1. Identification and Cloning of the Putative Ganglioside-Binding Proteins from Borrelia
burgdorferi From MlpD
Ryan L. Brown, Christopher W. Reid
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Department of Science and Technology, Bryant University, 1150 Douglas Pike Smithfield, RI 02917
BLAST algorithm in Borrelia
burgdorferi genomes
2nd BLAST of top results into
the B. burgdorferi sensu lato
family
Predict cell localization
The most likely candidate
was B. burgdorferi B31
(mlpD)
“Bait” proteins
Must be expressed on the
surface of the cell to
interact with the nerve cell
Must be conserved in
pathogenic strains
Figure 3: An agarose gel electrophoresis (1%)
confirming amplification of mlpD from B.
burgdorferi B31 genomic DNA
Marker mlpD
Match
Details
E-Value Identity PSORT
Location
(gram
positive)
PSORT
Location
(gram
negative)
Is T3
Secreted?
Is SEC
secreted?
(gram
positive)
Is SEC
Secreted?
(gram
negative)
Involve
ment in
infection?
BafACA1 V35
[Borrelia
afzelii ACA-1]
0.031 24% Cytoplasm Cytoplasm No No No N.d.
BAVAVS116
O0016
[Borrelia
valaisiana
VS116]
0.15 28% Unknown Unknown Yes No No N.d.
BVAVS116
N006 [Borrelia
valasiana
VS116]
0.52 40% Unknown Outer
membrane
No Yes Yes N.d.
mlpD
[Borrelia
burgdorferi
B31]
0.58 35% Unknown Unknown Yes Yes Yes* Yes
Table 1: Bioinformatics results for putative ganglioside-binding proteins in B. burgdorferi sensu lato
using botulinum toxin D.
Figure 2: The predicted 3D model of
MlpD, primarily alpha-helical
botulinum
toxin D
Literature search for reports
on involvement in
pathogenesis
Figure 1: Work flow of bioinformatic analysis of B. burgdorferi
1000 bp
750 bp
500 bp
We ultimately chose to examine MlpD based on several
lines of evidence:
• MlpD is localized to the outer membrane
• May be able to bind to gangliosides in the brain (see
panel A)
• Certain mlp genes are expressed during the growth of
B. burgdorferi and upregulated at 37°C in vitro and in
mammalian models [3].
Acknowlegements
In 2012 Lyme Disease was the seventh most common nationally
notifiable disease in addition to being the most commonly
reported vector borne illness [1]. The distinctive erythema migrans
or “bulls-eye” rash is widely reported but late stage neurological
disorders occur due to delayed diagnosis. Borrelia burgdorferi is
capable of inducing inflammation as well as apoptosis in dorsal
root ganglia, a contributing factor to the peripheral neuropathy in
lyme neuroborreliosis [2]. The purpose of this project is to identify,
clone and characterize potential ganglioside-binding proteins that
could be involved in establishing neuroborreliosis. Using a
bioinformatics approach we probed Borrelia genomes with
botulinum toxin D (a highly characterized ganglioside-binding
protein (GBP)) as “bait” to identify potential GBPs from B.
burgdorferi sensu lato.
MATERIALS AND METHODS
INTRODUCTION RESULTS DISCUSSION
ONGOING RESEARCH
• MlpD became a top candidate for study
after the initial bioinformatics screen
performed to identify potential
ganglioside-binding proteins
• mlpD is known to be SEC secreted in the
outer membrane
• Further research into the growth of the
mlp family in B. burgdorferi led us to
believe mlpD was a viable candidate for
involvement in neuroborreliosis
• Complete cloning
• Protein expression and purification
• Establish ganglioside-binding
assay
• Screen for ganglioside-binding
activity
References
1. Center for Disease Control. Lyme Disease Data. 2012. [Online].
2. Ramesh, G.Journal of Neuroinformation. 2013. Vol. 10:88. [Online].
3. Yang, Xiaofeng. Infection and Immunity. November 1999. Vol. 67(11): 6008-6018. [Online].
4. Schulze, RJ. Zucket, W.R. Molecular Microbiology. 2006. Vol. 59, Issue 5, 1473-1484. [Online].
Special thanks to the Rhode Island INBRE program for providing funding as well as Bryant
University, specifically Professor Christopher Reid, for support throughout the course of this
project.
*SEC secretion targets protein in B. burgdorferi to outer membrane [4].