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

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 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...

Haemophilus spills its guts to make a biofilm

“…The composition of the biofilm matrix differs with different organisms but most often contains extracellular DNA (eDNA), polysaccharides, and proteins. The article in PNAS by Jurcisek et al (3) demonstrates that nontypeable Haemophilus influenzae (Hflu) (4) uses components from two different secretion systems to release DNA and a biofilm-associated DNA-binding protein integration host factor (IHF) to allow efficient biofilm formation. This work shows that the secretion of DNA and DNABII proteins is an active process that uses components from two different apparatuses to enable robust biofilm formation.…”

“…The pilus is a dynamic fiber involved in adherence and motility, and the apparatus that produces the pilus can also be responsible for internalizing DNA for genetic transformation in many gram-negative bacteria (12). Jurcisek et al (3) asked whether the secretin protein that allows the pilus to transit the outer membrane of the envelope might also be the portal for DNA and DNABII protein secretion. Indeed, inactivating the comE gene encoding the secretin totally blocked the secretion of both DNA and IHF.…”

“…Finally, Jurcisek et al (3) show that inactivation of traCG or comE prevents normal biofilm formation and that the amount of DNA and the IHF protein in the biofilms formed by the mutants was drastically reduced. The traCG mutant had reduced biofilm formation relative to the parent strain but was still more competent for biofilm formation than the comE mutant.…”

“…The study by Jurcisek et al (3) shows that eDNA and at least one protein important for maintaining a biofilm are transported in a dedicated process that appears to use proteins normally associated with conjugation (ComC and ComG) and the ComE secretin that is involved in pilus expression and DNA transformation, but there are several next-step questions to be answered. It will be informative to determine which other known factors of the Tra system and the type IV pilus system are required for the secretion event and whether there are unrecognized factors unique to this secretion system.…”

“…The coupled complex envisions the TraCG, ComE, and other unknown factors forming a distinct transport complex that spans the envelope. The individual component model is the one preferred by Jurcisek et al (3), and suggests that the inner membrane complex and ComE secretin act separately. It is unlikely that the DNA and DNABII proteins in the chromosome and the eDNA are arranged identically as depicted.…”