# A Possible Link between Infection with Burkholderia Bacteria and Systemic Lupus Erythematosus Based on Epitope Mimicry

Authors: Wei Zhang; Morris Reichlin
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

We previously demonstrated that purified polyclonal and monoclonal anti-dsDNA antibodies bind a 15-mer peptide ASPVTARVLWKASHV in ELISA and Dot blot. This 15-mer peptide partial sequence ARVLWKASH shares similarity with burkholderia bacterial cytochrome B 561 partial sequence ARVLWRATH. In this study, we show that purified anti-dsDNA antibodies react with burkholderia fungorum bacterial cell lysates in Western blot. We used anti-dsDNA antibodies to make an anti-dsDNA antibodies affinity column and used this column to purify the burkholderia fungorum bacterial protein. Purified anti-dsDNA antibodies bind specifically to purified bacterial antigen and purified bacterial antigen blocked the anti-dsDNA antibodies binding to dsDNA antigen. Sera with anti-dsDNA antibodies bind specifically to purified bacterial antigen. We obtained protein partial sequence of RAGTDEGFG which is shared with burkholderia bacterial transcription regulator protein sequence. Sera with anti-dsDNA antibodies bind to RAGTDEGFG peptide better than control groups. These data support our hypothesis that the origin of anti-dsDNA antibodies in SLE may be associated with burkholderia bacterial infection.


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## Body

## 1. Introduction

Systemic lupus erythematosus (SLE)
is an autoimmune disease that affects multiple organ systems. Although the
etiology of SLE, and of autoimmunity in general remain unknown, considerable
evidence has been accumulated on the pathophysiologic mechanisms, which lead to the
failure of distinction between self and nonself and the production of autoantibodies.
Genetic, hormonal, and environmental factors have been attributed roles in the
etiology of autoimmunity.Antinuclear antibodies are a
hallmark of SLE, whereas anti-dsDNA antibodies are a very specific marker for
this disease. High-affinity anti-dsDNA antibodies correlate with disease
activity, especially with renal involvement. Over the past several years, it
has been clearly demonstrated that anti-dsDNA antibodies have pathogenic
potential. Clinical data demonstrate that anti-dsDNA antibody titers correlate
with disease activity in a significant number of patients with lupus nephritis,
and glomerular eluates from patients with active lupus nephritis contain
anti-dsDNA antibodies [1, 2].The mechanisms responsible for the
production of anti-dsDNA antibodies are not understood. The search for
crossreactive antigens continues, as these antigens may yield clues to the
origin and pathogenesis of anti-dsDNA antibodies.A major goal of understanding of
the structure, origin, and pathogenicity of anti-dsDNA antibodies is to develop
novel targeted treatments for SLE.In previous study, we used a
patient anti-dsDNA antibody to select phage clones and four positive clones
were found [3]. Those four clones sequence are clone B (ASPVTARVLWKASHV),
clone C (VSSLVLLSHGGPHSS), clone D (IMVLCPLWLGTTS), and clone E (AVAHVTSRRVPRWSAA). We demonstrated
that purified polyclonal anti-dsDNA antibodies and a monoclonal anti-dsDNA
antibody specifically bind a 15-mer clone B peptide ASPVTARVLWKASHV. This
chemically synthesized peptide could be recognized by anti-dsDNA antibodies in
ELISA and Dot blot [3].The sequences of the four clones
were loaded into the National center for Biotechnology Information (NCBI)
protein database to search for similarity to some protein sequence. It is of
interest to find that this 15-mer clone B peptide partial sequence ARVLWKASH
shares similarity with burkholderia bacterial cytochrome B 561 partial sequence
ARVLWRATH (including Burkholderia fungorum, Burkholderia dolosa,  Burkholderia cenocepacia, Burkholderia
mallei, Burkholderia pseudomallei, Burkholderia thailandensis and Burkholderia
cenocepaci). The clone C partial sequence LVLLSHGGPH shares sequence with burkholderia
bacterial transcriptional regulator partial sequence.In this study, we have examined
anti-dsDNA antibodies from SLE patient’s sera to see whether they can react
with Burkholderia bacterial protein. We have purified and isolated bacterial
protein and sequenced these proteins. Synthetic
peptides have been prepared to confirm that anti-dsDNA antibodies can bind
these antigens in vitro. A
possible link between an immune response to burkholderia and anti-dsDNA
antibody production in lupus patients has been investigated.


## 2. Materials and Methods

### 2.1. Patient Data

Human sera used in this study were
collected from SLE patients and CFII from Sigma (St. Louis, Mo, USA) was used as control antibody. All 30 SLE
patients satisfied American College of Rheumatology
revised criteria for the classification of SLE [4]. SLE sera were assayed for the quantity of
anti-dsDNA by ELISA and Crithidia luciliae anti-dsDNA antibody test kit from HELIX
diagnostic (Madison, WI). All
30 SLE patients have anti-dsDNA antibodies.


