Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo

Novotny, Laura A.; Jurcisek, Joseph A.; Goodman, Steven D.; Bakaletz, Lauren O.

doi:10.1016/j.ebiom.2016.06.022

Cited by 85 publications

(142 citation statements)

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“…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). It is expected that NTHI also establish biofilms rapidly in vivo, and as indicated by the data presented herein, the presence of IHF-specific antibody early in this process likely hindered the development of a robust biofilm structure, exposed NTHI to clearance by host immune mediators, and prevented the onset of OM.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Novotny

Clements

Goodman

2017

Clin Vaccine Immunol

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Otitis media (OM) is a common pediatric disease, and nontypeable Haemophilus influenzae (NTHI) is the predominant pathogen in chronic OM, recurrent OM, and OM associated with treatment failure. OM is also a polymicrobial disease, wherein an upper respiratory tract viral infection predisposes to ascension of NTHI from the nasopharynx, the site of colonization, to the normally sterile middle ear, resulting in disease. Using a clinically relevant viral-bacterial coinfection model of NTHIinduced OM, we performed transcutaneous immunization (TCI) via a band-aid delivery system to administer each of three promising NTHI vaccine candidates derived from bacterial adhesive proteins and biofilm mediators: recombinant soluble PilA (rsPilA), chimV4, and integration host factor. Each immunogen was admixed with the adjuvant LT(R192G/L211A), a double mutant of Escherichia coli heat-labile enterotoxin, and assessed for relative ability to prevent the onset of experimental OM. For each cohort, the presence of circulating immunogen-specific antibody-secreting cells and serum antibody was confirmed prior to intranasal NTHI challenge. After bacterial challenge, blinded video otoscopy and tympanometry revealed a significant reduction in the proportion of animals with signs of OM compared to levels in animals receiving adjuvant only, with an overall vaccine efficacy of 64 to 77%. These data are the first to demonstrate the efficacy afforded by TCI with a band-aid vaccine delivery system in a clinically relevant polymicrobial model of OM. The simplicity of TCI with a band-aid and the significant efficacy observed here hold great promise for reducing the global burden of OM in the pediatric population.KEYWORDS IHF, biofilms, chimV4, nontypeable Haemophilus influenzae, rsPilA T ranscutaneous immunization (TCI) is a noninvasive strategy to induce an immune response by engagement of the numerous antigen-presenting cells within the dermis and epidermis (1, 2). This regime results in both systemic and mucosal immune responses, important outcomes as the mucosae serve as critical defensive barriers to antigenic insult and bacterial disease (3, 4). TCI is needle free, which is expected to aid in patient compliance, to limit risks associated with both administration and waste, and to reduce costs related to formulation and delivery (5). Thus, TCI promises to facilitate greater vaccine distribution.Otitis media (OM) is a disease of the uppermost respiratory tract mucosa and is one of the most common bacterial diseases of childhood due to a multifactorial combination of anatomical, immunological, and environmental factors (6). OM is also a polymicrobial disease caused by one or more of several bacterial species that typically reside within the human nasopharynx. The ability of nontypeable Haemophilus influenzae (NTHI), Streptococcus pneumoniae, and Moraxella catarrhalis to ascend from the nasopharynx through the Eustachian tube and gain access to the normally sterile middle ear space is facilitated by perturbation of the physical and inn...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Novotny

Clements

Goodman

2017

Clin Vaccine Immunol

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“…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).…”

Section: Discussionmentioning

confidence: 98%

“…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).…”

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confidence: 99%

NontypeableHaemophilus influenzaereleases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

Jurcisek

Brockman

Novotny

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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The role of extracellular DNA in the formation, architecture, stability, and treatment of bacterial biofilms

Panlilio

Rice

2021

Biotech & Bioengineering

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Advances in biotechnology to treat and cure human disease have markedly improved human health and the development of modern societies. However, substantial challenges remain to overcome innate biological factors that thwart the activity and efficacy of pharmaceutical therapeutics. Until recently, the importance of extracellular DNA (eDNA) in biofilms was overlooked. New data reveal its extensive role in biofilm formation, adhesion, and structural integrity. Different approaches to target eDNA as anti-biofilm therapies have been proposed, but eDNA and the corresponding biofilm barriers are still difficult to disrupt. Therefore, more creative approaches to eradicate biofilms are needed. The production of eDNA often originates with the genetic material of bacterial cells through cell lysis.However, genomic DNA and eDNA are not necessarily structurally or compositionally identical. Variations are noteworthy because they dictate important interactions within the biofilm. Interactions between eDNA and biofilm components may as well be exploited as alternative anti-biofilm strategies. In this review, we discuss recent developments in eDNA research, emphasizing potential ways to disrupt biofilms. This review also highlights proteins, exopolysaccharides, and other molecules interacting with eDNA that can serve as anti-biofilm therapeutic targets.Overall, the array of diverse interactions with eDNA is important in biofilm structure, architecture, and stability. K E Y W O R D Santi-biofilm therapies, biofilms, eDNA, eDNA therapy, eDNA-interactions | BACKGROUNDA global health crisis is growing due to antibiotic resistance. Antibiotics were first prescribed to treat severe infections after Alexander Fleming discovered penicillin in the 1940's. Over time, various classes of antibiotics that target different bacterial machineries were introduced to the market. However, in recent years, the rate of discovery of new antibiotic classes has stagnated whereas the rate of antibiotic consumption has continued to increase (Silver, 2011). As a result, antibiotic resistance can emerge as bacteria respond to therapeutic treatments. The Centers for Disease Control and Prevention reports that at least 2.8 million people get infected and at least 35,000 die because of antibiotic resistant bacteria in the United States annually (CDC, 2019). In Europe, an estimated number of 25,000 deaths are associated to antibiotic resistant bacteria ("Annual report of the European Medicines Agency," 2010). Greater than 30,000 deaths per year are reported in countries like Thailand as well as increasing variants of antibiotic resistant bacteria emerging in South America, the Middle East, and Asia (Howell, 2013). In addition to these statistics, neonatal sepsis attributed to antibiotic resistance are increasing in Tanzania and Mozambique, and approximately 58,000 neonatal sepsis deaths related to antibiotic resistance are reported in India (Hellen et al., 2015

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Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo

Cited by 85 publications

References 45 publications

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

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

NontypeableHaemophilus influenzaereleases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

The role of extracellular DNA in the formation, architecture, stability, and treatment of bacterial biofilms

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