Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes
Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes
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DOI：
10.1128/jb.02053-14
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发表时间：
2014-11-01
影响因子：
3.2
通讯作者：
Portnoy, Daniel A.
中科院分区：
文献类型：
--
作者：
Burke, Thomas P.;Loukitcheva, Anastasia;Portnoy, Daniel A.
单核细胞增生李斯特菌是一种革兰氏阳性兼性细胞内病原体，对溶菌酶具有高度抗性，溶菌酶是先天免疫系统中普遍存在的酶，可降解细胞壁肽聚糖。两种肽聚糖修饰酶 PgdA 和 OatA 赋予单核细胞增生李斯特菌溶菌酶抗性；然而，这些酶在溶菌酶敏感的非病原体中也是保守的。我们试图找出导致单增李斯特菌溶菌酶抗性的其他因素。对溶菌酶敏感突变体的正向遗传筛选鉴定出 174 个转座子插入突变，这些突变映射到 13 个单独的基因。四种突变体仅被溶菌酶而非其他细胞壁靶向分子杀死，包括 pgdA 编码的肽聚糖脱乙酰酶、pbpX 编码的假定羧肽酶、degU 编码的孤儿反应调节剂以及 rli31 编码的高丰度非编码 RNA。 degU和rli31突变体均降低了pbpX和pgdA的表达，但DegU和Rli31并不相互调节。由于pbpX和pgdA也存在于溶菌酶敏感细菌中，这表明新酶的获得并不是造成溶菌酶抗性的原因，而是DegU和Rli31对保守酶的调节赋予了高度溶菌酶抗性。每种溶菌酶敏感突变体在小鼠中均表现出减弱的毒力，感染的时间过程显示，对溶菌酶最敏感的菌株在静脉感染后 30 分钟内被杀死，这种表型在纯化的血液中得到重现。总的来说，这些数据表明，溶菌酶抗性所需的基因是单核细胞增生利斯特氏菌发病机制高度上调的决定因素，是避免血液中溶菌酶的酶活性所必需的。
Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.