Nontypeable Haemophilus influenzae gene expression induced in vivo in a chinchilla model of otitis media
Nontypeable Haemophilus influenzae gene expression induced in vivo in a chinchilla model of otitis media
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DOI：
10.1128/iai.71.6.3454-3462.2003
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发表时间：
2003-06-01
影响因子：
3.1
通讯作者：
Bakaletz, LO
中科院分区：
文献类型：
--
作者：
Mason, KM;Munson, RS;Bakaletz, LO
革兰氏阴性细菌不可分型的流感嗜血杆菌（NTHI）是积液性慢性中耳炎的主要病原体，与肺炎链球菌和卡他莫拉菌一起，是急性中耳炎的病原体。为了确定潜在的毒力决定因素，在nthi诱导的中耳炎鼠模型的一个特定解剖位中，通过差异荧光诱导在疾病早期进展中监测细菌基因表达。NTHI菌株86-028NP基因组DNA片段克隆在无启动子gfpmut3基因上游。NTHI菌株86-028NP作为启动子陷阱文库的宿主。将2000个转化体分别接种于龙猫左右中耳腔内。在24和48 h用上耳膜穿刺回收中耳积液，用双色荧光激活细胞分选分离体内诱导产生绿色荧光蛋白的含有启动子元件的克隆。对插入DNA进行测序，并与流感嗜血杆菌Rd株的全基因组序列进行比较。在16,000个克隆的筛选中，我们分离出44个克隆，其中包含编码生物合成酶、代谢和调节蛋白以及功能未知的假设蛋白的独特基因片段。另外8个克隆含有NTHI分离物特有的基因片段。利用定量逆转录- pcr，我们证实了26个克隆在体内的基因表达相对于在体外的表达有所增加。这些数据提供了NTHI细菌在中耳炎早期事件中感知和响应中耳微环境时的反应。
The gram-negative bacterium nontypeable Raemophilus influenzae (NTHI) is the predominant pathogen in chronic otitis media with effusion and, with Streptococcus pneumoniae and Moraxella catarrhalis, is a causative agent of acute otitis media. To identify potential virulence determinants, bacterial gene expression was monitored by differential fluorescence induction during early disease progression in one specific anatomical niche of a chinchilla model of NTHI-induced otitis media. Genomic DNA fragments from NTHI strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene. NTHI strain 86-028NP served as the host for the promoter trap library. Pools of 2,000 transformants were inoculated into the left and right middle ear cavities of chinchillas. Middle ear effusions were recovered by epitympanic tap at 24 and 48 h, and clones containing promoter elements that were induced in vivo and producing green fluorescent protein were isolated by two-color fluorescence-activated cell sorting. Insert DNA was sequenced and compared to the complete genome sequence of H. influenzae strain Rd. In a screen of 16,000 clones, we have isolated 44 clones that contain unique gene fragments encoding biosynthetic enzymes, metabolic and regulatory proteins, and hypothetical proteins of unknown function. An additional eight clones contain gene fragments unique to our NTHI isolate. Using quantitative reverse transcription-PCR, we have confirmed that 26 clones demonstrated increased gene expression in vivo relative to expression in vitro. These data provide insight into the response of NTHI bacteria as they sense and respond to the middle ear microenvironment during early events of otitis media.