Bringing Antimicrobial Agents into the Light
OCTOBER 12, 2007
Electron density profile (top) of the 2-D unit cell and (bottom) a proposed model of the unit cell.
Improving and understanding antimicrobials—agents that help our bodies fight bacteria, fungi, or viruses at the original point of infection—are the focus of new research carried out by University of Illinois scientists using the U.S. Department of Energy-funded Argonne Advanced Photon Source (APS) and Stanford Synchrotron Radiation Laboratory (SSRL). Their findings could help cystic fibrosis patients breathe easier, and provide all of us with new, more potent antibiotics.
In the first study, by better understanding how antimicrobials bind and are then inactivated in the mucus of air passages, one group of researchers from the University of Illinois may have found a way to help cystic fibrosis patients fight off deadly infections. “While not a cure, this work has potential as a therapeutic strategy against bacterial infections in cystic fibrosis,” said Gerard Wong a professor of materials science and engineering, of physics, and of bioengineering at the U. of I., and a corresponding author of a paper published in the Proceedings of the National Academy of Sciences of the United States of America (PNAS).
Ordinarily, pulmonary passages are lined with a thin layer of mucus that traps bacteria and other pathogens. Moved along by the motions of countless cilia, the mucus also acts as a conveyor belt that disposes of the debris. In patients with cystic fibrosis, however, the mucus is more like molasses: thick and viscous. Because the cilia can no longer move the mucus, the layer becomes stuck, and the bacteria grow, multiply and colonize. Long-term bacterial infections are the primary cause of death in cystic fibrosis.
Using synchrotron x-ray techniques at X-ray Operations and Research beamline 12-ID-C at the APS and at an SSRL beamline, and molecular dynamics simulations, the researchers took a closer look at the mucous mess. Debris in the infected mucus includes negatively charged, long-chained molecules such as mucin, DNA, and actin (from dead white blood cells). It turns out most of the body’s antimicrobials, such as lysozyme, are positively charged.
“We found that actin and lysozyme – two of the most common components in infected mucus – form ordered bundles of aligned molecules, which is something you don’t expect in something as messy as mucus,” said Wong. “Held together tightly by the attraction of opposite charge, these bundles basically lock up the antimicrobials so that they are unable to kill bacteria.”
The researchers then developed a computational model to mimic the biological system. “The model accurately predicted the structure of the actin-lysozyme bundles, and agreed quantitatively with the small-angle x-ray scattering experiments,” said Erik Luijten, a professor of materials science and engineering at U.I.U.C., and of physics, as well as a researcher at the Beckman Institute and the other corresponding author of the PNAS paper.
The next step was to find a way to liberate the lysozyme, or prevent it from binding in the first place. Using their model, the researchers explored the consequences of varying the positive charge on the lysozyme. “When we reduced the charge, we found a huge effect in our model,” Luijten said. “The lysozyme would not bind to the actin. It floated around independently in the mucus.”
Then, through genetic engineering, the researchers made lysozyme with roughly half the normal charge. Experiments confirmed the simulations; the reduced charge prevented lysozyme from sticking to actin, without significantly reducing the all-important antimicrobial activity.
Synchrotron two-dimensional x-ray diffraction pattern of partially aligned actin–WT lysozyme bundles self- assembled in a solution containing 100mMNaCl.
Although much work remains, future cystic fibrosis patients might use an inhaler to deliver genetically modified charge-reduced antimicrobials to upper airways. There, these ‘non-stick’ antimicrobials would go to work killing bacteria, and mitigate against long-term infection.
The implications of this research extend into other areas as well. In water purification, for example, one of the steps involves putting positively charged molecules in the water to grab negatively charged pollutants. The resulting aggregates settle to the bottom of holding tanks and are removed from the water supply.
“A better understanding of how oppositely charged molecules bind in aqueous environments could lead to ways of removing emerging pathogens in water purification,” Wong said.
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The second study, by a different group of U. of I. researchers headed by Wong, helped scientists decipher the mechanism behind so-called antimicrobial “hole punchers.” The rapid development of bacterial resistance to conventional antibiotics (such as penicillin or vancomycin) has become a major public health concern. Because resistant strains of bacteria can arise faster than drug companies can create antibiotics, understanding how these molecules function could help companies narrow their focus on potential antibiotics and bring them to market sooner.
