Methods: Klebsiella pneumoniae (KP), a common cause of nosocomial infections was used for all BBs disruption assays. Human recombinant full length HMGB1 (rHMGB1; 1-215), a C45S mutation variant (mHMGB1) and the HMGB1 domains A Box (1-89), B Box (90-176), AB Boxes (1-176), B-linker Box (80-179), and B-linker Box C106S were expressed (in E.coli) and purified to >95%. To evaluate the effect of rHMGB1 and the various domains on established BBs, each protein species (200 nM) was added to pre-formed BBs at 24h. At 40h the BBs were washed, stained with LIVE/DEAD®, visualized via confocal laser scanning microscopy and images were analyzed by COMSTAT to calculate average thickness and biomass.
Results: Exogenous rHMGB1 and its individual domains, with the exception of A Box caused a significant reduction (p<0.05) in average thickness (AT) and biomass (BM) of KP biofilms as compared to untreated KP biofilms (%reduction mean ± SE in AT: 44%±0.33, 75%±0.04, 63%±0.1, 77%±0.03, 64%±0.08, 54%±0.15 and in BM: 61%± 0.01, 80%± 0.01, 68%± 0.02, 67%± 0.01, 73%± 0.02, 56%±0.02 induced by rHMGB1, mHMGB1, B-Box, B-linker Box, AB Boxes, and B-linker Box C106S, respectively).
Conclusion: Full length recombinant HMGB1 was able to significantly disrupt established KP biofilms as were all truncated HMGB1 forms containing the B Box domain and could potentially be used as a therapeutic treatment for BB-related infections.
J. Buzzo, ProclaRx: Collaborator , Research support .
S. Goodman, ProclaRx: Collaborator and Scientific Advisor , Research support .