### 2.2. Preparation of IgG Anti-dsDNA Antibody and Biopanning of Phage Display Random Peptide Libraries by Anti-dsDNA Antibodies

The IgG fraction from anti-dsDNA positive sera was
isolated by chromatography on DE-52 and protein A-sepharose (Sigma) [3]. Purified
IgG from either DE-52 or protein A was passed through a dsDNA affinity column
(Sigma). The column was washed with Tris buffered saline (PH 7.4), and bound protein was
eluted with 3 M MgCl2. The purified antibodies were assayed for quantity of anti-dsDNA by ELISA and
Crithidia luciliae anti-dsDNA antibody test kit (HELIX diagnostic).A 15-mer peptide library display on
gene VIII product of fd phage [5] was used in the study. This library was
generously provided by Dr. G. P. Smith (University of Missouri, Columbia, Mo, USA). The basic
methods of screening were adapted from the previous reports [6, 7]. Three
rounds of screening were performed with the biotinylated anti-dsDNA antibodies.
For the first round of selection, 10 ug of biotinylated anti-dsDNA antibodiesin 400 μL of 0.5 χTBST (25 mM Tris, 75 mM NaCl and
0.05% Tween 20, pH 7.5) containing 1mg/mL BSA were
immobilized onto a streptavidin-coated petri dish by incubation for 2 hours at
room temperature. The phage suspension library was added and incubated out 4 hours at room temperature. After the
removal of unbound phage by washing 10 times with 0.5 χTBST, the bound phage was
recovered using elution buffer (0.1 N HCl-glycine, pH 2.2,). The eluates were
immediately neutralized using 2 M Tris. Eluted phage was amplified in
Escherichia coli K91 Kan and used as input in the subsequent rounds of
selection. After three rounds of selection, phage clones harvested from the
third round output were randomly selected and propagated in 1.5 mL of terrific
broth [1.2% (w/v) bacto-tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v)
glycerol, 17 mM KH2PO4 and 72 mM K2HPO4] supplemented with 20 ug/mL tetracycline at 37°C for 20 hours. The
cultures were clarified at 2500 g for 10 minutes. The supernatants were
recovered for the selection of anti-dsDNA antibody binding clones and
subsequent DNA sequencing.


## 2.1. Patient Data

Human sera used in this study were
collected from SLE patients and CFII from Sigma (St. Louis, Mo, USA) was used as control antibody. All 30 SLE
patients satisfied American College of Rheumatology
revised criteria for the classification of SLE [4]. SLE sera were assayed for the quantity of
anti-dsDNA by ELISA and Crithidia luciliae anti-dsDNA antibody test kit from HELIX
diagnostic (Madison, WI). All
30 SLE patients have anti-dsDNA antibodies.


## 2.2. Preparation of IgG Anti-dsDNA Antibody and Biopanning of Phage Display Random Peptide Libraries by Anti-dsDNA Antibodies

The IgG fraction from anti-dsDNA positive sera was
isolated by chromatography on DE-52 and protein A-sepharose (Sigma) [3]. Purified
IgG from either DE-52 or protein A was passed through a dsDNA affinity column
(Sigma). The column was washed with Tris buffered saline (PH 7.4), and bound protein was
eluted with 3 M MgCl2. The purified antibodies were assayed for quantity of anti-dsDNA by ELISA and
Crithidia luciliae anti-dsDNA antibody test kit (HELIX diagnostic).A 15-mer peptide library display on
gene VIII product of fd phage [5] was used in the study. This library was
generously provided by Dr. G. P. Smith (University of Missouri, Columbia, Mo, USA). The basic
methods of screening were adapted from the previous reports [6, 7]. Three
rounds of screening were performed with the biotinylated anti-dsDNA antibodies.
For the first round of selection, 10 ug of biotinylated anti-dsDNA antibodiesin 400 μL of 0.5 χTBST (25 mM Tris, 75 mM NaCl and
0.05% Tween 20, pH 7.5) containing 1mg/mL BSA were
immobilized onto a streptavidin-coated petri dish by incubation for 2 hours at
room temperature. The phage suspension library was added and incubated out 4 hours at room temperature. After the
removal of unbound phage by washing 10 times with 0.5 χTBST, the bound phage was
recovered using elution buffer (0.1 N HCl-glycine, pH 2.2,). The eluates were
immediately neutralized using 2 M Tris. Eluted phage was amplified in
Escherichia coli K91 Kan and used as input in the subsequent rounds of
selection. After three rounds of selection, phage clones harvested from the
third round output were randomly selected and propagated in 1.5 mL of terrific
broth [1.2% (w/v) bacto-tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v)
glycerol, 17 mM KH2PO4 and 72 mM K2HPO4] supplemented with 20 ug/mL tetracycline at 37°C for 20 hours. The
cultures were clarified at 2500 g for 10 minutes. The supernatants were
recovered for the selection of anti-dsDNA antibody binding clones and
subsequent DNA sequencing.


## 3. Western Blot

Burkholderia fungorum bacterial
cells were purchased from ATCC. These bacteria in BBL trypticase soy broth (Becton,
Dickinson and Company, Sparks,
Md, USA) were cultured overnight at 37°C [8]. The bacterial cell lysates were applied to 12.5% SDS-PAGE gel. After
electrophoretic separation, the proteins were transferred to a nitrocellulose
membrane. The nitrocellulose membrane was used for screening antibodies
reactive with Burkholderia fungorum bacterial proteins.


### 3.1. Preparation of IgG
Anti-dsDNA Antibody Column and Purification of Proteins from Burkholderia Fungorum Bacterial
Cell Lysates

A purified IgG anti-dsDNA antibody
preparation which bound
to Burkholderia fungorum bacterial protein was used
to prepare an anti-dsDNA antibody affinity column. Purified anti-dsDNA
antibodies were coupled to CNBr-activated sepharose 4B [9]. The Burkholderia
fungorum bacterial lysates were passed through an anti-dsDNA antibody affinity
column. The column was washed with Tris buffered saline (PH 7.4), and bound
protein was eluted with 3M MgCl2.


