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December 14, 2012
The tango between the Legionella pneumophila bacterium and the alveolar macrophage is fraught with frustration: as Legionella struggles to grow and replicate, the host cell scrambles to eliminate the microbe. This complex push-and-pull is theoretically orchestrated by hundreds of proteins within numerous pathways in both cells, but little research has shown which, if any, proteins actually promote the microbe’s growth.
With a new method called insertional mutagenesis and depletion (iMAD), however, researchers have finally revealed that many of these proteins are key for growth, and exposed several of Legionella’s Achilles’ heels. The team says that the technique—described in a paper published December 13 in Science (1)—can help describe the interplay between any two close-knit organisms, and maybe even how better to stop infection. “This gives insight into how a pathogen is built,” said study author Ralph Isberg, professor at Tufts University School of Medicine. “That’s quite important for our understanding of how disease occurs.”
Legionella normally lives in murky pond water, invading and parasitizing amoeba cells. When a human breathes droplets of contaminated water, the lungs’ alveolar macrophages become the microbe’s new host. Once engulfed by a macrophage, Legionella shoots hundreds of proteins into the host cell through a channel called Dot/Icm. Scientists believed these proteins interact with each other and with proteins in the host cell to help the bacteria grow and reproduce, which causes the human to develop pneumonia.
No one really understood how these proteins worked to overcome host cell defenses, so Isberg tried eliminating one protein at a time to see if Legionella growth stopped. Every time, he said, “the bacterium still grew. It was like a very bad joke.”
This led the team to believe that the bacterium employed multiple redundant ways to survive and thrive inside a host cell: if one method malfunctioned, another quickly picked up the slack, “like backup systems in a space shuttle,” said Isberg. To stop Legionella, multiple pathways needed to be eliminated.
So, Isberg and colleagues attacked the problem from both sides: host and microbe. First, the researchers created a collection of insertion mutations in almost every Legionella gene. Next, using RNAi, they created five different cultured host cells (in this case, from Drosophila flies) depleted for a single gene from one of their pathways involved in macrophage growth. Next, they followed the behavior of thousands of mutants after infecting them into the Drosophila cells, creating a grid of experimental cells they examined for Legionella growth. This time, it worked: Isberg and colleagues found several pairs of mutated microbe genes and depleted host proteins that slowed the microbe’s growth.
Within this grid, the team identified 14 groups of Legionella mutants that showed similar growth patterns, suggesting the genes are part of the same pathway; when the team made double mutants of genes from selected non-redundant pathways, Legionella grew poorly.
After six years spent uncovering these genes, Isberg is now working to pin down their roles and predict their functions.
1. O’Conner, T.J., D. Boyd, M.S. Dorer, R.R. Isberg. 2012. Aggravating genetic interactions allow a solution to redundancy in a bacterial pathogen. Science 338:1440-1444.
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