Jacob Bailey and Faculty Mentor: Richard Robinson, Micro and Molecular Biology

The goal of this project was to determine if Borrelia Burgdorferi’s linear plasmid 36 is
required for intracellular localization in B lymphocytes. A more complete understanding
of the intracellular localization of B. burgdorferi may provide the means to effective
Lyme treatment.
It has been proposed that intracellular localization of Lyme spirochetes may be, at least
in part, responsible for Post Treatment Lyme Disease. As the spirochetes are safe
within human cells, Borrelia are able to evade host immune system responses and be
protected from antibiotic treatment. A more complete understanding of the intracellular
localization of B. burgdorferi may provide the means to better treat and clear Lyme
infection.
Data was collected by measuring the total GFP-tagged B. Burgdorferi associated with
APC labeled B lymphocytes by flow cytometry analysis following the Petzke lab
protocols. These protocols called for co-incubating human B cells with GFP-tagged B.
burgdorferi at a Multiplicty of Infection(MOI) of 10. Following co-incubation, the contents
of each well were transferred to 5 mL FACS tubes (with ∼2×106cells in each tube) and
were pelleted via centrifugation at 300 ×gravity for 5 min. Cells were washed twice with
1 mL HBSS to remove unattached spirochetes and once with 1 ml FACS stain buffer,
fixed with 3.2% formaldehyde in HBSS for 30 min, and were pelleted and re-suspended
in 0.5 mL FACS stain buffer (1X PBS, 0.2% bovine serum albumin, pH 7.4). To block Fc
receptors, cells were re-suspended in 70 μl stain buffer containing 1 μg/mL human
IgG1, kappa, purified myeloma protein (Sigma 5154). Antibody stain’s were then added
to each sample. Samples were mixed well for 5 minutes and then covered and
refrigerated at 4–8°C for 10 min. Cells were washed by adding 2 mL stain buffer
followed by centrifugation at 300 ×gravity for 10 min at 4 °C. Samples were run with a
minimum of 70,000 events per sample.
We ran this protocol multiple times and found contradicting results to our initial
hypothesis. Although we initially believed the isolate with the reinsertion of lp36 would
be required for increased intracellular invasion and attachment to human B
lymphocytes, the flow cytometry data revealed possible necessary modifications to our
initial hypothesis. Though further testing is needed to confirm current results, it appears
that Borrelia burgdorferi linear plasmid 36 deficient isolates attach to and are
internalized by human B lymphocytes more frequently than the wild type or the reinsert plasmid isolate.

 

 

 

 

 

 

 

 

 

 

 

 

 

Using ANOVA for statistical analysis we found that all sets are statistically significant
from one another using p<0.05 from controls to the other groups. Between groups the P
value was found to be 4.34475E-08 and thus statistically significant. The results and
data warrant further investigation into lp36 and whether or not lp36+ limits B.
burgdorferi’s ability to infect human B lymphocytes.
Further investigation will be carried out by fellow researcher and microbiology master
student Ann-Aubrey Reid. This will include further confocal microscopy analysis to verify
the data we analyzed using flow cytometry. Additional time will be required due to
difficulties with our -80 degree freezer. All of our Borrelia Burgdorferi stocks were lost
due to temperature inconsistencies. The problem has been mediated but we have to
wait for additional bacterial cultures and stocks to be sent. Mary Petzke’s lab has been
very considerate and generous in helping us continue research by providing additional
stocks. Findings from this project with be published and continue with the sustained
support from Dr. Robison’s lab.
The data and findings from this research so far signify a promising direction and set the
foundation for better understanding the pathogenesis and mechanism of Borrelia
burgdorferi intracellular localization.

Scholarly sources
Krupna-Gaylord Michelle A., Liveris Dionysios, Love Andrea CWormser Gary P., Schwarts Ira,Petzke Mary M.
Induction of Type I and Type III interferon&’s by Borrelia burgdorferi Correlates with Pathogenesis and requires
Linear Plasmid 36. Plos one. June 19, 2014.
Dorward DW, Fischer ER. Brooks DM. Invasion and Cytopathic Killing of Human Lymphocytes by Spirochetes
Causing Lyme Disease. Clinical Infectious Diseases. 1997; 25
Whitmire WE, Garon CF. Specific and non-specific response of murine B cells to membrane blebs of Borrelia
burgdorferi. Infect Immun 1993; References 61:1460–7

With a special thanks to Ann-Aubrey Reid and Dr. Robison for an extraordinary learning and research
experience.