### 3.2. Purified Human IgG Anti-dsDNA Antibodies Binding to Purified Burkholderia Fungorum Bacterial Antigen

IMMULON plates from Thermo Scientific (Milford, MA, USA)
were used to detect the binding of 5 affinity purified human IgG
anti-dsDNA
antibodies isolated from SLE patients sera.
In addition, one human IgG anti-dsDNA monoclonal antibody, and normal IgG as
control were assessed  for binding to the purified burkholderia fungorum bacterial antigen. The ELISA plate was coated  with
purified bacterial antigen (50 ug/mL) and
purified anti-dsDNA Ab (1 ug/100 uL) was applied to the plate. Binding of IgG was detected using  goat antihuman IgG Fc fragment specific
alkaline.


### 3.3. Inhibition of Anti-dsDNA Binding to dsDNA Antigen by the Purified
Bacterial Antigen

Reaction mixtures of anti-dsDNA
antibody at the same concentration and the purified bacterial antigen at
varying concentrations were prepared. The ELISA plate was coated with dsDNA (50 ug/mL). After the
purified bacterial antigen was incubated for two hours with anti-dsDNA
antibodies at RT, the reaction mixture was transferred to dsDNA-coated wells.
The amount of human antibodies bound to the plate was determined by using an antihuman IgG Fc specific alkaline
phosphatase conjugate (Sigma). Following another washing, the substrate was added to
the plate for color development.


### 3.4. Serum Binding to Purified Bacterial Antigen

IMMULON plates were used to detect three
groups of sera; 30 SLE patients’ sera, secondly 60 control sera that include 15
rheumatoid arthritis sera, 15 polymyositis dermatomyositis sera, 15 sclerodema
sera, 15 anti-Ro/anti-La positive sera, and thirdly 30 normal sera. The plates
were coated with purified bacterial antigen (50 ug/mL)
and incubated at 4°C overnight. The wells were washed and treated with blocking
solution. Sera diluted 1/100 with PBS were added to the peptide-coated wells
and incubated for 2 hours at room temperature. After
washing, binding of IgG was detected using  goat antihuman IgG Fc fragment specific
alkaline phosphatase conjugate (Sigma).


## 3.1. Preparation of IgG
Anti-dsDNA Antibody Column and Purification of Proteins from Burkholderia Fungorum Bacterial
Cell Lysates

A purified IgG anti-dsDNA antibody
preparation which bound
to Burkholderia fungorum bacterial protein was used
to prepare an anti-dsDNA antibody affinity column. Purified anti-dsDNA
antibodies were coupled to CNBr-activated sepharose 4B [9]. The Burkholderia
fungorum bacterial lysates were passed through an anti-dsDNA antibody affinity
column. The column was washed with Tris buffered saline (PH 7.4), and bound
protein was eluted with 3M MgCl2.


## 3.2. Purified Human IgG Anti-dsDNA Antibodies Binding to Purified Burkholderia Fungorum Bacterial Antigen

IMMULON plates from Thermo Scientific (Milford, MA, USA)
were used to detect the binding of 5 affinity purified human IgG
anti-dsDNA
antibodies isolated from SLE patients sera.
In addition, one human IgG anti-dsDNA monoclonal antibody, and normal IgG as
control were assessed  for binding to the purified burkholderia fungorum bacterial antigen. The ELISA plate was coated  with
purified bacterial antigen (50 ug/mL) and
purified anti-dsDNA Ab (1 ug/100 uL) was applied to the plate. Binding of IgG was detected using  goat antihuman IgG Fc fragment specific
alkaline.


## 3.3. Inhibition of Anti-dsDNA Binding to dsDNA Antigen by the Purified
Bacterial Antigen

Reaction mixtures of anti-dsDNA
antibody at the same concentration and the purified bacterial antigen at
varying concentrations were prepared. The ELISA plate was coated with dsDNA (50 ug/mL). After the
purified bacterial antigen was incubated for two hours with anti-dsDNA
antibodies at RT, the reaction mixture was transferred to dsDNA-coated wells.
The amount of human antibodies bound to the plate was determined by using an antihuman IgG Fc specific alkaline
phosphatase conjugate (Sigma). Following another washing, the substrate was added to
the plate for color development.


## 3.4. Serum Binding to Purified Bacterial Antigen

IMMULON plates were used to detect three
groups of sera; 30 SLE patients’ sera, secondly 60 control sera that include 15
rheumatoid arthritis sera, 15 polymyositis dermatomyositis sera, 15 sclerodema
sera, 15 anti-Ro/anti-La positive sera, and thirdly 30 normal sera. The plates
were coated with purified bacterial antigen (50 ug/mL)
and incubated at 4°C overnight. The wells were washed and treated with blocking
solution. Sera diluted 1/100 with PBS were added to the peptide-coated wells
and incubated for 2 hours at room temperature. After
washing, binding of IgG was detected using  goat antihuman IgG Fc fragment specific
alkaline phosphatase conjugate (Sigma).


## 4. Protein Sequencing

The eluate from the anti-dsDNA
antibody column was applied to 12.5% SDS-PAGE gel and then electrotransferred
to PVDF membranes [10]. Proteins bound to PVDF were stained by Coomassie Blue
R-250. The stained protein band was excised with a clean razor blade. The protein
sequence was determined by Oklahoma University Health Center Molecular Biology-Proteomics Facility.


### 4.1. Peptide Synthesis

A protein partial sequence of RAGTDEGFG from the bacterial protein band
in PVDF was obtained. The RAGTDEGFG sequence is shared with burkholderia
bacterial transcription regulator protein sequence. The peptide RAGTDEGFG and a scrambled form as control peptide
FGAGTRDGE were synthesized by the Molecular Biology Resource Facility, University of Oklahoma Health Sciences Center. The purity of the peptides
was=>90% by reverse phase HPLC and the compositions were
confirmed by amino acid analysis.


