Woods Emily C, Nawrocki Kathryn L, Suárez Jose M, McBride Shonna M
Department of Microbiology and Immunology, Emory Antibiotic Resistance Center, Emory University School of Medicine, Atlanta, Georgia, USA.
Department of Microbiology and Immunology, Emory Antibiotic Resistance Center, Emory University School of Medicine, Atlanta, Georgia, USA
Infect Immun. 2016 May 24;84(6):1902-1916. doi: 10.1128/IAI.00207-16. Print 2016 Jun.
Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of d-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σ(V) does not induce dlt expression in response to lysozyme, indicating that σ(V) is required for regulation of lysozyme-dependent d-alanylation of the cell wall. Using reporter gene fusions and 5' RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall d-alanylation in C. difficile is induced by lysozyme in a σ(V)-dependent manner and that this pathway impacts virulence in vivo.
艰难梭菌（也称为难辨梭状芽孢杆菌）是一种主要的医院病原体，也是全球抗生素相关性腹泻的主要病因。艰难梭菌引起疾病需要在肠道内定殖。宿主产生的抗菌蛋白（AMPs），如溶菌酶，存在于肠道中，可阻止许多细菌病原体的定殖，然而艰难梭菌在这些AMPs存在的情况下仍能在结肠中存活。我们之前的研究表明，Dlt途径通过向磷壁酸添加d - 丙氨酸来增加细菌的表面电荷，对艰难梭菌抵抗多种AMPs很重要。我们试图确定调控Dlt途径表达的遗传机制。在本研究中，我们表明dlt基因缺失突变体在溶菌酶中的生长严重减弱，并且dltDABC操纵子的表达会因溶菌酶而被诱导。此外，我们发现缺乏胞外功能（ECF）σ因子σ(V)的突变体不会因溶菌酶而诱导dlt表达，这表明σ(V)是细胞壁溶菌酶依赖性d - 丙氨酰化调控所必需的。使用报告基因融合和5' RACE（cDNA末端快速扩增）分析，我们确定了溶菌酶依赖性和非溶菌酶依赖性dlt表达所需的启动子元件。此外，我们观察到sigV突变体和dlt突变体在仓鼠感染模型中都更具毒力。这些发现表明，艰难梭菌中的细胞壁d - 丙氨酰化以σ(V)依赖性方式由溶菌酶诱导，并且该途径影响体内毒力。
