Biology subjects

Barquist, L.

Publications and source records attributed to Barquist, L..

31 records · Page 2Linked to original sources

bioRxiv2022

High-throughput transposon mutagenesis in the family Enterobacteriaceae reveals core essential genes and rapid turnover of essentiality

The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the gut commensal and model organism Escherichia coli, as well as several major human pathogens including multiple serovars of Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, with the E. coli Keio single-gene deletion library often regarded as a gold standard for gene essentiality studies. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of thirteen sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first benchmark a number of gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes, and reconstruct an ancestral essential gene set of 296 genes. Despite the presence of a large cohort of variably essential genes, surprisingly we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the {sigma}E extracytoplasmic stress response, which appears to have independently become essential multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts that have emerged from within the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival, and uncovers several instances of essential stress responses masked by redundancy in free-living bacteria.

microbiology↗

bioRxiv2022

Improved prediction of bacterial CRISPRi guide efficiency through data integration and automated machine learning

CRISPR interference (CRISPRi), the targeting of a catalytically dead Cas protein to block transcription, is the leading technique to silence gene expression in bacteria. However, design rules for CRISPRi remain poorly defined, limiting predictable design for gene interrogation, pathway manipulation, and high-throughput screens. Here we develop a best-in-class prediction algorithm for guide silencing efficiency by systematically investigating factors influencing guide depletion in multiple genome-wide essentiality screens, with the surprising discovery that gene-specific features such as transcriptional activity substantially impact prediction of guide activity. Accounting for these features as part of algorithm development allowed us to develop a mixed-effect random forest regression model that provides better estimates of guide efficiency than existing methods, as demonstrated in an independent saturating screen. We further applied methods from explainable AI to extract interpretable design rules from the model, such as sequence preferences in the vicinity of the PAM distinct from those previously described for genome engineering applications. Our approach provides a blueprint for the development of predictive models for CRISPR technologies where only indirect measurements of guide activity are available.

bioinformatics↗

bioRxiv2022

Predicting off-target effects of antisense oligomers targeting bacterial mRNAs with the MASON webserver

Antisense oligomers (ASOs) such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (Make AntiSense Oligomers Now), a webserver for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASONs off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq dataset from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed our findings are not unique to Salmonella. We believe that MASONs off-target assessment will improve the design of specific PNAs and other ASOs.

bioinformatics↗

bioRxiv2022

INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors

Ribosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has limitations. These include difficulties with detection of low abundance transcripts and analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developed in vitro Ribo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. Using Escherichia coli as an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.

microbiology↗

bioRxiv2022

Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli

Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.

microbiology↗

bioRxiv2021

A target expression threshold dictates invader defense and autoimmunity by CRISPR-Cas13

Immune systems must recognize and clear foreign invaders without eliciting autoimmunity. CRISPR-Cas immune systems in prokaryotes manage this task by following two criteria: extensive guide:target complementarity and a defined target-flanking motif. Here we report an additional requirement for RNA-targeting CRISPR-Cas13 systems: expression of the target transcript exceeding a threshold. This finding is based on targeting endogenous non-essential transcripts, which rarely elicited dormancy through collateral RNA degradation. Instead, eliciting dormancy required over-expressing targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as the principal determinant of cytotoxic autoimmunity and revealed that the threshold shifts with the guide:target pair. This expression threshold ensured defense against a lytic bacteriophage yet allowed tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as a third criterion for immune activation by RNA-targeting CRISPR-Cas systems, buffering against autoimmunity and distinguishing pathogenic and benign invaders. HIGHLIGHTSO_LICas13-induced dormancy requires RNA target levels to exceed an expression threshold C_LIO_LIThe expression threshold can prevent cytotoxic self-targeting for endogenous transcripts C_LIO_LIThe threshold shifts depending on the CRISPR RNA guide:target pair C_LIO_LIThe threshold allows cells to distinguish pathogenic and benign infections C_LI

molecular biology↗

bioRxiv2020

Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq

Posttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N binding sites at single-nucleotide resolution and mapped more than 500 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa. IMPORTANCEThe ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) two-component system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we identified hundreds of RsmA/N regulatory target RNAs and the consensus motifs for RsmA/N bindings by UV crosslinking in vivo. Moreover, our CLIP-seq revealed that RsmA/N posttranscriptionally regulate cell wall organization and exert feedback control on GacAS-RsmA/N systems. Many genes including small regulatory RNAs identified in this study are attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.

