Biology subjects

Lau, R. K.

Publications and source records attributed to Lau, R. K..

5 recordsLinked to original sources

bioRxiv2022

A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage

To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction/modification, CRISPR/Cas, and many others. Here we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS (Cyclic oligonucleotide Based Anti-phage Signaling System) immune systems, and demonstrate that this module drives expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is inactive except in the presence of single-stranded DNA, and CapP activity in cells is stimulated by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. In both their structures and mechanisms, CapH and CapP resemble regulators that drive increased expression of DNA damage response genes in radiation-resistant Deinococcus, and control the mobilization of prophages and mobile elements in response to DNA damage. We also identify CapH and CapP-related proteins associated with diverse known and putative bacterial immune systems, including DISARM and two uncharacterized operons encoding proteins related to eukaryotic ubiquitin signaling pathways. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal stress signal, potentially enabling multiple immune systems to mount a coordinated defensive effort against an invading pathogen.

biochemistry↗

bioRxiv2022

cGASylation by a bacterial E1-E2 fusion protein primes antiviral immune signaling

In all organisms, innate immune pathways sense viral infection and rapidly activate potent immune responses while maintaining a high degree of specificity to prevent inappropriate activation (autoimmunity). In humans, the innate-immune receptor cGAS detects viral infection to produce the nucleotide second messenger cGAMP, which initiates STING-dependent antiviral signaling. Bacteria encode predecessors of the cGAS-STING pathway, termed cyclic oliogonucleotide-based antiphage signaling systems (CBASS), and bacterial cGAS detects bacteriophage infection to produce cGAMP. How bacterial cGAS activation is controlled, however, remains unknown. Here, we show that the CBASS-associated protein Cap2 primes bacterial cGAS for activation through a ubiquitin transferase-like mechanism. A cryoelectron microscopy structure of the Cap2-cGAS complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling the eukaryotic E1 protein ATG7 and the E2 proteins ATG10 and ATG3. The structure captures a reactive-intermediate state with the cGAS C-terminus extending into the Cap2 E1 active site and conjugated to AMP. We find that Cap2 ligates the cGAS C-terminus to a target molecule in cells, a process we call cGASylation. cGASylation primes cGAS for a [~]50-fold increase in cGAMP production. We further demonstrate that Cap2 activity is balanced by a specific endopeptidase, Cap3, which deconjugates cGAS and antagonizes antiviral signaling. Our data demonstrate that bacteria control immune signaling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of E1 and E2 machinery across the kingdoms of life.

microbiology↗

bioRxiv2022

Control of bacterial anti-phage signaling by a WYL domain transcription factor

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how they are regulated to avoid cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW suggest that the protein switches from an unliganded, DNA binding-competent state to a ligand-bound state unable to bind DNA. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that phage infection causes increased CBASS expression in a CapW-dependent manner. Unexpectedly, this CapW-dependent increase in CBASS expression is not required for robust anti-phage activity, suggesting that CapW may mediate CBASS activation and cell death in response to a signal other than phage infection. Our results parallel concurrent reports on the structure and activity of BrxR, a transcription factor associated with the BREX anti-phage system, suggesting that CapW and BrxR are members of a family of universal defense signaling proteins.

biophysics↗

bioRxiv2019

Structure and mechanism of a cyclic trinucleotide-activated bacterial endonuclease mediating bacteriophage immunity

Bacteria possess an array of defenses against foreign invaders, including diverse nucleases that target and destroy the genome of an invading bacteriophage or foreign DNA element. A recently-described bacteriophage immunity pathway employs a cGAS/DncV-like nucleotidyltransferase to produce a cyclic tri-AMP second messenger, which activates the DNA endonuclease effector NucC. Here, we show that NucC is related to restriction enzymes but uniquely assembles into a homotrimer in solution. cAAA binding in a conserved allosteric pocket promotes assembly of two NucC trimers into a homohexamer competent for double-strand DNA cleavage. We propose that NucC mediates bacteriophage immunity either through global activation, causing altruistic cell death and abortive infection, or through local activation and targeted phage genome destruction. Finally, we identify NucC homologs in type III CRISPR-Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems effector complexes.

biochemistry↗

bioRxiv2019

HORMA domain proteins and a Pch2-like ATPase regulate bacterial cGAS-like enzymes to mediate bacteriophage immunity

Bacteria are continually challenged by foreign invaders including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including pathogenic E. coli and Pseudomonas aeruginosa. This pathway employs eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic tri-AMP (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and likely disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage {lambda} infection. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection, and negative regulation by a Pch2-like ATPase.

biochemistry↗