Biology subjects

Hobbs, S. J.

Publications and source records attributed to Hobbs, S. J..

7 recordsLinked to original sources

bioRxiv2026

Phage protease enzymes activate CBASS antiphage immunity

Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are immunity pathways in bacteria that use a cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme to sense phage infection and initiate antiviral defense. Bacteria encode thousands of diverse CBASS operons, demonstrating a critical role for CD-NTase activation in controlling the prokaryotic response to viral infection. Here we discover proteolytic cleavage by phage prohead proteases as a mechanism of CD-NTase activation and demonstrate that diverse CBASS operons function as molecular sensors of protease activity. We reconstitute CBASS recognition of phage T4 infection in vitro and identify proteolytic cleavage of a surface exposed CD-NTase activation loop as a trigger of enzyme catalysis and nucleotide immune signal synthesis. Phage prohead proteases are sufficient to activate CBASS in vivo and explain how immune signaling is initiated during late stages of viral infection. Combining biochemical and structure-based phylogenetic analyses, we map activation loops in Clade A, D, and G CD-NTase enzymes and define critical residues that control CBASS recognition of distinct phage families. Our results define CBASS recognition of phage protease activity as a widespread mechanism of antiviral defense.

microbiology↗

bioRxiv2024

A widespread family of viral sponge proteins reveals specific inhibition of nucleotide signals in anti-phage defense

Cyclic oligonucleotide-based antiviral signaling systems (CBASS) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 [A] crystal structure of Acb4 in complex with 3'3'-cGAMP reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo. Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.

microbiology↗

bioRxiv2023

Bacterial SEAL domains undergo autoproteolysis and function in regulated intramembrane proteolysis

Gram-positive bacteria use SigI/RsgI-family sigma factor/anti-sigma factor pairs to sense and respond to cell wall defects and plant polysaccharides. In Bacillus subtilis this signal transduction pathway involves regulated intramembrane proteolysis (RIP) of the membrane-anchored anti-sigma factor RsgI. However, unlike most RIP signaling pathways, site-1 cleavage of RsgI on the extracytoplasmic side of the membrane is constitutive and the cleavage products remain stably associated, preventing intramembrane proteolysis. The regulated step in this pathway is their dissociation, which is hypothesized to involve mechanical force. Release of the ectodomain enables intramembrane cleavage by the RasP site-2 protease and activation of SigI. The constitutive site-1 protease has not been identified for any RsgI homolog. Here, we report that RsgIs extracytoplasmic domain has structural and functional similarities to eukaryotic SEA domains that undergo autoproteolysis and have been implicated in mechanotransduction. We show that site-1 proteolysis in B. subtilis and Clostridial RsgI family members is mediated by enzyme-independent autoproteolysis of these SEA-like (SEAL) domains. Importantly, the site of proteolysis enables retention of the ectodomain through an undisrupted {beta}-sheet that spans the two cleavage products. Autoproteolysis can be abrogated by relief of conformational strain in the scissile loop, in a mechanism analogous to eukaryotic SEA domains. Collectively, our data support the model that RsgI-SigI signaling is mediated by mechanotransduction in a manner that has striking parallels with eukaryotic mechanotransducive signaling pathways. SIGNIFICANCESEA domains are broadly conserved among eukaryotes but absent in bacteria. They are present on diverse membrane-anchored proteins some of which have been implicated in mechanotransducive signaling pathways. Many of these domains have been found to undergo autoproteolysis and remain noncovalently associated following cleavage. Their dissociation requires mechanical force. Here, we identify a family of bacterial SEA-like (SEAL) domains that arose independently from their eukaryotic counterparts but have structural and functional similarities. We show these SEAL domains autocleave and the cleavage products remain stably associated. Importantly, these domains are present on membrane-anchored anti-sigma factors that have been implicated in mechanotransduction pathways analogous to those in eukaryotes. Our findings suggest that bacterial and eukaryotic signaling systems have evolved a similar mechanism to transduce mechanical stimuli across the lipid bilayer.

microbiology↗

bioRxiv2023

Convergent mutations in phage virion assembly proteins enable evasion of Type I CBASS immunity

CBASS is an anti-phage defense system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by viral DNA but the stage of phage replication which activates bacterial CBASS remains unclear. Here we define the specificity of Type I CBASS immunity using a comprehensive analysis of 975 operon-phage pairings and show that Type I CBASS operons composed of distinct CD-NTases, and Cap effectors exhibit striking patterns of defense against dsDNA phages across five diverse viral families. We demonstrate that escaper phages evade CBASS immunity by acquiring mutations in structural genes encoding the prohead protease, capsid, and tail fiber proteins. Acquired CBASS resistance is highly operon-specific and typically does not affect overall fitness. However, we observe that some resistance mutations drastically alter phage infection kinetics. Our results define late-stage virus assembly as a critical determinant of CBASS immune activation and evasion by phages.

microbiology↗

bioRxiv2023

Phages overcome bacterial immunity via diverse anti-defense proteins

It was recently shown that bacteria employ, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here, we analyzed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defense systems, and identified multiple families of anti-defense proteins that inhibit the Gabija, Thoeris, and Hachiman systems. We show that these proteins efficiently cancel the defensive activity when co-expressed with the respective defense system or introduced into phage genomes. Homologs of these anti-defense proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that an anti-Gabija protein, denoted Gad1, blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Our data further reveal an anti-Thoeris protein, denoted Tad2, which is a "sponge" that sequesters the immune signaling molecules produced by Thoeris TIR-domain proteins in response to phage. Our results demonstrate that phages encode an arsenal of anti-defense proteins that can disable a variety of bacterial defense mechanisms.

genetics↗

bioRxiv2022

Viruses inhibit TIR gcADPR signaling to overcome bacterial defense

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants, and animals. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signaling molecule whose molecular structure remained elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function. We identified a large family of phage-encoded proteins, denoted here Thoeris anti-defense 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are "sponges" that bind and sequester the immune signaling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. A high-resolution crystal structure of Tad1 bound to the signaling molecule revealed that its chemical structure is 1'-2' glycocyclic ADPR (gcADPR), a unique molecule not previously described in other biological systems. Our results define the chemical structure of a central immune signaling molecule, and reveal a new mode of action by which pathogens can suppress host immunity.

microbiology↗

bioRxiv2018

Network stress test reveals novel drug potentiators in Mycobacterium tuberculosis

Deciphering molecular stress response is highly relevant to studies of microbes such as Mycobacterium tuberculosis (MTB), the causative pathogen of tuberculosis (TB) which sickens 10 million people and kills 1.8 million each year1. Prolonged therapy and unfavorable outcomes arise partially because MTB has evolved stress responses to achieve tolerance, wherein MTB persists in otherwise inhibitory drug concentrations by means independent of heritable resistance mutations2-4. Understanding these adaptations and how they are regulated can reveal new biology, including unexplored drug targets and treatment-enhancing strategies. Here, we present a novel network-based genetic screening approach: the Transcriptional Regulator Induced Phenotype (TRIP) screen, which we used to identify previously uncharacterized MTB network adaptations to the first-line drug isoniazid (INH). We found regulators that alter IN ...

systems biology↗