### 4.2. Purified Human IgG Anti-dsDNA Antibodies Binding to Synthetic Peptide

IMMULON plates were used to detect the
binding of 5 affinity purified human IgG anti-dsDNA antibodies
isolated from SLE patients sera. In addition, one human IgG anti-dsDNA monoclonal
antibody, and  normal IgG as control were
assessed for binding to the synthetic
peptide (RAGTDEGFG) and control peptide (FGAGTRDGE). The ELISA plate was coated with synthetic peptide (50 ug/mL) and
purified anti-dsDNA antibody (1 ug/100 uL) was applied to the plate. Binding of IgG was detected using goat
antihuman IgG Fc fragment specific alkaline phosphatase conjugate (Sigma).


### 4.3. Inhibition

Inhibition of anti-dsDNA binding to
dsDNA antigen by the synthetic peptide.Reaction mixtures of anti-dsDNA
antibody at the same concentration and the synthetic peptide at varying
concentrations were prepared. The ELISA plate was coated with dsDNA (50 ug/mL).
After the synthetic peptide (RAGTDEGFG) was incubated for two hours with
anti-dsDNA antibodies at RT, the reaction mixture was transferred to dsDNA
coated wells. The amount of human antibodies bound to the plate was determined
by using an antihuman IgG Fc specific alkaline phosphatase conjugate (Sigma). Following
another washing, the substrate was added to the plate for color development.


### 4.4. Serum Binding to Synthetic Peptides

IMMULON plates were used to detect two
groups of sera; 30 SLE patients’ sera and 60 control sera that include 15 rheumatoid
arthritis, 15 polymyositis dermatomyositis, 15 sclerodema, 15 anti-Ro/anti-La
positive sera, and 30 normal sera. The plates were coated with peptide RAGTDEGFG (100 ug/mL) and incubated at 4°C overnight. The wells were washed and
blocked with blocking solution. Sera diluted 1/100 with PBS were added to the
peptide-coated wells and incubated for 2 hours at room temperature. After
washing, binding of IgG was detected using  goat antihuman IgG Fc fragment specific
alkaline phosphatase conjugate (Sigma).


## 4.1. Peptide Synthesis

A protein partial sequence of RAGTDEGFG from the bacterial protein band
in PVDF was obtained. The RAGTDEGFG sequence is shared with burkholderia
bacterial transcription regulator protein sequence. The peptide RAGTDEGFG and a scrambled form as control peptide
FGAGTRDGE were synthesized by the Molecular Biology Resource Facility, University of Oklahoma Health Sciences Center. The purity of the peptides
was=>90% by reverse phase HPLC and the compositions were
confirmed by amino acid analysis.


## 4.2. Purified Human IgG Anti-dsDNA Antibodies Binding to Synthetic Peptide

IMMULON plates were used to detect the
binding of 5 affinity purified human IgG anti-dsDNA antibodies
isolated from SLE patients sera. In addition, one human IgG anti-dsDNA monoclonal
antibody, and  normal IgG as control were
assessed for binding to the synthetic
peptide (RAGTDEGFG) and control peptide (FGAGTRDGE). The ELISA plate was coated with synthetic peptide (50 ug/mL) and
purified anti-dsDNA antibody (1 ug/100 uL) was applied to the plate. Binding of IgG was detected using goat
antihuman IgG Fc fragment specific alkaline phosphatase conjugate (Sigma).


## 4.3. Inhibition

Inhibition of anti-dsDNA binding to
dsDNA antigen by the synthetic peptide.Reaction mixtures of anti-dsDNA
antibody at the same concentration and the synthetic peptide at varying
concentrations were prepared. The ELISA plate was coated with dsDNA (50 ug/mL).
After the synthetic peptide (RAGTDEGFG) was incubated for two hours with
anti-dsDNA antibodies at RT, the reaction mixture was transferred to dsDNA
coated wells. The amount of human antibodies bound to the plate was determined
by using an antihuman IgG Fc specific alkaline phosphatase conjugate (Sigma). Following
another washing, the substrate was added to the plate for color development.


## 4.4. Serum Binding to Synthetic Peptides

IMMULON plates were used to detect two
groups of sera; 30 SLE patients’ sera and 60 control sera that include 15 rheumatoid
arthritis, 15 polymyositis dermatomyositis, 15 sclerodema, 15 anti-Ro/anti-La
positive sera, and 30 normal sera. The plates were coated with peptide RAGTDEGFG (100 ug/mL) and incubated at 4°C overnight. The wells were washed and
blocked with blocking solution. Sera diluted 1/100 with PBS were added to the
peptide-coated wells and incubated for 2 hours at room temperature. After
washing, binding of IgG was detected using  goat antihuman IgG Fc fragment specific
alkaline phosphatase conjugate (Sigma).


## 5. Results

### 5.1. Detection of Anti-dsDNA Antibody Reaction with Burkholderia Fungorum
Bacterial Cell Lysates by Western Blot

12 SLE patients sera with
anti-dsDNA antibodies were used as a source for the affinity purification of
antibodies. We purchased burkholderia fungorum bacterial cells from ATCC. We
tested 12 purified anti-dsDNA antibodies from SLE patients and found that seven
of these purified antibodies can bind the burkholderia fungorum bacterial protein
in Western Blot (Figure1).Figure 1
12 purified
anti-dsDNA antibodies from SLE patients. Seven of these purified
antibodies can bind a 35 KD burkholderia fungorum
bacterial protein in Western Blot.


