Data_Sheet_1_AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion.docx

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

本研究经鉴定发现，一株携带转座子插入至asnB基因5'非翻译区的单核细胞增生李斯特菌（Listeria monocytogenes）ScottA突变株，对抗菌剂反式肉桂醛（t-cinnamaldehyde）呈现高度敏感表型。本研究针对AsnB在肽聚糖（peptidoglycan, PG）修饰与胞内感染过程中的功能开展了表征分析。尽管李斯特菌的AsnB被注释为谷氨酰胺依赖型天冬酰胺合酶，但序列比对结果显示，该蛋白与一类可催化其他细菌物种肽聚糖中内消旋二氨基庚二酸（meso-diaminopimelic acid, mDAP）残基酰胺化的同源蛋白亚群具有紧密的进化相关性。通过对比asnB突变株、同基因野生型（wild-type, WT）及互补突变菌株的肽聚糖结构，研究证实AsnB可介导单核细胞增生李斯特菌中的mDAP酰胺化过程。mDAP酰胺化缺陷会使asnB突变株出现多种与肽聚糖及细胞表面相关的表型：包括细胞形态变短变粗、对溶菌酶敏感性升高、丧失鞭毛形成能力与运动能力，以及生物膜形成能力显著降低。此外，该突变株对人类上皮JEG-3和Caco-2细胞的侵袭能力也出现显著下降。免疫荧光显微镜分析显示，asnB基因失活会破坏入侵蛋白InlA在李斯特菌表面的正常展示——该蛋白通常可通过其LPXTG基序与mDAP发生交联。综上，本研究表明单核细胞增生李斯特菌的AsnB，与其相关革兰氏阳性菌中的多数同源蛋白一样，可介导肽聚糖中mDAP残基的酰胺化过程，进而影响多项细胞壁与细胞表面相关特性。本研究还首次证实，肽聚糖mDAP残基的酰胺化参与了InlA的细胞壁锚定以及细菌的毒力过程。