CRISPR-Cas systems are a family of adaptive immune systems that use small CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) nucleases to protect prokaryotes from invading plasmids and viruses (i.e., phages). Type III systems launch a multilayered immune response that relies upon both Cas and non-Cas cellular nucleases, and although the functions of Cas components have been well described, the identities and roles of non-Cas participants remain poorly understood. Previously, we showed that the type III-A CRISPR-Cas system in Staphylococcus epidermidis employs two degradosome-associated nucleases, PNPase and RNase J2, to promote crRNA maturation and eliminate invading nucleic acids (Chou-Zheng and Hatoum-Aslan, 2019). Here, we identify RNase R as a third ‘housekeeping’ nuclease critical for immunity. We show that RNase R works in concert with PNPase to complete crRNA maturation and identify specific interactions with Csm5, a member of the type III effector complex, which facilitate nuclease recruitment/stimulation. Furthermore, we demonstrate that RNase R and PNPase are required to maintain robust anti-plasmid immunity, particularly when targeted transcripts are sparse. Altogether, our findings expand the known repertoire of accessory nucleases required for type III immunity and highlight the remarkable capacity of these systems to interface with diverse cellular pathways to ensure successful defense.
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A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity
Just as humans are susceptible to viruses, bacteria have their own viruses to contend with. These viruses – known as phages – attach to the surface of bacterial cells, inject their genetic material, and use the cells’ enzymes to multiply while destroying their hosts. To defend against a phage attack, bacteria have evolved a variety of immune systems. For example, when a bacterium with an immune system known as CRISPR-Cas encounters a phage, the system creates a ‘memory’ of the invader by capturing a small snippet of the phage’s genetic material. The pieces of phage DNA are copied into small molecules known as CRISPR RNAs, which then combine with one or more Cas proteins to form a group called a Cas complex. This complex patrols the inside of the cell, carrying the CRISPR RNA for comparison, similar to the way a detective uses a fingerprint to identify a criminal. Once a match is found, the Cas proteins chop up the invading genetic material and destroy the phage. There are several different types of CRISPR-Cas systems. Type III systems are among the most widespread in nature and are unique in that they provide a nearly impenetrable barrier to phages attempting to infect bacterial cells. Medical researchers are exploring the use of phages as alternatives to conventional antibiotics and so it is important to find ways to overcome these immune responses in bacteria. However, it remains unclear precisely how Type III CRISPR-Cas systems are able to mount such an effective defense. Chou-Zheng and Hatoum-Aslan used genetic and biochemical approaches to study the Type III CRISPR-Cas system in a bacterium called Staphylococcus epidermidis. The experiments showed that two enzymes called PNPase and RNase J2 played crucial roles in the defense response triggered by the system. PNPase helped to generate CRISPR RNAs and both enzymes were required to help to destroy genetic material from invading phages. Previous studies have shown that PNPase and RNase J2 are part of a machine in bacterial cells that usually degrades damaged genetic material. Therefore, these findings show that the Type III CRISPR-Cas system in S. epidermidis has evolved to coordinate with another pathway to help the bacteria survive attack from phages. CRISPR-Cas immune systems have formed the basis for a variety of technologies that continue to revolutionize genetics and biomedical research. Therefore, along with aiding the search for alternatives to antibiotics, this work may potentially inspire the development of new genetic technologies in the future.
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- Award ID(s):
- 1749886
- NSF-PAR ID:
- 10096811
- Date Published:
- Journal Name:
- eLife
- Volume:
- 8
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.45393
