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Microbial murder mystery solved
Immune cells called "killer cells" target bacteria invading our body's cells. But how do they do this so effectively? Bacteria can quickly evolve resistance against antibiotics, yet it seems they are not easily able to evade killer cells. This ability captures researchers interest in finding the exact mechanism killer cells use to destroy bacterial invaders.
Although one way killer cells trigger bacterial death is by inflicting oxidative damage, how do they destroy bacteria in environments without oxygen?
Now, for the first time, researchers have caught killer cells red-handed in the act of microbial murder, observing as they systematically kill three strains of microbes: E. coli and bacteria responsible for Listeria and tuberculosis. The findings, published in Cell, reveal that killer cells act methodically, shooting deadly enzymes into bacteria to "program" a complete internal breakdown and cell death.
Researchers, from Boston Children's Hospital, the Wistar Institute and the University of Michigan (U-M), used an equally systematic approach to make their discovery.
"We took three bacteria that are very different - and to see which proteins were destroyed by killer cells - we measured their protein levels before, during and after they were attacked," says Judy Lieberman, MD, PhD, of Boston Children's Program in Cellular and Molecular Medicine (PCMM), who is co-senior author on the study.
Proteins are critical to life because they direct the use of nutrients and production of cellular machinery that bacteria need to survive.
"Each strain of bacteria has about 3,000 proteins and we saw that - in all three bacterial species - about five to 10 percent of those proteins were slashed by the killer cells' death-inducing enzyme, called granzyme B," Lieberman explains. "If you made a list of the proteins that bacteria absolutely needed to survive, it would be a small list - interestingly, this seems to be identical to granzyme B's hit list."
To deliver the fatal injection of granzyme B, killer cells seek out surface markers on cell surfaces that might indicate a bacterial invader has taken up residence inside. They then latch onto the infected cell and use an enzyme to create a small pore (hole) in the cell's surface through which they inject granzyme B. Once inside the invading bacterium, granzyme B destroys proteins critical to it's survival as well as its ribosomes, or protein making machines.
"The bacteria's ribosomes fall apart and stop functioning," says Lieberman, also a professor of pediatrics at Harvard Medical School. It's as if the bacteria's internal factory of life not only loses the blueprints for the parts it needs to make, but also suffers a catastrophic mechanical failure of its assembly line.
"By discovering the bacterial proteins that killer cells 'take out,' we have identified potential therapeutic targets that could pave the way for a new class of antimicrobial drugs," Lieberman suggests. "We have a huge crisis of antibiotic resistance right now in that most drugs that treat diseases like tuberculosis or listeria, or pathogens like E.coli, are not effective," adds Sriram Chandrasekaran, PhD, co-senior author of the study. "So, there is a huge need for figuring out how the immune system does its work. We hope to design a drug that goes after bacteria in a similar way."
Importantly, no matter how many times the researchers exposed the bacteria to granzyme B, the bacteria did not develop resistance to its fatal attack. It's possible that the only way bacteria can survive is to camouflage themselves so that killer cells cannot identify them to inject granzyme B.
Lieberman is now searching for the specific mechanisms by which bacteria might evade killer cells. She's also investigating how similar "death pathways" take effect in fungi and parasites, such as those causing malaria.
• Granzyme B activates a multipronged program of cell death in bacteria
• Granzyme B cleaves vital biosynthetic and metabolic pathway enzymes
• 20 common orthologous groups are shared E. coli, listeria, and mycobacteria targets
• Like antibiotics, Granzyme B disrupts protein synthesis, folding, and degradation
Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack.
Authors: Farokh Dotiwala, Farokh Dotiwala, Sumit Sen Santara, Andres Ariel Binker-Cosen, Bo Li, Sriram Chandrasekaran, Judy Lieberman
In addition to Lieberman and Chandrasekaran, other authors on the paper are Farokh Dotiwala (Wistar Institute), Sumit Sen Santara (PCMM), Andres Ariel Binker-Cosen (Harvard University) and Bo Li (Harvard University and Broad Institute).
This work was supported the National Institutes of Health (RO1 AI23265, T32 HL066987) and the Harvard Society of Fellows.
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This cartoon: "Monkey Wrench", portrays bacterial protein synthesis as a machine and granzyme B as a group of disruptive monkeys. Granzyme B monkeys shut down essential systems in bacteria, causing catastrophe. Art by Sylvie Shaffer of the Wistar Institute.