ribosome display of combinatorial an tibody libraries de...

Title: Ribosome Display of Combinatorial Antibody Libraries Derived From Mice 1

Immunised With Heat-Killed Xylella fastidiosa and the Selection of MopB-Specific 2

Single-Chain Antibodies. 3

4

Running Title: Anti-Xylella fastidiosa Antibody Libraries 5

Authors: Armaghan Azizi1, Arinder Arora2,3, Anatoliy Markiv1, David J. Lampe4, Thomas 6

Miller2 and Angray S. Kang1,5,* 7

8

1University of Westminster, School of Life Sciences, 115 New Cavendish St, London, 9

W1W 6UW, UK. 10

2University of California, Riverside, Department of Entomology, Riverside, CA 92521, 11

USA. 12

3University of New Mexico, Department of Biology, Albuquerque, NM 87131, USA. 13

4Duquesne University, Department of Biological Sciences, 600 Forbes Ave., Pittsburgh, 14

PA 15282, USA. 15

5Queen Mary University of London, Barts & The London School of Medicine and 16

Dentistry, Institute of Dentistry, London, E1 2AT, UK. 17

18

*Correspondent 19

Dr Angray S Kang 20

Queen Mary University of London, Barts & The London School of Medicine and 21

Dentistry, Institute of Dentistry, London E1 2AT, UK. 22

Tel: +44(0)207 882 7158, Fax: +44(0)207 882 7137, Email: [email protected] 23

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.07807-11 AEM Accepts, published online ahead of print on 10 February 2012

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Abstract 24

Pierce's disease is a devastating lethal disease of Vitus vinifera grapevines caused by 25

the bacterium Xylella fastidiosa. There is no cure for Pierce’s disease and control is 26

achieved predominantly by suppressing transmission of the glassy winged sharpshooter 27

insect vector. We present a simple robust approach for the generation of panels of 28

recombinant single chain antibodies against the surface exposed elements of X. 29

fastidiosa that may have potential use in diagnosis and/or disease transmission blocking 30

studies. In vitro combinatorial antibody ribosome display libraries were assembled from 31

immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed 32

X. fastidiosa. The libraries were used in a single round of selection against an outer-33

membrane protein MopB, resulting in the isolation of a panel of recombinant antibodies. 34

The potential use of selected anti-MopB antibodies was demonstrated by the successful 35

application of the 4XfMopB3 antibody in an ELISA, western blot and 36

immunofluorescence assay. These immortalised in vitro recombinant single chain 37

antibody libraries generated against heat killed X. fastidiosa are a resource for the 38

Pierce’s disease research community that may be readily accessed for the isolation of 39

antibodies against a plethora of X. fastidiosa surface exposed antigenic molecules. 40

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Introduction 42

Xylella fastidiosa includes a group of closely related pathogens each affecting a 43

specific plant target. One subspecies affects grapevines, but little else, another 44

subspecies affects almonds and grapevines and still another affects only oleanders (1). 45

The subspecies Xylella fastidiosa subsp. fastidiosa (Xanthomonadales: 46

Xanthomonadaceae) is the pathogen of the grapevine (14) an economically important 47

crop in California. This bacterium is the causative agent of Pierce’s disease (PD) of 48

grapevines and is vectored by the leaf hopper Homalodisca vitripennis (Germar) 49

(Hemiptera: Cicadellidae) (formerly H. coagulate) also known as the glassy-winged 50

sharp-shooter (GWSS) (39). The precise pathogenic mechanisms resulting in disease 51

are not clearly understood, however the ability of the pathogen to colonize in both the 52

xylem of the grapevine and in the foregut of the GWSS vector is well established (11). 53

The X. fastidiosa genome has been sequenced and comparative analysis has provided 54

valuable information of genes, metabolic pathways and potential virulence factors that 55

may be involved in pathogenicity (43). 56

Investigating the interfaces between the plant-pathogen-insect interactions may 57

reveal sites for molecular interventions that could confer resistance or reduce 58

transmission of the pathogen. It is possible to predict and explore the surface exposed 59

components that may potentially play a role in bacterial virulence and/or be involved in 60

attachment or biofilm formation in either the plant or arthropod. The function of surface 61

displayed targets may also be probed using lectins or polyclonal antibodies (29). 62

However, if monoclonal antibodies tools were available they would allow a more 63


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detailed study of the assembly, distribution, function and the role in plant-vector 64

interactions. 65

The desire for the efficient rapid generation of monoclonal antibodies to 66

biologically important protein antigens over the past two decades has driven the 67

development of a range of in vitro technologies based around combinatorial 68

immunoglobulin repertoire cloning (26), high throughput screening, phage display (4, 69

