Like humans, bacteria have evolved various immune systems to fight viruses. One such system is CBASS (cyclic oligonucleotide-based anti-phage signaling system), wherein an infected bacterium senses infection by a virus called a bacteriophage and kills itself, likely to prevent infection from spreading throughout the community. Scientists have recently learned much about how CBASS systems function, but how they are regulated remains a mystery. Now, a team of scientists carrying out studies at two U.S. Department of Energy x-ray light sources, including the Advanced Photon Source (APS), has discovered a set of proteins that regulate CBASS in response to DNA damage. Their findings have clinical applications and contribute to a fundamental understanding of bacteria, viruses, and immune systems, including ours.
CBASS is one of several immune systems used by bacteria to kill themselves when they become infected with a virus. CBASS comes in different varieties—over 6500, in fact. While all CBASS systems sense infection and direct the death of the cell using cyclic oligonucleotide-based signals, they encode a variety of different regulatory proteins that likely help these systems sense and respond to different stimuli.
Since CBASS is lethal to the host cell when activated, it’s heavily regulated. In three papers published this year, this team of scientists describe their discovery of four proteins that regulate CBASS system expression in response to stress signals including DNA damage. DNA damage is a universal stress signal in bacteria that can be caused either by viral infection or by other external stresses.
The team’s first paper, published in The EMBO Journal [1], describes the discovery of two proteins, dubbed CapH and CapP, that activate expression of their linked CBASS systems in response to DNA damage. In a healthy bacterium, CapH binds CBASS’s promoter region, turning CBASS off. DNA damage activates the protease activity of CapP, which cleaves CapH to release it from DNA, activating CBASS expression.
The team made their deductions from crystal structures of CapH’s C terminal domain, which they solved at 1.26-Å resolution using the Northeastern Collaborative Access Team’s (NE-CAT’s) x-ray beamline 24-ID-C at the APS, a Department of Energy Office of Science user facility at Argonne National Laboratory. The crystal structures revealed how CapH forms homodimeric and homotetrameric complexes, enabling the team to understand how it binds the CBASS promoter region and giving them a structural model for where it is cleaved by CapP. The team also solved crystal structures of CapH’s N terminal domain and CapP at other facilities.
The next study, published in Nucleic Acids Research [2], addressed the function of another regulator called CapW, which is associated with a minority of CBASS systems. They found that CapW binds to CBASS’s promoter region, much as CapH does, and suppresses expression of the system. When a cell is infected by a bacteriophage, CapW senses the production of an unidentified ligand—possibly a product of DNA damage—and alters its conformation so that it can no longer bind DNA.
The team contrasted crystal structures of two CapW proteins from different species, one from P. aeruginosa determined at NE-CAT’s 24-ID-E beamline and a second from S. maltophilia structures obtained at the Advanced Light Source BCSB beamline 5.0.2, an Office of Science user facility at Lawrence Berkeley National Laboratory. These two structures showed strikingly different conformations of the protein’s DNA binding domains. The structure determined at the APS was critical in developing the model of how conformational changes regulate DNA binding by the protein.
Finally, a third study, published in Protein Science [3], revealed a protein that may regulate these regulators. Dubbed Cap18, this protein is found in more than half of CBASS systems that encode CapH/CapP or CapW—an extremely high correlation. The authors found that Cap18 degrades single-stranded DNAand hypothesize that Cap18 limits inappropriate activation of CBASS by degrading the molecules that activate CapP and possibly also CapW.
For this research, they determined crystal structures of a Cap18 protein from E. coli on NE-CAT beamline 24-ID-C (Fig.1). The structure of Cap18 enabled the team to design structure-based mutants to confirm that Cap18 specifically degrades single-stranded DNA.
The team is now researching why CBASS systems that encode these regulatory proteins are activated by DNA damage. Their work is part of a broader effort to understand how bacterial antiviral immune systems are regulated, what signals they respond to, and whether they cooperate with each other rather than work independently. The research described in these three papers represents a foundational step in that understanding.
This research also has potentially life-saving clinical applications. As more bacteria become resistant to antibiotics, a treatment known as phage therapy is reemerging. In phage therapy, doctors kill a drug-resistant bacterium not with a drug but with a bacteriophage that infects and kills that bacterium. Phage therapy is not currently approved by the FDA—phages are difficult to prepare, and identifying the right phage, dose, and length of treatment is difficult. Nevertheless, phage therapy has saved the lives of people with dangerous multidrug resistant infections.
