Poster presented at Banff Conference on Infectious Diseases 2014. This poster received Roche-BCID 2014 award for the best poster.
Copyright@ Devender Kumar, The University of Calgary, 2014
Poster presented at Banff Conference on Infectious Diseases 2014. This poster received Roche-BCID 2014 award for the best poster.
Copyright@ Devender Kumar, The University of Calgary, 2014
Poster presented at Banff Conference on Infectious Diseases 2014. This poster received Roche-BCID 2014 award for the best poster.
Copyright@ Devender Kumar, The University of Calgary, 2014
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. 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 bulls- eye manifested at the site of a tick bite. Source: http://phil.cdc.gov/phil/details.asp?pid=9875 Photo credit: CDC/ James Gathany Fig. 2. Establishment of Borrelia burgdorferi infection and Lyme disease pathology in humans. Modified from Coburn et al., (2013). SUMMARY 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 post- infection 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. 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). Intravenous Injection of 4x10 8 B. burgdorferi in CD1d -/- mice Intravital Spinning Disk Microscopy of knee joint at 24 hours post-infection Engineered, GFP+ B. burgdorferi grown in Barbour-Stoenner-Kelly media + 1% mouse blood 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. RESULTS Infectious GFP-expressing B. burgdorferi transmigrate out of blood vessels at 24h 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. 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. 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, 4x10 8 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. AIM To examine the role of adhesin P66 in vascular transmigration of B. burgdorferi in live mice using intravital microscopy METHODS CONCLUSION P66 is essentially involved in the vascular transmigration of B. burgdorferi. FUTURE WORK Confirmation of P66s role by using Itgb -/- mice Confirmation of P66s role by using integrin blockers. Study the mechanisms of vascular transmigration and dissemination REFERENCES 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. ACKNOWLEDGEMENTS 1h 2h 3h 4h 6h 7h 8h 12h 16h 20h CD31 B. burgdorferi CD31 B. burgdorferi CD31 B. burgdorferi CD31 B. burgdorferi CD31 B. burgdorferi 1A 1B Borrelia burgdorferi uses P66 adhesin, a 3-integrin ligand, for vascular transmigration in vivo. Devender Kumar 1 , Meiqing Shi 2 , Laura Ristow 3 , Woo-Yong, Lee 1 , Paul Kubes 1 , Jenifer Coburn 3 , George Chaconas 1 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 Connect on 5 60 5 60 5 60 0 20 40 60 80 100 Time post-infection (minutes) %
[ B .
b u r g d o r f e r i ] i n
b l o o d wt p66 p66/comp B .
b u r g d o r f e r i /
F O V wt p66 p66/comp 0 2 4 6 p<0.0001 **** ns p<0.0001 **** 1 h 2 h 3 h 4 h 6 h 7 h 8 h 1 2 h 1 6 h 2 0 h 2 4 h 0 5 10 15 20 Hours post-infection T r a n s m i g r a t e d