Novel Bacterial Immune Weapon that Starves Viral Invaders

Every living organism must defend itself against potential threats; bacteria are no exception. Despite their apparent simplicity, bacteria deploy highly sophisticated defense mechanisms against viral invaders. The most well-known of these is CRISPR-Cas9, which has been adapted for human use as the first FDA-approved genome editing tool.

Over the last year, researchers at Rockefeller's Laboratory of Bacteriology, led by Luciano Marraffini, and the Structural Biology Laboratory at MSKCC, led by Dinshaw Patel, have been investigating key immune components of certain CRISPR systems known as CARF effectors.

These newly discovered effectors employ varied mechanisms to achieve a common outcome: they halt cellular activity to prevent viral replication and the spread of infection within the bacterial population.

In a recent study published in Science, the team describes a newly identified CARF effector, named Cat1. This protein exhibits a highly intricate molecular structure that enables it to deplete a metabolite essential for cellular function. Deprived of this critical resource, the infected cell becomes a dead end for the virus, halting further propagation.

The collective work of the labs is revealing just how effective and different these CARF effectors are. The range of their molecular activities is quite amazing.

Luciano Marraffini, Professor, Laboratory of Bacteriology, The Rockefeller University

Multiple Defense Systems

The adaptive immune systems of bacteria and other single-celled organisms rely on a process called CRISPR to defend against viruses known as phages. All six types of CRISPR systems operate on a shared principle: when a CRISPR RNA detects foreign genetic material, it activates a Cas enzyme that initiates an immune response, often by cutting the invading genetic sequence.

However, a growing amount of data suggests that CRISPR systems use a wide range of defense mechanisms in addition to genetic scissors. Much of this research has been spearheaded by Marraffini’s lab. They have specifically been researching a class of molecules in CRISPR-Cas10 systems known as CARF effectors, which are proteins that are triggered when a bacterium is infected with a phage.

CARF effectors are thought to defend the cell by creating conditions that inhibit viral replication. For instance, the Cam1 effector induces membrane depolarization in infected cells, while Cad1 triggers a kind of molecular fumigation, saturating the cell with toxic compounds.

Metabolic Freeze

The current study focused on identifying additional CARF effectors. To do this, the researchers used Foldseek, a powerful tool for structural homology searches, which led them to the discovery of Cat1.

They found that Cat1 detects viral infection by binding to a secondary messenger molecule called cyclic tetra-adenylate (cA4). This interaction activates the enzyme, which then depletes a critical cellular metabolite known as NAD+.

Once a sufficient amount of NAD⁺ is cleaved, the cell enters a growth-arrest state. 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.

Christian Baca, Study Co-First Author and Graduate Student, The Rockefeller University

Unique Complexity

However, while Cat1’s immune strategy resembles that of other CARF effectors, its structure is distinct. Co-first author Puja Majumder, a postdoctoral research scholar in the Patel Lab, uncovered this through detailed structural analysis using cryo-electron microscopy (cryo-EM).

Her findings revealed that Cat1 forms a surprisingly complex architecture. Upon viral infection, Cat1 dimers are linked together by cA4 signaling molecules, assembling into extended filaments that trap NAD+ metabolites within adhesive molecular pockets.

Once the NAD⁺ metabolite is cleaved by Cat1 filaments, it is not available for the cell to use.

Puja Majumder, Postdoctoral Research Scholar, The Rockefeller University

But the protein's unique structural intricacy does not end there, she says.

“The filaments interact with each other to form trigonal spiral bundles, and these bundles can then expand to form pentagonal spiral bundles,” Majumder added.

It is still unknown what these structural elements are used for.

The fact that Cat1 frequently appears to function alone is also peculiar.

Baca added, “Normally in type III CRISPR systems, you have two activities that contribute to the immunity effect. However, most of the bacteria that encode Cat1 seem to primarily rely on Cat1 for their immunity effect.”

According to Marraffini, these results raise fascinating new questions.

“While I think we have 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,” Marraffini concluded.

Video Credit: The Rockefeller University