Lazarus Jacob E, Waldor Matthew K, Hooper David C
Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA.
Microbiol Spectr. 2025 Jan 7;13(1):e0194024. doi: 10.1128/spectrum.01940-24. Epub 2024 Dec 10.
Beta-lactam antibiotics are often the treatment of choice for serious bacterial infections. In a previous screen for novel genetic mediators affecting beta-lactam susceptibility, we discovered that deletion of , a conserved gene of unknown function, leads to increased resistance to beta-lactams, as well as increased susceptibility to detergent compounds. Here, we further characterize YdgH in , and using a combination of biochemical and cell biological approaches. We find that YdgH fractionates with periplasmic proteins, and this periplasmic localization is necessary for its function. Using purified recombinant protein, we demonstrate that YdgH is a relatively compact, globular monomer. The YdgH polypeptide contains three tandem DUF1471 domains. In a Δ background, overexpression of polypeptides containing both the second and the third, but not the first DUF1471 domain, is necessary to rescue the deletion phenotype. To determine how YdgH function influences beta-lactam and detergent susceptibility, we tested several targeted hypotheses. We found that deletion of neither affects or transcript levels nor does it alter the processing of lipopolysaccharide, nor does it activate the sigma E regulon alone or in combination with mutations in other periplasmic proteins. Finally, we delineate the results of a genetic screen for spontaneous mutants that complement the detergent susceptibility phenotype, the results of which may fuel the further studies that are necessary to determine the precise role YdgH plays in bacterial physiology.IMPORTANCEBeta-lactams such as penicillins and cephalosporins are the most commonly prescribed antibiotics for serious bacterial infections. Increasing antibiotic resistance threatens their effectiveness. We previously identified the uncharacterized gene as a modifier of beta-lactam susceptibility in Gram-negative bacteria. To begin to understand the specific role of YdgH, in this study, we perform initial characterizations of this protein. We also test hypotheses as to how the function of YdgH contributes to beta-lactam physiology.
β-内酰胺类抗生素通常是严重细菌感染的首选治疗药物。在先前针对影响β-内酰胺敏感性的新型遗传介质进行的筛选中，我们发现，一个功能未知的保守基因的缺失会导致对β-内酰胺类药物的耐药性增加，以及对去污剂化合物的敏感性增加。在此，我们使用生化和细胞生物学方法相结合的方式，进一步对大肠杆菌中的YdgH进行表征。我们发现YdgH与周质蛋白一起分级分离，这种周质定位对其功能是必需的。使用纯化的重组蛋白，我们证明YdgH是一种相对紧凑的球状单体。YdgH多肽包含三个串联的DUF1471结构域。在Δ背景下，过表达包含第二个和第三个但不包含第一个DUF1471结构域的多肽对于挽救缺失表型是必需的。为了确定YdgH功能如何影响β-内酰胺和去污剂敏感性，我们测试了几个有针对性的假设。我们发现，缺失既不影响转录水平，也不改变脂多糖的加工过程，也不会单独或与其他周质蛋白中的突变一起激活σE调控子。最后，我们描述了对补充去污剂敏感性表型的自发突变体进行遗传筛选的结果，其结果可能会推动进一步的研究，以确定YdgH在细菌生理学中的确切作用。重要性青霉素和头孢菌素等β-内酰胺类药物是治疗严重细菌感染最常用的抗生素。抗生素耐药性的增加威胁着它们的有效性。我们之前在革兰氏阴性细菌中鉴定出未表征的基因作为β-内酰胺敏感性的调节因子。为了开始了解YdgH的具体作用，在本研究中，我们对该蛋白进行了初步表征。我们还测试了关于YdgH功能如何影响β-内酰胺生理学的假设。
