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）构成的外层和磷脂构成的内层组成。脂多糖层作为分子筛限制扩散到小溶质。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、细胞膜、周质和外膜中。为了适应环境的变化或压力，细菌通过用不同的基团修饰LPS的结构，共价修饰LPS的结构。LPS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线索作出反应。共价修饰的一个例子包括通过称为pEtN转移酶的酶将磷酸乙醇胺（pEtN）从磷脂磷脂酰乙醇胺转移到LPS。LPS的几个结构域可以被特定的pEtN转移酶修饰。例如，PmrC （EptA）将pEtN添加到脂质A的磷酸基团上，而CptA将pEtN添加到我在LPS内核中发现的庚糖的磷酸基团上。最初，与pEtN修饰的LPS与宿主阳离子抗菌肽的耐药性有关。在这项提议中，我们假设pEtN修饰具有新的功能，可以极大地影响外膜响应应力（如过量的铁）的功能。鼠柠檬酸杆菌（Citrobacter rodentium）是肠杆菌科（Enterobacteriaceae）的革兰氏阴性大菌群，之所以选择它作为模式生物，是因为：(a)鼠柠檬酸杆菌的PmrAB信号独立于PhoPQ，因为鼠柠檬酸杆菌的基因组中没有pmrD基因。(b) C. rodentium不具有负责向脂质A添加4-氨基糖的臂操纵子，这是另一种干扰pEtN修饰的LPS修饰。通过研究pEtN修饰在C. rodentium中的作用，我们发现当细菌在铁存在下生长时，缺乏pEtN修饰会影响外膜的通透性和完整性。我们的短期目标是描述由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LPS向外膜的转运。与野生型菌株相比，在铁存在下生长的DeltapmrC-DeltacptA啮齿鼠菌株产生的LPS量增加，并在质周空间积累。该菌株与pmrC或cptA的互补促进了LPS向外膜的运输。(2)在发现DeltapmrC-DeltacptA C. rodentium菌株在环境中LPS的脱落急剧减少后，我们将评估PmrC和CptA介导的pEtN修饰是否会影响含有LPS的外膜囊泡的释放。(3)除了pmrC和cptA外，啮齿鼠基因组还包含两个编码pEtN转移酶的基因。这两个额外的pEtN转移酶的作用将被确定。总的来说，提出的研究计划可能会揭示影响细菌生理的C. rodentium LPS的pEtN修饰的新功能。这些发现很可能也适用于肠杆菌科的其他物种。我们的长期目标是通过使用特定抑制剂靶向pEtN转移酶的活性来控制外膜通透性。

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.