Staphylococcus aureus is a Gram-positive pathogen responsible for numerous antibiotic-resistant infections. Identifying vulnerabilities in S. aureus is crucial for developing new antibiotics to treat these infections. With this in mind, we probed the function of GdpS, a conserved Staphylococcal membrane protein containing a cytoplasmic GGDEF domain. These domains are canonically involved in cyclic-di-GMP signaling processes, but S. aureus is not known to make cyclic-di-GMP. Using a transposon screen, we found that loss of GdpS is lethal when combined with disruption in synthesis of the glycolipid anchor of a cell surface polymer called lipoteichoic acid (LTA) or with deletion of genes important in cell division. Taking advantage of a small molecule that inhibits LTA glycolipid anchor synthesis, we selected for suppressors of Δ gdpS lethality. The most prevalent suppressors were hypermorphic alleles of dgkB , which encodes a soluble diacylglycerol (DAG) kinase required to recycle DAG to phosphatidylglycerol. By following up on these suppressors, we found that the GGDEF domain of GdpS interacts directly with DgkB, orienting its active site at the membrane to promote DAG recycling. DAG kinase hypermorphs also suppressed the lethality caused by combined loss of gdpS and cell division factors, highlighting the importance of lipid homeostasis for cell division. GdpS’ positive regulation of DAG kinase function was dependent on the GGDEF domain but not its catalytic residues. As the sole conserved GGDEF-domain protein in Staphylococci, GdpS promotes an enzymatic process independent of cyclic-di-GMP signaling, revealing a new function for the ubiquitously conserved GGDEF domain.

中文翻译：

---

金黄色葡萄球菌使用 GGDEF 蛋白将甘油二酯激酶募集到膜上以进行脂质回收


金黄色葡萄球菌是一种革兰氏阳性病原体，可导致多种抗生素耐药性感染。识别金黄色葡萄球菌的脆弱性对于开发治疗这些感染的新抗生素至关重要。考虑到这一点，我们探测了 GdpS 的功能，GdpS 是一种包含细胞质 GGDEF 结构域的保守葡萄球菌膜蛋白。这些结构域通常参与 cyclic-di-GMP 信号转导过程，但尚不清楚金黄色葡萄球菌会产生 cyclic-di-GMP。使用转座子筛选，我们发现当 GdpS 的丢失与称为脂磷壁酸 （LTA） 的细胞表面聚合物的糖脂锚合成中断或与细胞分裂中重要的基因缺失相结合时，GdpS 的丢失是致命的。利用抑制 LTA 糖脂锚合成的小分子，我们选择了 Δ gdpS 致死率的抑制因子。最普遍的抑制因子是 dgkB 的异形等位基因，它编码将 DAG 回收为磷脂酰甘油所需的可溶性甘油二酯 （DAG） 激酶。通过跟踪这些抑制因子，我们发现 GdpS 的 GGDEF 结构域直接与 DgkB 相互作用，将其活性位点定位在膜上以促进 DAG 循环。DAG 激酶物还抑制了 GDPS 和细胞分裂因子联合损失引起的致死性，突出了脂质稳态对细胞分裂的重要性。GdpS 对 DAG 激酶功能的正调节依赖于 GGDEF 结构域，但不依赖于其催化残基。作为葡萄球菌中唯一保守的 GGDEF 结构域蛋白，GdpS 促进了独立于环状 di-GMP 信号传导的酶促过程，揭示了普遍保守的 GGDEF 结构域的新功能。