Abstract
Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.
Highlights
Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease
Extracellular DNA is a major constituent of the extrapolymeric substance (EPS) formed by multiple human pathogens [5,6,7] and serves diverse roles within the bacterial biofilm. 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]
Our laboratory demonstrated that DNABII proteins bind at the vertices of crossed eDNA strands and act as lynchpin-like molecules to stabilize the structure of eDNA within the biofilm matrix formed by nontypeable Haemophilus influenzae (NTHI) and multiple other human pathogens in vitro [25,26,27,28,29,30,31, 37, 38]
Summary
Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. We describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. Our laboratory demonstrated that DNABII proteins bind at the vertices of crossed eDNA strands and act as lynchpin-like molecules to stabilize the structure of eDNA within the biofilm matrix formed by nontypeable Haemophilus influenzae (NTHI) and multiple other human pathogens in vitro [25,26,27,28,29,30,31, 37, 38]. Knowledge of how and when DNA and DNABII proteins are released into the extracellular milieu for integration into the biofilm matrix will further our understanding of biofilm formation and maturation and, in turn, guide development of directed therapies for diseases with a biofilm etiology
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