Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.
Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.
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DOI：
10.1371/journal.ppat.1009612
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发表时间：
2021-07
期刊：
影响因子：
6.7
通讯作者：
Giacani L
中科院分区：
文献类型：
--
作者：
Romeis E;Tantalo L;Lieberman N;Phung Q;Greninger A;Giacani L
尽管经过了一个多世纪的研究，对梅毒螺旋体亚种的基因操纵。梅毒的病原体苍白菌（T. pallidum）尚未成功。基因工程工具的缺乏严重限制了人们对白僵菌成功作为病原体背后机制的理解。然而，最近描述的一种体外培养苍白球绦虫的方法，使得在这种病原体中进行转化和选择方案的实验成为可能。在这里，我们描述了一种方法，成功地用卡那霉素抗性（kanR）盒替换了SS14 T. pallidum菌株中的tprA （tp0009）假基因。利用pUC57质粒骨架构建自杀载体。在载体中，kanR基因克隆在tp0574基因启动子的下游。然后将tp0574prom-kanR盒置于与tprA假基因上游和下游序列相同的两个1 kbp同源臂之间。为了诱导kanR盒同源重组并整合到T. pallidum染色体上，将体外培养的SS14株螺旋体暴露在基于cacl2的转化缓冲液中，并在添加含卡那霉素的选择性培养基之前恢复24小时。通过定性PCR、液滴数字PCR （ddPCR）和全基因组测序（WGS）在体外和/或体内繁殖的转化密螺旋体，证明了kanR盒的整合性。RNA的ddPCR分析和质谱分析证实了在体外繁殖的密螺旋体中kanR信息和蛋白的表达。此外，tprA敲除（tprAko-SS14）密螺旋体在卡那霉素浓度下生长，比野生型SS14 （wt-SS14）菌株的MIC高64倍，在卡那霉素处理的感染家兔中生长。我们证明了T. pallidum的基因操作是可以实现的。这一发现将允许应用功能遗传学技术来研究梅毒的发病机制和改进梅毒疫苗的开发。梅毒在许多低收入和中等收入国家仍然是一种地方病，近20年来在高收入国家又死灰复燃。在流行地区，梅毒引起显著的发病率和死亡率，特别是当其病原体梅毒螺旋体亚种。苍白菌（T. pallidum）在怀孕期间传染给胎儿。更好地了解梅毒螺旋体生物学和梅毒发病机制将有助于制定更好的控制策略。研究苍白球绦虫的局限性之一是，我们无法从基因上改变这种病原体，以评估编码毒力因子的基因的功能，或者在使用该疾病的兔模型进行研究时，无法产生可为疫苗开发提供信息的减毒菌株。在这里，我们报告了一种转化方案，使我们能够将T. pallidum基因组中含有假基因（即非功能基因）的特定区域替换为稳定整合的卡那霉素抗性基因。据我们所知，这是第一次报道一种方法来实现转基因T. pallidum菌株。
Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development. Syphilis is still an endemic disease in many low- and middle-income countries, and it has been resurgent in high-income nations for almost two decades. In endemic areas, syphilis causes significant morbidity and mortality, particularly when its causative agent, the spirochete Treponema pallidum subsp. pallidum (T. pallidum) is transmitted to the fetus during pregnancy. A better understanding of T. pallidum biology and syphilis pathogenesis would help devise better control strategies for this infection. One of the limitations associated with working with T. pallidum was our inability to genetically alter this pathogen to evaluate the function of genes encoding virulence factors or create attenuated strains that could be informative for vaccine development when studied using the rabbit model of the disease. Here, we report a transformation protocol that allowed us to replace a specific region of the T. pallidum genome containing a pseudogene (i.e., a non-functional gene) with a stably integrated kanamycin resistance gene. To our knowledge, this is the first-ever report of a method to achieve a genetically modified T. pallidum strain.
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DOI：
10.1128/genomea.00333-14
发表时间：
2014-04-17
期刊：
Genome announcements
影响因子：
--
作者：
Giacani L;Iverson-Cabral SL;King JC;Molini BJ;Lukehart SA;Centurion-Lara A
通讯作者：
Centurion-Lara A
影响因子：
16.6
作者：
Costa A;Hood IV;Berger JM
通讯作者：
Berger JM
影响因子：
6.4
作者：
Edmondson DG;DeLay BD;Kowis LE;Norris SJ
通讯作者：
Norris SJ
影响因子：
168.9
作者：
Gerbase, AC;Rowley, JT;Mertens, TE
通讯作者：
Mertens, TE
影响因子：
11.4
作者：
Jin, FY;Prestage, GP;Grulich, AE
通讯作者：
Grulich, AE