Identification and Cloning of the Putative Ganglioside-Binding Proteins from Borrelia burgdorferi From MlpD

Ryan L. Brown, Christopher W. ReidInclude in the format of your notation to any graphic elements, the appropriate identifiers. For example:

Figure 1. Caption in Arial, at 24 points in Bold. Department of Science and Technology, Bryant University, 1150 Douglas Pike Smithfield, RI 02917

BLAST algorithm in Borrelia burgdorferi genomes2nd BLAST of top results into the B. burgdorferi sensu lato family Predict cell localizationThe most likely candidate was B. burgdorferi B31 (mlpD)

Bait proteinsMust be expressed on the surface of the cell to interact with the nerve cell

Must be conserved in pathogenic strainsFigure 3: An agarose gel electrophoresis (1%) confirming amplification of mlpD from B. burgdorferi B31 genomic DNA

MarkermlpD

Match DetailsE-ValueIdentityPSORT Location (gram positive)PSORT Location (gram negative)Is T3 Secreted?Is SEC secreted? (gram positive)Is SEC Secreted? (gram negative)Involvement in infection?BafACA1 V35 [Borrelia afzelii ACA-1]0.03124%CytoplasmCytoplasmNoNoNoN.d.BAVAVS116 O0016 [Borrelia valaisiana VS116]0.1528%UnknownUnknownYesNoNoN.d.BVAVS116 N006 [Borrelia valasiana VS116] 0.5240%UnknownOuter membraneNoYesYesN.d.mlpD [Borrelia burgdorferi B31] 0.5835%UnknownUnknownYesYesYes*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 DLiterature search for reports on involvement in pathogenesis

Figure 1: Work flow of bioinformatic analysis of B. burgdorferi

1000 bp750 bp500 bp

We ultimately chose to examine MlpD based on several lines of evidence:

MlpD is localized to the outer membraneMay 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 37C 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

INTRODUCTIONRESULTSDISCUSSIONONGOING RESEARCH

MlpD became a top candidate for study after the initial bioinformatics screen performed to identify potential ganglioside-binding proteinsmlpD 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 cloningProtein expression and purification Establish ganglioside-binding assayScreen for ganglioside-binding activity

References

Center for Disease Control. Lyme Disease Data. 2012. [Online]. Ramesh, G.Journal of Neuroinformation. 2013. Vol. 10:88. [Online].Yang, Xiaofeng. Infection and Immunity. November 1999. Vol. 67(11): 6008-6018. [Online].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].