27, 37) and ribosome display (22, 23, 36). For ribosome display immunoglobulin mRNA 70

transcripts can be isolated from antibody producing cells converted to cDNA and 71

assembled in vitro to create linear DNA templates encoding libraries of single chain 72

fragment variable antibodies (scFv’s), which can be transcribed in vitro into mRNA that 73

lack a termination codon. Translation of mRNA templates in the absence of a stop 74

codon results in each ribosome stalling at the last codon and retaining the correctly 75

folded antibody polypeptide and the mRNA, creating tri-partite antibody-ribosome-76

mRNA complexes (ARMs) as shown in Figure 2. The ARMs library can then be affinity 77

enriched to select the desired ARM for recovery. The essence of the approach is the 78

linking of the recognition to the replication functions, i.e., linking the phenotype to the 79

genotype (27). Recombinant protein display technologies allows the ready access to 80

genetically encoded ligands or receptors against defined targets, an approach 81

pioneered more than 25 years ago (44). 82

The aim of this study was to create an antibody resource from mice immunized 83

with X. fastidiosa that would permit the isolation of recombinant antibodies against 84

surface accessible molecules on X. fastidiosa and to validate the approach by selecting 85

scFv’s against the outer membrane protein MopB (8). 86


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87

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Materials and methods 89

Materials 90

Bacterial strains and plasmids 91

The oligonucleotide primers used in this study are listed in Table S1 in the supplemental 92

material. The pSANG10-3F vector (42) was provided by Prof. John McCafferty 93

(University of Cambridge) and used with XL1-Blue E. coli strain (Stratagene) for 94

plasmid construction and BL21(DE3) or Rosetta gamiB (DE3) strain E. coli (Novagen, 95

UK) for recombinant protein expression. Dr. David Lampe provided the pMALc2x_mopB 96

plasmid. Plasmid pAHAHis for scFv bacterial cytoplasmic expression was based on 97

modified pET32a (unpublished). The plasmid pFab1-PhoA-H (47) was provided by 98

Prof. Masataka Takekoshi (Tokai University, Japan). Anti-NANP repeat monoclonal 99

antibody 2A10 (38, 49) VH/VL sequence was provided by Prof. Anthony A. James 100

(University of California Irvine). Xylella fastidiosa Temecula1 strain was prepared at the 101

University of California Riverside in the laboratory of Prof. Thomas A. Miller. 102

Methods 103

MopB 104

The complete amino acid sequence of X. fastidiosa Temecula1 strain (NCBI 105

NP_779898.1) MopB was submitted to web based protein prediction programs, SIG-106

Pred: signal peptide prediction (http://bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html ) 107

(6) and to the PRED TMBB which is a Hidden Markov Model method, capable of 108

predicting transmembrane beta-strands of outer membrane proteins of gram negative 109


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bacteria (http://biophysics.biol.uoa.gr/PRED-TMBB/) (2, 3, 45) to identify the signal 110

peptide and the putative surface exposed domains respectively. The predicted encoded 111

mature (MopB 354 amino acids (aa)) and surface exposed domain (MopB 182 aa) 112

were PCR amplified using primers designed to include NdeI (5′) site encoding an in-113

frame methionine start codon and NotI (3′) site encoding in-frame triple alanine (primers 114

are listed in Table S1. The MopB encoding inserts were PCR amplified using 115

pMALc2x_mopB template. The PCR products were restricted with NdeI and NotI, and 116

inserted into pSANG 10-3F vector to produce proteins with in-frame C-terminal 117

sequence (AAASA(H)6 KLDYKDHDGAYKDHDIAYK(D)4K. The molecular weight and 118

isoelectric points were predicted using ExPASy bioinformatics resource portal 119

(http://web.expasy.org/compute_pi/) (20). The plasmids were used to transform XL1-120

Blue E. coli cells, and confirmed by DNA sequencing. 121

Bacterial expression of recombinant MopB 122

Plasmids encoding recombinant MopB were transformed into BL21 (DE3) E. coli cells 123

(Novagen) and grown in 200 mL of LB medium containing 30 µg/mL kanamycin at 37 °C 124

with shaking at 250 rpm until optical density of 0.4-0.6 at 600 nm. Protein expression 125

was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, final 126

concentration 0.3 mM), incubation resumed for 3 h before harvesting by centrifugation 127

5000 rpm, at 4 °C for 20 min (using Sorvall Super T 21 bench top centrifuge, with SL-128

250T rotor). Cell pellets were frozen and stored at -80 °C. 129

Recombinant MopB extraction 130


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The cell pellet obtained from 200 mL culture was re-suspended in 5 mL denaturing lysis 131

buffer (6 M guanidine hydrochloride, 10 mM Tris–HCl, 500 mM NaCl, 10 mM imidazole, 132

pH 8.0). The cells were lysed by sonication on ice (6×30 sec pulses), in an ultrasonic 133

cell disruptor; and centrifuged at 16,000 rpm for 45 min to pellet the cellular debris. The 134

supernatant containing soluble material was retained for the subsequent purification 135

using Ni2+-NTA agarose affinity resin. One mL of nickel chelating agarose slurry (G-136

Biosciences) was applied to a 10 mL column and equilibrated with 5 mL wash buffer (8 137

M Urea, 10 mM Tris–HCl, 100 mM NaH2PO4, pH 8.0). The supernatant containing the 138

hexa-histidine tagged protein was loaded onto the resin. The flow through was collected 139

and the resin washed with 5 mL of wash buffer (8 M Urea, 10 mM Tris–HCl, 100 mM 140

