Protein-protein interaction essential for bacterial growth and virulence

描述(申请人提供)：虽然许多抗菌剂的目标是合成细菌肽聚糖，但我们仍然需要详细的分子理解这种动态结构是如何形成和调节的。这将揭示控制细菌感染的新的更好的方法，以绕过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，负责延长肽链(转糖基酶)和形成交联链(转肽酶)，也是β-内酰胺类抗生素(阿莫西林、头孢菌素等)的主要靶点之一。在大肠杆菌中，需要一个外膜蛋白LPOA(YraM)与MRCA结合并激活其转肽酶。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。流感嗜血杆菌会导致儿童的耳朵感染，并加剧慢性阻塞性肺疾病(COPD)，这是一种主要的死亡原因，特别是在海外。奇异变形杆菌是大多数复杂的尿路感染的原因。这项研究的长期目标是了解LPOA是如何调节MRCA功能的，并找出阻止MRCA激活的抑制剂。提出了三个具体的目标：(1)证明这两种病原体的LPOA具有与大肠杆菌蛋白相似的功能，并进一步表征其在奇异P.mirabilis致病中的作用。(2)澄清与MRCA的结合是关键激活剂，还是LPOA与另一分子结合。抑制这种相互作用并阻止细胞生长的突变体将验证未来对抑制剂的寻找。(2)从两种生物中培养MRCA-LPOA复合体的晶体，以确定相互作用表面并了解对MRCA反肽酶活性的影响。Saper基团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结合的保守结合裂隙。由于LPOA基因只存在于特定的革兰氏阴性菌家族中，LPOA-MRCA的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将与β-内酰胺类药物互补，但不同。

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.