### 5.2. Purified Bacterial Protein from Burkholderia Fungorum

We used one of the SLE patients
anti-dsDNA antibodies that reacts with the burkholderia fungorum bacterial
protein to make an anti-dsDNA antibody affinity column. We used this affinity
column to purify the burkholderia fungorum bacterial protein. We found that
purified burkholderi fungorum bacterial protein contains two major bands in 12.5% SDS gel. One
is about 35 KD and another one is about 25 KD, as seen in Figure2. We tested 4
purified anti-dsDNA antibodies for binding with the burkholderia fungorum
bacterial protein by ELISA. All 4 anti-dsDNA antibodies were reactive with the burkholderia
fungorum bacterial protein, and the control IgG was very weakly reactive (Figure 3). The burkholderia fungorum bacterial
protein was studied for the ability to inhibit anti-dsDNA antibody activity. As
seen in Figure 4, anti-dsDNA antibody preparations bound to dsDNA antigen can
be inhibited by the bacterial protein. We tested the SLE sera, control sera,
and normal sera. As seen in Figure 5, the burkholderia fungorum bacterial
protein was reactive with SLE sera with anti-dsDNA antibodies. The reaction can
be blocked by dsDNA antigen. In control sera, some rheumatoid arthritis sera
with anti-ssDNA antibodies were reactive to burkholderia fungorum bacterial
protein. We picked these sera and did inhibition by ssDNA and synthesized
peptide. The result shows that reaction can be blocked by ssDNA antigen. The
differences in means of OD410 reading between the SLE sera
(mean = 0.7617, N=30) and normal sera (mean = 0.2320, N=30) when they bound to
the bacterial antigen were statistically compared by t-test (using GraphPad
prism), and the differences were very significant (P<.0001).Figure 2
Burkholderia fungorum bacterial protein
purified by anti-dsDNA antibodies affinity column. The eluate
from the column was examined by 12.5% SDS gel. The result shows that purified
proteins contain two major bands, one is 35 KD bacterial protein and the other
one is 25 KD bacterial protein.Figure 3
Testing 4 affinity purified human IgG anti-dsDNA antibodies in binding to the purified bacterial antigen in ELISA. ELISA plates were coated with
the purified bacterial antigen (50μg/mL). Lanes 1–4 contain
purified anti-dsDNA antibodies (1 μg/100 μL) binding to purified bacterial antigen
and lane 5 has CF II as control binding to purified
bacterial antigen. Anti human IgG Fc fragment specific
alkaline phosphatase conjugate was used to detect anti-dsDNA antibody binding. The data were expressed as the mean ± SD.Figure 4
Inhibition of anti-dsDNA antibodies binding to dsDNA antigen by purified bacterial antigen.
ELISA plates were coated with dsDNA (50μg/mL).
Lane 1, 2, and 3 are three different patients anti-dsDNA
antibodies. The light column shows the patient purified anti-dsDNA
antibodies (1.25 μg/100 uL) binding to dsDNA antigen, and
dark column shows the patient purified anti-dsDNA
antibodies (1.25 μg/100 uL) mixed with purified bacterial antigen(10 μg/100 uL).
The data were expressed as the mean ± SD.Figure 5
Reaction of SLE sera and control
sera with the purified bacterial antigen. The sera were 1/100
in PBS and added into purified bacterial antigen-coated
ELISA plates and the reaction of sera antibodies with the purified
bacterial antigen were detected by goat antihuman IgG Fc fragment
specific alkaline phosphatase conjugate. The 30 SLE sera and the
control sera include 15 rheumatoid arthritis, 15 polymyositis
dematomyositis, 15 sclerodema, 15 Ro/La positive sera, and 30 normal sera. The
mean reading of 30 SLE sera bound with  purified bacterial antigen was statistically
different from that of control sera (P<.0001 by
unpaired t-test).


### 5.3. Sequence Analysis of 25 KD and 35 KD Bacterial Proteins

The electrophoresed purified burkholderia
fungorum proteins from an anti-dsDNA affinity column were transferred on to
polyvinyl difluoride (PVDF) membranes. The bacterial proteins were screened by  Western Blot using anti-dsDNA antibodies. The two immunoreactive bands (25 KD and 35 KD) were
excised from the PVDF and sequenced. A protein partial sequence of RAGTDEGFG
from the protein band is shared with burkholderia bacterial
transcription regulator protein sequence.


### 5.4. Confirming the Binding of Anti-dsDNA Antibodies to the Peptide by Chemical Synthesis of Peptides

RAGTDEGFG peptide which is shared
with burkholderia bacterial transcription regulator protein sequence was
synthesized. We tested 4 purified anti-dsDNA antibodies for binding with the RAGTDEGFG peptide
by ELISA. All 4 anti-dsDNA antibodies were reactive with the synthesized
peptide, and the control IgG was very weakly reactive (Figure6). We also
compared RAGTDEGFG peptide with scramble control peptide FGAGTRDGE. Anti-dsDNA antibodies were more
strongly reactive with RAGTDEGFG peptide than scrambled control peptide FGAGTRDGE.Figure 6
Testing 4 affinity purified human IgG anti-dsDNA antibodies in binding to the synthetic
peptide in ELISA. ELISA plates
were coated with synthetic peptide (100μg/mL). Lanes 1–4 contain purified
anti-dsDNA antibodies (1 μg/100 μL) binding to
synthetic peptide and lane 5 has CF II as control binding to synthetic
peptide. Antihuman IgG Fc fragment specific alkaline phosphatase conjugate was used to detect
anti-dsDNA antibody binding. The data were expressed as the mean ± SD.