NaH2PO4, pH 6.3). The bound recombinant MopB fusion proteins were eluted in 5 x 1 141

mL of elution buffer (8 M Urea, 10 mM Tris–HCl, 100 mM NaH2PO4, pH 4.5). Fractions 142

were analyzed by SDS–PAGE (27) using 12 % Tris-Glycine gels and visualized using a 143

fast microwave assisted Coomassie stain technique described elsewhere (34). Protein 144

concentrations were estimated by the Bradford dye binding assay (6). 145

Mouse immunisation 146

X. fastidiosa Temecula strain was grown in PD3 media (13) 100 mL in a 0.5 L flask at 28 147

°C at 180 rpm for 10 days until OD 0.5 at 600 nm. The bacteria were pelleted and re-148

suspending in 100 mL PBS (2x) and the final pellet wet weight determined, then re-149

suspended in PBS at 10 mg/mL w/v and 1 mL aliquot prepared. An aliquot of the X. 150

fastidiosa was incubated at 28 °C (viability, positive control) and the remainder heat 151

treated at 55 °C for 1 h and checked for viability by plating on PD3 agar and incubated 152

for 15 days at 28 °C, the remainder of the bacterial vials were frozen at -80 °C. Once it 153


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had been confirmed that no viable bacteria were present in the heat-treated ampules, 154

aliquots were sent to ProSci Inc (Poway, CA, USA) for immunisation of Balb/c mice 155

(n=5). Initial immunisation (0.5 mL/mouse) was followed by boosting at weeks 4 and 8 156

and 12. Test bleeds were taken a week after the third boosting for ELISA and dot blot 157

analysis. Following testing, mice were boosted and three days later underwent a 158

splenectomy, each spleen was placed in 10 mL of TRIzol (Invitrogen, CA, USA) for use 159

in RNA isolation. 160

MopB ELISA 161

Recombinant MopB 10 µg/mL or BSA 10 μg/mL in PBS pH 7.4; 0.1 mL/well were used 162

to coat 96-well plate (NUNC) at 4 °C overnight and blocked with 0.2 mL of 2 % BSA in 163

PBS at room temperature for 1 h. Polyclonal sera from mice (n=5) previously immunized 164

with whole heat-killed X. fasitidosa were diluted to 1/20,000 in PBS containing 0.05 % 165

Tween-20 (PBS-T) added in duplicate to the wells (0.1 mL/well) and incubated at 37 ºC 166

for 2 h. The wells were washed with PBS-T and 0.1 mL/well of rabbit anti-mouse 167

antibody (SIGMA-Aldrich) diluted 1:20,000 in 1 % BSA in PBS-T was added and 168

incubated at 37 °C for 2 h. The plates were washed as before and 0.1 mL/well goat-anti-169

rabbit IgG alkaline phosphatase conjugate (SIGMA-Aldrich, diluted 1:40,000 in 1% BSA 170

in PBS-T) was added and incubated at 37 °C for 2 h. The plates were washed as before 171

with an additional final wash with PBS alone and developed with p-nitrophenyl 172

phosphate 1mg/mL in 0.2 M Tris buffer pH 8.0, 0.1 mL/well at 37 °C for 30 min. The 173

absorbance was measured at 405 nm on an ELISA plate reader (VersaMax, Molecular 174

Devices, UK). 175


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Bacterial dot blot 176

Strips of nitrocellulose ~5 mm wide were spotted with 5 µL of 5 % w/v skimmed milk 177

powder in PBS, 5 µL of bacterial suspension of E. coli and 5 µL X. fastidiosa Temecula1 178

strain, allowed to air dry, blocked with 1 % BSA in PBS and incubated with immune sera 179

(bleed 3) diluted 1/20,000 in PBS-T with 1 % BSA for 1 h. Following washing with PBS-180

T incubated with rabbit anti-mouse alkaline phosphatase antibody (Sigma-Aldrich), 181

diluted 1:20,000 in 1 % BSA in PBS-T for 1 h, washed as before and developed with 182

0.02 %, 5-bromo-4-chloro-3-indolylphosphate (BCIP) and 0.03 % nitroblue tetrazolium 183

(NBT) in 10 mL 100 mM Tris, 100 mM NaCl, 5 mM MgCl2, 0.05 % Tween 20, at pH 9.5. 184

Mouse immunoglobulin library assembly 185

The mouse spleens, each in 10 mL TRIzol (Invitrogen) were shipped on dry ice and 186

stored at -20 °C prior to processing. Upon thawing at room temperature each sample 187

was macerated using an IKA T8 Ultra Turrrax homogeniser and processed following the 188

instructions for total RNA isolation (TRIzol, Invitrogen). The resulting RNA pellets were 189

dissolved in 50 µL RNase-free water and analyzed using a NanoDrop (Thermo-Fisher, 190

UK) to determine relative purity, RNA concentration and stored at -20 °C until required. 191

First strand cDNA was synthesised from ~1 µg of total RNA using ProtoScript® First 192

Strand cDNA Synthesis Kit (M-MuLV) Reverse Transcriptase (New England Biolabs) 193

with Oligo-dT29VN for light chains and MVHLink2 for the heavy chains (all primers are 194

listed in Table 1) following the instructions provided. For antibody library construction 195

the PCR primers were based on published sequences (26, 28) with minor modifications 196

(Table S1). The primers were designed to introduce in-frame NcoI and NotI restriction 197


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sites to the 5’ end of the VL and to the 3’ end of the VH respectively as shown in Figure 198

