Center of Clinical Laboratory, Zhongshan Hospital, School of Medicine, Xiamen University, Xiamen, China.
Institute of Infectious Disease, School of Medicine, Xiamen University, Xiamen, China.
PLoS Negl Trop Dis. 2019 Feb 21;13(2):e0006855. doi: 10.1371/journal.pntd.0006855. eCollection 2019 Feb.
The highly variable tprK gene of Treponema pallidum has been acknowledged to be one of the mechanisms that causes persistent infection. Previous studies have mainly focused on the heterogeneity in tprK in propagated strains using a clone-based Sanger approach. Few studies have investigated tprK directly from clinical samples using deep sequencing.
METHODS/PRINCIPAL FINDINGS: We conducted a comprehensive analysis of 14 primary syphilis clinical isolates of T. pallidum via next-generation sequencing to gain better insight into the profile of tprK in primary syphilis patients. Our results showed that there was a mixture of distinct sequences within each V region of tprK. Except for the predominant sequence for each V region as previously reported using the clone-based Sanger approach, there were many minor variants of all strains that were mainly observed at a frequency of 1-5%. Interestingly, the identified distinct sequences within the regions were variable in length and differed by only 3 bp or multiples of 3 bp. In addition, amino acid sequence consistency within each V region was found among the 14 strains. Among the regions, the sequence IASDGGAIKH in V1 and the sequence DVGHKKENAANVNGTVGA in V4 showed a high stability of inter-strain redundancy.
The seven V regions of the tprK gene in primary syphilis infection demonstrated high diversity; they generally contained a high proportion sequence and numerous low-frequency minor variants, most of which are far below the detection limit of Sanger sequencing. The rampant variation in each V region was regulated by a strict gene conversion mechanism that maintained the length difference to 3 bp or multiples of 3 bp. The highly stable sequence of inter-strain redundancy may indicate that the sequences play a critical role in T. pallidum virulence. These highly stable peptides are also likely to be potential targets for vaccine development.
苍白密螺旋体的高度可变 tprK 基因已被认为是导致持续性感染的机制之一。先前的研究主要集中在使用基于克隆的 Sanger 方法对增殖株中 tprK 的异质性上。很少有研究直接从临床样本中使用深度测序来研究 tprK。
方法/主要发现：我们通过下一代测序对 14 例原发性梅毒临床分离的苍白密螺旋体进行了全面分析，以更好地了解原发性梅毒患者 tprK 的特征。我们的结果表明，在 tprK 的每个 V 区都存在不同序列的混合物。除了先前使用基于克隆的 Sanger 方法报道的每个 V 区的主要序列外，所有菌株都存在许多主要频率为 1-5%的次要变体。有趣的是，在所鉴定的 V 区的不同序列在长度上是可变的，仅相差 3bp 或 3bp 的倍数。此外，在 14 株中发现每个 V 区的氨基酸序列一致性。在这些区域中，V1 区的序列 IASDGGAIKH 和 V4 区的序列 DVGHKKENAANVNGTVGA 显示出菌株间冗余的高度稳定性。
原发性梅毒感染中 tprK 基因的 7 个 V 区显示出高度多样性；它们通常包含高比例的序列和许多低频的次要变体，其中大多数都远低于 Sanger 测序的检测限。每个 V 区的猖獗变异受严格的基因转换机制调控，保持长度差异为 3bp 或 3bp 的倍数。菌株间冗余的高度稳定序列可能表明这些序列在苍白密螺旋体毒力中起着关键作用。这些高度稳定的肽也可能成为疫苗开发的潜在目标。
