The peptidoglycan (PG) cell wall has long been an attractive target for antibiotic intervention since the late stages of its synthesis take place on he solvent accessible surface of bacterial cells. This essential macromolecule defines bacterial size and shape and provides cells with mechanical strength to resist cell envelope breakdown. Additionally, recent research has demonstrated the importance of the spatiotemporal coordination of PG biosynthesis for bacterial growth, revealing a vulnerability that can be exploited for the development of new antibiotics. The development of methods to enable spatiotemporal tracking of PG synthesis in live bacterial cells is critical to advancing the understanding of the mechanisms of PG synthesis dynamics. In the absence of such methods, identification of new antibiotic targets or identification of novel antibiotic agents will remain elusive. This project has three specific aims that are focused on a long-term goal of elucidating the mechanisms of PG synthesis dynamics. The first Specific Aim seeks to design and develop a series of D-amino acid- and dipeptide-based fluorogenic probes, with optimized photophysical properties, that when coupled with integrated nanochannel and microfluidic devices, and automated image analysis tools, will propel the study of PG dynamics to an unprecedented level of spatiotemporal resolution. The subsequent specific aims will utilize these tools and approaches to analyze the mechanisms of PG dynamics in bacterial model systems with differing cell shapes and cell envelope architectures.
In Specific Aim 2, the probes and methods developed under specific aim 1 will be employed to test two major and long-standing hypotheses regarding the spatiotemporal coordination of the elongation and division PG synthesis machineries as well as the coordination between PG hydrolysis and synthesis in the principal model for ovoid-shaped cells, Streptococcus pneumoniae.
In Specific Aim 3, PG spatiotemporal dynamics will be examined at an unprecedented resolution for the major model species for rod-shaped Gram negative bacteria with a thin layer of PG, E. coli, and for rod-shaped Gram-positive bacteria with a thick layer of PG in the model organism, B. subtilis. Furthermore, Aim 3 will leverage a high-throughput microscopy screening platform, the availability of a comprehensive strain collection in which each gene has been separately deleted, and the powerful genetics of both species, to systematically and randomly screen for genes involved in PG synthesis dynamics. The comparative analysis of the three model systems will identify the core principles of PG dynamics and how they can be modified to yield different outcomes in dynamics, cell shape and cell envelope architecture.
Aims 2 and 3 will feed back into Aim 1 and lead to the design of improved probes and nanochannel configurations. The highly integrated approach, coupled with the individual expertise of the investigators, will provide an unprecedented understanding of PG synthesis and dynamics that can be used to uncover new antibacterial targets, an important step toward addressing the critical need for the discovery of new antibiotics.

Public Health Relevance

Bacterial resistance to currently available therapeutic agents represents a significant threat to public health. The research proposed in this application seeks to continue the development of fluorescent D-amino acid (FDAA) probes for detailed study of the dynamics of peptidoglycan (PG) synthesis. Use of these probes, in conjunction with microfluidic devices and advanced image analysis tools, will provide an unprecedented opportunity for study of the key steps and genes involved in PG synthesis and will unveil new targets for antibiotic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM113172-02
Application #
9005872
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Marino, Pamela
Project Start
2015-02-05
Project End
2018-11-30
Budget Start
2015-12-01
Budget End
2016-11-30
Support Year
2
Fiscal Year
2016
Total Cost
$766,702
Indirect Cost
$251,888
Name
Indiana University Bloomington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
Radkov, Atanas D; Hsu, Yen-Pang; Booher, Garrett et al. (2018) Imaging Bacterial Cell Wall Biosynthesis. Annu Rev Biochem 87:991-1014
Peters, Katharina; Pazos, Manuel; Edoo, Zainab et al. (2018) Copper inhibits peptidoglycan LD-transpeptidases suppressing ?-lactam resistance due to bypass of penicillin-binding proteins. Proc Natl Acad Sci U S A 115:10786-10791
Weaver, Anna I; Murphy, Shannon G; Umans, Benjamin D et al. (2018) Genetic Determinants of Penicillin Tolerance in Vibrio cholerae. Antimicrob Agents Chemother 62:
Pende, Nika; Wang, Jinglan; Weber, Philipp M et al. (2018) Host-Polarized Cell Growth in Animal Symbionts. Curr Biol 28:1039-1051.e5
Caccamo, Paul D; Brun, Yves V (2018) The Molecular Basis of Noncanonical Bacterial Morphology. Trends Microbiol 26:191-208
Monteiro, João M; Pereira, Ana R; Reichmann, Nathalie T et al. (2018) Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis. Nature 554:528-532
Rued, Britta E; Zheng, Jiaqi J; Mura, Andrea et al. (2017) Suppression and synthetic-lethal genetic relationships of ?gpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin-binding protein interactions in Streptococcus pneumoniae D39. Mol Microbiol 103:931-957
Sharifzadeh, Shabnam; Boersma, Michael J; Kocaoglu, Ozden et al. (2017) Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in Streptococcus pneumoniae. ACS Chem Biol 12:2849-2857
Zheng, Jiaqi J; Perez, Amilcar J; Tsui, Ho-Ching Tiffany et al. (2017) Absence of the KhpA and KhpB (JAG/EloR) RNA-binding proteins suppresses the requirement for PBP2b by overproduction of FtsA in Streptococcus pneumoniae D39. Mol Microbiol 106:793-814
Mura, Andrea; Fadda, Daniela; Perez, Amilcar J et al. (2017) Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae. J Bacteriol 199:

Showing the most recent 10 out of 39 publications