High Mobility Group Box 1 (HMGB1) is a 215-amino acid protein that plays multiple roles in humans.
Intracellularly, HMGB1 is chromatin-associated and involved in virtually all types of DNA metabolism (e.g.
replication, repair, recombination), primarily through its ability to bind with high affinity and specificity to
various DNA structures. Extracellularly, HMGB1 is the prototypical damage-associated molecular pattern
molecule (DAMP) with strong pro-inflammatory functions. Here, we have discovered, and will show, that
endogenous HMGB1 has a heretofore unknown function in its ability to control bacteria that cause chronic
and recurrent infections, which thereby contributes to the delicate balance of host-pathogen interactions. For
bacteria to enter a chronic infection state, they must assume a community architecture called a biofilm,
replete with a self-made extracellular matrix commonly composed of scaffolded extracellular DNA (eDNA)
that is highly resistant to clearance by both the host immune system and antimicrobials. We have previously
shown that this eDNA-dependent structure is stabilized by the DNABII family of bacterial proteins, that when
added exogenously can drive free-living (planktonic) bacteria into a biofilm. Unlike these proteins, we show
that HMGB1 destabilizes the eDNA structure and drives biofilm-resident bacteria into the planktonic,
vulnerable state. The DNABII family and HMGB1 have similar DNA structure binding preferences in vitro
despite a lack of primary amino acid sequence identity and secondary structure. We therefore hypothesize
that despite their similar DNA structure binding preferences, these proteins facilitate converse reactions.
Further, the fact that endogenous native HMGB1 steady state levels restrict, but fail to clear, chronic
infections suggests a balance with HMGB1’s needed pro-inflammatory functions, i.e. release of bacteria
from biofilms under strong inflammatory conditions could lead to sepsis. Herein, we will work under the
scientific premise that eDNA-binding is essential for HMGB1 to disrupt bacterial biofilms and further, that it
will be possible to separate its anti-biofilm activity from pro-inflammatory functions. Indeed, we have
truncated HMGB1 to 97 amino acids, a form which still retains full anti-biofilm activity but without pro-
inflammatory functions, thereby likely able to tip the host-pathogen interaction in favor of the host. Through
the completion of 3 highly integrated specific aims, we will determine the capacity of this HMGB1 derived
97-mer to act as an anti-biofilm agent on biofilms formed by diverse human pathogens in vitro as well as
biofilms within polymicrobial clinical samples, assayed ex-vivo (to determine the breadth of activity and
support our overarching hypothesis; AIM 1), the anti-biofilm mechanism of action, through a process of DNA
binding (AIM 2), and the therapeutic efficacy in two distinct animal models of biofilm infections (AIM 3).