Quantifying the Size of Holes Antibacterial Compounds Create in Cell Walls

Research has shown that unconventional antimicrobials such as PlyC can effectively kill bacteria. However, essential questions remain about how bacteria respond to the holes that these therapeutics make in their cell wall and what size holes bacteria can withstand before degrading. Solving those problems could improve the efficacy of current antibacterial drugs and begin to develop new ones.

Researchers from the Georgia Institute of Technology (Atlanta, USA) and the University of Maryland (College Park, USA) recently conducted a study to try to answer those questions. The researchers created a biophysical model of the response of a Gram-positive bacterium to the formation of a hole in its cell wall. Then they used experimental measurements to validate the theory, which predicted that a hole in the bacteria cell wall larger than 15–24 nm in diameter would cause the cell to lyse, or burst. These small holes are approximately one-hundredth the diameter of a typical bacterial cell.

“Our model correctly predicted that the membrane and cell contents of Gram-positive bacteria cells explode out of holes in cell walls that exceed a few dozen nanometers. This critical hole size, validated by experiments, is much larger than the holes Gram-positive bacteria use to transport molecules necessary for their survival, which have been estimated to be less than 7 nanometers in diameter,” said Dr. Joshua Weitz, an associate professor in the School of Biology at Georgia Tech.

The study’s findings were published online on January 9, 2013, in the Journal of the Royal Society Interface. Common Gram-positive bacteria that infect humans include Streptococcus, which causes strep throat; Staphylococcus, which causes impetigo; and Clostridium, which causes botulism and tetanus. Gram-negative bacteria include Escherichia, which causes urinary tract infections; Vibrio, which causes cholera; and Neisseria, which causes gonorrhea.

Gram-positive bacteria are different from Gram-negative bacteria in the structure of their cell walls. The cell wall comprises the outer layer of Gram-positive bacteria, whereas the cell wall lies between the inner and outer membrane of Gram-negative bacteria and is therefore protected from direct exposure to the environment.

Georgia Tech biology graduate student Gabriel Mitchell, Georgia Tech physics professor Dr. Kurt Wiesenfeld, and Dr. Weitz developed a biophysical theory of the response of a Gram-positive bacterium to the formation of a hole in its cell wall. The model detailed the effect of pressure, stretching and bending forces on the altering configuration of the cell membrane due to a hole. The force associated with bending and stretching pulls the membrane inward, while the pressure from the inside of the cell pushes the membrane outward through the hole.

“We found that bending forces act to keep the membrane together and push it back inside, but a sufficiently large hole enables the bending forces to be overpowered by the internal pressure forces and the membrane begins to escape out and the cell contents follow,” said Dr. Weitz.

The balance between the bending and pressure forces led to the model prediction that holes 15–24 nm in diameter or larger would cause a bacteria cell to burst. To assess the hypothesis, Dr. Daniel Nelson, an assistant professor at the University of Maryland, utilized transmission electron microscopy images to gauge the size of holes created in lysed Streptococcus pyogenes bacteria cells following PlyC exposure.

Dr. Nelson discovered holes in the lysed bacteria cells that ranged in diameter from 22–180 nm, with a mean diameter of 68 nm. These experimental measurements agreed with the researchers’ theoretic calculation of hole sizes that cause bacterial cell death. According to the researchers, their theoretic model is the first to consider the effects of cell wall thickness on lysis. “Because lysis events occur most often at thinner points in the cell wall, cell wall thickness may play a role in suppressing lysis by serving as a buffer against the formation of large holes,” said Mr. Mitchell.

The combination of research and theory used in this study provided clues into the effect of defects on a cell’s viability and the processes employed by enzymes to interrupt homeostasis and cause bacteria cell death. To additionally determine the processes behind enzyme-induced lysis, the researchers plan to measure membrane dynamics as a function of hole geometry in the future.

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Georgia Institute of Technology
University of Maryland