Multidrug-resistant bacteria are very tough to fight. If the team can understand better how bacteria and phages interact by studying these defense systems, perhaps they can help design or choose the right phage for a particular bacterial infection to make phage therapy more effective. ― Judy Myers
See:
[1] Rebecca K Lau, Eray Enustun, Yajie Gu, Justin V. Nguyen, and Kevin D Corbett*, “A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage,” EMBO J. 41, e111540 (published on line September 26, 2022). DOI 10.15252/embj.2022111540
Author affiliation: University of California, San Diego
Correspondence: * [email protected]
K.D.C. acknowledges support from the National Institutes of Health (R35 GM144121). R.K.L. was supported by the UCSD Quantitative and Integrative Physiology Training Grant (NIH T32 GM127235) and an individual National Institutes of Health Predoctoral Fellowship (F31 GM137600). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
[2] Chelsea L. Blankenship, Justin V. Nguyen, Rebecca K. Lau, Qiaozhen Ye, Yajie Gu, and Kevin D. Corbett*, “Control of bacterial immune signaling by a WYL domain transcription factor,” Nucleic Acids Res. 50(9), 5239 (2022). DOI: 10.1093/nar/gkac343
Author affiliation: University of California, San Diego
Correspondence: * [email protected]
K.D.C. acknowledges support from UC San Diego and NIH/NIAID [R21 AI148814]; C.L.B. was supported by the UCSD Molecular Biophysics Training Grant (NIH) [T32 GM139795]; R.K.L. was supported by the UCSD Quantitative and Integrative Physiology Training Grant [NIH T32 GM127235] and an NIH/NIGMS individual predoctoral fellowship [F31 GM137600]. Funding for open access charge: National Institutes of Health. The Advanced Light Source is a Department of Energy (DOE) Office of Science User Facility under Contract No. DE-AC02- 05CH11231. The Pilatus detector on 5.0.1. was funded under NIH grant S10OD021832. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169. NE-CAT is funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on the 24-ID-E beamline is funded by a NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC02- 06CH11357.
[3] Qishan Liang, Sara T. Richey‡, Sarah N. Ur‡‡, Qiaozhen Ye, Rebecca K. Lau, and Kevin D. Corbett*, “Structure and activity of a bacterial defense-associated 3¢-5¢ exonuclease,” Prot. Sci. 31, e4374 (2022). DOI: 10.1002/pro.4374
Author affiliation: University of California, San Diego Present addresses: ‡The Scripps Research Institute, ‡‡Vividion Therapeutics
Correspondence: * [email protected]
K.D.C. acknowledges support from the National Institutes of Health, (R21 AI148814 and R35 GM144121). R.K.L. was supported by the UCSD Quantitative and Integrative Physiology Training Grant (NIH T32 GM127235) and an individual National Institutes of Health Predoctoral Fellowship (F31 GM137600). S.N.U. was supported by the UCSD Molecular Biophysics Training Grant (T32 GM008326) and a National Science Foundation Graduate Research Fellowship. NE-CAT is funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
The U.S. Department of Energy's APS at Argonne National Laboratory is one of the world’s most productive x-ray light source facilities. Each year, the APS provides high-brightness x-ray beams to a diverse community of more than 5,000 researchers in materials science, chemistry, condensed matter physics, the life and environmental sciences, and applied research. Researchers using the APS produce over 2,000 publications each year detailing impactful discoveries and solve more vital biological protein structures than users of any other x-ray light source research facility. APS x-rays are ideally suited for explorations of materials and biological structures; elemental distribution; chemical, magnetic, electronic states; and a wide range of technologically important engineering systems from batteries to fuel injector sprays, all of which are the foundations of our nation’s economic, technological, and physical well-being.
The Advanced Light Source is a U.S. Department of Energy scientific user facility at Lawrence Berkeley National Laboratory. Our mission is to advance science for the benefit of society by providing our world-class synchrotron light source capabilities and expertise to a broad scientific community.
Argonne National Laboratory seeks solutions to pressing national problems in science and technology. The nation's first national laboratory, Argonne conducts leading-edge basic and applied scientific research in virtually every scientific discipline. Argonne researchers work closely with researchers from hundreds of companies, universities, and federal, state, and municipal agencies to help them solve their specific problems, advance America's scientific leadership and prepare the nation for a better future. With employees from more than 60 nations, Argonne is managed by UChicago Argonne, LLC, for the U.S. DOE Office of Science.
The U.S. Department of Energy's Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time. For more information, visit the Office of Science website.