1. Members of the light chain families were individually amplified by PCR using Oligo-199

dT29VN cDNA template using combinations of MVKF1-7 with MVKR. Members of the 200

heavy chain families were individually amplified by PCR using MVHLink2 cDNA 201

templates using combinations of MVHF1-10 and MVHR1.1. The amplified light chain 202

products were purified and pooled and an aliquot subject to another round of PCR 203

amplification using MVKFLink and MVKRLink to introduce part of the T7 site and Kozak 204

sequence (32) on the 5’ end and an overlap extension on the 3’ end to facilitate joining 205

to the variable heavy chain libraries. The amplified heavy chain products were 206

processed and modified in a similar manner using primers MVHFLink and MVHR1.1. 207

The modified variable light and heavy chain products were combined and amplified 208

using MVKFLink and MVHR1.1. A synthetic mouse kappa constant (MKC) domain 209

(optimised for E.coli codon usage, synthesised by Epoch Biolabs, Tx USA) was 210

amplified using MKNotCF and MKRev. The MKC was joined to the VL-link-VH 211

combinations by a PCR overlap extension reaction using MVKFLink and MKRev (Figure 212

1). Finally the PCR product encoding all the variable light chains and heavy chain 213

combinations were amplified with primers RDT7 and MKRev to produce the DNA 214

encoding the anti-X. fastidiosa immunoglobulin scFv libraries. The initial PCR 215

amplification reactions were performed at 50 °C annealing temperature with 30 cycles 216

and subsequent library assembly step used 16 cycles with Taq DNA polymerase and 20 217

pmol of each primer pair per reaction. DNA fragments were resolved by gel 218

electrophoresis on 2 % (w/v) agarose gels. DNA isolation from agarose gels was carried 219


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out following Qiagen DNA Gel-purification kit instructions. In total five combinatorial 220

scFv antibody DNA libraries were constructed. 221

Antibody ribosome display 222

To select for single-chain antibodies a modified eukaryotic ribosome display (23) was 223

used as outlined in Figure 2. The PCR generated DNA library of antibody coding genes 224

derived from mouse #4 were expressed in a coupled rabbit reticulocyte lysate system 225

(Promega; TNT quick coupled transcription/translation system). An in vitro coupled 226

transcription/translation was set up in a 0.5 mL tube as follow: to 40 µL TNT T7 Quick 227

Master Mix, 2 µL of DNA library (0.1-1.0 μg), 1 µL (1 mM) methionine, 1 µL DNA 228

enhancer and 6 µL water were added and incubated at 30 °C for 90 min. Then, 6 µL of 229

DNase I, RNase free (10,000 u/mL, Roche) was added and incubated for 20 min at 30 230

°C (to degrade input DNA). To select specific antibody fragments a 0.5 mL PCR tube 231

was coated with 0.1 mL of 10 µg/mL of recombinant truncated MopB in PBS at room 232

temperature for 1 h, washed with PBS and blocked with 100 μL of molecular biology 233

grade BSA in PBS (10 mg/mL) (New England Biolabs) for 1 h. The 234

translation/transcription mixture, containing the antibody-ribosome-mRNA complexes 235

was added to MopB-coated tube and incubated on ice for 1 h. The tube was washed by 236

filling once with PBS (0.5 mL) and decanting and carefully removing the residual liquid 237

using a sterile pipette tip. The retained RNA was recovered using RNeasy (350 µL) mini 238

protocol following the manufacturer’s instructions and used in One-step™ (Qiagen) RT-239

PCR with MVKFLink and MKRD2 primers to amplify the antibody encoding DNA in a 50 240

µL reaction, using 6 µL RNA template and 5 µL of each primer (20 pM). The mixture 241

was cycled as follows: 50 ˚C for 30 min, 95 ˚C for 15 min, then 35 cycles 94 ˚C for 30 242


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sec, 52 ˚C for 30 sec, 72 ˚C for 1 min, 72 ˚C for 5 min and finally kept at 8 ˚C. The RT-243

PCR product from a single round of ribosome display was purified by agarose gel 244

electrophoresis and cloned into pCR®IITOPO vector (TOPO TA Cloning® Kits, 245

Invitrogen) according to manufacturer’s instructions and used to transform XL1-Blue 246

competent cells. Plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside 247

(X-gal) were used to select for disruption of the β-galactosidase. Four white colonies 248

were randomly selected and inoculated in 10 mL of LB media with 100 μg/mL of 249

carbenicillin; plasmid DNA was isolated from E. coli using standard procedures using a 250

QIAprep® Spin Miniprep Kit (Qiagen) following the manufacturer’s instructions, and 251

DNA sequenced at the core facility at The Wolfson Institute for Biomedical Research, 252

University College London. 253

ScFv expression 254

A scFv antibody cytoplasmic expression vector with a carboxyl terminal tag peptide 255

sequence derived from influenza hemagglutinin (HA) (Asp Val Pro Asp Tyr Ala Ser: 256

DVPDYAS) followed by a hexa-histidine tag designated pAHAHis was constructed 257

based on pET32a. The recovered single chain antibody encoding sequences were 258

inserted into pAHAHis plasmid as NcoI/NotI fragments for cytoplasmic expression in 259

Rosetta gami B (DE3) E. coli. The pAHAHis encoding the scFv transformed into Rosetta 260

gami B (DE3) E. coli cells (Novagen), inoculated into 200 mL of LB medium containing 261

