Preventing biofilm assembly or facilitating biofilm dispersal would expedite treatment of biofilm-related infections. We hypothesize that penicillin-binding proteins (PBPs) are responsible for non-canonical D-amino acid incorporation into the peptidoglycan of Bacillus subtilis and, once incorporated, play a role in biofilm stability. Throug this proposed work, we expect to show that non-canonical D-amino acids, specifically D-Tyr, are incorporated into the pentapeptide of B. subtilis peptidoglycan in vivo using a combination of enzymatic degradation, chromatography, mass spectrometry, and comparison to chemically synthesized standards. We will also show that B. subtilis PBP 1a and 1b can perform this function in vitro by utilizing chemically synthesized lipid II substrate and cloned PBP1a and 1b from B. subtilis to assess their incorporation efficiency. Mutations in a single gene influence the disassembly of biofilms of Bacillus subtilis. We expect that recently identified mutations in ponA, the gene encoding for PBP 1a and 1b will give rise to mutant PBPs that are inefficient in incorporating D-Tyr into peptidoglycan. We will clone, over express, and purify the mutant B. subtilis PBPs and test incorporation efficiency to further validate our hypothesis that cell wall tailoring is responsible for biofilm regulation. This work will provide the foundation for elucidatng the mechanism of D- amino acid triggered biofilm disassociation. Demonstrating biofilm regulation by PBPs will allow for the development of new strategies toward treatment of biofilm related infections.

Public Health Relevance

Infectious bacteria form biofilms that are a serious threat to public health. The research proposed here is directed toward understanding the mechanism of biofilm dispersal. A better understanding of how these biofilms dissociate might lead to therapeutic strategies to better combat bacterial infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM103056-02
Application #
8514399
Study Section
Special Emphasis Panel (ZRG1-F04-K (09))
Program Officer
Barski, Oleg
Project Start
2012-08-01
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
2
Fiscal Year
2013
Total Cost
$52,190
Indirect Cost
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Lebar, Matthew D; May, Janine M; Meeske, Alexander J et al. (2014) Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis. J Am Chem Soc 136:10874-7
Qiao, Yuan; Lebar, Matthew D; Schirner, Kathrin et al. (2014) Detection of lipid-linked peptidoglycan precursors by exploiting an unexpected transpeptidase reaction. J Am Chem Soc 136:14678-81
Sham, Lok-To; Butler, Emily K; Lebar, Matthew D et al. (2014) Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis. Science 345:220-2
Lupoli, Tania J; Lebar, Matthew D; Markovski, Monica et al. (2014) Lipoprotein activators stimulate Escherichia coli penicillin-binding proteins by different mechanisms. J Am Chem Soc 136:52-5
Leiman, Sara A; May, Janine M; Lebar, Matthew D et al. (2013) D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis. J Bacteriol 195:5391-5