It’s been said that the bigger they come the harder they fall, an adage that turns out to be uncannily accurate when it comes to finding new ways to treat antibiotic-resistant bacterial infections.
Researchers in the lab of K.C. Huang, associate professor of bioengineering, have found that a single mutation to an important protein in E. coli—the bacteria you hope never to find in your vegetables—can cause these cells to bloat up by more than 500 percent. If the same were true of humans, a typical 150-pound adult might find that their nearly identical twin—the one with the single mutation that affected the size of every cell in its body—would weigh as much as a grizzly bear.
And the best part, says Huang, is that the bigger bacteria come, the harder they fall. For E. coli cells, which look like microscopic hot dogs, as they become longer and plumper they also become more susceptible to certain antibiotics.
“Most strategies to killing bacteria are linear: You find a very specific target and block it with a drug.”
“These findings point in the direction of totally orthogonal therapies, in which you predispose cells to death by tweaking a global property like their size,” Huang said.
Remarkably, researchers in the Huang lab found a genetic “tuning knob” that allowed them to make both subtle and extreme modifications to the shape and size of bacterial mutants. By twisting this dial the team could study how the dimensions of the bacteria impacted their lifestyle. The immediate application was to find that fattening up bacteria prepared for the kill. But as a basic research tool this “dial the size” technique allowed the team to ask questions about the evolution of life.
The findings in the paper are motivated by questions from the science of cellular biophysics. Much as astrophysics examines the forces that push and pull the stars and planets and galaxies, and thereby give shape to the universe, biophysics explores how forces and physical principles affect the basic building blocks of life, and thereby give shape to cells and organisms.
Inside cells, the fundamental units of life, lies a menagerie of DNA, RNA and proteins that collectively encode the programming instructions necessary for all cellular processes. To many of us, proteins are simply material to be consumed as part of our diet, but proteins are the heroes of all life as we know it. They make up the magnificent machines that perform complex tasks such as making ATP (the fuel that powers cells), dividing cells in two and building the home (the cell itself) in which they live. In fact, the surface of the cell is far more than just a boundary between inside and outside, it defines the geometry in which all proteins organize and the concentrations of all cellular components.
The number of proteins inside a typical cell is in the millions, with many thousands of different varieties. Given this complexity, understanding how cells are constructed would appear to be a daunting task. One of the surprises that emerges from this paper is that a single protein called MreB serves as a master regulator of cell size. MreB acts as a platform that spatially coordinates the machinery that builds the cell wall, which ultimately dictates the size and shape of every cell. Huang’s team, led by graduate student Handuo Shi, discovered that changing just one out of the 347 letters in MreB’s genetic code was a prescription to bring about dramatic increases in size and, consequently, antibiotic susceptibility that has the researchers so excited.
Like all proteins, MreB is a macromolecule: A long chain of smaller molecules, known as amino acids. To make any protein, the cellular machinery reads off its instructions, three letters at a time, from the four letters—A, T, G and C—of the genetic code. This simple set of instructions explains all of life on our planet. Remarkably, the long, gangly chain of amino acids produced by genetic coding somehow folds up into a perfectly shaped protein machine. All of the atoms and molecules in the protein machine engage in minute pushing and pulling that enable this macromolecule to precisely arrange itself and move with a purpose. From a biophysics perspective, life is composed of the interactions of gazillions of protein machines, with all their composite atoms interacting with each other according to mathematical rules that researchers aim to make as predictable as the movements of the stars and planets (perhaps the big breakthrough will require an apple to fall on the head of a modern-day Newton).
Rather than waiting for anything to hit them in the head, the Huang lab wondered just how much a key protein like MreB could tolerate change. You might think that a protein that is absolutely required for survival would be evolutionarily frozen, like the great white sharks prowling the ocean for hundreds of millions of years. Russell Monds, a former postdoctoral scholar in Huang’s lab, identified a strain of E. coli that had a mutation in MreB that made cells bigger and also better at dealing with starvation. He then devised an experiment to see what would happen if just one amino acid at a time across all of the 347 amino acids in MreB was subtly changed: Which mutants would survive? To do so, the team manufactured many copies of E. coli’s DNA, each one with a change to a randomly selected amino acid in the gene that codes for MreB, using a process called error-prone PCR.
Shi and fellow teammates Alexandre Colavin, Marty Bigos and Carolina Tropini then developed a method that used a cell sorter to pick out individual cells with different sizes, identifying the bigger and smaller needles in the haystack of cells. Armed with this library, they were able to able to ask fundamental questions about whether and how cells care about their size.
Huang said this was conceived as a basic research study into the ways cell grow and how bacterial populations evolve, but he also highlighted the medicinal benefits.
“We’ve discovered a single tuning knob that can enlarge or shrink bacteria across a large range,” he said. “While we don’t yet know how to twist this bacterial size dial in patients, it’s good to have such an exciting new therapeutic approach as antibiotic resistance becomes increasingly prevalent.”
Read more in their scientific paper: “Deep phenotype mapping of bacterial cytoskeletal mutants reveals physiological robustness to cell size.”