100 μg/mL of carbenicillin and 34 μg/mL of chloramphenicol, grown at 37 °C with 262

shaking at 250 rpm until optical density reached 0.4-0.6 at 600 nm. Protein expression 263

induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, final 264

concentration 1 mM) and cells then incubated for 20 h at 20 °C with shaking at 250 rpm. 265


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Cells were harvested in 4 x 50 mL tubes by centrifugation 5000 rpm, 4 °C for 20 min 266

(using Hettich Universal 320 Centrifuge, 1617 rotor). Cell pellets were frozen and stored 267

at -80 °C before undergoing further processing. The cell pellet was re-suspended in 3 268

mL of native lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 0.15 Triton 269

X100, pH 8.0 and 250 µL lysozyme at 1 mg/mL). The cells were lysed by sonication on 270

ice as described earlier and centrifuged at 18000 rpm for 45 min. The supernatant 271

containing the soluble fraction was retained for affinity purification. One mL of nickel 272

chelating resin suspension (G-Biosciences, Mo, USA) was packed in a micro spin 273

column and equilibrated with 600 µL native lysis buffer. The supernatants containing the 274

hexa-histidine-tagged recombinant antibodies were loaded onto the equilibrated 1 mL 275

Ni2+-NTA spin column and centrifuged (using Hettich Universal Centrifuge 320R, 1689-276

A rotor) at 1600 rpm for 5 min. The flow through was collected and the Ni2+-NTA column 277

washed two times with 600 µL of wash buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM 278

imidazole, pH 8.0) at 2900 rpm for 2 min. The bound recombinant antibodies were 279

eluted into 100 µL fractions using 500 µL of elution buffer (20 mM Tris-HCl, 500 mM 280

NaCl, 500 mM imidazole, pH 8.0) at 2900 rpm for 2 min and the purified scFv stored at 281

4 °C. 282

Functional assays 283

Recombinant scFv binding to MopB was examined by western blot, ELISA and 284

Immunofluorescence analysis. The scFv antibody binding to recombinant MopB, was 285

detected via the DVPDYAS peptide tag using a secondary recombinant antibody scFv 286

with a (Gly4Ser)3 linker based on VH and VL sequences of the mouse monoclonal 287

antibody 26/9 anti-DVPDYAS (12) fused to alkaline phosphatase fusion (PhoA) (46) 288


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(anti-HA scFv-AP) in both the ELISA and western blot. ELISA with either full-length 289

mature, truncated MopB was as described earlier with a modification using 5 % w/v milk 290

in TBS containing 0.1 % NaN3 as a negative control. Purified antibodies, selected scFv 291

antibody for MopB and negative control anti-NANP repeat scFv based on 2A10 mAb 292

diluted 1 in10 using TBS/NaN3 were incubated for 6 h at 8 ˚C. After washing with 293

TBST/NaN3, anti-HA scFv-AP antibody (1:10 diluted in 1 % w/v milk/TBS NaN3) was 294

added and incubated overnight at 8 ˚C, washed as before and developed with p-295

nitrophenyl phosphate as described earlier. 296

For the western blot recombinant MopB proteins were resolved by SDS-PAGE as 297

described earlier and blotted onto polyvinylidene fluoride (PVDF) membrane, blocked in 298

5 % w/v milk in TBS at room temperature for 1 h, then incubated with the scFv 299

4XfMopB3 as the primary antibody (diluted 1:10 in 1 % w/v milk/TBS) for 6 h at 8 ˚C. 300

After washing with TBST incubated with the secondary antibody anti-HA scFv-AP 301

antibody (1:10 diluted in 1 % w/v milk/TBS, 0.1 % NaN3) at room temperature for 1 h on 302

a rocking platform followed by washes as before and a final rinse with TBS (5 min) prior 303

to the addition of substrate BCIP/NBT as described earlier. 304

Heat-killed X. fasitidosa was air dried, on glass slides, blocked with 2 % w/v milk in PBS 305

for 30 min. Purified scFv antibodies (selected scFv antibody for MopB and negative 306

control scFv 2A10 antibody) were diluted 1 in 10 with 1 % w/v milk/PBS. The slides 307

were incubated with either the XfMopB3 (anti-MopB), 2A10 (non-specific) or no scFv for 308

1 h. After washing with PBS anti-poly-histidine-FITC conjugate (AbCam, UK) (diluted 309

1:10,000 in 1 % w/v milk/PBS) was added. The slides were incubated for 1 h at ambient 310

temperature in the dark. Slides were washed with PBS and viewed under oil immersion 311


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on a fluorescence microscope Axioscop 50 (Zeiss). Images were captured using a CCD 312

camera (PowerShot digital camera, Canon) and AxioVision software (Zeiss). 313

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Results 315

MopB bioinformatic analysis 316

The X. fastidiosa Temecula1 MopB protein sequence downloaded from NCBI data 317

base was analyzed using SIG-PRED to predict the putative signal sequences, Met1-318

Ala36 and Met15-Ala36 were identified (Figure 3A). The β-barrel transmembrane 319

domain identification using PRED TMBB was determined to be between Trp43-Tyr177. 320

