Borrelia burgdorferi uses P66 adhesin, a 3-integrin ligand,

for vascular transmigration in vivo.

Devender

1
Kumar ,

Meiqing

2
Shi ,

Laura

3
Ristow ,

Woo-Yong,

1
Lee ,

Paul

1
Kubes ,

Jenifer

3
Coburn ,

George

1
Chaconas

1- Department of Microbiology, Immunology, & Infectious Diseases, The University of Calgary, Calgary, AB T2N 4N1
2- Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742, USA
3- Center for Infectious Disease Research, Medical College of Wisconsin, Milwaukee, WI 53226, USA
RESULTS

INTRODUCTION

1A

Infectious GFP-expressing B. burgdorferi transmigrate out of

blood vessels at 24h post-infection
1h

2h

100

% [B. burgdorferi]
in blood

Lyme disease is a zoonotic, an extremely debilitating and

multisystemic infection caused by Borrelia burgdorferi and
transmitted by ticks of Ixodes genus.
After a tick bite, B. burgdorferi multiplies locally, colonizes, and
disseminates to various sites of predilection through vascular
adhesion and transmigration.
1B

p66/comp

p66

wt

80
60
40
20
0

CD31
B. burgdorferi
3h

4h

Fig. 1A. Adult deer tick, Ixodes scapularis.

Source: http://www.ars.usda.gov/is/graphics/photos/mar98/k8002-3.htm
Photo credit: USDA-ARS/Scott Bauer.
Fig. 1B. The pathognomonic erythematous rash in the pattern of a bullseye manifested at the site of a tick bite.
Source: http://phil.cdc.gov/phil/details.asp?pid=9875
Photo credit: CDC/ James Gathany

60

60

60

Time post-infection (minutes)

Fig. 5. The effect of p66 deletion on vascular clearance of B. burgdorferi in
CD1d-/- mice. Blood was withdrawn at 5 and 60 minutes post-infection. Blood
cells were allowed to settle overnight and spirochetes in the plasma were
directly counted by dark-field microscopy. The change in spirochete
concentration between 5 and 60 minutes was determined for each mouse as
the percentage of spirochetes present at 60 minutes relative to the initial
5 minute time-point.

ns
CD31
B. burgdorferi
6h

7h

p<0.0001

p<0.0001

****

****

B. burgdorferi / FOV

After entry into a mammalian host, B. burgdorferi adapts to its

new host environment by differentially expressing mammalian
cell surface protein-binding adhesins P66 (Cugini et al., 2003).
P66 is an outer membrane protein and is encoded on a linear
chromosome by gene bb0603 and has an integrin binding activity
(Bunikis et al., 1995).
P66 has been shown to be responsible for transmigration across
a monolayer of endothelial cells in an in vitro transwell assay
(Ristow et al., 2013).

CD31
B. burgdorferi
8h

12h

p66
p66/comp
Fig. 6. The effect of p66 deletion on vascular transmigration in CD1d-/- mice.
GFP-expressing B. burgdorferi strains, infectious wt, a p66 deletion strain and a
strain where the wt p66 gene was reintroduced into the p66 mutant were
injected into the tail vein of CD1d-/- mice (n=10/group, 4x108 spirochetes per
mouse). After 24 hours, vascular transmigration was scored in the knee joint in
the living mice by intravital microscopy using a spinning disk laser confocal
microscope. Counts were done in at least five fields of view per mouse. Data
was analysed for statistical significance using the nonparametric Kruskal-Wallis
test and error bars were plotted as standard deviations.
wt

FUTURE WORK
CD31
B. burgdorferi
16h

20h

Confirmation of P66s role by using Itgb-/- mice

Confirmation of P66s role by using integrin blockers.
Study the mechanisms of vascular transmigration and dissemination

Fig. 2. Establishment of Borrelia burgdorferi infection and Lyme disease

pathology in humans.
Modified from Coburn et al., (2013).

CONCLUSION
P66 is essentially involved in the vascular transmigration of
B. burgdorferi.

SUMMARY

AIM
To examine the role of adhesin P66 in vascular transmigration of
B. burgdorferi in live mice using intravital microscopy

METHODS

REFERENCES
CD31
B. burgdorferi
24h

Fig. 3. Visualization of
transmigrated infectious wt
Borrelia burgdorferi in knee
joints of CD1d-/- mice at various
time points post-infection by
spinning disk laser confocal
microscope. Blood vessels were
stained with PE-conjugated
CD31 antibody and spirochetes
are fluorescent green.

20

Transmigrated Bb/FOV

The aim of this work was to study the earlier host-pathogen

interactions by evaluating the role of bacterial adhesin P66 in
vascular transmigration in vivo. Firstly, we showed that wild-type
B. burgdorferi expressing P66 transmigrates from the blood vessels
at 24 hours post-infection. Secondly, we studied kinetics of vascular
transmigration of a wild-type B. burgdorferi strain expressing P66,
a P66 strain, and a complemented P66 strain at 24 hours postinfection in CD1d KO mice. We found that at 24 hours post-infection,
a significant number of the wild-type bacteria had transmigrated to
the tissues around the knee joint of the mice, whereas the P66
strain did not transmigrate to the same tissues. Furthermore,
bacterial transmigration was restored to wild-type levels in the
complemented P66 strain. These in vivo vascular adhesion and
transmigration experiments reiterate that P66 is involved in the
vascular escape of the B. burgdorferi in the tissues. Further studies
are required to define the mechanisms of B. burgdorferi vascular
escape and dissemination to tissues in vivo.

Bunikis, J., Noppa, L., & Bergstrm, S. (1995). Molecular analysis of

a 66kDa protein associated with the outer membrane of Lyme
disease borrelia. FEMS Microbiology Letters, 131(2), 139-145.
Coburn, J., Leong, J., & Chaconas, G. (2013). Illuminating the roles
of the Borrelia burgdorferi adhesins. Trends in Microbiology, 21(8),
372-379.
Cugini, C., Medrano, M., Schwan, T. G., & Coburn, J. (2003).
Regulation of expression of the Borrelia burgdorferi beta(3)-chain
integrin ligand, P66, in ticks and in culture. Infection and Immunity,
71(2), 1001-1007.
Ristow, L., Kumar, D., Shi, M., Chaconas, G., & Coburn, J. (2013,
September). Analysis of the role of P66, Borrelia burgdorferis 3
integrin ligand, in mammalian infection. Poster session presented
at Microbial Pathogenesis and Host Response, Cold Spring Harbor,
NY.

15

ACKNOWLEDGEMENTS
10
5

Engineered, GFP+
B. burgdorferi grown in
Barbour-Stoenner-Kelly
media + 1% mouse
blood

Intravital Spinning Disk

Microscopy of knee joint
at 24 hours
post-infection

12
h
16
h
20
h
24
h

8h

7h

6h

4h

3h

Intravenous Injection of
4x108 B. burgdorferi in
CD1d-/- mice

2h

1h

Hours post-infection
Fig. 4. Kinetics of transmigration by B. burgdorferi in
CD1d-/- mice. Vascular transmigration was scored in the knee
joint in the living mice by intravital microscopy using a
spinning disk laser confocal microscope. Spirochetes outside
of vasculature were counted in at least five fields of view per
Mouse. Error bars are standard deviations.

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