### 5.5. Inhibition Assay

The synthesized peptide (RAGTDEGFG)
was studied for the ability to inhibit anti-dsDNA antibody activity. As seen in
Figure7, anti-dsDNA antibody preparations bound to dsDNA antigen can be
inhibited by the synthetic peptide.Figure 7
Inhibition of anti-dsDNA antibodies binding to dsDNA antigen by synthetic
peptide (RAGTDEGFG). ELISA plates were coated with
dsDNA (50μg/mL). Lane 1, 2, and 3 are three different patients
anti-dsDNA antibodies. The light column shows the patient purified
anti-dsDNA antibodies (1.25 μg/100 uL) bind to dsDNA antigen, and
dark column shows the patient purified anti-dsDNA
antibodies (1.25 μg/100 uL) mixed with synthetic peptide (3.4 μg/100 uL). The data were expressed as the mean ± SD.


### 5.6. Binding of SLE Sera with Synthesized Peptide RAGTDEGFG in ELISA

We tested the SLE sera, control
sera, and normal sera, as seen in Figure8. The synthetic peptide was reactive
with SLE sera with anti-dsDNA antibodies. The reaction can be blocked by dsDNA
antigen. In control sera, some sera with anti-ssDNA antibodies were reactive to
synthesized peptide. We picked these sera and did inhibition by ssDNA and
synthesized peptide. The results show that reaction can be blocked by ssDNA
antigen. The differences in means of OD410 reading between the SLE
sera (mean = 1.015, N=30) and normal sera (mean = 0.2513, N=30) when they bound to the synthetic peptide were statistically compared by t-test
(using GraphPad prism), and the differences were very significant (P<.0001).
The differences in means of OD410 reading between the SLE sera (mean
= 1.015, N=30) and control sera (mean = 0.3383, N=60) when they bound to the
synthetic peptide were statistically compared by t-test (using GraphPad prism),
and the differences were very significant (P<.0001).Figure 8
Reaction of SLE sera and control sera with the peptide. The
sera were diluted 1/100 in PBS and added to peptide-coated ELISA plates and the reaction of sera
antibodies with the peptide were
detected by goat antihuman IgG Fc fragment specific alkaline phosphatase conjugate. The 30 SLE sera and the
control sera include 14 rheumatoid
arthritis, 12 polymyositis dematomyositis, 14 sclerodema, 15 Ro/La positive sera, and 30 normal sera. The
mean reading of 30 SLE sera bound
with  peptide was statistically different
from that of control sera (P<.0001 by unpaired t-test).


## 5.1. Detection of Anti-dsDNA Antibody Reaction with Burkholderia Fungorum
Bacterial Cell Lysates by Western Blot

12 SLE patients sera with
anti-dsDNA antibodies were used as a source for the affinity purification of
antibodies. We purchased burkholderia fungorum bacterial cells from ATCC. We
tested 12 purified anti-dsDNA antibodies from SLE patients and found that seven
of these purified antibodies can bind the burkholderia fungorum bacterial protein
in Western Blot (Figure1).Figure 1
12 purified
anti-dsDNA antibodies from SLE patients. Seven of these purified
antibodies can bind a 35 KD burkholderia fungorum
bacterial protein in Western Blot.


## 5.2. Purified Bacterial Protein from Burkholderia Fungorum

We used one of the SLE patients
anti-dsDNA antibodies that reacts with the burkholderia fungorum bacterial
protein to make an anti-dsDNA antibody affinity column. We used this affinity
column to purify the burkholderia fungorum bacterial protein. We found that
purified burkholderi fungorum bacterial protein contains two major bands in 12.5% SDS gel. One
is about 35 KD and another one is about 25 KD, as seen in Figure2. We tested 4
purified anti-dsDNA antibodies for binding with the burkholderia fungorum
bacterial protein by ELISA. All 4 anti-dsDNA antibodies were reactive with the burkholderia
fungorum bacterial protein, and the control IgG was very weakly reactive (Figure 3). The burkholderia fungorum bacterial
protein was studied for the ability to inhibit anti-dsDNA antibody activity. As
seen in Figure 4, anti-dsDNA antibody preparations bound to dsDNA antigen can
be inhibited by the bacterial protein. We tested the SLE sera, control sera,
and normal sera. As seen in Figure 5, the burkholderia fungorum bacterial
protein was reactive with SLE sera with anti-dsDNA antibodies. The reaction can
be blocked by dsDNA antigen. In control sera, some rheumatoid arthritis sera
with anti-ssDNA antibodies were reactive to burkholderia fungorum bacterial
protein. We picked these sera and did inhibition by ssDNA and synthesized
peptide. The result shows that reaction can be blocked by ssDNA antigen. The
differences in means of OD410 reading between the SLE sera
(mean = 0.7617, N=30) and normal sera (mean = 0.2320, N=30) when they bound to
the bacterial antigen were statistically compared by t-test (using GraphPad
prism), and the differences were very significant (P<.0001).Figure 2
Burkholderia fungorum bacterial protein
purified by anti-dsDNA antibodies affinity column. The eluate
from the column was examined by 12.5% SDS gel. The result shows that purified
proteins contain two major bands, one is 35 KD bacterial protein and the other
one is 25 KD bacterial protein.Figure 3
Testing 4 affinity purified human IgG anti-dsDNA antibodies in binding to the purified bacterial antigen in ELISA. ELISA plates were coated with
the purified bacterial antigen (50μg/mL). Lanes 1–4 contain
purified anti-dsDNA antibodies (1 μg/100 μL) binding to purified bacterial antigen
and lane 5 has CF II as control binding to purified
bacterial antigen. Anti human IgG Fc fragment specific
alkaline phosphatase conjugate was used to detect anti-dsDNA antibody binding. The data were expressed as the mean ± SD.Figure 4
Inhibition of anti-dsDNA antibodies binding to dsDNA antigen by purified bacterial antigen.
ELISA plates were coated with dsDNA (50μg/mL).
Lane 1, 2, and 3 are three different patients anti-dsDNA
antibodies. The light column shows the patient purified anti-dsDNA
antibodies (1.25 μg/100 uL) binding to dsDNA antigen, and
dark column shows the patient purified anti-dsDNA
antibodies (1.25 μg/100 uL) mixed with purified bacterial antigen(10 μg/100 uL).
The data were expressed as the mean ± SD.Figure 5
Reaction of SLE sera and control
sera with the purified bacterial antigen. The sera were 1/100
in PBS and added into purified bacterial antigen-coated
ELISA plates and the reaction of sera antibodies with the purified
bacterial antigen were detected by goat antihuman IgG Fc fragment
specific alkaline phosphatase conjugate. The 30 SLE sera and the
control sera include 15 rheumatoid arthritis, 15 polymyositis
dematomyositis, 15 sclerodema, 15 Ro/La positive sera, and 30 normal sera. The
mean reading of 30 SLE sera bound with  purified bacterial antigen was statistically
different from that of control sera (P<.0001 by
unpaired t-test).


