New ammo in arms race against antibiotic- resistance

NIH provides grant to Indiana University to build knowledge base about key bacterial cell processes

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BLOOMINGTON, Ind.—A team of chemists and microbiologists at Indiana University (IU) announced earlier this year that it will receive a $3.3-million grant from the National Institutes of Health (NIH) that will assist the researchers in developing new and improved tools for studying the formation and structure of cell walls in bacteria, a line of research with the potential to uncover a multitude of targets for new compounds to combat strains of bacteria that have become resistant to existing antibiotics.
IU’s Clyde Culbertson Professor of Biology Dr. Yves Brun and associate professor of chemistry Dr. Michael VanNieuwenhze, two of the co-authors of the grant proposal, also led an IU team in 2012 that discovered a nanoscale, fluorescent chemical probe that pinpoints where bacterial cells build a compound called peptidoglycan (PG), which is a mesh-like polymer that gives shape and structure to cell walls.
The grant will allow principal investigators Brun, VanNieuwenhze and IU professor of biology Dr. Malcolm Winkler to undertake a four-year project to improve upon previously discovered methods of investigating the peptidoglycan building process.
Co-investigators for the research will be associate professor of biology Daniel Kearns, chemistry professor Stephen Jacobson, and associate professor of biology Sidney Shaw. The grant will also allow the team to hire eight to 10 additional research personnel and purchase the necessary reagents and lab supplies for the proposed research.
The research will consist of three main facets: creating a second-generation probe that will improve upon the probe discovered in 2012, testing two long-standing hypotheses about PG synthesis in ovoid bacteria (Streptococcus pneumoniae), and systematically screening for genes responsible for key functions of PG dynamics and coordination in model species Escherichia coli and Bacillus subtilis.
The improved probes will be designed to observe PG synthesis in action, capturing imaging of PG construction in E. coli and B. subtilis in greater resolution than previously possible, and in real time. The probes are called FDAAs for the fluorescent D-amino acids that are used.
“We will synthesize probes with a variety of properties (e.g. sizes, colors) with the emphasis on improved brightness and photostability,” says Brun. “These improved properties will enhance the utility of these probes in microscopy applications to analyze the dynamics of PG synthesis.”
Using the newly optimized probes in concert with the existing ones, the team will analyze the mechanisms of PG dynamics in bacterial model systems for two different cell shapes and two cell envelope architectures.
“We will test two major long-standing hypotheses about the spatiotemporal coordination of the elongation and the division PG synthesis machineries and about the coordination between PG hydrolysis and synthesis in the major model for ovoid-shaped cells, the pathogen S. pneumonia”, says Brun. “We will also analyze PG spatiotemporal dynamics at super-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 (B. subtilis).
Finally, the team will take advantage of high-throughput screening technology and extensive genetic data relating to these model bacteria species to randomly and systematically screen for genes that may be involved in PG dynamics and coordination in bacterial cell walls.
“We will further take advantage of our recent development of a high-throughput microscopy screening platform, the availability of comprehensive strain collection in which each gene has been separately deleted, and the powerful genetics of both species,” says Brun.
The goal of this multifaceted is to 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. Understanding PG synthesis can lead to new treatments and targets for new antibiotics active across diverse bacterial groups, including antibiotic-resistant strains.
“At the end of the funding period, we expect that we will have generated a powerful and unprecedented pipeline for the high-throughput isolation of loss of function mutants and conditional alleles defective in PG synthesis, and for the single-cell quantitative analysis of their effect on PG spatiotemporal dynamics,” says Brun. “Not only will we gain insight onto the molecular mechanism of PG synthesis and dynamics in well-established model organisms, but the pipeline can be applied to any bacterial species, including pathogens, limited only by the ability to be grown in the laboratory.”
Widely considered an emerging and urgent health threat worldwide, more than 2 million Americans alone are infected by antibiotic-resistant bacteria each year.

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