Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute , The University of Melbourne , Parkville , Victoria 3010 , Australia.
J Proteome Res. 2019 Apr 5;18(4):1567-1581. doi: 10.1021/acs.jproteome.8b00860. Epub 2019 Feb 26.
The identification and localization of outer membrane proteins (Omps) and lipoproteins in pathogenic treponemes such as T. denticola (periodontitis) and T. pallidum (syphilis) has been challenging. In this study, label-free quantitative proteomics using MaxQuant was applied to naturally produced outer membrane vesicles (OMVs) and cellular fractions to identify 1448 T. denticola proteins. Of these, 90 proteins were localized to the outer membrane (OM) comprising 59 lipoproteins, 25 β-barrel proteins, and six other putative OM-associated proteins. Twenty-eight lipoproteins were localized to the inner membrane (IM), and 43 proteins were assigned to the periplasm. The signal cleavage regions of the OM and IM lipoprotein sequences were different and may reveal the signals for their differential localization. Proteins significantly enriched in OMVs included dentilisin, proteins containing leucine-rich repeats, and several lipoproteins containing FGE-sulfatase domains. Blue native PAGE analysis enabled the native size of the dentilisin complex and Msp to be determined and revealed that the abundant β-barrel Omps TDE2508 and TDE1717 formed large complexes. In addition to the large number of integral Omps and potentially surface-located lipoproteins identified in T. denticola, many such proteins were also newly identified in T. pallidum through homology, generating new targets for vaccine development in both species.
鉴定和定位包括牙周病病原体牙龈卟啉单胞菌（T. denticola）和梅毒病原体苍白密螺旋体（T. pallidum）在内的致病性疏螺旋体中的外膜蛋白（Omps）和脂蛋白一直具有挑战性。在这项研究中，采用 MaxQuant 的无标记定量蛋白质组学方法对天然产生的外膜囊泡（OMVs）和细胞部分进行了分析，共鉴定出 1448 种牙龈卟啉单胞菌蛋白。其中，90 种蛋白定位于外膜（OM），包括 59 种脂蛋白、25 种β桶蛋白和 6 种其他可能与 OM 相关的蛋白。28 种脂蛋白定位于内膜（IM），43 种蛋白被分配到周质空间。OM 和 IM 脂蛋白序列的信号切割区不同，这可能揭示了它们差异定位的信号。在 OMVs 中显著富集的蛋白包括牙龈蛋白酶、富含亮氨酸重复的蛋白和几种含有 FGE-硫酸酯酶结构域的脂蛋白。蓝色 native PAGE 分析能够确定牙龈蛋白酶复合物和 Msp 的天然大小，并揭示了丰富的β桶 Omps TDE2508 和 TDE1717 形成了大型复合物。除了在牙龈卟啉单胞菌中鉴定出大量的完整 Omps 和潜在的表面定位脂蛋白外，通过同源性还在苍白密螺旋体中也新鉴定出许多此类蛋白，为这两个物种的疫苗开发提供了新的靶标。
