Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205.
Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH 43210.
Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):E6632-E6641. doi: 10.1073/pnas.1705508114. Epub 2017 Jul 10.
Biofilms formed by nontypeable (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.
非定型（NTHI）形成的生物膜是导致多种人类呼吸道疾病（包括中耳炎、慢性鼻窦炎和囊性纤维化和慢性阻塞性肺疾病恶化）慢性化、复发和治疗耐药的核心。细胞外 DNA（eDNA）和相关的 DNAII 蛋白是 NTHI 及迄今为止所有其他测试过的细菌病原体形成的生物膜的整体结构和结构完整性所必需的。虽然细胞裂解和外膜囊泡挤出是这些典型的细胞内成分可能释放到细胞外环境中并整合到生物膜基质中的可能途径，但我们假设 NTHI 还使用了一种主动 DNA 释放机制。在此，我们描述了一种机制，通过该机制，DNA 和相关的 DNAII 蛋白通过与 IV 型分泌样系统同源的内膜孔复合物（TraC 和 TraG）从细菌细胞质转运到周质。这些成分通过 ComE 孔离开细菌细胞，NTHI 的 IV 型菌毛通过该孔表达。所描述的机制独立于爆炸性细胞裂解或细胞死亡，并且 DNA 的释放仅限于离散的亚极位置，这表明了一种从有活力的 NTHI 释放 DNA 的新形式。鉴定机制并确定关键生物膜基质稳定成分释放的动力学将有助于设计针对由 NTHI 和许多其他已知将 eDNA 和 DNAII 蛋白整合到其生物膜基质中的人类病原体引起的疾病的新型生物膜靶向治疗和预防策略。
