一种新颖的基于生长的选择策略，确定了单核细胞增生李斯特菌中主要毒力调节剂PrfA的新组成型活性变体。,Journal of Bacteriology

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一种新颖的基于生长的选择策略，确定了单核细胞增生李斯特菌中主要毒力调节剂PrfA的新组成型活性变体。
Journal of Bacteriology ( IF 2.7 ) Pub Date : 2020-05-11 , DOI: 10.1128/jb.00115-20
Sabine Hansen _{1,

2} , Michael Hall _{2,

3} , Christin Grundström _{2,

3} , Kristoffer Brännström ₄ , A Elisabeth Sauer-Eriksson _{2,

5} , Jörgen Johansson _{2,

6,

7}

Affiliation

单核细胞增生李斯特菌是能够引起严重人类感染的革兰氏阳性病原体。它的主要毒力调节剂是转录激活剂PrfA，它是Crp / Fnr转录调节剂家族的成员。要建立成功的单核细胞增生李斯特氏菌感染，PrfA蛋白必须处于活性构型，方法是结合同源诱导物谷胱甘肽（GSH）或通过氨基酸取代使蛋白具有组成型活性（PrfA *）。通过一个未知的机制，磷酸转移酶系统（PTS）糖抑制PrfA的活性。因此，我们采用了基于转座子的方法来确定PTS糖抑制PrfA活性的机制。为此，我们筛选了一个转座子突变体库，以鉴定能够在6-磷酸葡萄糖作为唯一碳源的情况下生长的克隆。出奇，大多数分离的转座子突变体在PrfA中也带有氨基酸取代。在无转座子的菌株中，PrfA氨基酸取代突变体表现出生长，毒力因子表达，感染性和DNA结合，与先前鉴定的PrfA *突变体一致。因此，在分离的克隆中观察到的初始生长表型是由于PrfA中的氨基酸取代，与转座子突变体失活的基因座无关。最后，我们提供了存在中间激活的PrfA状态的结构性证据，这为PrfA蛋白激活提供了新的见解。重要提示革兰氏阳性单核细胞增生性李斯特菌是一种人类病原体，主要影响老年人，免疫力低下的人群和孕妇。它可能导致脑膜脑炎，败血病和流产。单核细胞增生李斯特氏菌的主要毒力调节剂是PrfA蛋白，一种转录激活因子。使用基于增长的选择策略，我们确定了PrfA蛋白中的突变，导致组成性活性毒力因子表达。我们为中间激活的PrfA状态的存在提供结构证据，这为PrfA蛋白激活提供了新的见解。

"点击查看英文标题和摘要"

A Novel Growth-Based Selection Strategy Identifies New Constitutively Active Variants of the Major Virulence Regulator PrfA in Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive pathogen able to cause severe human infections. Its major virulence regulator is the transcriptional activator PrfA, a member of the Crp/Fnr family of transcriptional regulators. To establish a successful L. monocytogenes infection, the PrfA protein needs to be in an active conformation, either by binding the cognate inducer glutathione (GSH) or by possessing amino acid substitutions rendering the protein constitutively active (PrfA*). By a yet unknown mechanism, phosphotransferase system (PTS) sugars repress the activity of PrfA. We therefore took a transposon-based approach to identify the mechanism by which PTS sugars repress PrfA activity. For this, we screened a transposon mutant bank to identify clones able to grow in the presence of glucose-6-phosphate as the sole carbon source. Surprisingly, most of the isolated transposon mutants also carried amino acid substitutions in PrfA. In transposon-free strains, the PrfA amino acid substitution mutants displayed growth, virulence factor expression, infectivity, and DNA binding, agreeing with previously identified PrfA* mutants. Hence, the initial growth phenotype observed in the isolated clone was due to the amino acid substitution in PrfA and unrelated to the loci inactivated by the transposon mutant. Finally, we provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.IMPORTANCE The Gram-positive bacterium Listeria monocytogenes is a human pathogen affecting mainly the elderly, immunocompromised people, and pregnant women. It can lead to meningoencephalitis, septicemia, and abortion. The major virulence regulator in L. monocytogenes is the PrfA protein, a transcriptional activator. Using a growth-based selection strategy, we identified mutations in the PrfA protein leading to constitutively active virulence factor expression. We provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.

更新日期：2020-05-11

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