Evaluation of the kinetics and mechanism of action of anti‐integration host factor‐mediated disruption of bacterial biofilms

Abstract

Summary The extracellular polymeric substance produced by many human pathogens during biofilm formation often contains extracellular DNA (eDNA). Strands of bacterial eDNA within the biofilm matrix can occur in a lattice-like network wherein a member of the DNABII family of DNA-binding proteins is positioned at the vertex of each crossed strand. To date, treatment of all biofilms tested with antibodies directed against one DNABII protein, Integration Host Factor (IHF), results in significant disruption. Here, using nontypeable Haemophilus influenzae as a model organism, we report that this effect was rapid, IHF-specific and mediated by binding of transiently dissociated IHF by anti-IHF even when physically separated from the biofilm by a nucleopore membrane. Further, biofilm disruption fostered killing of resident bacteria by previously ineffective antibiotics. We propose the mechanism of action to be the sequestration of IHF upon dissociation from the biofilm eDNA, forcing an equilibrium shift and ultimately, collapse of the biofilm. Further, antibodies against a peptide positioned at the DNA-binding tips of IHF were as effective as antibodies directed against the native protein. As incorporating eDNA and associated DNABII proteins is a common strategy for biofilms formed by multiple human pathogens, this novel therapeutic approach is likely to have broad utility.

Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model

“…The mechanism for biofilm disruption is the antibody-mediated sequestration of DNABII proteins from the extracellular milieu as the proteins rapidly cycle between eDNA-bound and -free states. The resulting equilibrium imbalance promotes dissociation of additional DNABII proteins from the eDNA matrix, destabilization of the eDNA lattice, and subsequent collapse of the biofilm structure (17). Earlier, we showed that therapeutic TCI with IHF promotes rapid clearance of preformed NTHI biofilms from the middle ears of chinchillas (39), as did infusion of the NTHI-challenged chinchilla middle ear with IgG-enriched polyclonal serum against IHF or monoclonal antibodies directed against the DNA-binding tips of IHF (16).…”

“…Moreover, it has shown utility to test preclinically the robustness of various vaccine candidates in a polymicrobial disease model system (25-27, 29, 30). Therefore, we employed this viral-bacterial coinfection model to assess the efficacy afforded by TCI with three lead vaccine candidates under development by our lab: rsPilA, which targets the majority subunit of NTHI Tfp (14); chimV4, which is directed against both Tfp and a protective B-cell epitope of OMP P5 (14); and IHF, which is a DNABII protein family member that plays an important role in the structural integrity of bacterial biofilms, including those formed by NTHI (15)(16)(17).…”

“…We have developed three immunogens that demonstrate efficacy against NTHI both in vitro and preclinically in animal models of disease. These include the following: an NTHI type IV pilus (Tfp)-targeted recombinant protein called rsPilA, for recombinant soluble PilA, designed to inhibit NTHI adherence, twitching motility, and biofilm formation (14); a chimeric immunogen that targets both NTHI outer membrane protein (OMP) P5 and Tfp, called chimV4, designed to block adherence and pathogenesis of NTHI as mediated by two important adhesive proteins/virulence determinants (14); and integration host factor (IHF), a DNABII protein family member that serves as a critical structural element to the extracellular DNA scaffold within the extracellular polymeric substance incorporated into biofilms formed by many bacterial species (15)(16)(17).…”

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

“…Extracellular DNA protects bacteria within the biofilm from host defenses via the chelation of antimicrobial peptides and divalent cations (14,64). In addition, we have shown that eDNA is an important structural component of the biofilm matrix and that the DNABII proteins IHF and HU are required to stabilize the eDNA scaffold that provides structural support to biofilms formed by NTHI and many additional human pathogens (7,13,25,27,28,30,(37)(38)(39).…”

“…We also show that biofilms formed within the chinchilla middle ear during experimental otitis media, as well as those within clinical specimens recovered from children with chronic posttympanostomy tube otorrhea and pediatric cystic fibrosis patients infected with Burkholderia cenocepacia, are stabilized by these proteins (13,26,38,39). Further, we show that targeting the DNABII proteins with IHF-and/or HU-specific antibodies induces catastrophic collapse of the biofilm structure and subsequent release of resident bacteria that are now significantly more susceptible to the action of traditional antibiotics (13,28,39). Moreover, in a rat model of periodontitis (oral osteolytic infection) due to the oral pathogen Significance Extracellular DNA and DNABII proteins are essential structural components of the extracellular polymeric substance, or matrix, of the nontypeable Haemophilus influenzae biofilm; however, the mechanisms by which these elements are released from the bacterial cell for incorporation into the biofilm matrix are not yet characterized.…”