PDF(Pubmed)
Abstract：
Oral spirochetes are among a small group of keystone pathogens contributing to dysregulation of tissue homeostatic processes that leads to breakdown of the tissue and bone supporting the teeth in periodontal disease. Additionally, our group has recently demonstrated that Treponema are among the dominant microbial genera detected intracellularly in tumor specimens from patients with oral squamous cell carcinoma. While over 60 species and phylotypes of oral Treponema have been detected, T. denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is regularly performed. Thus, T. denticola is a key model organism for studying spirochete metabolic processes, interactions with other microbes, and host cell and tissue responses relevant to oral diseases, as well as venereal and nonvenereal treponematoses whose agents lack workable genetic systems. We previously demonstrated improved transformation efficiency using an Escherichia coli-T. denticola shuttle plasmid and its utility for expression in T. denticola of an exogenous fluorescent protein that is active under anaerobic conditions. Here, we expand on this work by characterizing T. denticola Type I and Type II restriction-modification (R-M) systems and designing a high-efficiency R-M-silent \"SyngenicDNA\" shuttle plasmid resistant to all T. denticola ATCC 35405 R-M systems. Resequencing of the ATCC 33520 genome revealed an additional Type I R-M system consistent with the relatively low transformation efficiency of the shuttle plasmid in this strain. Using SyngenicDNA approaches, we optimized shuttle plasmid transformation efficiency in T. denticola and used it to complement a defined T. denticola ΔfhbB mutant strain. We further report the first high-efficiency transposon mutagenesis of T. denticola using an R-M-silent, codon-optimized, himarC9 transposase-based plasmid. Thus, use of SyngenicDNA-based strategies and tools can enable further mechanistic examinations of T. denticola physiology and behavior.
摘要：
口腔螺旋体是导致组织稳态过程失调的一小部分关键病原体之一，导致牙周病中支撑牙齿的组织和骨骼破裂。此外,我们研究小组最近证明,在口腔鳞状细胞癌患者的肿瘤标本中,螺旋体属是细胞内检测到的优势微生物属之一.虽然已经检测到超过60种口腔密螺旋体的种类和类型，T.denticola是少数可以在培养中生长的一种，也是唯一定期进行遗传操作的一种。因此，T.denticola是研究螺旋体代谢过程的关键模式生物，与其他微生物的相互作用，以及与口腔疾病相关的宿主细胞和组织反应，以及缺乏可行遗传系统的性病和非性病螺旋体病。我们先前证明了使用大肠杆菌T的转化效率提高。Denticola穿梭质粒及其在Denticola中表达在厌氧条件下具有活性的外源荧光蛋白的用途。这里,我们通过表征I型和II型限制性修饰（R-M）系统并设计对所有T.denticolaATCC35405R-M系统具有抗性的高效R-M沉默的“SyngenicDNA”穿梭质粒来扩展这项工作。ATCC33520基因组的重新测序揭示了额外的I型R-M系统，与该菌株中穿梭质粒的相对较低的转化效率一致。使用SyngenicDNA方法，我们优化了T.denticola的穿梭质粒转化效率，并用它来补充确定的T.denticolaΔfhbB突变株。我们进一步报道了使用R-M沉默的T.denticola的首次高效转座子诱变，密码子优化，基于himarC9转座酶的质粒。因此，使用基于SyngenicDNA的策略和工具可以进一步对T.denticola生理和行为进行机械检查。