As reported in the Journal of the American Chemical Society (JACS), the researchers have now deciphered the molecular mechanism behind selective antimicrobial activity for a prototypical class of synthetic compounds.
The compounds, which mimic antimicrobial peptides found in biological immune systems, “function as molecular ‘hole punchers,’ punching holes in the membranes of bacteria,” said Wong, also a corresponding author on the JACS paper. “It’s a little like shooting them with a hail of nanometer-sized bullets – the perforated membranes leak and the bacteria consequently die.”
The researchers also determined why some compounds punch holes only in bacteria, while others kill everything within reach, including human cells.
“We can use this as a kind of Rosetta stone to decipher the mechanisms of much more complicated antimicrobial molecules,” said Wong. “If we can understand the design rules of how these molecules work, then we can assemble an arsenal of killer molecules with small variations, and no longer worry about antimicrobial resistance.”
The researchers first synthesized a prototypical class of antimicrobial compounds, then used synchrotron small-angle x-ray scattering, again at 12-ID-C at the APS, and at an SSRL beamline, to examine the structures made by the synthetic compounds and cell membranes. Composed of variously shaped lipids, including some that resemble traffic cones, the cell membrane regulates the passage of materials in and out of the cell. In the presence of the researchers’ antimicrobial molecules, the cone-shaped lipids gather together and curl into barrel-shaped openings that puncture the membrane. Cell death soon follows.
The effectiveness of an antimicrobial molecule depends on both the concentration of cone-shaped lipids in the cell membrane, and on the shape of the antimicrobial molecule, Wong said. For example, by slightly changing their synthetic molecule’s length, the researchers created antimicrobial molecules that would either kill nothing, kill only bacteria, or kill everything within reach.
“By understanding how these molecules kill bacteria, and how we can prevent them from harming human cells, we can provide a more direct and rational route for the design of future antibiotics,” Wong said.
“How antimicrobials bind”:
Contact: Erik Luijten, luijten@uiuc.edu, Gerard C. L. Wong, gclwong@uiuc.edu
See: LoriK. Sanders, Wujing Xian, Camilo Guáqueta, Michael J. Strohman, Chuck R. Vrasich, Erik Luijten, and Gerard C. L. Wong, “Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media,” Proc. Nat. Acad. Sci. USA 104(41), 15994 (October 9, 2007). DOI: 10.1021/ja072310o S0002-7863(07)02310-4
The original news release can be found at www.news.uiuc.edu/news/07/0924cysticfibrosis.html.
This material is based on work supported by the National Institutes of Health under Grant 1R21DK6843-01 (to G.C.L.W.), the Cystic Fibrosis Foundation (G.C.L.W.), and the National Science Foundation under Grants DMR-0346914 (to E.L.), DMR-0409769 (to G.C.L.W.), and CTS-0120978 (to E.L. and G.C.L.W.) via the WaterCAMPWS Science and Technology Center. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory and at the Advanced Photon Source. The Stanford Synchrotron Radiation Laboratory Structural Molecular Biology Program is supported by the U.S. Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357.
“The mechanism behind… antimicrobial ‘hole punchers’ ”:
Contact: Gregory N. Tew, tew@mail.pse.umass.edu, Gerard C. L. Wong gclwong@uiuc.edu
The original news release can be found at: www.eurekalert.org/pub_releases/2007-09/uoia-sdm092007.php.
See: Lihua Yang, Vernita D. Gordon, Abhijit Mishra, Abhigyan Som, Kirstin R. Purdy, Matthew A. Davis, Gregory N. Tew, and Gerard C. L. Wong, “Synthetic Antimicrobial Oligomers Induce a Composition-Dependent Topological Transition in Membranes,” J. Am. Chem. Soc. 129, 12141-12147 (2007).
DOI: 10.1021/ja072310o
This work was supported in part by the NSF (DMR-0409769 and the CAMPWS STC), the Petroleum Research Fund (PRF-41352-AC7), the NIH (R21-DK68431-01, RO1-GM-65803), and the ONR (N00014-03-1-0503). Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357.
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