Antibiotic resistance is a global problem. Today, routine surgeries can be complicated by infections that are seemingly impossible to treat with the available drugs. There are many ways that bacteria can become resistant to antibiotics, but a common way is to produce an enzyme that will actively degrade antibiotics as they enter the bacterial cell. A particular prevalent cluster of these enzymes are called carbapenemases, which are enzymes that break down or hydrolyse a clinically important group of antibiotics called cephalosporins.
Individual strains of the hospital pathogen Klebsiella pneumoniae can produce different carbapenemases that have unique activity against a range of cephalosporins. Some of these carbapenemases are more problematic than others as they have a broad spectrum of activity, meaning that the bacteria producing them will be able to hydrolyse a greater range of cephalosporins, making them more resistant to treatments. Until recently we didn’t understand why that was, but an international group of scientists used beamlines I04 and I24 in the Macromolecular Crystallography (MX) group at Diamond to investigate what molecular mechanisms were causing different carbapenemase enzymes to be more or less active against a wider range of antibiotics.
To study this, the research team focused on two different cephalosporins, cefotaxime and ceftazidime. While these are very similar, carbapenemases responded very differently and many couldn’t hydrolyse both, meaning that there was always a viable option in the clinic. However, new strains emerged with carbapenemases that could hydrolyse both cefotaxime and ceftazidime. The researchers identified and isolated a carbapenemase that could hydrolyse only cefotaxime, as well as a carbapenemase that could hydrolyse both cefotaxime and ceftazidime.
Using the MX beamlines at Diamond and Alba in Barcelona, researchers studied how different carbapenemases interacted with the two different antibiotics. They discovered that a single change in one of the amino acids making up the carbapenemase enzyme, was responsible for a broader spectrum of activity. In the carbapenemase without the mutation, cefotaxime sat perfectly in the active site and could be easily hydrolysed. However, ceftazidime with a different structure was awkward to fit in the active site and so could not be cleaved properly by the enzyme. But with a single mutation in one of the amino acids in a structure of the carbapenemase know as a Ω-loop, it could more easily accommodate ceftazidime, which could then be effectively hydrolysed and lose its ability to kill the bacteria.
Work such as this illustrates the power of bringing together different techniques to tackle the grand challenges in science. The models determined from diffraction data collected at Diamond provide a strong experimental basis for understanding how these enzymes interact with antibiotics.
Robin Owen, Principal Beamline Scientist on I24.
Knowing the molecular details of antibiotic resistance is important if we are to reduce the threat caused by bacterial infections with no known cure. Understanding why certain antibiotics are targeted and broken down can help inform treatment in the clinic, and in the long run, also help us to develop new treatments that can overcome the bacteria’s natural defences. This could be brand new antibiotics or, as is more frequently used, drugs used in conjunction with antibiotics that overcome resistance mechanisms, such as a carbapenemase inhibitor.
The researchers who published their findings in the Journal of Biological Chemistry ran computer simulations based on the experimental data they obtained using X-ray crystallography. With the exponential increase in computing power that we have seen over recent decades, smart design of new antibiotics could be a viable option. However, for this to work, it is essential to have strong experimental data on which to base any computer models. The research performed at Diamond aids our understanding of the molecular mechanisms of antibiotic resistance, which will bring us one step closer to finding new antibiotics that can be used to save lives.
Catherine L. Tooke et al. Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance. Journal Of Biological Chemistry 296, 100126 (January 2021). DOI: 10.1074/jbc.RA120.016461
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