Scientists have discovered a previously unknown strategy that phages – viruses that infect bacteria – use to disable bacterial defence systems.
The strategy relies on T7 kinase, a phage enzyme that sets off an explosion of protein modifications inside infected bacteria, shutting down their defence systems.
Molecular arms race
Like all viruses, phages exist in a perpetual molecular arms race with their hosts. Bacteria evolve mechanisms to defend against infecting phages, while phages evolve anti-defence systems to shut down or evade these immune mechanisms. The new study demonstrates, for the first time, how a single phage protein can set off a wave of molecular events that can disarm multiple bacterial defences.
These findings, published in Nature, resulted from a long-standing collaboration between two research groups at EMBL Heidelberg – the Typas Group, which specialises in high-throughput studies of bacterial interactions, and the Savitski Team, who are experts in cutting-edge proteomics technologies.
“Phage research has led to a lot of exciting developments, the CRISPR-Cas9 gene editing system among them,” said Mikhail Savitski, Senior Scientist and Head of Proteomics Core Facility at EMBL Heidelberg. “Using the sensitive technologies we had available in the lab, we wanted to understand in an unbiased way how phages affect bacterial proteins during infection.”
A loose cannon inside the cell
For this, the researchers used a well-known model system – E. coli, a rod-shaped bacterium that lives in our guts, and T7 phage, a virus that infects E. coli. The team decided to look closely at protein phosphorylation in phage-infected bacteria. Phosphorylation is a kind of rapid chemical modification of proteins which can change their function, e.g. by activating or inactivating them.
Surprisingly, the scientists found that phage infection resulted in almost every single bacterial protein getting phosphorylated within minutes, at least for a fraction of their population within the cell. The likely culprit was T7 kinase, a phage enzyme first discovered in the 1970s. However, the scale of phosphorylation was unlike anything previously observed. In fact, the list of phosphorylation targets for the T7 kinase surpassed what’s known so far for any kinase in nature, leading the researchers to dub it a ‘loose cannon’.
“We realised that we were seeing a quite unprecedented molecular event: a catastrophic phosphorylation across the entire proteome in a completely nonspecific manner,” said Savitski. “That had never been seen before, and it was fascinating that there was also no pattern to it.”
Shutting down bacterial defences
However, this discovery posed a new question. Previous studies had shown that deleting T7 kinase from the phage’s DNA doesn’t really affect the infection process. “As puzzles go, it leaves you a bit flabbergasted,” said Savitski. “You have a kinase with apparently no phenotype that seems to phosphorylate everything in the proteome.”
The researchers confirmed that the activity of the T7 kinase was short-lived – as previously reported, it inactivates itself within 5-6 minutes post infection. Examining the structure of T7 kinase yielded another important clue: one section of the kinase, called the shutoff domain, was not needed for its phosphorylation activity but was rich in chemical features that might help it bind DNA. This led the team to hypothesise that this domain might help the kinase attach to DNA and subsequently come close to other DNA-binding proteins.
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“Methodologically, it is not easy to test such things, but we designed an elegant experiment that could measure exactly how much of a protein population is phosphorylated inside a cell,” said Tara Bartolec, postdoc at EMBL Heidelberg and one of the first authors of the paper. Using this, the researchers found that the T7 kinase preferentially targeted DNA-binding bacterial proteins, phosphorylating and presumably inactivating them.
DNA-binding proteins are often the bedrock of bacterial defence systems, helping them detect and destroy phage DNA inside the cell. And indeed, the researchers found that the kinase could help the virus infect strains of bacteria that had such defence systems.
By also comparing kinases across different types of phages, the researchers believe they may have hit upon an evolutionarily conserved mechanism that certain phages use to deactivate bacterial immune systems.
Phage therapy
In the future, the researchers plan to look at other protein modifications and the role they might play in phage infections. The study also opens up the possibility of novel bioengineering approaches that use these new insights into phage biology to design or predict the effectiveness of phage therapies.
”To be effective for therapy, phages should be capable of infecting diverse versions (strains) of the same pathogen,” said Typas. “Interestingly, pathogenic strains are exquisitely diverse in their immune repertoire, and can always pick up new systems. So engineering phages with broad anti-defence systems, such as the T7 kinase, might be key in this quest for effective phage therapies. We identified the first one here, but we are sure there are many more out there.”
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