Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae.
Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae.
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DOI：
10.1128/mbio.01466-17
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发表时间：
2017-12-19
期刊：
影响因子：
6.4
通讯作者：
Bakaletz LO
中科院分区：
文献类型：
--
作者：
Das J;Mokrzan E;Lakhani V;Rosas L;Jurcisek JA;Ray WC;Bakaletz LO
不可分型流感嗜血杆菌（NTHI）在中耳形成的生物膜是中耳炎（OM）慢性化、复发和屈光性的核心。然而，特定NTHI生物膜结构出现的机制尚不清楚。我们结合了计算分析工具和计算机建模植根于统计物理与共聚焦成像的NTHI生物膜在体外形成的静态培养过程中，以确定机制，引起区分形态特征。我们使用平行于基底的连续平面内局部细菌密度的成对相关性对NTHI菌株86- 028 NP形成的生物膜的共聚焦图像进行分析，显示存在短长度尺度（≤10 μm）的分形结构。计算机模拟显示，细胞外DNA（eDNA）和IV型菌毛（Tfp）表达在产生分形结构中起着重要作用，并使我们能够预测这些结构在等基因突变体（ΔcomE）中的实质性减少，该突变体将eDNA释放到生物膜基质中的能力显著受损，并且Tfp功能受损。通过分析体外ΔcomE菌株生物膜的共聚焦图像证实了这一预测。分形结构潜在地通过显著增加生物膜与周围环境的接触面积、促进营养交换以及通过产生对群体信号传导的空间正反馈来产生用于NTHI在敌对中耳微环境中存活的小生境。NTHI是OM的主要细菌病原体，OM是全球儿童常见的耳部感染。慢性OM与中耳细菌生物膜形成有关;因此，了解NTHI生物膜形成的机制对于制定NTHI相关OM的治疗策略非常重要。我们使用共聚焦成像的NTHI生物膜在体外形成的数学工具，成对的密度相关性和代理为基础的建模分析的组合方法显示，eDNA和Tfp表达的NTHI生物膜的分形结构的发展中的重要因素。这些结构可以帮助NTHI在恶劣的环境中生存，例如中耳。我们的计算机模拟模型可与实验室或动物建模研究结合使用，以进一步确定OM期间NTHI生物膜形成的机制，从而指导实验室和临床前研究的合理设计，并优化时间和成本。
Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.