Emerging roles for phosphoethanolamine modification of bacterial lipopolysaccharide

革兰氏阴性菌的外膜是不对称的，由脂多糖(LPS)组成的外层和磷脂组成的内层组成。脂多糖层起到分子筛的作用，将扩散限制在小的溶质中。脂多糖的生物合成是一个复杂的过程，需要多种蛋白质分布在细胞质、内膜、周质和外膜上。为了适应环境的变化或压力，细菌通过用不同的基团修饰脂多糖来共价修饰脂多糖的结构。脂多糖的修饰通常由双组分系统调节，如PhoPQ和PmrAB，它们对包括镁和铁在内的各种环境信号做出反应。共价修饰的一个例子包括通过称为PETN转移酶的酶将磷脂乙醇胺(PETN)从磷脂磷脂酰乙醇胺转移到脂多糖。脂多糖的几个结构域可以被特定的PETN转移酶修饰。例如，PMRC(EPTA)将PETN添加到脂A的磷酸基团中，而CPTA将PETN添加到在脂多糖核心中发现的七糖的磷酸中。最初，用PETN修饰内毒素与对宿主阳离子抗菌肽的抗性有关。 在这个提议中，我们假设PETN修饰具有新的功能，极大地影响外膜的功能，以响应诸如过量铁的压力。轮状柠檬酸杆菌是肠杆菌科的革兰氏阴性大肠菌群，被选为模式菌的原因如下：(A)轮状柠檬酸杆菌PmrAB信号不依赖于PhoPQ，因为轮状柠檬酸杆菌基因组中不存在pmrD基因。(B)轮状梭菌不具有负责将4-氨基阿拉伯糖加到脂A中的ARN操纵子，这是另一种干扰PETN修饰的脂多糖修饰。通过研究PETN修饰在轮状芽孢杆菌中的作用，我们发现当细菌在铁存在的情况下生长时，缺乏PETN修饰会影响细菌外膜的通透性和完整性。 我们的短期目标是描述PMRC和CPTA介导的PETN修饰的新功能。(1)我们的初步数据表明，PETN修饰促进了内毒素向外膜的转运。与野生型菌株相比，在铁存在的情况下生长的DelapmrC-DeltakptA C.rolinum菌株产生更多的内毒素，积聚在周质空间中。该菌株与PMRC或CPTA互补可促进内毒素向外膜的转运。(2)在Delap mrC-DeltakptA C.rodium菌株中发现环境中内毒素的释放显著减少，我们将评估PMRC和CPTA介导的PETN修饰是否影响含内毒素的外膜囊泡的释放。(3)除PMRC和CPTA外，轮齿冠藻基因组中还含有两个编码PETN转移酶的基因。这两个额外的PETN转移酶的作用将被确定。 总体而言，拟议的研究计划可能会揭开影响细菌生理的轮状芽胞杆菌脂多糖的PETN修饰的新功能。这些发现似乎很可能适用于肠杆菌科的其他物种。我们的长期目标是通过用特定的抑制剂靶向PETN转移酶的活性来控制外膜的通透性。

The outer membrane of Gram-negative bacteria is asymmetric and consists of an outer layer made of lipopolysaccharide (LPS) and an inner layer made of phospholipids. The LPS layer acts as a molecular sieve restricting the diffusion to small solutes. LPS biosynthesis is a complex process that requires multiple proteins located in the cytoplasm, inner membrane, periplasm and outer membrane. To adapt to environmental changes or stresses, bacteria covalently modify the structure of LPS by decorating it with various moieties. LPS modifications are commonly regulated by two-component systems, such as PhoPQ and PmrAB that respond to various environmental cues including magnesium and iron. One example of covalent modification consists of the transfer of phosphoethanolamine (pEtN) from the phospholipid phosphatidylethanolamine to LPS by enzymes known as pEtN transferases. Several domains of LPS can be modified by specific pEtN transferases. For example, PmrC (EptA) adds pEtN to the phosphate groups of lipid A, whereas CptA adds pEtN to the phosphate of heptose I found in the LPS inner core. Initially, LPS modifications with pEtN have been associated with resistance to host cationic antimicrobial peptides. In this proposal, we hypothesize that pEtN modifications have novel functions that greatly impact the function of the outer membrane in response to stresses like excess of iron. Citrobacter rodentium, which is a Gram-negative coliform bacterium of the Enterobacteriaceae family, was chosen as a model organism for the following reasons: (a) C. rodentium PmrAB signals independently of PhoPQ, since the pmrD gene is absent from the C. rodentium genome. (b) C. rodentium does not possess the arn operon that is responsible for the addition of 4-aminoarabonose to lipid A, another LPS modification that interferes with pEtN modifications. By investigating the role of pEtN modifications in C. rodentium, we found that the absence of pEtN modifications affects the permeability and integrity of the outer membrane when bacteria were grown in the presence of iron. Our short-term objective is to describe novel functions for pEtN modifications mediated by PmrC and CptA. (1) Our preliminary data suggest that pEtN modifications promote LPS transport to the outer membrane. In contrast to the wild-type strain, a DeltapmrC-DeltacptA C. rodentium strain grown in the presence of iron produces increased amounts of LPS that accumulate in the periplasmic space. Complementation of this strain with either pmrC or cptA promotes the transport of the LPS to the outer membrane. (2) Having found that shedding of LPS in the environment is drastically reduced in the DeltapmrC-DeltacptA C. rodentium strain, we will evaluate whether pEtN modifications mediated by PmrC and CptA influence the release of LPS-containing outer-membrane vesicles. (3) In addition to pmrC and cptA, the C. rodentium genome contains two additional genes encoding pEtN transferases. The roles of these two additional pEtN transferases will be determined. Overall, the proposed research plan is likely to unravel novel functions for pEtN modifications of the C. rodentium LPS that impact bacterial physiology. It appears most likely that these findings can apply to other species of the Enterobacteriaceae family. Our long-term objective is to control outer-membrane permeability by targeting the activity of pEtN transferases with specific inhibitors.