Protein-protein interaction essential for bacterial growth and virulence

描述（由申请人提供）：尽管许多抗菌剂靶向细菌肽聚糖的合成，但我们仍然需要详细了解这种动态结构是如何形成和调节的。这将揭示新的和更好的方法来控制细菌感染，以规避对当前抗菌药物日益增长的耐药性。肽聚糖是与短肽交联以形成细胞壁的多糖，并且是生长所必需的。MrcA（PBP 1A）是一种双功能酶，负责延长肽聚糖链（转糖基酶）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）。在大肠在大肠杆菌中，需要外膜蛋白LpoA（YraM）来结合MrcA并激活其转肽酶。与E. coliK 12中，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，并使慢性阻塞性肺病（COPD）恶化，这是导致死亡的主要原因，尤其是在海外。奇异变形杆菌是最复杂的尿路感染的原因。该研究的长期目标是了解LpoA如何调节MrcA功能，并确定阻止MrcA激活的抑制剂。本研究的目的有三：（1）证明两种病原菌的LpoA具有与大肠杆菌相似的功能; coli蛋白，并进一步研究其在奇异变形杆菌致病性中的作用。(2)澄清是否与MrcA结合是关键激活剂，或者LpoA是否与另一种分子结合。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抑制剂的研究。(2)从两种生物体中生长MrcA-LpoA复合物晶体，以确定相互作用表面并了解对MrcA转肽酶活性的影响。Saper小组已经确定了H的晶体结构。流感病毒LpoA，其特征在于可以结合MrcA的保守结合裂缝。由于lpoA基因仅存在于选定的革兰氏阴性细菌家族中，因此LpoA-MrcA的抑制剂可能不会干扰宿主微生物组。此外，这种抑制剂的作用模式将与？内酰胺。 公共卫生相关性：随着耐药细菌的增加，开发不选择耐药性的新抗菌药物是一个高度优先事项。在医院发生的大多数复杂的尿路感染是由于奇异变形杆菌感染。无法分型的流感嗜血杆菌会导致儿童耳部感染，并加剧肺部疾病，特别是在发展中国家。在这两种病原体中，LpoA蛋白对于生长或毒力是必需的，并且刺激细菌细胞壁合成。拟议的研究将确定与酶MrcA结合的LpoA的三维结构，以了解该复合物如何激活细胞壁合成。虽然MrcA是青霉素类药物的靶点，但发现阻断LpoA结合的分子将是互补的，可能更不易受到耐药性的影响。

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. PUBLIC HEALTH RELEVANCE: With drug-resistant bacteria on the rise, developing new antimicrobials that don't select for resistance is a high priority. Most complicated urinary tract infections that occur in hospitals are due to Proteus mirabilis infections. And the nontypeable Haemophilus influenzae causes children's ear infections and exacerbates lung diseases, especially in the developing world. In both of these pathogens, the LpoA protein is essential for growth or virulence, and stimulating bacterial cell wall synthesis. The proposed research will determine three-dimensional structures of LpoA bound to the enzyme MrcA to understand how the complex activates cell wall synthesis. Although MrcA is the target for penicillin-like drugs, finding molecules that block LpoA binding will be complementary and perhaps less susceptible to drug resistance.