Every living creature on Earth needs to protect itself from things that would do it harm. Bacteria are no different. And despite their relative simplicity, they deploy remarkably savvy defensive strategies against viral invaders. The most well-known is CRISPR-Cas9, adapted for human use as the first FDA-approved genetic editing technique.
In the past year, researchers at Rockefeller's Laboratory of Bacteriology, headed by Luciano Marraffini, and at the MSKCC's Structural Biology Laboratory, headed by Dinshaw Patel, have been studying key immune components of some CRISPR systems called CARF effectors. These newly discovered weapons take different approaches to achieving the same goal: arresting cellular activity, which prevents a virus from spreading through the rest of the bacterial population.
In a recent publication in Science, the scientists announce the newest CARF effector they've discovered, which they coined Cat1. Thanks to an unusually complex molecular structure, this protein can deplete a metabolite essential for cellular function. Left without fuel, the viral invader's plans for a further onslaught are brought to a grinding halt.
"The collective work of our labs is revealing just how effective-and different-these CARF effectors are," says Marraffini. "The range of their molecular activities is quite amazing."
Multiple defense systems
CRISPR is a mechanism in the adaptive immune systems of bacteria and other certain single-cell organisms that offers protection against viruses, called phages. The six types of CRISPR systems work roughly the same way: A CRISPR RNA identifies foreign genetic code, which triggers a cas enzyme to mediate an immune response, often snipping off the invader material.
But an increasing body of evidence indicates that CRISPR systems deploy a wide variety of defensive strategies beyond genetic scissors. Marraffini's lab has led the way on much of this research. In particular, they have been studying a class of molecules in CRISPR-Cas10 systems called CARF effectors, which are proteins that are activated upon phage infection of a bacterium.
CARF effector immunity is believed to work by creating an inhospitable environment for viral replication. For example, the Cam1 CARF effector causes membrane depolarization of an infected cell, while Cad1 triggers a sort of molecular fumigation, flooding an infected cell with toxic molecules.
Metabolic freeze
For the current study, the researchers wanted to try to identify additional CARF effectors. They used Foldseek, a powerful structural homology search tool, to find Cat1.
They found that Cat1 is alerted to the presence of a virus by the binding of secondary messenger molecules called cyclic tetra-adenylate, or cA4, which stimulate the enzyme to cleave an essential metabolite in the cell called NAD+.
"Once a sufficient amount of NAD+ is cleaved, the cell enters a growth-arrest state," says co-first author Christian Baca, a TPCB graduate student in the Marraffini lab. "With cellular function on pause, the phage can no longer propagate and spread to the rest of the bacterial population. In this way, Cat1 is similar to Cam1 and Cad1 in that they all provide population-level bacterial immunity."
Unique complexity
But while its immune strategy may be similar to these other CARF effectors, its form is not, as co-first author Puja Majumder, a postdoctoral research scholar in the Patel Lab, revealed through detailed structural analysis using cryo-EM.
She found that the Cat1 protein has a surprisingly complex structure in which Cat1 dimers are glued by cA4 signal molecule, forming long filaments upon viral infection, and trap the NAD+ metabolites within sticky molecular pockets. "Once the NAD+ metabolite is cleaved by Cat1 filaments, it's not available for the cell to use," Majumder explains.
But the protein's singular structural complexity doesn't stop there, she adds. "The filaments interact with each other to form trigonal spiral bundles, and these bundles can then expand to form pentagonal spiral bundles," she says. The purpose of these structural components remains to be investigated.
Also unusual is the fact Cat1 often seems to work alone. "Normally in type III CRISPR systems, you have two activities that contribute to the immunity effect," Baca says. "However, most of the bacteria that encode Cat1 seem to primarily rely on Cat1 for their immunity effect."
Marraffini says these findings pose intriguing new questions. "While I think we've proven the big picture-that CARF effectors are great at preventing phage replication-we still have a lot to learn about the details of how they do it. It will be fascinating to see where this work leads us next."
