Jonathan O. Wright and Dr. Paul Savage, Department of Chemistry and Biochemistry
The project that I have worked on this past year has been focused on a novel group of antibiotics developed in the lab of Dr. Savage. The antibiotics, called ceragenins or cationic steroid antibiotics (CSA’s), are molecules synthesized from cholic acid, a common bile acid, and they mimic the structure and functionality of antimicrobial peptides found in nature. Although the CSA’s are much smaller than the peptides, they have similar structural aspects, a hydrophobic side as well as a cationic side, and are believed to have a similar mechanism of action. They, like the antimicrobial peptides, have been found to be effective against a broad spectrum of bacteria, Gram-positive as well as Gram-negative, but with much lower required concentrations than the peptides. They have also been found to be effective against strains of antibiotic-resistant bacteria, and some of the compounds have also been found to be active against membrane-enclosed viruses, including HIV and herpes.
Recently, there has been much interest in antimicrobial peptides and similarly functioning molecules, because even though many antimicrobial peptides are found in the innate immune systems of most organisms and bacteria have been exposed to them for thousands of years, bacteria generally remain susceptible to these compounds. This is of much interest as strains of bacteria are becoming more and more resistant to current antibiotics. Therefore, several cationic peptide antibiotics (CPAs) have been developed in recent years. These CPAs, however, are very expensive to synthesize, and some bacteria have been able to overcome their effects by secreting proteases. The CSAs developed in our lab overcome both the expense and the protease issues, making medical applications much more realistic.
However, despite the development of these CSAs and their successful testing against infectious agents, much about their properties still needs to be uncovered. Currently, there are two main theories proposed to explain the bactericidal activity of antimicrobial peptides which may also apply to CSAs. The first is that these molecules function by permeabilizing bacterial membranes, and the second is that they inhibit polymerization steps in the cell wall biosynthesis.3 Not clearly understanding the mechanism of action of these antibiotics has made it difficult to understand ways to make them more effective against prokaryotic cells while still maintaining a low level of toxicity for eukaryotic cells, allowing for medical applications.
The main thrust of my project was to find evidence supporting one of these models. Previous experiments resulted in data which supported the conclusion that the CSAs were permeabilizing the cells, so my project was aimed at finding evidence for this model.
I began by finding the minimum bactericidal concentrations (MBC) – the concentration at which we see a complete eradication of bacteria – of three different for many strains of Gram-positive and Gram-negative bacteria. With that information, I began running experiments using a fluorometer to measure membrane depolarization of the different strains of bacterial at several concentrations of the CSAs above and below the MBC values. I began with Gram-positive strains, and those assays resulted in data which greatly support the permeabilization theory. Membrane depolarization occurred rapidly after addition of the CSAs, and greater depolarization was seen at CSA concentrations above the MBC than at those below the MBC, suggesting that membrane depolarization could be a factor in cell death.
After completing assays using several Gram-positive strains of bacteria, I began running similar experiments with Gram-negative bacteria. Initial assays did not show the results that we were expecting, but I noticed some interesting trends. Follow-up experiments exploring these trends helped shed even further light on the mechanism of action of these CSAs. The data suggest that at concentrations below the MBC, the CSAs permeabilize the outer membrane without greatly affecting the inner membrane of the bacterium. However, at concentrations near and above the MBC, the data suggests that following permeabilization of the outer membrane, the antibiotic is able to penetrate to the inner membrane and cause its depolarization. This interpretation of the data also provides an explanation for the synergistic affects the CSAs have with other antibiotics.
As of now, I am gathering the last bits of data which are needed to conclude this study. We plan to publish the results of the study early next year in peer-reviewed journals, possibly with other studies performed by collaborators at McMaster University in Ontario, Canada which support the same conclusions. We have also considered future kill kinetic experiments which could correlate the level of membrane depolarization at the time of cell death. These experiments would further support the conclusions we have made based on the data already gathered.
Overall, this has been a great learning opportunity for me. I have learned much about experimental design and research techniques which will benefit me as I go on to pursue an MD/PhD. The studies have also been quite successful, shedding more light onto the mechanism of action of these CSAs. This data will help in future development of the antibiotics and may also be beneficial as FDA approval is sought to get these antibiotics into medical applications where they will be able to help.
References
- Schmidt, E, Boswell, J, Walsh, J, Schellenberg, M, Winter, T, Chunhong, L, Allman, G, Savage, P (2001). Activities of cholic acid-derived antimicrobial agents against multidrug-resistant bacteria. Journal of Antimicrobial Chemotherapy 47, 671-674.
- Chin, J, Rybak, M, Cheung, C, Savage, P. (2007). Antimicrobial Activities of Ceragenins against Clinical isolates of resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, Apr. 2007, 1268-1273.
- Bambeke, F, Mingeot-Leclercq, M, Stuelens, M, Tulkens, P. (2007). The bacterial envelope as a target for novel anti-MRSA antibiotics. Trends in Pharmacological Sciences 29, 124-134.