Preciado Diego, Poley Marian, Tsai Stephanie, Tomney Amarel, Brown Kristy, Val Stephanie
Sheihk Zayed Institute, Washington, DC, United States; Division of Pediatric Otolaryngology-Head and Neck Surgery, Washington, DC, United States.
Sheihk Zayed Institute, Washington, DC, United States; Division of Pediatric Otolaryngology-Head and Neck Surgery, Washington, DC, United States; Center for Genetic Medicine at Children's National Medical Center, Washington, DC, United States.
Int J Pediatr Otorhinolaryngol. 2016 Jan;80:8-16. doi: 10.1016/j.ijporl.2015.11.016. Epub 2015 Nov 24.
Non-typeable Haemophilus influenzae (NTHi) is a ubiquitous bacterial pathogen which accounts for a majority of human upper respiratory tract infections. Laboratory lysate preparations from this bacterium are commonly utilized to investigate the promulgation of inflammatory responses in respiratory and middle ear epithelium both in vivo and in vitro. We undertook an unbiased proteomics based analysis of NTHi lysate preps to: (a) identify abundant bacterial proteins present in these lysates that could play a role in NTHi biological effects and (b) determine the protein content variability in different lysate prep batches from the same NTHI strain.
Proteomic analysis of laboratory NTHi lysate preparations from clinical strain 12.
NTHi lysates were denatured, gel-fractionated, digested by trypsin and the generated peptides were identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS). Western blot analyses for the important proinflammatory enhancer, outer membrane protein 6 (OMP6), was performed to validate the MS findings. Luciferase assays for NF-kB activation were used to measure the pro-inflammatory biologic effects from each NTHi lysate preparation.
The MS identified 793 unique NTHi proteins. Most common and abundant proteins found have been described to either contribute to biofilm formation, elude the innate immune system, or activate epithelial pro-inflammatory pathways such as Toll Like Receptor 2 (TLR-2) signaling and NF-kB transcription factor. Strong positive signal for OMP6 was found in each of the NTHi lysate preparations. Significant NF-kB promoter response activation as expected with NTHi stimulation over control was also noted for each NTHi lysate preparation.
Proteomics was a successful technique to broadly define the protein content of NTHi lysates. This is the first report of the proteome of NTHi lysates widely used in laboratories to study the biological effect of NTHi. Despite the variability of the protein composition from different preps, all the batches of NTHi lysates induced similar NFκB activation.
NA.
不可分型流感嗜血杆菌（NTHi）是一种普遍存在的细菌病原体，在大多数人类上呼吸道感染中起作用。该细菌的实验室裂解物制剂通常用于研究体内和体外呼吸道及中耳上皮中炎症反应的传播。我们对NTHi裂解物制剂进行了基于蛋白质组学的无偏分析，以：（a）鉴定这些裂解物中存在的丰富细菌蛋白，这些蛋白可能在NTHi生物学效应中起作用；（b）确定来自同一NTHI菌株的不同裂解物制剂批次中的蛋白质含量变异性。
对临床菌株12的实验室NTHi裂解物制剂进行蛋白质组学分析。
将NTHi裂解物变性、凝胶分离、用胰蛋白酶消化，然后使用液相色谱串联质谱（LC-MS/MS）鉴定产生的肽段。对重要的促炎增强子外膜蛋白6（OMP6）进行蛋白质印迹分析以验证质谱结果。使用针对NF-κB激活的荧光素酶测定法来测量每种NTHi裂解物制剂的促炎生物学效应。
质谱鉴定出793种独特的NTHi蛋白。发现最常见和丰富的蛋白质要么有助于生物膜形成、逃避先天免疫系统，要么激活上皮促炎途径，如Toll样受体2（TLR-2）信号传导和NF-κB转录因子。在每种NTHi裂解物制剂中均发现OMP6的强阳性信号。每种NTHi裂解物制剂也均观察到与对照相比，NTHi刺激预期的显著NF-κB启动子反应激活。
蛋白质组学是广泛定义NTHi裂解物蛋白质含量的成功技术。这是关于实验室广泛用于研究NTHi生物学效应的NTHi裂解物蛋白质组的首次报告。尽管不同制剂的蛋白质组成存在变异性，但所有批次的NTHi裂解物均诱导了相似的NFκB激活。
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