A two dimensional representation of the result show the exposed loops with the last 321

loop being the longest (Figure 3B). The recombinant mature and truncated MopB 322

expressed in pSANG 10-3F were predicted to have a pI of 6.36, Mwt of 42.98 kD and pI 323

of 6.79 and Mwt of 24.64 kDa respectively. 324

Expression and purification of the full length mature and truncated MopB protein 325

The gene encoding full length mature Met-Ala36-Asn389 and truncated Met-Ala36-326

Gly252 MopB from X. fastidiosa were PCR amplified and cloned into the pSANG10-3F 327

vector and verified by DNA sequencing. Proteins analyzed by SDS–PAGE, revealed 328

abundant full length and truncated MopB at 3 h at 37 °C post induction with IPTG. The 329

SDS–PAGE indicated proteins with an apparent molecular weight of about 43 kDa for 330

full length MopB and 25 kDa for truncated MopB (Figure 4A,B). The recoveries of 331

recombinant mature and truncated MopB proteins were estimated to be approximately 332

50 mg/L of culture. 333

Sera ELISA and Dot Blot 334

The ELISA with immobilised recombinant truncated MopB protein and 1/20,000 dilution 335


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of sera from bleed 3 of the mice immunized with whole heat-killed X. fastidiosa indicated 336

that 4 out of 5 mice produced antibodies that recognise recombinant surface exposed 337

portion of MopB with little or no cross-reactivity with the control protein BSA (Figure 5A). 338

The immune sera from bleed 3 was also evaluated in a dot blot against whole intact E. 339

coli and X. fastidiosa Temecula strain (Figure 5B). Mouse sera #1, 2, 4 and 5 had clear 340

antibody binding to X. fastidiosa at a dilution of 1/20,000 whereas mouse #3 serum did 341

not. The antibody activity against milk proteins was absent or very weak in mouse sera 342

#2 and #4. 343

Mouse Immunoglobulin Library Assembly and Ribosome Display 344

The recovery and purity of total RNA isolated for each of the mice varied with a range of 345

200-50 µg as shown in Table S2 in the supplemental material, providing ample material 346

for the subsequent cDNA reactions. The immunoglobulin libraries were assembled as 347

outlined in the schematic, the PCR amplification results and steps of the library 348

construction using mouse 4# spleen derived cDNA are shown in Figure 1. Mouse Vk6 349

and VH4 and 8 were present at lower levels, all other immunoglobulin families were 350

readily amplified. The final DNA template encoding the library flanked by a T7 site and a 351

synthetic mouse kappa constant chain was used in an in vitro ribosome display with a 352

single selection step with truncated MopB as outlined in Figure 2. The affinity enriched 353

mRNA’s were amplified by RT-PCR and cloned into pCR®IITOPO vector. From 354

approximately one hundred white colonies four were randomly selected to prepare 355

plasmids for sequencing. Three with confirmed intact in-frame VL and VH combinations 356

were sub-cloned into pAINFHis for scFv expression and characterization (4XfMopB1-3). 357

Analysis of the variable region sequences (Table S3 in the supplemental material) 358


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shows that different gene segments of the antibody sequences are present in the 359

selected recombinant antibody clones. Antibody 4XfMopB3 scFv detected recombinant 360

mature and truncated MopB in the western blot and ELISA (Figure 6) whereas clone 361

4XfMopB1 detected MopB in the western blot only (data not shown). Furthermore the 362

Xf4MopB3 scFv was used to image X. fastidiosa by immunofluorescence microscopy as 363

shown in Figure 7. GenBank accession numbers for 4XfMopB1 and 4XfMopB3 scFv’s 364

are JQ606804 and JQ606805 respectively. 365

366


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Discussion 367

Investigations of plant-pathogen and vector-pathogen interfaces in PD would be 368

greatly facilitated with the availability of highly specific monoclonal antibodies. Although 369

it is possible to use conventional hybridoma technology (30) to obtain monoclonal 370

antibodies and then clone and reassemble the corresponding antigen binding site 371

genes, recombinant antibody display technology is a more cost-effective option for the 372

non-specialized laboratory, requiring general molecular biology skills/reagents and 373

techniques to build and access antibody libraries. Moreover, genetically encoded scFv’s 374

are amenable to further engineering to optimize the desired characteristics, such as 375

affinity, specificity (21), formation of multimers (25) or as fluorescent molecules (34, 35). 376

When using a complex immunogen (i.e., whole bacteria) a mixture of antibodies 377

against a mixture of targets (on the cell surfaces) (5), results in combinatorial complexity 378

and invariably requires de-convolution at some point (50). When initially validating a 379

technique, a simpler approach is to anchor one component and search through a 380

complex mixture of potential partners. In this study we assembled combinatorial libraries 381

of recombinant antibodies from mice immunized with whole heat-killed X. fastidiosa. To 382

de-convolute the mixture we chose a surface exposed molecule MopB as bait. 383

Although, the role of MopB on X. fastidiosa is not known, it is an abundant surface 384

protein and may potentially be a target for bacterial agglutination via engineered 385

recombinant antibodies. Earlier reports describing attempts to isolate natural MopB and 386

to make recombinant MopB protein had met with limited success (7, 8). An in silico 387

analysis of the target identified key features that assisted in the design of an appropriate 388

recombinant bait molecule for use in antibody selection. MopB has a characteristic 389


