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。

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.