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.
more »
« less
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.
more »
« less
- 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
More Like this
-
-
Hatfull, Graham F. (Ed.)ABSTRACT Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria and use them as replication factories to assemble progeny phages. Bacteria have evolved powerful defense mechanisms to destroy the invading phages by severing their genomes soon after entry into cells. We discovered a counterdefense mechanism evolved by phage T4 to stitch back the broken genomes and restore viral infection. In this process, a small amount of genetic material is deleted or another mutation is introduced, making the phage resistant to future bacterial attack. The mutant virus might also gain survival advantages against other restriction conditions or DNA damaging events. Thus, bacterial attack not only triggers counterdefenses but also provides opportunities to generate more fit phages. Such defense and counterdefense mechanisms over the millennia led to the extraordinary diversity and the greatest abundance of bacteriophages on Earth. Understanding these mechanisms will open new avenues for engineering recombinant phages for biomedical applications.more » « less
-
ABSTRACT Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. QS relies on the cell-density-dependent production, accumulation, and receptor-mediated detection of extracellular signaling molecules called autoinducers (AIs). Gram-negative bacteria commonly use N -acyl homoserine lactones (AHLs) as their AIs, and they are detected by LuxR-type receptors. Often, LuxR-type receptors are insoluble when not bound to a cognate AI. In this report, we show that LuxR-type receptors are encoded on phage genomes, and in the cases we tested, the phage LuxR-type receptors bind to and are solubilized specifically by the AHL AI produced by the host bacterium. We do not yet know the viral activities that are controlled by these phage QS receptors; however, our observations, coupled with recent reports, suggest that their occurrence is more widespread than previously appreciated. Using receptor-mediated detection of QS AIs could enable phages to garner information concerning the population density status of their bacterial hosts. We speculate that such information can be exploited by phages to optimize the timing of execution of particular steps in viral infection. IMPORTANCE Bacteria communicate with chemical signal molecules to regulate group behaviors in a process called quorum sensing (QS). In this report, we find that genes encoding receptors for Gram-negative bacterial QS communication molecules are present on genomes of viruses that infect these bacteria. These viruses are called phages. We show that two phage-encoded receptors, like their bacterial counterparts, bind to the communication molecule produced by the host bacterium, suggesting that phages can “listen in” on their bacterial hosts. Interfering with bacterial QS and using phages to kill pathogenic bacteria represent attractive possibilities for development of new antimicrobials to combat pathogens that are resistant to traditional antibiotics. Our findings of interactions between phages and QS bacteria need consideration as new antimicrobial therapies are developed.more » « less
-
ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) systems are adaptive defense systems that protect bacteria and archaea from invading genetic elements. In Pseudomonas aeruginosa , quorum sensing (QS) induces the CRISPR-Cas defense system at high cell density when the risk of bacteriophage infection is high. Here, we show that another cue, temperature, modulates P. aeruginosa CRISPR-Cas. Increased CRISPR adaptation occurs at environmental (i.e., low) temperatures compared to that at body (i.e., high) temperature. This increase is a consequence of the accumulation of CRISPR-Cas complexes, coupled with reduced P. aeruginosa growth rate at the lower temperature, the latter of which provides additional time prior to cell division for CRISPR-Cas to patrol the cell and successfully eliminate and/or acquire immunity to foreign DNA. Analyses of a QS mutant and synthetic QS compounds show that the QS and temperature cues act synergistically. The diversity and level of phage encountered by P. aeruginosa in the environment exceed that in the human body, presumably warranting increased reliance on CRISPR-Cas at environmental temperatures. IMPORTANCE P. aeruginosa is a soil dwelling bacterium and a plant pathogen, and it also causes life-threatening infections in humans. Thus, P. aeruginosa thrives in diverse environments and over a broad range of temperatures. Some P. aeruginosa strains rely on the CRISPR-Cas adaptive immune system as a phage defense mechanism. Our discovery that low temperatures increase CRISPR adaptation suggests that the rarely occurring but crucial naive adaptation events may take place predominantly under conditions of slow growth, e.g., during the bacterium’s soil dwelling existence and during slow growth in biofilms.more » « less
-
Pfeiffer, Julie K. (Ed.)ABSTRACT The interplay between defense and counterdefense systems of bacteria and bacteriophages has been driving the evolution of both organisms, leading to their great genetic diversity. Restriction-modification systems are well-studied defense mechanisms of bacteria, while phages have evolved covalent modifications as a counterdefense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant, which contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR escape mutations, allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential countermechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counterdefense mechanisms exhibited by bacteria and phages, while affording protection against one another, also provide evolutionary benefits for both. IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well accepted that T4 and other phages have evolved counterdefense mechanisms to protect their genomes from R-M cleavage by covalent modifications, such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRISPR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and it is also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counterdefense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.45393
