PORTLAND, Ore. – A discovery at Oregon Health & Science University is giving researchers detailed, visual clues into how gram-positive bacteria, including those that cause life-threatening diseases, can stay alive in adverse environmental conditions.
The findings, published recently in the journal Cell, could someday lead to the development of a new class of drugs that disrupt a crucial mechanism that bacteria use to adapt to available energy sources, and essentially starve these pathogens to death.
Using X-ray crystallography to examine the three-dimensional structure of proteins in atomic-level detail, scientists in the Department of Biochemistry and Molecular Biology, OHSU School of Medicine, uncovered the mechanism behind the ability of "gram-positive" bacteria to switch their metabolic programs to take advantage of the best-available carbon source for energy.
Gram-positive bacteria include those that cause staph infections, strep throat, pneumonias, botulism, toxic shock syndrome and anthrax.
"What we did is solve the crystal structure of a protein-phosphoprotein-DNA complex," said Professor Richard Brennan, Ph.D., whose lab completed the work in collaboration with Assistant Professor Maria Schumacher, Ph.D., also at OHSU, and Professor Wolfgang Hillen at the University of Erlangen, Germany.
"The nice thing about crystallography is you have a very high-resolution picture that gives you a detailed view of the molecules that ultimately cause specific biological responses."
The research team knew that infectious bacteria are highly resilient when hunting down and using other sources of carbon for energy when their staple supplies, such as the carbohydrate glucose, are low.
"Bacteria are out there in the real world fighting each other for limited sources of food. They also run into a whole bunch of different types of sugars, carbohydrates," Brennan said. "If they don't have glucose and they have access to another sugar, such as xylose, they can use this carbohydrate as a carbon source and meet their energy needs this way. Interestingly, if you put them in with glucose and xylose, they'll use glucose. They do have strong preference for which sugars they want to consume."
The survival technique, carbon catabolite repression (CCR), is one of the most fundamental and oldest mechanisms used for environmental sensing and signalling in bacteria, and it's critical for successful competition in diverse and frequently changing conditions.
As a neutral molecule, glucose is able to pass freely in and out of a bacterium cell's membrane. When glucose is available as the energy source, the cell traps the molecule inside its walls by adding a phosphate – called "phosphorylation" – with the help of a phospho-relay system that requires a protein known as HPr.
HPr can itself be phosphorylated in two ways, with one form binding the CCR master regulatory protein, CcpA, and allowing them to then bind a large number of specific DNA sites on the bacterial chromosome. Such binding, in turn, redirects the metabolic program of the cell so only proteins that are needed for glucose metabolism are made.
When glucose is not present, HPr is not phosphorylated properly for CcpA binding, leading to CcpA falling off the DNA. This allows the cell to make other proteins that can break down alternative carbohydrates that are present in the environment.
"If you don't have any glucose, it's time to upregulate genes involved in the catabolism of secondary carbon sources," Brennan said.
Brennan said developing a visualization of a protein's structure through X-ray crystallography helps all biomedical scientists to better understand its function.
"Our major focus is to determine structures of proteins and their biologically relevant complexes so we can understand the biology more completely," he said. "My lab focuses on bacterial systems. If you can understand the function of the players, which control critical basic functions in bacteria, they offer the opportunity to develop novel antibiotics, which disrupt those functions."
In the case of the Cell study, an antibiotic could be developed to disrupt the HPr-CcpA interface, rendering the bacteria dysfunctional – but not necessarily dead.
"They would then be at a great disadvantage," Brennan said. "They would be less competitive and not as robust, growing very slowly, if at all. Often times, that's all you need for the human body to mount a counter-attack to overcome a bacterial infection."
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