Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA.
Department of Microbiology, College of Arts and Sciences, University of Georgia, Athens, Georgia, USA.
J Bacteriol. 2023 May 25;205(5):e0006723. doi: 10.1128/jb.00067-23. Epub 2023 Apr 18.
Gram-negative bacteria have a unique cell surface that can be modified to maintain bacterial fitness in diverse environments. A well-defined example is the modification of the lipid A component of lipopolysaccharide (LPS), which promotes resistance to polymyxin antibiotics and antimicrobial peptides. In many organisms, such modifications include the addition of the amine-containing constituents 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN). Addition of pEtN is catalyzed by EptA, which uses phosphatidylethanolamine (PE) as its substrate donor, resulting in production of diacylglycerol (DAG). DAG is then quickly recycled into glycerophospholipid (GPL) synthesis by the DAG kinase A (DgkA) to produce phosphatidic acid, the major GPL precursor. Previously, we hypothesized that loss of DgkA recycling would be detrimental to the cell when LPS is heavily modified. Instead, we found that DAG accumulation inhibits EptA activity, preventing further degradation of PE, the predominant GPL of the cell. However, DAG inhibition of pEtN addition results in complete loss of polymyxin resistance. Here, we selected for suppressors to find a mechanism of resistance independent of DAG recycling or pEtN modification. Disrupting the gene encoding the adenylate cyclase, , fully restored antibiotic resistance without restoring DAG recycling or pEtN modification. Supporting this, disruptions of genes that reduce CyaA-derived cAMP formation (e.g., ) or disruption of the cAMP receptor protein, Crp, also restored resistance. We found that loss of the cAMP-CRP regulatory complex was necessary for suppression and that resistance arises from a substantial increase in l-Ara4N-modified LPS, bypassing the need for pEtN modification. Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance.
革兰氏阴性菌具有独特的细胞表面，可通过修饰来维持其在不同环境中的生存能力。一个明确的例子是脂多糖（LPS）中脂质 A 成分的修饰，这有助于提高对抗多粘菌素抗生素和抗菌肽的抗性。在许多生物体中，这种修饰包括添加含胺成分 4-氨基-4-去氧-L-阿拉伯糖（L-Ara4N）和磷酸乙醇胺（pEtN）。pEtN 的添加由 EptA 催化，EptA 以磷脂酰乙醇胺（PE）为底物供体，产生二酰基甘油（DAG）。然后，DAG 激酶 A（DgkA）迅速将其回收用于甘油磷脂（GPL）合成，产生磷脂酸，这是主要的 GPL 前体。此前，我们假设当 LPS 严重修饰时，DgkA 回收的丧失对细胞是有害的。相反，我们发现 DAG 的积累抑制了 EptA 的活性，从而阻止了 PE 的进一步降解，PE 是细胞中主要的 GPL。然而，DAG 抑制 pEtN 的添加会导致多粘菌素抗性完全丧失。在这里，我们选择了抑制剂，以找到一种独立于 DAG 回收或 pEtN 修饰的抗性机制。破坏编码腺苷酸环化酶的基因 ，完全恢复了抗生素抗性，而没有恢复 DAG 回收或 pEtN 修饰。支持这一点的是，减少 CyaA 衍生 cAMP 形成的基因（例如 ）的破坏或 cAMP 受体蛋白 Crp 的破坏也恢复了抗性。我们发现，cAMP-CRP 调节复合物的缺失对于抑制是必要的，并且抗性的产生来自于 LPS 中 L-Ara4N 修饰的大量增加，从而无需进行 pEtN 修饰。革兰氏阴性菌可以改变其 LPS 的结构，以促进对抗阳离子抗菌肽的抗性，包括多粘菌素抗生素。多粘菌素被认为是治疗多重耐药革兰氏阴性菌的最后手段抗生素。在这里，我们探讨了一般代谢和碳分解代谢物阻遏途径的变化如何改变 LPS 结构并影响多粘菌素抗性。
