Structural and Functional Studies of Lipoprotein Signal Peptidase (LspA) from Methicillin-resistant Staphylococcus aureus(MRSA)

Antimicrobial resistance has drawn more and more attention from the world public health community and global leaders in the past decade. Staphylococcus aureus is one of the most threatening clinical pathogens and has caused global problems due to antimicrobial resistance. There is an urgent need for new drug targets and the development of new antibiotics. Bacterial lipoproteins are primarily peripherally anchored membrane proteins that play various roles in bacterial physiology and virulence both for Gram-positive and Gram-negative bacteria. In this thesis, I have studied the biosynthesis pathway of lipoproteins to understand more about this important class of proteins. The hypothesis is that targeting one of the biosynthesis pathway enzymes may cause a lack of essential lipoproteins in bacteria and influence bacteria growth and their interaction with host cells. I have worked on the structural and functional studies of one specific enzyme involved in the lipoprotein biosynthesis pathway, the lipoprotein signal peptidase (LspA), aiming to develop drugs that kill or weaken the pathogens targeting this enzyme. LspA is essential in Gram-negative bacteria and serves as an important virulence factor in Gram- positive bacteria. Several advantages make LspA a promising target to pursue the development of novel antibiotics. Two naturally occurring antibiotics globomycin and myxovirescin were reported to inhibit LspA activity. The first crystal structure of LspA (from Pseudomonas aeruginosa) was published in complex with globomycin in 2016 (PDB: 5dir). There is a paucity of information regarding LspA from other bacteria species and other forms of crystal structures. This thesis demonstrates the structural and functional studies of LspA from methicillin-resistant Staphylococcus aureus (LspMrs) to gain further information on this enzyme for antibiotics development. Three crystal structures of LspMrs in complex with globomycin (1.9 ?), LspMrs in complex with myxovirescin A1 (2.3 ?), and LspMrs in complex with myxovirescin M1 (2.1 ?) were obtained using the in meso crystallization method. The crystallization work on pursuing the apo-form LspA structure was carried out resulted in several potential protein crystals. LspMrs-globomycin and the LspPae-globomycin complex structures shared very high similarity with a Ca r.m.s.d. value over 155 residues of 0.713 ?. Different structure and surface electrostatics were observed at the end of the β-cradle region and the extracellular loop (EL) region of LspMrs and LspPae. The LspMrs-globomycin and LspMrs-myxovirescin complex structures also showed distinct structure and surface electrostatics in the EL region. Although globomycin and myxovirescin molecules positioned at the catalytic site of LspA on the right side and the left side, a striking backbone structure contained nineteen atoms were obtained from the two antibiotics that appeared at the catalytic site and coordinated with LspA in almost the same way. This discovery may provide a structural guide for developing antibiotics targeting LspA. The functional study of LspA was performed using two activity assays (the gel-shift assay and the F?rster resonance energy transfer assay) developed in our lab. The enzyme kinetic study of LspMrs and LspPae includes the enzyme concentration-dependent test, substrate concentration-dependent test (Michaelis-Menten curve), the dose-response research and type iii of inhibition study for globomycin and myxovirescin. LspPae showed three to ten times higher activity than LspMrs in the assays. Type of inhibition test indicated that globomycin and myxovirescin are both tight-binding inhibitors for LspA. I also performed the activity study of nineteen LspMrs mutants. The catalytic dyad residues D118 and D136 were proved to be essential for the enzyme activity. The highly conserved residues (N106, N133, N52, G54, and R110) are crucial for the function of LspA. Besides LspMrs and LspPae, I have also studied LspA from Streptomyces hagronensis and Myxococcus xanthus which are the two bacteria that produce globomycin and myxovirescin, respectively. Unlike most bacteria that only have one lspA gene (such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa), Streptomyces hagronensis has two lspA genes and Myxococcus xanthus has four lspA genes. The study of these six LspA proteins is aimed to investigate whether they have built resistance to globomycin and myxovirescin. Recombinant constructs of all six proteins were built in this study with reasonable expression level. Purification protocol was developed for one LspA protein from Streptomyces hagronensis. In summary, this thesis has provided information on structural and functional studies of LspA as well as the inhibition study of globomycin and myxovirescin. LspA proteins from four organisms (Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, Streptomyces hagronensis, and Myxococcus xanthus) are included in this project. The crystal structures and kinetic data can be used in intelligent drug design to identify biological molecules that inhibit LspA activity as new antibiotics