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首页> 外文期刊>Infection and immunity >Nontypeable Haemophilus influenzae Gene Expression Induced In Vivo in a Chinchilla Model of Otitis Media
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Nontypeable Haemophilus influenzae Gene Expression Induced In Vivo in a Chinchilla Model of Otitis Media

机译：不可诱导的流感嗜血杆菌基因表达在中耳炎龙猫模型中体内诱导。

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The gram-negative bacterium nontypeable Haemophilus 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.
机译：革兰氏阴性细菌不可分型的流感嗜血杆菌（NTHI）是慢性中耳炎伴渗出液的主要病原体，并与肺炎链球菌和卡他莫拉菌是急性中耳炎的病原体。为了确定潜在的毒力决定因素，在疾病早期发展过程中，通过NTHI诱导的中耳炎龙猫模型的一个特定解剖位中的差异荧光诱导监测细菌基因的表达。 NTHI菌株86-028NP的基因组DNA片段被克隆到无启动子的 gfpmut3 基因的上游。 NTHI菌株86-028NP充当启动子陷阱文库的宿主。将2,000个转化体的池接种到龙猫的左右中耳腔中。在24和48小时通过鼓室拍打恢复中耳积液，并通过双色荧光激活细胞分选分离出含有在体内被诱导并产生绿色荧光蛋白的启动子元件的克隆。对插入物DNA进行测序，并与 H的完整基因组序列进行比较。流感菌株在16,000个克隆的筛选中，我们分离了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。另外八个克隆包含我们的NTHI分离株特有的基因片段。使用定量逆转录-PCR，我们已经证实，相对于体外表达，26个克隆证明了体内基因表达的增加。这些数据提供了对NTHI细菌在中耳炎早期事件中感知并响应中耳微环境的反应的见解。

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