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. The conserved whiB7 stress response reduces the effectiveness of antibiotic therapy by activating several intrinsic antibiotic resistance mechanisms. Despite our comprehensive biochemical understanding of WhiB7, the complex set of signals that induce whiB7 expression remain less clear. We employed a reporter-based, genome-wide CRISPRi epistasis screen to identify a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 expression. We show that whiB7 expression is determined by the amino acid composition of the 5' regulatory uORF, thereby allowing whiB7 to sense amino acid starvation. Although deprivation of many amino acids can induce whiB7, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. These findings describe a metabolic function for whiB7 and help explain its evolutionary conservation across mycobacterial species occupying diverse ecological niches.

Bacterial cGAS senses a viral RNA to initiate immunity.

Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.

Ubiquitin-targeted bacterial effectors: rule breakers of the ubiquitin system.

Regulation through post-translational ubiquitin signaling underlies a large portion of eukaryotic biology. This has not gone unnoticed by invading pathogens, many of which have evolved mechanisms to manipulate or subvert the host ubiquitin system. Bacteria are particularly adept at this and rely heavily upon ubiquitin-targeted virulence factors for invasion and replication. Despite lacking a conventional ubiquitin system of their own, many bacterial ubiquitin regulators loosely follow the structural and mechanistic rules established by eukaryotic ubiquitin machinery. Others completely break these rules and have evolved novel structural folds, exhibit distinct mechanisms of regulation, or catalyze foreign ubiquitin modifications. Studying these interactions can not only reveal important aspects of bacterial pathogenesis but also shed light on unexplored areas of ubiquitin signaling and regulation. In this review, we discuss the methods by which bacteria manipulate host ubiquitin and highlight aspects that follow or break the rules of ubiquitination.

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.

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 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.

The intestinal environment as an evolutionary adaptation to mouthbrooding in the Astatotilapia burtoni cichlid.

Many of the various parental care strategies displayed by animals are accompanied by a significant reduction in food intake that imposes a substantial energy trade-off. Mouthbrooding, as seen in several species of fish in which the parent holds the developing eggs and fry in the buccal cavity, represents an extreme example of reduced food intake during parental investment and is accompanied by a range of physiological adaptations. In this study we use 16S sequencing to characterize the gut microbiota of female Astatotilapia burtoni cichlid fish throughout the obligatory phase of self-induced starvation during the brooding cycle in comparison to stage-matched females that have been denied food for the same duration. In addition to a reduction of gut epithelial turnover, we find a dramatic reduction in species diversity in brooding stages that recovers upon release of fry and refeeding that is not seen in females that are simply starved. Based on overall species diversity as well as differential abundance of specific bacterial taxa, we suggest that rather than reflecting a simple deprivation of caloric intake, the gut microbiota is more strongly influenced by physiological changes specific to mouthbrooding including the reduced epithelial turnover and possible production of antimicrobial agents.