular basis on the pathogenesis of this novel Lyme disease 370
spirochete. 371
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Acknowledgements
We thank Christa Hanssen-Hübner and Jane Herrlich for skillful and expert technical assistance,
and Brian Stevenson for critical reading of the manuscript. This work was funded by the
Deutsche Forschungsgemeinschaft DFG, Project Kr3383/1-1.
This work forms part of the MD thesis of P.H.
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Figure Legends
Figure 1: Serum susceptibility among B. spielmanii isolates.
Growth inhibition assay was applied to investigate serum susceptibility to human serum of B.
spielmanii isolates A14S (A), PC-Eq17 (B), PMai (C), PHap (D), PMew (E) and serum-sensitive
B. garinii isolate G1 (F). Spirochetes were incubated in either 50% NHS or 50% heat-inactivated
NHS over a cultivation period of 10 days at 33°C, respectively. Color changes were monitored
by measurement of the absorbance at 562/630 nm. All experiments were performed three times
in which each test was done fivefold with very similar results. For clarity only data from
representative experiments are shown. Error bars represent ± SEM.
Figure 2: Deposition of complement components C3, C6 and C5b-9 on the surface of B.
spielmanii.
Complement components deposited on B. spielmanii isolates A14S, PC-Eq17, and PMew as well
as serum-sensitive B. garinii isolate G1 were detected by indirect immunofluoresecence
microscopy. Spirochetes were incubated with either 25% NHS of hiNHS for 30 min at 37°C with
gentle agitation and bound C3, C6, C5b-9 were analyzed with specific antibodies against each
component and appropriate Alexa 488-conjugated secondary antibodies. For visualization of the
spirochetes in a given microscopic field, the DNA-binding dye DAPI was used. The spirochetes
were observed at a magnification of 100 × objective. The data were recorded via a DS-5Mc CCD
camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Panels shown are
representative for at least 20 microscope fields.
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Figure 3: Binding of complement regulator factor H and FHL-1 by different B. spielmanii
isolates.
B. spielmanii isolates A14S, PC-Eq17, PMai, PHap, and PMew incubated in NHS-EDTA were
extensively washed with PBSA containing 0.05% Tween-20 and bound proteins were eluted
using 0.1 M glycin (pH 2.0). Both the last wash (w) and the eluate (e) fractions obtained from
each strain were separated in a non-reducing conditions 12.5 % SDS-PAGE gel, transferred to
nitrocellulose and probed with either mAb VIG8 specific for SCR 20 of factor H or mAb B22 for
SCR5 of factor H and FHL-1.
Figure 4: Detection of factor H/FHL-1 on the surface of intact cells.
Serum-resistant isolates A14S, PC-Eq17, and serum-sensitive B. garinii isolate G1 were
incubated with NHS-EDTA. Bound proteins were detected by immunofluorescence microscopy
after incubation with mAb B22 for factor H and FHL-1. For counterstining, the DNA-binding
dye DAPI was used to identify cells in a given microscopic field. The spirochetes were observed
at a magnification of 100 × objective. The data were recorded via a DS-5Mc CCD camera
(Nikon) mounted on an Olympus CX40 fluorescence microscope. Panels shown are
representative for at least 20 microscope fields.
Figure 5: Analysis of functional activity of factor H and FHL-1 bound to B. spielmanii.
Cofactor activity of factor H and FHL-1 bound to spirochetes was analyzed by measuring factor
I-mediated conversion of C3b to iC3b. B. spielmanii isolates PC-Eq17, A14S, PMai, PHap, and
PMew were incubated with either factor H (A) or purified FHL-1 (3 µg/ml each) (B) for 60 min
at RT. For control purposes cells were incubated without factor H. After extensive washing with
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PBS, C3b (Calbiochem, Darmstadt, Germany) (10 µg/ ml) and factor I (Calbiochem, Germany)
(50 µg/ ml) were added and the mixture was incubated for 30 min at 37°C. Subsequently, the
probes were boiled for 5 min, subjected to 12.5 % SDS-PAGE and transferred onto a
nitrocellulose membrane. The various C3b degradation products were visualized by Western
blotting using a polyclonal goat anti-human C3 antiserum (Calbiochem). As a positive control,
purified factor H or FHL-1 (50ng each) was added to the reaction mixture and as negative
control C3b and factor I were incubated in the absence of complement regulators.
Figure 6: Identification of factor H and FHL-1 binding proteins expressed within B.
spielmanii isolates.
