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.
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.