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signal leader sequence Met15-Ala36 with a classical cleavage motif Ala Ser Ala, 390

followed by a transmembrane β-barrel (with surface exposed loops) Trp43-Tyr177 and a 391

periplasmic domain Arg178-Asn389 (Figure. 3). The protein is extremely toxic to the 392

heterologous host E. coli when inserted into the outer membrane, either using its natural 393

leader sequence or an alternative such as OmpA. A leaderless construct results in 394

proteins not traversing or inserting in the inner membrane but being retained in the 395

bacterial cytoplasm and aggregating due to the hydrophobic nature of the trans-396

membrane β-sheets. Full length mature (43 kDa) and truncated MopB (25 kDa) 397

recombinant proteins were both efficiently expressed without a leader sequence. 398

Extraction using denaturing conditions released the recombinant MopB into the soluble 399

fraction. SDS-PAGE analysis tracking the expression and purification revealed that the 400

recombinant proteins were enriched and the major products eluting from the affinity 401

resin corresponded to the predicted sizes for the mature and truncated MopB (Figure 4), 402

with yield of ~50 mg/L of bacterial culture. 403

Sera from the X. fastidiosa immune mice #1, 2, 4 and 5 diluted 1/20,000 had 404

activity against truncated MopB protein and X. fastidiosa in the ELISA and immuno-dot 405

blot respectively. Mouse #3 serum produced the lowest signal in both assays (Figure 406

5A, B). The very high dilution used in both the evaluations of the sera suggests a very 407

robust response to the whole bacterial antigen preparation. 408

Five antibody libraries were assembled following the steps outlined in Figure 1, 409

and mouse 4# DNA antibody library was used in the subsequent selection procedure. 410

After a single enrichment step using ribosome display, scFv specific for MopB were 411

readily isolated. This rapid enrichment may be a common feature of antibody libraries 412


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constructed from intentionally immunized or naturally immune sources and is in 413

agreement with our findings when using antibody ribosome display to access human 414

scFv’s against HIV-1 gp120 constructed from an HIV-1 positive donors (48), where only 415

a single round of selection was required. 416

One scFv 4XfMopB3 recognized the mature and truncated recombinant MopB 417

proteins in both the ELISA and western blot (Figure 6) and heat killed X. fastidiosa by 418

IFA (Figure 7) suggesting that the epitope is a non-conformational and possibly one of 419

the three predicted loops exposed on the surface of the bacteria. These results provide 420

evidence that immunization with whole heat-killed X. fastidiosa clearly induces 421

antibodies against surface exposed antigenic molecules such as MopB, indicating that 422

the mild heat treatment used to kill the bacteria, preserved the surface accessible 423

protein immunogenicity. We speculate that the immune sera may also contains 424

antibodies against other putative surface exposed molecules possibly associated with 425

attachment in either the GWSS or the V. vinifera grapevine (31). 426

The in vitro anti-X. fastidiosa scFv libraries generated in this study and the strategy 427

for preparing recombinant putative membrane proteins provides approaches for rapidly 428

discovering additional scFv’s against surface components involved in aggregation (19) 429

and/or motility (15, 24, 33). The anti-MopB or other anti-X. fastidiosa scFv’s molecules 430

may also have use in diagnostic applications for pathogen surveillance and could be 431

assembled with in-built fluorophores as described recently (34, 35). 432

With the availability of genetically encoded molecules that target the surface of X. 433

fastidiosa alternative ways to investigate the biology of X. fastidiosa host vector 434

interactions may be explored to develop interventions. One such approach is 435


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paratransgenesis, which in concept involves the genetic manipulation of vector’s 436

naturally occurring microorganisms such as bacteria, fungi or viruses to compromise 437

competence to transmit a particular agent. 438

Engineering symbiotic bacteria to express and secrete recombinant molecules 439

inside an arthropod was initially demonstrated using the bacteria Rhodococcus rhodnii 440

to express cecropin (16) in the kissing bug Rhodnius prolixus, the vector of Chagas’ 441

disease and subsequently to express a scFv (17). Recently scFv’s that target 442

Trypanosoma cruzi epimastigotes glycans have been developed for use in this 443

paratransgenic application (34). This strategy may also be implemented with symbionts 444

or commensal organism of the GWSS (40). Alternatively, other ‘Trojan horse’ vehicles 445

may be developed. 446

Recently, a scFv against Plasmodium falciparum sporozoite surface protein 447

isolated from a malaria immune individual (10) and shown to inhibit sporozoite invasion 448

of human hepatocytes (9) has been used to reduce P. falciparum sporozoite levels in 449

Anopheles gambiae via an engineered entomopathogenic fungus (18), basically using 450

an infection to fight an infection (41). In the malaria study the recombinant antibody 451

PfNPNA-1 targeted a conserved surface accessible repetitive target on the highly 452

abundant circumsporozoite protein and was engineered to agglutinate the parasite 453

preventing migration to and invasion of the salivary gland. Such an approach is 454

independent of disrupting host cell specific interactions and may be a general strategy 455

against targets that are present at high density on the pathogen surface. Recombinant 456

antibodies against MopB and other abundant surface exposed molecules on X. 457

fastidiosa could also be engineered in a similar way to agglutinate the bacteria and 458


