Hall Cherisse L, Tschannen Michael, Worthey Elizabeth A, Kristich Christopher J
Department of Microbiology and Molecular Genetics.
Antimicrob Agents Chemother. 2013 Dec;57(12):6179-86. doi: 10.1128/AAC.01472-13. Epub 2013 Sep 30.
Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections, in part due to its intrinsic resistance to cephalosporins. The mechanism that confers intrinsic cephalosporin resistance in enterococci remains incompletely defined. Previously, we have shown that the Ser/Thr protein kinase and phosphatase pair IreK and IreP act antagonistically to regulate cephalosporin resistance in E. faecalis. We hypothesize that IreK senses antibiotic-induced cell wall damage and activates a signaling pathway leading to antibiotic resistance. However, the factors downstream of IreK have not yet been identified. To discover such factors, suppressor mutations that restored resistance to a ΔireK kinase mutant were identified. Mutations were found in IreB, a highly conserved gene of unknown function that is widespread among low-GC Gram-positive bacteria. We show that IreB plays a negative regulatory role in cephalosporin resistance and is an endogenous substrate of both IreK and IreP. IreB is phosphorylated on conserved threonine residues, and mutations at these sites impair cephalosporin resistance. Our results are consistent with a model in which the activity of IreB is modulated by IreK-dependent phosphorylation in a signaling pathway required for cephalosporin resistance and begin to shed light on the function of this previously uncharacterized protein.
粪肠球菌是一种革兰氏阳性细菌，是医院获得性感染的主要原因之一，部分原因是其对头孢菌素具有内在抗性。肠球菌中赋予内在头孢菌素抗性的机制仍未完全明确。此前，我们已经表明，丝氨酸/苏氨酸蛋白激酶和磷酸酶对IreK和IreP起拮抗作用，以调节粪肠球菌对头孢菌素的抗性。我们假设IreK感知抗生素诱导的细胞壁损伤并激活导致抗生素抗性的信号通路。然而，IreK下游的因子尚未被鉴定出来。为了发现这些因子，我们鉴定了恢复对ΔireK激酶突变体抗性的抑制突变。在IreB中发现了突变，IreB是一个功能未知的高度保守基因，在低GC革兰氏阳性细菌中广泛存在。我们表明，IreB在头孢菌素抗性中起负调节作用，并且是IreK和IreP的内源性底物。IreB在保守的苏氨酸残基上被磷酸化，这些位点的突变会损害头孢菌素抗性。我们的结果与一个模型一致，在该模型中，IreB的活性在头孢菌素抗性所需的信号通路中通过IreK依赖性磷酸化进行调节，并开始揭示这种以前未被表征的蛋白质的功能。
