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How an antibiotic combats multi-resistant bacteria and hospital pathogens

Munich, 08/27/2008

Hopes were high in 2002 as the active agent linezolid was approved for use in Germany: for the first time in 20 years, a truly a new class of antibiotic entered the market. Finally there was a chance to finish-off, once and for all, the so-called "super-bugs" that had already become resistant to conventional antibiotics and were the bane of hospitals and retirement houses. Six years later, this agent is still the last resort when other antibiotics no longer offer any help against infections of gram-positive pathogens, such as those that cause pneumonia or infections of the skin and soft tissue, or accumulation of bacteria in the blood (bacteremia), the outcome of which can be fatal. And yet bacteria have again proven all too resourceful with the first cases of resistance having already appeared. "That makes it all the more important to know exactly how linezolid works. Only then can the antibiotic be rationally improved" says Dr. Daniel Wilson of the Gene Center of LMU Munich and the "Center for Integrated Protein Science Munich" (CIPSM). Using X-ray crystallography, his team and a group working under Professor Paola Fucini of Frankfurt University have demonstrated where linezolid docks at the active center of the ribosome – the cell's protein factory – and thereby interrupts protein synthesis in bacteria. The researchers published their results in Proceedings of the National Academy of Science USA (PNAS, 18/8/2008).

In order to survive and reproduce, every bacteria cell has tens of thousands of tiny protein factories, called ribosomes. The heart of the protein factory is the catalytic or so-called peptidyl transferase center (PTC). Here, the amino acid building blocks are brought together sequentially to generate proteins. A new protein leaves this production plant every minute. Messenger ribonucleic acid (mRNA) is sped through the ribosome as if on a production line. In its base sequence, this threadlike molecule carries a form of the DNA blueprint for the proteins, and is read piece-by-piece at two sites by transfer ribonucleic acids (tRNA). To do this, a tRNA molecule first docks onto the mRNA at the A site based on complementary of the position. The protein component (the amino acid) that it transports thereupon combines with the existing chain of amino acids, elongating it. The tRNA next migrates with its entire chain to the P-site; the production line jogs one step along. This process repeats until the protein is completed.

“We have demonstrated that the antibiotic linezolid blocks a part of the A-site in the catalytic center, so that the tRNA can no longer dock into it properly,” explains Wilson. The result is that no more amino acids can be added to the existing chain, and protein synthesis is interrupted. This observation was made using X-ray crystallography. Firstly, bacterial ribosomes were crystallized, soaked in linezolid, and then these ribosome crystals were exposed to X-rays. From the pattern of rays deflected by the atoms, the researchers could create three-dimensional images of linezolid bound at the active site of the ribosome.

Similarly to linezolid, it is also possible for other environmental influences to cause stress in a ribosome, disrupting the flow of the production line in the protein factory: either an incorrectly encoded amino acid is incorporated, or the ribosome is brought to a complete standstill. In order to prevent this, the production line in the ribosomes of bacteria has a kind of rewind function that is activated by retro-translocation factor LepA. As a result, another attempt can be made in the ribosome to finish the process of building the protein properly according to the existing blueprint.

In Nature Structural & Molecular Biology (NSMB), the groups of Wilson and Fucini, together with researchers Professor Christian Spahn of the Charité Berlin and Professor Knud Nierhaus of the Max-Planck Institute for Molecular Genetics in Berlin report that they have demonstrated in a second study how this rewind takes place. They discovered that during the rewind process, LepA stabilizes the tRNA in a novel position on the ribosome. This site appears to resemble a step during the accommodation of a tRNA into the A-site. “When linezolid blocks a part of the A-site, the tRNA also resembles the incompletely accommodated tRNA,” says Wilson. “That explains why LepA recognizes and binds to those ribosomes that have previously been jammed by linezolid.”

“The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning”
Daniel N. Wilson, Frank Schlünzen, Jörg M. Harms, Agata L. Starosta, Sean R. Connell, and Paola Fucini
Proceedings of the National Academy of Sciences (PNAS)
doi: 10.1073/pnas.0804276105

“A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation”
Sean R. Connell, Maya Topf, Yan Qin, Daniel N. Wilson, Thorsten Mielke, Paola Fucini, Knud H. Nierhaus & Christian M T Spahn
Nature Structural & Molecular Biology
doi: 10.1038/nsmb.1469

Dr. Daniel Wilson
Gene Center
Feodor-Lynen-Str. 25
81377 Munich
Tel.: ++49 (0)89-2180 76902


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