Protein extracts (15 µg each) obtained from B. burgdorferi s.s. LW2, B. afzelii FEM1-D15, B.
garinii G1, and B. spielmanii PC-Eq17, A14S, PMai, PHap, and PMew were separated by 10%
Tris-Tricine SDS-PAGE and transferred to nitrocellulose. The membranes were incubated with
either NHS as source for factor H (A) or FHL-1 (B) and binding of the proteins was detected
with mAb VIG8 specific for SCR 20 of factor H or polyclonal serum specific for SCRs 1-4 of
FHL-1. For detection of FlaB as a control, mAb L41 1C11 was applied. The identified CRASP
proteins are indicated on the right and the mobility of the marker proteins (in kilodalton) is
indicated on the left.
Figure 7: Protease treatment affects surface expression of native BsCRASP-1 and
BsCRASP-2 and binding to factor H and FHL-1.
B. spielmanii A14S cells were incubated with the indicated concentrations of proteinase K or
trypsin. After 2 h of incubation cells were lysed by sonication and each protein lysate was
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35
subjected to 10% Tris/Tricine SDS-PAGE. BsCRASP-1 and BsCRASP-2 were identified using
recombinant FHL-1 and polyclonal antibody αSCR1-4 (dilution 1/1000) specific for the N-
terminus of FHL-1/factor H by ligand affinity analysis (A). Flagellin (FlaB) was detected with
mAbs L41 1C11 (dilution 1/1000) by Western blotting (B). A part of a Coomassie-stained 10 %
Tris-Tricine SDS-polyacrylamide gel was shown to demonstrated susceptibility of OspA, and
OspB to proteolytic degradation (C).
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0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
1 2 3 4 5 6 7 8 9 10
NHS
hiNHS
0,00
0,50
1,00
1,50
2,002,50
3,00
3,50
4,00
4,50
1 2 3 4 5 6 7 8 9 10
Time (d)
NHS
hiNHS
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
4,50
1 2 3 4 5 6 7 8 9 10
Time (d)
NHS
hiNHS
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
1 2 3 4 5 6 7 8 9 10
Time (d)
NHS
hiNHS
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
1 2 3 4 5 6 7 8 9 10
Time (d)
NHS
hiNHS
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.5
4.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.5
4.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.5
4.0
Figure 1
A B
Ab
so
rban
ce a
t 56
2/6
30n
mA
bso
rban
ce a
t 56
2/6
30n
m
Ab
so
rban
ce a
t 56
2/6
30n
m
Ab
so
rban
ce a
t 56
2/6
30n
m
C D
E F
0,00
0,50
1,00
1,50
2,00
2,50
3,00
1 2 3 4 5 6 7 8 9 10
NHS
hiNHS
Ab
so
rban
ce a
t 56
2/6
30n
m
0.5
1.0
1.5
2.0
2.5
3.0
0
Time (d)
0Ab
so
rban
ce a
t 56
2/6
30n
m
Time (d)
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.5
4.0
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C3 DAPI C6 DAPI C5b-9 DAPI
NHS
hiNHS
NHS
hiNHS
NHS
hiNHS
NHS
hiNHS
A14S
PC-Eq17
PMew
G1
Figure 2 revised
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Figure 3
A14S
PC
-Eq
17
PH
ap
PM
ai
PM
ew
w e w e w e w e w e
Factor H
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Figure 4 revised
FH DAPI
A14S
PC-Eq17
G1ACCEPTED
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po
s.co
ntr
ol
A14S
PC
-Eq
17
PH
ap
PM
ai
PM
ew
Figure 5
α´-chain
β -chainα´-68 kDa
α´-46 kDa
α´-43 kDa
α´-chain
β -chainα´-68 kDa
α´-46 kDa
α´-43 kDa
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ - + - + - + - + - + -
C3b
FI
FH/FHL-1
A
B
neg
. co
ntr
ol
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B.b
urg
do
rferi
LW
2B
.afz
eliiF
EM
1-D
15
B.g
ari
nii
G1
B.s
pie
lman
ii A
14S
B.s
pie
lman
ii P
C-E
q17
B.s
pie
lman
ii P
Hap
B.s
pie
lman
ii P
Mai
B.s
pie
lman
ii P
Mew
B.b
urg
do
rferi
LW
2B
.afz
eliiF
EM
1-D
15
B.g
ari
nii
G1
B.s
pie
lman
ii A
14S
B.s
pie
lman
ii P
C-E
q17
B.s
pie
lman
ii P
Hap
B.s
pie
lman
ii P
Mai
B.s
pie
lman
ii P
Mew
Figure 6
25
20
kDa
BsCRASP-1BsCRASP-2
BsCRASP-3
FlaB
A B
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Figure 7
FlaB
FlaBOspB
OspA
BsCRASP-1BsCRASP-2
0 12.5 25 50 100 0 12.5 25 50 100 µg/ml
Proteinase K Trypsin
A
B
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