Biology subjects

Roberts, C. G.

Publications and source records attributed to Roberts, C. G..

5 recordsLinked to original sources

bioRxiv2025

Cap1 forms a cyclic tetra-adenylate-induced membrane pore during the type III-A CRISPR-Cas immune response

Type III CRISPR-Cas systems synthesize cyclic oligoadenylates (cOAs) signaling molecules upon RNA-guided recognition of viral (phage) transcripts. cOAs bind to and activate CARF effectors, many of which contain transmembrane domains that cause membrane depolarization to prevent phage propagation. How ligand binding induces the activity of these effectors is poorly understood at the molecular level. Here we report the structure and function of the Cap1 effector, composed of a pair of transmembrane helices (TM1/2), a CARF-like (CARFL) domain and a domain of unknown function (DUF4579). In vivo, Cap1 activation results in membrane depolarization, a growth arrest of the bacterial host and the abrogation of the viral lytic cycle. Cryo-EM studies on apo- and cOA-bound states of Cap1 in glyco-diosgenin detergent revealed the formation of tetrameric complexes in both states, with one cOA molecule bound in a pocket composed of four CARFL domains. Ligand binding triggers conformational changes that widen an otherwise narrow transmembrane pore formed by the four TM1/2 domains. Our findings reveal at the atomic level the mechanism of pore opening and membrane depolarization mediated by cyclic nucleotide signaling during the type III CRISPR-Cas response.

microbiology↗

bioRxiv2024

A functional screen for ubiquitin regulation identifies an E3 ligase secreted by Pseudomonas aeruginosa

Ubiquitin signaling controls many aspects of eukaryotic biology, including targeted protein degradation and immune defense. Remarkably, invading bacterial pathogens have adapted secreted effector proteins that hijack host ubiquitination to gain control over host responses. These ubiquitin-targeted effectors can exhibit, for example, E3 ligase or deubiquitinase activities, often without any sequence or structural homology to eukaryotic ubiquitin regulators. Such convergence in function poses a challenge to the discovery of additional bacterial virulence factors that target ubiquitin. To overcome this, we have developed a workflow to harvest natively secreted bacterial effectors and functionally screen them for ubiquitin regulatory activities. After benchmarking this approach on diverse ligase and deubiquitinase activities from Salmonella Typhimurium, Enteropathogenic Escherichia coli, and Shigella flexneri, we applied it to the identification of a cryptic E3 ligase activity secreted by Pseudomonas aeruginosa. We identified an unreported P. aeruginosa E3 ligase, which we have termed Pseudomonas Ub ligase 1 (PUL-1), that resembles none of the other E3 ligases previously established in or outside of the eukaryotic system. Importantly, in an animal model of P. aeruginosa infection, PUL-1 ligase activity plays an important role in regulating virulence. Thus, our workflow for the functional identification of ubiquitin-targeted effector proteins carries promise for expanding our appreciation of how host ubiquitin regulation contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2023

Beyond antibiotic resistance: the whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria

Pathogenic mycobacteria are a significant cause of morbidity and mortality worldwide. These bacteria are highly intrinsically drug resistant, making infections challenging to treat. The conserved whiB7 stress response is a key contributor to mycobacterial intrinsic drug resistance. Although we have a comprehensive structural and biochemical understanding of WhiB7, the complex set of signals that activate whiB7 expression remain less clear. It is believed that whiB7 expression is triggered by translational stalling in an upstream open reading frame (uORF) within the whiB7 5 leader, leading to antitermination and transcription into the downstream whiB7 ORF. To define the signals that activate whiB7, we employed a genome-wide CRISPRi epistasis screen and identified a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 activation. Many of these genes encode amino acid biosynthetic enzymes, tRNAs, and tRNA synthetases, consistent with the proposed mechanism for whiB7 activation by translational stalling in the uORF. We show that the ability of the whiB7 5 regulatory region to sense amino acid starvation is determined by the coding sequence of the uORF. The uORF shows considerable sequence variation among different mycobacterial species, but it is universally and specifically enriched for alanine. Providing a potential rationalization for this enrichment, we find that while deprivation of many amino acids can activate whiB7 expression, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. Our results provide a holistic understanding of the biological pathways that influence whiB7 activation and reveal an extended role for the whiB7 pathway in mycobacterial physiology, beyond its canonical function in antibiotic resistance. These results have important implications for the design of combination drug treatments to avoid whiB7 activation, as well as help explain the conservation of this stress response across a wide range of pathogenic and environmental mycobacteria.

microbiology↗

bioRxiv2023

Bacterial cGAS senses a viral RNA to initiate immunity

CBASS immunity protects prokaryotes from viral (phage) attack through the production of cyclic dinucleotides which activate effector proteins that trigger the death of the infected host. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a highly structured 400-nt RNA, termed CBASS-activating bacteriophage RNA (cabRNA), that binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP. Phages that escape CBASS immunity harbor mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. Since the mammalian cyclase OAS1 also binds viral dsRNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defense pathways.

microbiology↗

bioRxiv2020

Identification and characterization of diverse OTU deubiquitinases in bacteria

Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The Ovarian Tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co-opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin-bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and to recognize a ubiquitin substrate. With these new examples, we perform the first cross-kingdom structural analysis of the OTU fold that highlights commonalities among distantly-related OTU deubiquitinases.

biochemistry↗