Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA
Infect Immun. 2019 Mar 25;87(4). doi: 10.1128/IAI.00884-18. Print 2019 Apr.
Adaptation of to host microenvironments during chronic infection involves spontaneous mutations, yet changes underlying adaptive phenotypes remain incompletely explored. Here, we employed artificial selection and whole-genome sequencing to better characterize spontaneous chromosomal mutations that alter two pathogenicity phenotypes relevant to chronic infection in : intracellular invasiveness and intracellular cytotoxicity. We identified 23 genes whose alteration coincided with enhanced virulence, 11 that were previously known and 12 (52%) that had no previously described role in pathogenicity. Using precision genome editing, transposon mutants, and gene complementation, we empirically assessed the contributions of individual genes to the two virulence phenotypes. We functionally validated 14 of 21 genes tested as measurably influencing invasion and/or cytotoxicity, including 8 newly implicated by this study. We identified inactivating mutations (, , and a hypothetical membrane protein) and gain-of-function mutations ( Thr182Ile, Thr74Ile, and Asp486Glu in a hypothetical peptidase) in previously unrecognized virulence genes that enhance pathogenesis when introduced into a clean genetic background, as well as a novel activating mutation in the known virulence regulator gene (Ala106Thr). Investigation of potentially epistatic interactions identified a mutation (Ala271Val) that enhances virulence only in the context of purine operon repressor gene () inactivation. This project reveals a functionally diverse range of genes affected by gain- or loss-of-function mutations that contribute to adaptive virulence phenotypes. More generally, the work establishes artificial selection as a means to determine the genetic mechanisms underlying complex bacterial phenotypes relevant to adaptation during infection.
在慢性感染过程中，宿主微环境的适应涉及自发突变，但适应性表型变化的基础仍未得到充分探索。在这里，我们采用人工选择和全基因组测序来更好地描述改变与慢性感染相关的两种致病性表型的自发染色体突变，这两种表型分别是细胞内侵袭性和细胞内细胞毒性。我们确定了 23 个基因的改变与毒力增强相关，其中 11 个是以前已知的，12 个（52%）在 致病性中没有以前描述的作用。使用精确的基因组编辑、转座子突变体和基因互补，我们通过经验评估了个别基因对两种毒力表型的贡献。我们对 21 个经过测试的基因中的 14 个进行了功能验证，这些基因可测量地影响侵袭和/或细胞毒性，包括本研究新涉及的 8 个基因。我们鉴定了失活突变（、、和一个假设的膜蛋白）和功能获得性突变（ Thr182Ile、 Thr74Ile 和 Asp486Glu 在一个假设的肽酶中）在以前未被识别的 毒力基因中，当引入干净的遗传背景时会增强发病机制，以及已知毒力调节基因 （Ala106Thr）中的一个新的激活突变。对潜在上位性相互作用的研究发现，一个新的激活突变（Ala106Thr）在已知毒力调节基因 （Ala106Thr）中，只有在嘌呤操纵子阻遏基因（）失活的情况下，突变（Ala271Val）才会增强毒力。该项目揭示了一系列受功能获得或功能丧失突变影响的功能多样化基因，这些突变有助于 适应性毒力表型。更普遍地说，这项工作确立了人工选择作为确定与感染过程中适应相关的复杂细菌表型遗传机制的一种手段。
