Mokrzan Elaine M, Ward Michael O, Bakaletz Lauren O
Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA.
Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio, USA
J Bacteriol. 2016 Sep 9;198(19):2619-30. doi: 10.1128/JB.01022-15. Print 2016 Oct 1.
Nontypeable Haemophilus influenzae (NTHI), a commensal of the human nasopharynx (hNP), is a common cause of biofilm-associated diseases of the respiratory tract. However, NTHI biofilm biology at the average hNP temperature, i.e., 34°C, has not been well studied. Here we grew NTHI biofilms at 34°C and 37°C, to evaluate relative biofilm growth, expression, and function of the type IV pilus (Tfp), a critical adhesin important for NTHI biofilm formation. The kinetics and regulation of Tfp expression in NTHI biofilms are unclear, especially at 34°C. Tfp expression, as estimated by pilA promoter activity, was distributed throughout the biofilms, with a unique pattern that was dependent on temperature, time in culture, and position within the maturing biofilm. Tfp expression was required for the formation of the characteristic tower structures of NTHI biofilms and was significantly upregulated in NTHI biofilms formed at 34°C versus 37°C. This increase correlated with significantly greater twitching motility at 34°C than at 37°C. Treatment with antisera targeting the major subunit of Tfp (PilA) significantly inhibited NTHI biofilm formation at both temperatures, confirming the importance of this critical adhesin in biofilm formation. Additionally, treatment of preestablished biofilms with antisera against PilA significantly decreased biofilm biomass and mean thickness at both temperatures. These results demonstrated a pivotal role for Tfp in NTHI biofilm formation and stability at the temperature of the hNP, and they underscore the utility of PilA as a vaccine candidate for treatment and/or prevention of NTHI biofilm-associated diseases.
NTHI is an important cause of chronic respiratory tract infections, including otitis media, chronic rhinosinusitis, and exacerbations of chronic obstructive pulmonary disease and cystic fibrosis. The chronic and recurrent nature of these diseases is attributed to the presence of bacterial biofilms, which are highly resistant to antimicrobials. We characterized NTHI biofilm growth and expression of PilA, the major subunit of the Tfp, at the temperature of the hNP, which is the commensal habitat of NTHI. Our results expand the current understanding of the role of Tfp during biofilm formation and maturation at the temperature of both the hNP and the middle ear, and they strengthen support for PilA as a vaccine candidate for the prevention and treatment of NTHI biofilm-associated diseases.
不可分型流感嗜血杆菌（NTHI）是人类鼻咽部（hNP）的一种共生菌，是呼吸道生物膜相关疾病的常见病因。然而，在hNP的平均温度即34°C下NTHI生物膜生物学尚未得到充分研究。在此，我们在34°C和37°C下培养NTHI生物膜，以评估生物膜的相对生长、IV型菌毛（Tfp）的表达及功能，Tfp是NTHI生物膜形成的一种关键黏附素。NTHI生物膜中Tfp表达的动力学和调控尚不清楚，尤其是在34°C时。通过pilA启动子活性估计的Tfp表达在整个生物膜中分布，具有独特模式，该模式取决于温度、培养时间以及成熟生物膜内的位置。NTHI生物膜特征性塔状结构的形成需要Tfp表达，并且在34°C形成的NTHI生物膜中Tfp表达相对于37°C时显著上调。这种增加与34°C时比37°C时显著更高的颤动运动性相关。用靶向Tfp主要亚基（PilA）的抗血清处理在两个温度下均显著抑制NTHI生物膜形成，证实了这种关键黏附素在生物膜形成中的重要性。此外，用抗PilA抗血清处理预先形成的生物膜在两个温度下均显著降低生物膜生物量和平均厚度。这些结果证明了Tfp在hNP温度下NTHI生物膜形成和稳定性中的关键作用，并且强调了PilA作为治疗和/或预防NTHI生物膜相关疾病的疫苗候选物的效用。
NTHI是慢性呼吸道感染的重要病因，包括中耳炎、慢性鼻窦炎以及慢性阻塞性肺疾病和囊性纤维化的加重。这些疾病的慢性和复发性归因于细菌生物膜的存在，其对抗菌药物具有高度抗性。我们在hNP的温度下（hNP是NTHI的共生栖息地）对NTHI生物膜生长和Tfp主要亚基PilA的表达进行了表征。我们的结果扩展了当前对Tfp在hNP和中耳温度下生物膜形成和成熟过程中作用的理解，并且加强了对PilA作为预防和治疗NTHI生物膜相关疾病的疫苗候选物的支持。
