Non-typeable Haemophilus influenzae contains an N6-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.
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 outermembrane 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 biofilmtargeted 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.eDNA | Tra | secretin | IHF | HU B iofilms contribute substantially to the chronic and recurrent nature of many bacterial diseases of humans and animals, including those of the airway, urogenital tract, skin, and oral cavity (1-3). Bacteria within a biofilm are protected from environmental stresses by a self-produced extracellular matrix, called the extrapolymeric substance (EPS), whose composition varies greatly depending upon the microbes that produce it and the environment in which it is formed. Despite vast compositional variability, the EPS is typically composed of a complex mixture of lipopolysaccharides, proteins, lipids, glycolipids, and nucleic acids (4). Extracellular DNA (eDNA) is a major constituent of the EPS formed by multiple human pathogens (5-7) 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). Members of the DNABII family of DNA-binding proteins, integ...
Middle ear infections (or otitis media [OM]) are highly prevalent among children worldwide and present a tremendous socioeconomic challenge for health care systems. More importantly, this disease diminishes the quality of life of young children. OM is often chronic and recurrent, due to the presence of highly antibiotic-resistant communities of bacteria (called biofilms) that persist within the middle ear space. To combat these recalcitrant infections, new and powerful biofilm-directed approaches are needed. Here, we describe the ability to disrupt a biofilm formed by the two most common bacteria that cause chronic and recurrent OM in children, via an approach that combines the power of vaccines with that of traditional antibiotics. An outcome of this strategy is that antibiotics can more easily kill the bacteria that our vaccine-induced antibodies have released from the biofilm. We believe that this approach holds great promise for both the prevention and treatment of OM.
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