Most bacteria surround themselves with a crosslinked polysaccharide polymer called peptidoglycan (PG) that is critical for the maintenance of cell shape and integrity. Because of its essentiality, surface exposure, and uniqueness to bacteria, the PG synthetic pathway has historically been an effective target for many of our most important antibacterial treatments like penicillin and vancomycin. Penicillin targets the PG synthases called the penicillin binding proteins (PBPs). These enzymes come in several varieties, but the major cellular PG synthases are thought to be the bi-functional PBPs because they possess both the transglycosylase and transpeptidase activities needed to synthesize the glycan strands of PG and crosslink them, respectively. Despite their prominence as antibiotic targets, we still do not understand how the bi-functional PBPs assemble the cell-shaped PG meshwork or what additional factors might help them accomplish this task. One of the principle reasons for this has been an over-reliance on penicillin and other antibiotics as probes for the identification of important PG assembly factors. To extend our experimental reach beyond the "crutch" of antibiotic probes, we developed a genetic approach to identify factors needed for proper PBP function in vivo using E. coli as a model system. E. coli encodes three bi-functional PBPs: PBP1A, PBP1B, and PBP1C. Each one is individually dispensable, but the simultaneous inactivation of both PBP1A and PBP1B leads to rapid cell lysis. Based on the essentiality of the PBP1A/PBP1B combination, we reasoned that factors required to promote PBP1A activity could be identified by screening for mutants synthetically lethal with the loss of PBP1B (slb mutants) and vice versa. Using this approach, we have implicated several known division proteins and a lipoprotein of unknown function in the assembly of PG by PBP1A. In the first two aims of this proposal we describe genetic, cell biological, and biochemical experiments intended to investigate the connection between PBP1A and these factors. These studies will help us determine whether or not the Slb factors are directly interacting with and/or influencing either of the two enzymatic activities of PBP1A. In related work, we discovered that the EnvC protein is likely to be an activator of the PG hydrolases (amidases) AmiA and AmiB that stimulates their activity to bring about daughter cell separation during cytokinesis.
Specific Aim 3 seeks to determine how EnvC and the amidases cooperate to perform such a delicate operation without causing a lethal breach in the PG layer. We will begin addressing this by defining the mechanism by which EnvC might activate the amidases and identifying regulators of this activation activity. The long term goal of our work is to develop a molecular understanding of PG assembly by the PBPs and how it is remodeled in a controlled fashion by PG hydrolases. By gaining this understanding we hope to uncover new ways to disrupt the cellular balance between PG synthesis and hydrolysis for the development of novel classes of lytic antibiotics.

Public Health Relevance

Bacterial cells are typically fortified by a layer of tough polymer meshwork called peptidoglycan. The pathway for peptidoglycan synthesis an excellent antibiotic target because this layer is unique to bacteria and essential for their integrity. The focus of this research proposal is to identify and characterize the full set of cellular factors that build the peptidoglycan layer to help us uncover additional ways to disrupt the pathway for the future development of new antibiotic therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083365-03
Application #
8204857
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Korpela, Jukka K
Project Start
2010-01-01
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
3
Fiscal Year
2012
Total Cost
$413,749
Indirect Cost
$166,249
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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
Paradis-Bleau, Catherine; Kritikos, George; Orlova, Katya et al. (2014) A genome-wide screen for bacterial envelope biogenesis mutants identifies a novel factor involved in cell wall precursor metabolism. PLoS Genet 10:e1004056
Peters, Nick T; Morlot, Cecile; Yang, Desiree C et al. (2013) Structure-function analysis of the LytM domain of EnvC, an activator of cell wall remodelling at the Escherichia coli division site. Mol Microbiol 89:690-701
Cho, Hongbaek; Bernhardt, Thomas G (2013) Identification of the SlmA active site responsible for blocking bacterial cytokinetic ring assembly over the chromosome. PLoS Genet 9:e1003304
Meisner, Jeffrey; Montero Llopis, Paula; Sham, Lok-To et al. (2013) FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis. Mol Microbiol 89:1069-83
Peters, Nick T; Dinh, Thuy; Bernhardt, Thomas G (2011) A fail-safe mechanism in the septal ring assembly pathway generated by the sequential recruitment of cell separation amidases and their activators. J Bacteriol 193:4973-83
Cho, Hongbaek; McManus, Heather R; Dove, Simon L et al. (2011) Nucleoid occlusion factor SlmA is a DNA-activated FtsZ polymerization antagonist. Proc Natl Acad Sci U S A 108:3773-8
Yang, Desiree C; Peters, Nick T; Parzych, Katherine R et al. (2011) An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring. Proc Natl Acad Sci U S A 108:E1052-60
Uehara, Tsuyoshi; Bernhardt, Thomas G (2011) More than just lysins: peptidoglycan hydrolases tailor the cell wall. Curr Opin Microbiol 14:698-703
Dinh, Thuy; Bernhardt, Thomas G (2011) Using superfolder green fluorescent protein for periplasmic protein localization studies. J Bacteriol 193:4984-7

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