Protein-protein interaction essential for bacterial growth and virulence

描述（由申请人提供）：尽管许多抗菌剂都是针对细菌肽聚糖的合成，但我们仍然需要对这种动态结构是如何形成和调节的详细分子理解。这将揭示新的和更好的方法来控制细菌感染，以避免对当前抗菌素日益增长的耐药性。肽聚糖是一种与短肽交联形成细胞壁的多糖，是生长所必需的。MrcA （PBP1A）是一种双功能酶，负责延长肽聚糖链（转糖基化酶）和形成交联（转肽酶），是？内酰胺类抗生素（阿莫西林、头孢菌素等）。在大肠杆菌中，需要外膜蛋白LpoA （YraM）结合MrcA并激活其转肽酶。与大肠杆菌K12不同，至少有两种病原体需要LpoA才能产生毒力。流感嗜血杆菌引起儿童耳部感染，并加剧慢性阻塞性肺疾病（COPD），这是导致死亡的主要原因，特别是在海外。神奇变形杆菌是大多数复杂尿路感染的罪魁祸首。该研究的长期目标是了解LpoA如何调节MrcA的功能，并确定阻止MrcA激活的抑制剂。提出了三个具体目标：(1)证明来自两种病原体的LpoA的功能与大肠杆菌蛋白相似，并进一步表征其在P. mirabilis致病性中的作用。(2)明确是否与MrcA结合是关键的激活剂，或者LpoA是否与其他分子结合。抑制这种相互作用并阻止细胞生长的突变体将在未来验证对抑制剂的研究。(2)从两种生物体中培养MrcA- LpoA复合物的晶体，以确定相互作用表面并了解对MrcA反式肽酶活性的影响。Saper小组已经确定了流感嗜血杆菌LpoA的晶体结构，并表征了可能与MrcA结合的保守结合裂缝。由于lpoA基因仅存在于革兰氏阴性菌的特定家族中，因此lpoA - mrca抑制剂可能不会干扰宿主微生物群。此外，这种抑制剂的作用方式与-内酰胺互补，但又不同。

DESCRIPTION (provided by applicant): Although many antimicrobials are targeted towards the synthesis of bacterial peptidoglycan, we still need a de- tailed molecular understanding of how this dynamic structure is made and regulated. This will reveal new and better approaches to bacterial infection control to circumvent the growing resistance to current antimicrobials. Peptidoglycan is a polysaccharide cross-linked with short peptides to form a cell wall and is necessary for growth. MrcA (PBP1A) is a bifunctional enzyme responsible for extending the peptidoglycan chain (transglyco- sylase) and forming the cross-links (transpeptidase) and one of the major targets of ?-lactam antibiotics (amox- icillin, cephalosporin, etc). In E. coli an outer membrane protein LpoA (YraM) is required to bind MrcA and acti- vate its transpeptidase. Unlike E. coli K12, at least two pathogens require LpoA for virulence. Haemophilus in- fluenzae causes children's ear infections and exacerbates chronic obstructive pulmonary disorder (COPD), a leading cause of death especially overseas. Proteus mirabilis is responsible for most complicated urinary tract infections. The long-term goal of the research is to understand how LpoA regulates MrcA function, and identify inhitors that prevent activation of MrcA. Three specific aims are proposed: (1) Demonstrate that LpoA from the two pathogens function like the E. coli protein, and further characterize its role in P. mirabilis pathogenicity. (2) Clarify if biding to MrcA is the key activator, or if LpoA binds another molecule. Mutants that inhibit this inter- action and prevent cell growth will validate a search for inhibitors in the future. (2) Grow crystas of an MrcA- LpoA complex from both organisms to define the interacting surfaces and understand effects on MrcA trans- peptidase activity. The Saper group has determined crystal structures of H. influenzae LpoA and characterized a conserved binding cleft that may bind MrcA. Since the lpoA gene is present only in select families of Gram- negative bacteria, an inhibitor of LpoA-MrcA may not disturb the host microbiome. Moreover, the mode of ac- tion of such an inhibitor would be complementary with, but different from, ?-lactams.