Localization of Outer Membrane Proteins in Treponema denticola by Quantitative Proteome Analyses of Outer Membrane Vesicles and Cellular Fractions

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.

对外膜蛋白（Omps）和脂蛋白（Lipoproteins）在致病螺旋体如T. denticola（牙周炎）和T. pallidum（梅毒）中的识别与定位一直是一项颇具挑战性的任务。在本研究中，通过MaxQuant技术，采用无标记定量蛋白质组学方法对自然产生的外膜囊泡（OMVs）和细胞组分进行了分析，以识别1448种T. denticola蛋白。其中，90种蛋白定位于外膜（OM），包括59种脂蛋白、25种β-桶状蛋白以及6种其他可能的外膜相关蛋白。28种脂蛋白定位于内膜（IM），而43种蛋白被分配至周质。外膜和内膜脂蛋白序列的信号切割区域存在差异，这或许揭示了它们不同定位的信号。在OMVs中显著富集的蛋白包括dentilisin、富含亮氨酸重复序列的蛋白以及含有FGE-硫酸酯酶域的多种脂蛋白。通过蓝原生电泳分析，确定了dentilisin复合物和Msp的天然大小，并揭示了丰富的β-桶状Omps TDE2508和TDE1717形成了大复合体。除了在T. denticola中鉴定出的大量整合型Omps和可能位于表面的脂蛋白外，通过同源性分析，在T. pallidum中也新发现了许多此类蛋白，为这两种物种的疫苗研发提供了新的靶点。