Regions of active cell wall synthesis shown by high-resolution imaging of the bacterial pathogen Streptococcus pneumoniae. Long-term labeling is revealed with blue FDAA and short-time labeling with red FDAA. Photo by Michael Boersma, Indiana University
The alarming increase of antibiotic-resistant bacteria poses health and economic threats worldwide, with more than 2 million Americans infected by the bacteria each year. Now, a team of Indiana University chemists and biologists has been awarded a major grant to develop and use a chemical tagging method to better understand how bacteria build their cell wall, which is still the best target for new antibiotics.
About two years ago, an IU team led by chemist Michael VanNieuwenhze and microbiologist Yves Brun discovered what they saw as a new weapon in the arms race against antibiotic-resistant bacteria: a nanoscale, fluorescent chemical probe that pinpoints where bacterial cells build their peptidoglycan, the mesh-like polymer that provides shape and strength to cell walls.
Now the National Institutes of Health has agreed with their assessment and awarded them $3.3 million to form a team with four other IU chemists and biologists who plan to improve upon their method of exploring the dynamics of the peptidoglycan building process.
“If you can see how something is being built, you have a better chance of being able to develop ways to interfere with the building process,” VanNieuwenhze said. “Much like building a house, the various steps of building the peptidoglycan have to be coordinated in space and time; otherwise, the structure crumbles. Our new probes will allow us to see at what time different parts of the cell wall are being built.”
These second-generation probes -- called FDAAs for the fluorescent D-amino acids that are used -- are expected to allow imaging of peptidoglycan construction with greater resolution and in real time in two of the most studied bacterial organisms, Escherichia coli and Bacillus subtilis.
“We use these so-called model bacterial species because they represent major types of bacterial cell wall growth, and knowledge gained about their mechanisms can be applied to other bacteria, including pathogens,” Brun says.
The team will also study the pathogen Streptococcus pneumoniae, which causes 4 million infections -- including 1.2 million that are drug-resistant -- and 22,000 deaths per year in the U.S. and over 1 million deaths per year worldwide, according to Malcolm Winkler, a professor in the IU Bloomington College of Arts and Sciences’ Department of Biology whose work focuses on the physiology, genetics and pathogenesis of S. pneumoniae.
Winkler joins Brun and VanNieuwenhze as a principal investigator on the four-year project. Brun is the Clyde Culbertson Professor of Biology, and his work primarily focuses on bacterial differentiation; VanNieuwenhze is an associate professor in the College’s Department of Chemistry who specializes in cell wall and peptidoglycan biosynthesis.
The grant will allow the team to create new and better probes, test hypotheses on the synthesis of peptidoglycan in ovoid bacteria (S. pneumoniae) and identify genes that control peptidoglycan dynamics in rod-shaped bacteria (E. coli and B. subtilis).
“We anticipate filling in major gaps in our understanding of peptidoglycan construction mechanics and identifying potential new antibacterial targets and strategies,” Winkler says. “We think the overall impact of this approach on the knowledge base required to develop new antibiotics will be profound.”
Joining the three as co-investigators are associate professor of biology Daniel Kearns, a B. subtilis specialist and expert geneticist, and chemistry professor Stephen Jacobson, whose work focuses on the miniaturization of analytical instruments like the micro- and nanofluidic devices to be used in this project. Associate professor of biology Sidney Shaw will contribute his expertise in advanced microscopy methods and their use to study biological mechanisms. Shaw is technical director of the IU Light Microscopy Imaging Center and oversees the high-resolution microscopes essential for many of the proposed experiments.
The team will work to develop new probes of various sizes and colors that mimic amino acids already in the peptidoglycan peptide sequence and deliver nontoxic fluorescent dyes that prefer to label sites where peptidoglycan is built. Using high-resolution imaging processes called structured illumination microscopy and stochastic optical reconstruction microscopy, the researchers can create a chronological account of shifts in peptidoglycan synthesis in individual cells over time. The result is a series of multicolored, sequential images of bacterial peptidoglycan construction, much like a movie.
They also want to implement new molecular rotor-based probes that would eliminate the need to wash away the unincorporated fluorescent probe prior to microscopy imaging, a process that currently decreases temporal resolution during experiments. Other goals will be to create nanochannels that improve the ability to contain and track cell lineages over long periods of time and development of new microfluidic devices that speed delivery of probes to the bacterial cells.
In September, Brun and VanNieuwenhze were one of two teams of IU Bloomington faculty to receive the inaugural Outstanding Faculty Collaborative Research Award from the Office of the Provost and the Office of the Vice Provost for Research in recognition of their collaborative accomplishments in research, scholarship and creative activities.
“We created an integrated research proposal with multiple investigators that sets expectations that would not have been possible for a single laboratory to achieve,” Brun says. “Building this team and obtaining the preliminary data that were essential to making this grant proposal successful were greatly helped by the vision and seed funding of IU’s Vice President for Research Jorge José and the Dean of the College of Arts and Sciences Larry Singell.”
Source: Indiana University