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Study Shows How Bacteria Become Antibiotic Resistant

The Enzyme PpnN Binds pppGpp (IMAGE)
The figure shows how the enzyme PpnN binds pppGpp and speeds up conversion of guanylate (GMP) to its constituents ribose-5-phosphate and guanine. This enables bacteria to balance their tolerance towards antibiotics with the fitness requirements for survival (below). [Ditlev E. Brodersen/AU]

Scientists from Denmark and Switzerland say they have shown that bacteria produce a stress molecule, divide more slowly, and thus save energy when they are exposed to antibiotics. The new knowledge is expected to form the basis for the development of a new type of antibiotic, according to the researchers.

In a paper—“(p)ppGpp Regulates a Bacterial Nucleosidase by an Allosteric Two-Domain Switch”—published in Molecular Cell, a team from Aarhus University, the University of Copenhagen, and the technical university ETH Zürich in Switzerland demonstrated that bacteria quickly reduce their rate of cell division when exposed to antibiotics in order to maintain the highest possible tolerance, but rapidly start growing again when the substances are removed.

“The stringent response alarmones pppGpp and ppGpp are essential for rapid adaption of bacterial physiology to changes in the environment. In Escherichia coli, the nucleosidase PpnN (YgdH) regulates purine homeostasis by cleaving nucleoside monophosphates and specifically binds (p)ppGpp. Here, we show that (p)ppGpp stimulates the catalytic activity of PpnN both in vitro and in vivo causing accumulation of several types of nucleobases during stress. The structure of PpnN reveals a tetramer with allosteric (p)ppGpp binding sites located between subunits. pppGpp binding triggers a large conformational change that shifts the two terminal domains to expose the active site, providing a structural rationale for the stimulatory effect. We find that PpnN increases fitness and adjusts cellular tolerance to antibiotics and propose a model in which nucleotide levels can rapidly be adjusted during stress by simultaneous inhibition of biosynthesis and stimulation of degradation, thus achieving a balanced physiological response to constantly changing environments,” the investigators wrote.

The bacterial enzyme PpnN was shown to be capable of saving molecular energy in the form of constituents (nucleotides) of cellular DNA which can be used for rapid regrowth when antibiotic treatment is ceased. When the bacteria are exposed to antibiotics, they immediately start breaking down nucleotides into smaller parts that are then stored in the cell.

Bacteria also produce a specific stress molecule called (p)ppGpp upon exposure to antibiotics that makes PpnN more active. This means that the saving of energy happens very quickly when the bacteria are exposed to stress, according to the researchers.

Using x-ray crystallography, the team has been able to generate detailed 3D pictures of the enzyme, both in its normal state and when bound to the stress molecule. The results show that the enzyme opens up when the stress hormone is present, and thus functions much more efficiently because the nucleotides can more easily access the active site where the breakdown process takes place.

It is expected that the new knowledge about the molecular basis for the reaction of bacteria to antibiotics can be used to develop a new type of antibiotic that prevents bacteria from saving energy and thus adapt to the treatment.