Department of Microbiology and Immunology, Center for Infectious Disease Research, Medical College of Wisconsingrid.30760.32, Milwaukee, Wisconsin, USA.
J Bacteriol. 2022 Oct 18;204(10):e0030422. doi: 10.1128/jb.00304-22. Epub 2022 Sep 12.
Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired mechanisms of resistance to commonly used antibiotics, including cephalosporins. The transmembrane serine/threonine PASTA kinase, IreK, is an important determinant of enterococcal cephalosporin resistance. Upon exposure to cephalosporins, IreK becomes autophosphorylated, which stimulates its kinase activity to phosphorylate downstream substrates and drive cephalosporin resistance. However, the molecular mechanisms that modulate IreK autophosphorylation in response to cell wall stress, such as that induced by cephalosporins, remain unknown. A cytoplasmic protein, GpsB, promotes signaling by PASTA kinase homologs in other bacterial species, but the function of enterococcal GpsB has not been previously investigated. We used and approaches to test the hypothesis that enterococcal GpsB promotes IreK signaling in response to cephalosporins to drive cephalosporin resistance. We found that GpsB promotes IreK activity both and . This effect is required for cephalosporins to trigger IreK autophosphorylation and activation of an IreK-dependent signaling pathway, and thereby is also required for enterococcal intrinsic cephalosporin resistance. Moreover, analyses of GpsB mutants and a Δ double mutant suggest that GpsB has an additional function, beyond regulation of IreK activity, which is required for optimal growth and full cephalosporin resistance. Collectively, our data provide new insights into the mechanism of signal transduction by the PASTA kinase IreK and the mechanism of enterococcal intrinsic cephalosporin resistance. Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired resistance to commonly used antibiotics. In particular, enterococci are intrinsically resistant to cephalosporin antibiotics, a trait that requires the activity of a transmembrane serine/threonine kinase, IreK, which belongs to the bacterial PASTA kinase family. The mechanisms by which PASTA kinases are regulated in cells are poorly understood. Here, we report that the cytoplasmic protein GpsB directly promotes IreK signaling in enterococci to drive cephalosporin resistance. Thus, we provide new insights into PASTA kinase regulation and control of enterococcal cephalosporin resistance, and suggest that GpsB could be a promising target for new therapeutics to disable cephalosporin resistance.
肠球菌是一种机会致病菌，可导致严重的细菌感染。这些感染的治疗具有挑战性，因为肠球菌具有内在和获得性机制来抵抗常用抗生素，包括头孢菌素。跨膜丝氨酸/苏氨酸 PASTA 激酶 IreK 是肠球菌头孢菌素耐药性的重要决定因素。暴露于头孢菌素后，IreK 自身磷酸化，这刺激其激酶活性磷酸化下游底物并驱动头孢菌素耐药性。然而，调节 IreK 自身磷酸化以响应细胞壁应激（例如由头孢菌素诱导的应激）的分子机制尚不清楚。细胞质蛋白 GpsB 促进其他细菌物种中 PASTA 激酶同源物的信号传导，但尚未研究肠球菌 GpsB 的功能。我们使用 和 方法来检验假设，即肠球菌 GpsB 促进 IreK 信号传导以响应头孢菌素来驱动头孢菌素耐药性。我们发现 GpsB 既促进 IreK 的 又促进 IreK 的 。这种作用是头孢菌素触发 IreK 自身磷酸化和 IreK 依赖性信号通路激活所必需的，因此也是肠球菌固有头孢菌素耐药性所必需的。此外，对 GpsB 突变体和 Δ 双突变体的分析表明，GpsB 具有除调节 IreK 活性之外的额外功能，这对于最佳生长和完全头孢菌素耐药性是必需的。总的来说，我们的数据提供了 PASTA 激酶 IreK 信号转导机制和肠球菌固有头孢菌素耐药性机制的新见解。
