Singh Preeti, Bankhead Troy
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America.
PLoS Pathog. 2025 Jan 10;21(1):e1012871. doi: 10.1371/journal.ppat.1012871. eCollection 2025 Jan.
Host-pathogen interactions represent a dynamic evolutionary process, wherein both hosts and pathogens continuously develop complex mechanisms to outmaneuver each other. Borrelia burgdorferi, the Lyme disease pathogen, has evolved an intricate antigenic variation mechanism to evade the host immune response, enabling its dissemination, persistence, and pathogenicity. Despite the discovery of this mechanism over two decades ago, the precise processes, genetic elements, and proteins involved in this system remain largely unknown. The vls locus, which is the site of antigenic variation, has been notoriously challenging to manipulate genetically due to its highly conserved structural features, even with significant advancements in molecular biology and genetic engineering for this highly segmented pathogen. Our study highlights the pivotal role of plasmid topology in facilitating in trans gene recombination. We demonstrate that gene conversion can occur in trans when a copy of vlsE gene is present on a linear plasmid, contrary to previous observations suggesting a cis arrangement is required for vlsE recombination. Significantly, employing this in trans gene conversion strategy with a linear plasmid, we have, for the first time, achieved targeted genetic mutation of putative cis-acting elements in the native vlsE gene. This has unveiled a potentially crucial role for the 17 bp direct repeats that flank the central variable cassette region of vlsE. Furthermore, we validated the reliability and reproducibility of our mutational approach by successfully inserting stop codons at two distinct sites within the central variable cassette of vlsE. Thus, this study presents a significant methodological innovation enabling the direct manipulation of the vls locus and lays the groundwork for systematic exploration of specific mutations affecting the mechanism of antigenic variation. As a result, it creates new avenues for research and raises intriguing questions that could guide the development of novel methods to explore host-pathogen interactions of the agent of Lyme disease.
宿主-病原体相互作用是一个动态的进化过程，在此过程中，宿主和病原体都不断发展出复杂的机制来战胜对方。莱姆病病原体伯氏疏螺旋体进化出了一种复杂的抗原变异机制，以逃避宿主的免疫反应，从而实现其传播、持续存在和致病性。尽管二十多年前就发现了这种机制，但该系统中涉及的精确过程、遗传元件和蛋白质在很大程度上仍然未知。可变淋巴细胞表面抗原（vls）位点是抗原变异的发生部位，由于其高度保守的结构特征，即使在针对这种高度分段的病原体的分子生物学和基因工程取得了重大进展的情况下，对其进行基因操作仍然极具挑战性。我们的研究突出了质粒拓扑结构在促进反式基因重组中的关键作用。我们证明，当vlsE基因的一个拷贝存在于线性质粒上时，基因转换可以反式发生，这与之前认为vlsE重组需要顺式排列的观察结果相反。重要的是，通过将这种反式基因转换策略应用于线性质粒，我们首次实现了对天然vlsE基因中假定的顺式作用元件的靶向基因突变。这揭示了vlsE中央可变盒区域两侧的17 bp直接重复序列可能具有至关重要的作用。此外，我们通过在vlsE的中央可变盒内的两个不同位点成功插入终止密码子，验证了我们突变方法的可靠性和可重复性。因此，本研究提出了一项重大的方法创新，能够直接操纵vls位点，并为系统探索影响抗原变异机制的特定突变奠定了基础。结果，它开辟了新的研究途径，并提出了有趣的问题，这些问题可以指导开发探索莱姆病病原体宿主-病原体相互作用的新方法。
