Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction

Tripathi, Surya; Voogdt, Carlos Geert Pieter; Bassler, Stefan Oliver; Anderson, Mary; Huang, Po-Hsun; Sakenova, Nazgul; Capraz, Tümay; Jain, Sunit; Koumoutsi, Alexandra; Bravo, Afonso Martins; Trotter, Valentine; Zimmerman, Michael; Sonnenburg, Justin L.; Buie, Cullen; Typas, Athanasios; Deutschbauer, Adam M.; Shiver, Anthony L.; Huang, Kerwyn Casey

doi:10.1016/j.celrep.2023.113517

Cited by 3 publications

(1 citation statement)

References 130 publications

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“…This review summarized the host-related in vivo platforms of genetic-level techniques and in vitro platforms of organoid or bionic system which are applicable to host-microbe interaction study, and discussed their implementation in exploring the detailed molecular mechanism of host responses regulated by gut microbiota. Complementing the techniques we summarized, the utilization of engineered microbes and bacteriophages provided an additional avenue to modulate the functional capacities and diversity of the gut microbiota, enabling researchers to manipulate the microbial community and understand its functional consequences ( Inda et al, 2019 ; Tripathi et al, 2024 ).…”

Section: Discussion and Prospectmentioning

confidence: 99%

Recent advances in host-focused molecular tools for investigating host-gut microbiome interactions

Wang,

Gong,

Xiao

et al. 2024

Front. Microbiol.

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Microbial communities in the human gut play a significant role in regulating host gene expression, influencing a variety of biological processes. To understand the molecular mechanisms underlying host-microbe interactions, tools that can dissect signaling networks are required. In this review, we discuss recent advances in molecular tools used to study this interplay, with a focus on those that explore how the microbiome regulates host gene expression. These tools include CRISPR-based whole-body genetic tools for deciphering host-specific genes involved in the interaction process, Cre-loxP based tissue/cell-specific gene editing approaches, and in vitro models of host-derived organoids. Overall, the application of these molecular tools is revolutionizing our understanding of how host-microbiome interactions contribute to health and disease, paving the way for improved therapies and interventions that target microbial influences on the host.

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Section: Discussion and Prospectmentioning

confidence: 99%

Recent advances in host-focused molecular tools for investigating host-gut microbiome interactions

Wang,

Gong,

Xiao

et al. 2024

Front. Microbiol.

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Systematic mapping of antibiotic cross-resistance and collateral sensitivity with chemical genetics

Sakenova,

Cacace,

Orakov

et al. 2024

Preprint

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By acquiring or evolving resistance to one antibiotic, bacteria can become resistant to a second one, due to shared underlying mechanisms. This is called cross-resistance (XR) and further limits therapeutic choices. The opposite scenario, in which initial resistance leads to sensitivity to a second antibiotic, is termed collateral sensitivity (CS) and can inform cycling or combinatorial treatments. Despite their clinical relevance, our current knowledge of such interactions is limited, mostly due to experimental constraints in their assessment and lack of understanding of the underlying mechanisms. To fill this gap, we used published chemical genetic data on the impact of all Escherichia coli non-essential genes on resistance/sensitivity to 40 antibiotics, and devised a metric that robustly discriminates between known XR and CS antibiotic interactions. This metric, based on chemical genetic profile (dis)similarity between two drugs, allowed us to infer 404 XR and 267 CS interactions, thereby expanding the number of known interactions by 3-fold and reclassifying 116 previously reported interactions. We benchmarked our results by validating 55 out of 59 inferred interactions via experimental evolution. By identifying mutants driving XR and CS interactions in chemical genetics, we recapitulated known and uncovered previously unknown mechanisms, and demonstrated that a given drug pair can exhibit both interactions depending on the resistance mechanism. Finally, we applied CS drug pairs in combination to reduce antibiotic resistance development in vitro. Altogether, our approach provides a systematic framework to map XR/CS interactions and their mechanisms, paving the way for the development of rationally-designed antibiotic combination treatments.

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Physiological Roles of anAcinetobacter-specific σ Factor

Bacon,

Myers,

Iruegas-López

et al. 2024

Preprint

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The Gram-negative pathogenAcinetobacter baumanniiis considered an “urgent threat” to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used byA. baumanniito mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress.Acinetobacterspecies contain the poorly characterized ECF “SigAb;” however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique toAcinetobacterspecies and distinct from RpoE. We combine promoter mutagenesis, motif scanning, and ChIP-seq to define the direct SigAb regulon, which consists ofsigAbitself, the stringent response mediator,relA, and the uncharacterized small RNA, “sabS.” However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown ofsigAborsabSresulted in increased copper sensitivity and excess copper induced SigAb-dependent transcription. Further, we found that two uncharacterized genes in thesigAboperon, “aabA” and “aabB”, have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show thatsigAb,aabA, andaabBare important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture ofA. baumannii.ImportanceAcinetobacter baumanniiis a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding howA. baumanniisenses and responds to stress may uncover novel routes to treat infections. Here, we examine how theAcinetobacter-specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically-relevant contexts.

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Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction

Cited by 3 publications

References 130 publications

Recent advances in host-focused molecular tools for investigating host-gut microbiome interactions

Recent advances in host-focused molecular tools for investigating host-gut microbiome interactions

Systematic mapping of antibiotic cross-resistance and collateral sensitivity with chemical genetics

Physiological Roles of anAcinetobacter-specific σ Factor

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