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evaluated in the GWSS via paratransgenic organisms such as engineered commensals 459

or symbionts or by infection with Metarrhizium spp or Beauvaria bassiana providing 460

novel alternative platforms to investigate in the control of PD. 461

Acknowledgments. 462

This work was supported by a Sub-Award of USDA APHIS Pierces Disease; Symbiotic 463

Control of Pierce’s Disease, #06-8500-0510-GR CA by Thomas A. Miller at UC 464

Riverside to ASK and was in part conducted within the EU FA COST Action FA0701 465

remit, Arthropod Symbiosis: From Fundamental Studies to Pest and Disease 466

Management. We thank Howard Boland for assistance with microscopy. 467

468


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Legends 469

Figure 1. An illustration of the mouse immunoglobulin DNA library assembly process 470

showing the PCR amplification and assembly steps. Corresponding representative 471

agarose gel resolved PCR products for each of the steps generated using mouse #4 472

spleen total RNA. 473

474

Figure 2. A schematic of the steps involved in in vitro antibody ribosome display. 1: The 475

scFv antibody library DNA template was prepared flanked with a T7 sequence and 476

mouse kappa constant sequence. 2: The antibody library DNA was transcribed to 477

mRNA and translated to form antibody-ribosome-mRNA complex. 3: The mixture 478

incubated with immobilized truncated MopB. 4: The unbound components removed by 479

washing. 5: The retained complexes released. 6: The mRNA reverse transcribed and 480

amplified by PCR. 7: The PCR products cloned into TOPO vectors and recovered 481

plasmids sequenced. Full-length in-frame scFv sequences inserted into pAHAHis vector 482

for recombinant antibody production. 483

484

Figure 3. The amino acid sequence of Xylella fastidiosa temecula1 MopB and the 485

predicted model for the B-barrel outer membrane domain. (A) Underlined black putative 486

leader sequence starting at Met1, overlined black in gray highlight is also a shorter but 487

potential leader sequence starting at Met15 (previously identified by Buening et al.,(8). 488

The arrow between Ala36 and Gln37 denotes signal peptide cleavage site, grey 489

periplasmic regions, blue membrane spanning regions and red surface exposed loops. 490

The yellow highlighted region corresponds to the truncated MopB. Sequence scored a 491


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value of 2.929, which is lower than the threshold value of 2.965. The difference between 492

the value and the threshold indicates the possibility of the protein being an outer 493

membrane protein. (B) The 2D figure of the predicted MopB transmembrane β-barrel 494

with the protruding surface exposed loops generated using PRED TMBB. The amino 495

acid numbers of the beginning and end of the β-strand traversing the outer membrane 496

are in green. 497

498

Figure 4. SDS-PAGE analysis of (A) Denaturing conditions mature full-length MopB 499

expression and purification. Lane 1, protein molecular weight markers; Lane 2, non 500

induced; Lane 3, induced; Lane 4, final wash; Lanes 5-6, elution 2-3. (B) Denaturing 501

conditions truncated MopB expression and purification. Lane 1: protein marker, Lane 2: 502

uninduced, Lane 3: induced, Lane 4: soluble fraction , Lane 5: flow through, Lanes 6: 503

final wash, lanes 7-10 elution 1 to 4. 504

505

Figure 5. Mouse immune sera evaluation by ELISA and immunodot blot. (A) Mouse 1-5 506

sera from bleed 3 diluted 1/20,000 in the ELISA on truncated MopB (10µg/mL) and the 507

BSA (10µg/mL) , blank, rabbit anti-mouse (1/20,000) + anti-rabbit alkaline phosphatase 508

and anti-rabbit alkaline phosphatase (1/40,000) without anti-mouse. (B) Immunodot blot 509

screen with 1/20,000 dilution of bleed 3 mouse 1-5 sera on 5% w/v milk protein control, 510

or 5µL of E.coli or X.fastidiosa spotted directly onto nitrocellulose strips and probed with 511

rabbit anti-mouse alkaline phosphatase and developed with NBT/BCIP. 512

513


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Figure 6. Functional Analysis of 4XfMopB3 INF scFv by ELISA and western blot. (A) 514

The ELISA with full-length and truncated MopB and 5% milk protein as a control. 2A10 515

scFv expressed in pAHAHis control antibody (against NANP repeat) was used as an 516

irrelevant negative control scFv. The detection anti-HA- alkaline phosphatase without a 517

first antibody and the substrate alone were used as additional controls. (B) MopB 518

western blot using 4XfMopB3 INF scFv with mature full-length and truncated MopB on 519

nitrocellulose membrane and detected with anti-HA- alkaline phosphatase. 520

521

Figure 7. Immunofluorescence detection of X. fastidiosa with Xf4MopB3 scFv. Panels 522

(A,C & E) viewed in bright field and (B,D & F) and with fluorescence microscopy. (A&B) 523

X. fastidiosa with 1° 4XfMopB3 scFv probed with 2° anti-polyhistidine FITC. (C&D) X. 524

fastidiosa with 1°anti-NPNA scFv probed with 2°anti-polyhistidine FITC and (E&F) X. 525

fastidiosa probed with 1°anti-polyhistidine FITC only. The size bars shown in panels A & 526

B are 5µm. 527

528

529


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http://aem.asm.org/

ribosome display of combinatorial an tibody libraries de...

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