## 5.3. Sequence Analysis of 25 KD and 35 KD Bacterial Proteins

The electrophoresed purified burkholderia
fungorum proteins from an anti-dsDNA affinity column were transferred on to
polyvinyl difluoride (PVDF) membranes. The bacterial proteins were screened by  Western Blot using anti-dsDNA antibodies. The two immunoreactive bands (25 KD and 35 KD) were
excised from the PVDF and sequenced. A protein partial sequence of RAGTDEGFG
from the protein band is shared with burkholderia bacterial
transcription regulator protein sequence.


## 5.4. Confirming the Binding of Anti-dsDNA Antibodies to the Peptide by Chemical Synthesis of Peptides

RAGTDEGFG peptide which is shared
with burkholderia bacterial transcription regulator protein sequence was
synthesized. We tested 4 purified anti-dsDNA antibodies for binding with the RAGTDEGFG peptide
by ELISA. All 4 anti-dsDNA antibodies were reactive with the synthesized
peptide, and the control IgG was very weakly reactive (Figure6). We also
compared RAGTDEGFG peptide with scramble control peptide FGAGTRDGE. Anti-dsDNA antibodies were more
strongly reactive with RAGTDEGFG peptide than scrambled control peptide FGAGTRDGE.Figure 6
Testing 4 affinity purified human IgG anti-dsDNA antibodies in binding to the synthetic
peptide in ELISA. ELISA plates
were coated with synthetic peptide (100μg/mL). Lanes 1–4 contain purified
anti-dsDNA antibodies (1 μg/100 μL) binding to
synthetic peptide and lane 5 has CF II as control binding to synthetic
peptide. Antihuman IgG Fc fragment specific alkaline phosphatase conjugate was used to detect
anti-dsDNA antibody binding. The data were expressed as the mean ± SD.


## 5.5. Inhibition Assay

The synthesized peptide (RAGTDEGFG)
was studied for the ability to inhibit anti-dsDNA antibody activity. As seen in
Figure7, anti-dsDNA antibody preparations bound to dsDNA antigen can be
inhibited by the synthetic peptide.Figure 7
Inhibition of anti-dsDNA antibodies binding to dsDNA antigen by synthetic
peptide (RAGTDEGFG). ELISA plates were coated with
dsDNA (50μg/mL). Lane 1, 2, and 3 are three different patients
anti-dsDNA antibodies. The light column shows the patient purified
anti-dsDNA antibodies (1.25 μg/100 uL) bind to dsDNA antigen, and
dark column shows the patient purified anti-dsDNA
antibodies (1.25 μg/100 uL) mixed with synthetic peptide (3.4 μg/100 uL). The data were expressed as the mean ± SD.


## 5.6. Binding of SLE Sera with Synthesized Peptide RAGTDEGFG in ELISA

We tested the SLE sera, control
sera, and normal sera, as seen in Figure8. The synthetic peptide was reactive
with SLE sera with anti-dsDNA antibodies. The reaction can be blocked by dsDNA
antigen. In control sera, some sera with anti-ssDNA antibodies were reactive to
synthesized peptide. We picked these sera and did inhibition by ssDNA and
synthesized peptide. The results show that reaction can be blocked by ssDNA
antigen. The differences in means of OD410 reading between the SLE
sera (mean = 1.015, N=30) and normal sera (mean = 0.2513, N=30) when they bound to the synthetic peptide were statistically compared by t-test
(using GraphPad prism), and the differences were very significant (P<.0001).
The differences in means of OD410 reading between the SLE sera (mean
= 1.015, N=30) and control sera (mean = 0.3383, N=60) when they bound to the
synthetic peptide were statistically compared by t-test (using GraphPad prism),
and the differences were very significant (P<.0001).Figure 8
Reaction of SLE sera and control sera with the peptide. The
sera were diluted 1/100 in PBS and added to peptide-coated ELISA plates and the reaction of sera
antibodies with the peptide were
detected by goat antihuman IgG Fc fragment specific alkaline phosphatase conjugate. The 30 SLE sera and the
control sera include 14 rheumatoid
arthritis, 12 polymyositis dematomyositis, 14 sclerodema, 15 Ro/La positive sera, and 30 normal sera. The
mean reading of 30 SLE sera bound
with  peptide was statistically different
from that of control sera (P<.0001 by unpaired t-test).


