Structural Stability of Burkholderia cenocepacia Biofilms Is Reliant on eDNA Structure and Presence of a Bacterial Nucleic Acid Binding Protein

Abstract

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.

Hyaluronan Modulation Impacts Staphylococcus aureus Biofilm Infection

“…This pathogen might contain surface proteins that bind HA and facilitate its incorporation into the biofilm structure. Heparin and eDNA binding proteins have been identified in a number of bacteria, including S. aureus (45)(46)(47)(48)(49)(50), lending support to this idea. Due to the high degree of HA association with the individual cell surfaces in S. aureus biofilm (Fig.…”

Biofilm Matrix Proteins

“…DNABII family proteins, members of the nucleoid-associated protein superfamily, can be grouped into two subtypes: HU (histone-like protein from E. coli strain U39), which is ubiquitous in Eubacteria, and IHF (integration host factor), which is only found in bacteria within the α- and γ-proteobacteria genera (113, 116). Extracellular-localized nucleoid-binding proteins have been found in association with eDNA in the biofilm matrix from sputum samples of cystic fibrosis patients (114, 115). eDNA contributes to biofilm stability in many bacterial species, including P. aeruginosa PAO1 (58).…”

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

“…Extracellular DNA and DNABII proteins are required for biofilm structure and stability. The targeted removal of eDNA and/or DNABII proteins has been shown to cause catastrophic biofilm collapse in vitro and disruption of biofilms in three distinct animal models of disease induced by three important human pathogens of the middle ear, oral cavity, and lung (13,26,29). Here we show that release of DNA and DNABII proteins via a mechanism that involves both the Tra inner-membrane complex and the ComE pore is critical for biofilm formation in vitro.…”

“…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).…”

“…eDNA provides structural stability to the matrix, acts as a sink for antimicrobial peptides, protects resident bacteria from the host immune response, provides a source of "common goods" for the resident bacteria, acts as a universal structural material conducive to microbial community architecture, and facilitates the uptake of genetic material between bacterial species via a process known as horizontal gene transfer (5,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Members of the DNABII family of DNA-binding proteins, integration host factor (IHF) and histone-like protein (HU), are known for their strong structural influences on DNA intracellularly (18-24), and are also critical to the stability of the lattice-like 3D DNA structure found in biofilm matrices extracellularly (13,(25)(26)(27)(28)(29)(30)(31). To date, the presence of bacterial eDNA within biofilms has been attributed to release via various mechanisms, including cell lysis (autolysis or phagemediated) (12,32,33), or active secretion through type IV secretion systems (T4SSs) (34,35).…”