Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni
Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni
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DOI：
10.1074/mcp.ra118.001199
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发表时间：
2019-04-01
影响因子：
7
通讯作者：
Cordwell, Stuart J.
中科院分区：
文献类型：
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作者：
Cain, Joel A.;Dale, Ashleigh L.;Cordwell, Stuart J.
空肠弯曲杆菌是一种主要的胃肠道病原体，通常通过食用未经充分准备的家禽获得。由pgl基因簇编码的N-连接蛋白糖基化靶向>80种膜蛋白，并且是无症状的鸡定殖和完全的人毒力所需的。尽管如此，N-糖基化的生物学功能仍然未知。我们检测了pgl基因缺失对C.利用标记液相色谱/串联质谱（LC-MS/MS）和数据独立采集（DIA-SWATH-MS）验证空肠蛋白质组。我们定量了1359个蛋白质，对应于84%的C。pgIB寡糖基转移酶（Δ pgIB）的缺失导致185种蛋白质的丰度的显著变化，其中137种蛋白质通过重新引入pgIB（Δ pgIB：：Δ pgIB）恢复到其野生型水平。pgIB的缺失与pgl靶标的丰度显著降低和应激相关蛋白质（包括ClpB、GroEL、GroES、GrpE和DnaK）的增加相关。与野生型C相比，pgIB突变体在温度（4 ℃和46 ℃）和渗透压（150 mm NaCl）休克后存活率降低，生物膜表型改变。空肠。靶向代谢组学研究证实pgl阴性C.空肠从天冬氨酸（Asp）转换为脯氨酸（Pro）摄取，并积累与蛋白质组变化相关的细胞内琥珀酸，包括升高的PutP/PutA（脯氨酸转运和利用）和降低的DctA/DcuB（分别为天冬氨酸输入和琥珀酸输出）。对某些底物（天冬氨酸、谷氨酸、琥珀酸和α-酮戊二酸）的Δ pgIB趋化性降低，并与转导子样（Tip）蛋白的丰度改变相关。糖基化阴性C.空肠耗尽了所有呼吸相关蛋白质，这些蛋白质允许在低氧下使用替代电子受体。我们首次证明，N-糖基化是必需的特定的酶活性（NAP硝酸还原酶），这是与减少丰度的NapAB糖蛋白。这些数据表明C.空肠生理学
Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry. N-linked protein glycosylation encoded by the pgl gene cluster targets >80 membrane proteins and is required for both nonsymptomatic chicken colonization and full human virulence. Despite this, the biological functions of N-glycosylation remain unknown. We examined the effects of pgl gene deletion on the C. jejuni proteome using label-based liquid chromatography/tandem mass spectrometry (LC-MS/MS) and validation using data independent acquisition (DIA-SWATH-MS). We quantified 1359 proteins corresponding to similar to 84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIASWATH-MS. Deletion of the pgIB oligosaccharyltransferase (Delta pgIB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pgIB (Delta pgIB::Delta pgIB)Deletion of pgIB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including CIpB, GroEL, GroES, GrpE and DnaK. pgIB mutants demonstrated reduced survival following temperature (4 degrees C and 46 degrees C) and osmotic (150 mm NaCl) shock and altered biofilm phenotypes compared with wild-type C. jejuni. Targeted metabolomics established that pgl negative C. jejuni switched from aspartate (Asp) to proline (Pro) uptake and accumulated intracellular succinate related to proteome changes including elevated PutP/PutA (proline transport and utilization), and reduced DctA/DcuB (aspartate import and succinate export, respectively). Delta pgIB chemotaxis to some substrates (Asp, glutamate, succinate and alpha-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tip) proteins. Glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen. We demonstrate for the first time that N-glycosylation is required for a specific enzyme activity (Nap nitrate reductase) that is associated with reduced abundance of the NapAB glycoproteins. These data indicate a multifactorial role for N-glycosylation in C. jejuni physiology.