## 6. Discussion

Environmental pathogens have long
been suspected to be inducers of autoimmune disease. Autoimmune NZB mice raised
in a germ-free environment develop reduced titers of auto-antibodies,
and elevated titers of anti-dsDNA antibodies can be found in patients with
microbial infections, suggesting that microbial stimulation of the immune system helps stimulate the
induction of auto-antibodies in some way [11, 12]. Anti-dsDNA antibodies
derived from naïve lupus prone mice can bind to the cell surfaces of murine
endogenous microbial flora [13]. Monoclonal anti-dsDNA antibodies derived from
mice and humans with SLE bind to the glycolipid components of the mycobacterial cell wall [14], suggesting that
bacterial antigens may elicit these antibodies. This crossreactivity, known as molecular
mimicry, offers a potential explanation for the development of autoimmunity.In a previous study, we
demonstrated that purified anti-dsDNA antibodies specifically bind a 15-mer
peptide ASPVTARVLWKASH [3], and the 15-mer peptide partial sequence ARVLWKASH is
shared with burkholderia bacterial cytochromeB 561 partial sequence ARVLWRATH. Burkholderia
bacteria are rod-shaped, motile, gram-negative bacteria that includes both human
and plant pathogens as well as environmentally important bacteria. The genus
Burkholderia currently comprises 19 named species [8]. The clinical
significance of Burkholderia cepacia, originally described as a plant pathogen
in the 1950s [15], increased in the 1980s as it emerged as an opportunistic
pathogen, colonizing the lungs of cystic fibrosis patients [16–18], and immunocompromised
individuals [19–21]. At its peak incidence in the mid 1980s, 20% of colonized
patients suffered from severe progressive respiratory failure with bacteremia
which was often fatal [16, 22].In this study, we have demonstrated
that purified anti-dsDNA antibodies from lupus patients can react with burkholderia
fungorum bacterial cell lysates in Western blot. We used one of the SLE patients
anti-dsDNA antibodies that reacts with the burkholderia fungorum bacterial protein to make an
anti-dsDNA antibodies affinity column. We used this affinity column to purify the burkholderia
fungorum bacterial protein. We found that purified burkholderi fungorum
bacterial protein contains two major bands in 12.5% SDS gel, one is about 35 KD
and the other one is about 25 KD. We tested purified human anti-dsDNA antibodies binding to
purified bacterial antigen compared with normal IgG binding. We also used
purified bacterial antigen to block the anti-dsDNA antibodies binding to dsDNA
antigen. The results show that anti-dsDNA antibody binding to purified bacterial
antigen is specific. We tested 3 groups of sera for binding studies with the
purified bacterial antigen. The results show that sera with anti-dsDNA
antibodies bind to purified bacterial antigen more strongly than other control
groups. We obtained protein partial sequence of RAGTDEGFG which is shared with
burkholderia bacterial transcription regulator protein
sequence. We tested 3 groups of sera for binding studies with the RAGTDEGFG
peptide. The results show that sera with anti-dsDNA antibodies bind to
RAGTDEGFG peptide and are
higher than other control groups. In a previous study, we used a patient
anti-dsDNA antibody to select phage clones and four positive clones were found
[3]. Those four clones
sequences are clone
B (ASPVTARVLWKASHV), clone C (VSSLVLLSHGGPHSS), clone D
(IMVLCPLWLGTTS), and clone E (AVAHVTSRRVPRWSAA). The clone B partial sequence ARVLWKASH
is shared with burkholderia bacterial cytochromeB 561 partial sequence. The
clone C partial sequence LVLLSHGGPH is shared with burkholderia bacterial
transcriptional regulator partial sequence. We used the same patient’s
anti-dsDNA antibody to make an antibody affinity column for purified
burkholderia bacterial protein. We obtained protein partial sequence of
RAGTDEGFG which is shared with burkholderia bacterial transcription regulator
protein sequence, but we did not obtain burkholderia bacterial cytochrome B561
protein sequence from purified bacterial antigen. It is possible that the anti-dsDNA
antibody affinity column can not efficiently isolate enough burkholderia
bacterial cytochrome B561 protein for sequencing. We also compared burkholderia
bacterial cytochrome B561 protein (186 amino acids) with burkholderia bacterial
transcription regulator (308 amino acids) protein sequence. The two proteins
sequences share 29 amino acids and 20 amino acid positions have the same charge.
These data support our hypothesis that the origin of anti-dsDNA antibodies in
SLE may be associated with burkholderia bacterial infection. The burkholderia
bacterial infection is an environmental risk factor that may be most closely
associated with SLE. The development of auto-antibodies is a common feature
in autoimmune diseases. Interactions between auto-antigens and auto-antibodies
have to date mainly been analyzed by truncated subclones or synthetic peptides
mimicking the proposed target epitopes. However, recent findings and reviews
have implied that most autoepitopes may be conformation-dependent [23], thus
stressing the need to develop procedures to analyze in detail which functional
domain of the auto-antigen could be immunodominant. Immunization of
non-autoimmune mice with Burkholderia antigens and its selected peptides would
support our hypothesis if autoimmunity is induced.Clearly, other environmental
factors such as Epstein-barr virus infection [24, 25] as well as genetic and
immunologic factors may also be important. Continued efforts in this sphere
include the possible development of an animal model that could provide fruitful
areas of investigation.



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*Source: 683489-2008-08-03.xml*