microbiology↗

bioRxiv2020

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important Gram-positive bacterium

The Gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, little is known about the bacteriums transcriptome architecture and mechanisms of post-transcriptional control. Here, we have applied a suite of RNA-centric techniques, including transcription start site mapping, transcription termination mapping and Hfq RIP-seq, to generate a single-nucleotide resolution RNA map of C. difficile 630. Our transcriptome annotation provides information about 5 and 3 untranslated regions, operon structures and non-coding regulators, including 42 sRNAs. These transcriptome data are accessible via an open-access browser called Clost-Base. Our results indicate functionality of many conserved riboswitches and predict novel cis-regulatory elements upstream of MDR-type ABC transporters and transcriptional regulators. Recent studies have revealed a role of sRNA-based regulation in several Gram-positive bacteria but their involvement with the RNA-binding protein Hfq remains controversial. Here, sequencing the RNA ligands of Hfq reveals in vivo association of many sRNAs along with hundreds of potential target mRNAs in C. difficile providing evidence for a global role of Hfq in post-transcriptional regulation in a Gram-positive bacterium. Through integration of Hfq-bound transcripts and computational approaches we predict regulated target mRNAs for the novel sRNA AtcS encoding several adhesins and the conserved oligopeptide transporter oppB that influences sporulation initiation in C. difficile. Overall, these findings provide a potential mechanistic explanation for increased biofilm formation and sporulation in an hfq deletion strain and lay the foundation for understanding clostridial ribo regulation with implications for the infection process.

microbiology↗

bioRxiv2020

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalogue of microbial RBPs.

microbiology↗

bioRxiv2020

A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence

Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (TraDIS), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella {Delta}mgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available "omics" datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria.

microbiology↗

bioRxiv2019

Dual RNA-seq provides insight into the biology of the neglected intracellular human pathogen Orientia tsutsugamushi

Emerging and neglected diseases pose challenges as their biology is frequently poorly understood, and genetic tools often do not exist to manipulate the responsible pathogen. Organism agnostic sequencing technologies offer a promising approach to understand the molecular processes underlying these diseases. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium and the causative agent of the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning, we investigated the transcriptional architecture of Ot, including operon structure and non-coding RNAs, and found evidence for wide-spread post-transcriptional antisense regulation. We compared the host response to two clinical isolates and identified distinct immune response networks that are up-regulated in response to each strain, leading to predictions of relative virulence which were confirmed in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.

microbiology↗

bioRxiv2018

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggests a differential effect on twitching motility by flagellum components based upon their cellular location. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

genomics↗

bioRxiv2017

Machine learning identifies signatures of host adaptation in the bacterial pathogen Salmonella enterica

Emerging pathogens are a major threat to public health, however understanding how pathogens adapt to new niches remains a challenge. New methods are urgently required to provide functional insights into pathogens from the massive genomic data sets now being generated from routine pathogen surveillance for epidemiological purposes. Here, we measure the burden of atypical mutations in protein coding genes across independently evolved Salmonella enterica lineages, and use these as input to train a random forest classifier to identify strains associated with extraintestinal disease. Members of the species fall along a continuum, from pathovars which cause gastrointestinal infection and low mortality, associated with a broad host-range, to those that cause invasive infection and high mortality, associated with a narrowed host range. Our random forest classifier learned to perfectly discriminate long-established gastrointestinal and invasive serovars of Salmonella. Additionally, it was able to discriminate recently emerged Salmonella Enteritidis and Typhimurium lineages associated with invasive disease in immunocompromised populations in sub-Saharan Africa, and within-host adaptation to invasive infection. We dissect the architecture of the model to identify the genes that were most informative of phenotype, revealing a common theme of degradation of metabolic pathways in extraintestinal lineages. This approach accurately identifies patterns of gene degradation and diversifying selection specific to invasive serovars that have been captured by more labour-intensive investigations, but can be readily scaled to larger analyses.

genomics↗