Improved RNA stability estimation through Bayesian modeling reveals most Salmonella transcripts have subminute half-lives.

RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in posttranscriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 min, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with cross-linking and immunoprecipitation followed by RNA sequencing (CLIP-seq) revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3' untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for ProQ in the oxidative stress response. Our findings provide insights into posttranscriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression.

An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity.

Plasticity in gene expression allows bacteria to adapt to diverse environments. This is particularly relevant in the dynamic niche of the human intestinal tract; however, transcriptional networks remain largely unknown for gut-resident bacteria. Here we apply differential RNA sequencing (RNA-seq) and conventional RNA-seq to the model gut bacterium Bacteroides thetaiotaomicron to map transcriptional units and profile their expression levels across 15 in vivo-relevant growth conditions. We infer stress- and carbon source-specific transcriptional regulons and expand the annotation of small RNAs (sRNAs). Integrating this expression atlas with published transposon mutant fitness data, we predict conditionally important sRNAs. These include MasB, which downregulates tetracycline tolerance. Using MS2 affinity purification and RNA-seq, we identify a putative MasB target and assess its role in the context of the MasB-associated phenotype. These data-publicly available through the Theta-Base web browser ( http://micromix.helmholtz-hiri.de/bacteroides/ )-constitute a valuable resource for the microbiome community.

Non-professional efferocytosis of Salmonella-infected intestinal epithelial cells in the neonatal host.

The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal intestine. Instead, here, we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighboring epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid cocultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis, and identified the involvement of the "eat-me" signals and adaptors phosphatidylserine and C1q as well as the "eat-me" receptors integrin-αv (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighboring epithelial cells may represent a previously unrecognized mechanism of neonatal antimicrobial host defense to maintain barrier integrity.

CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron.

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.

CRISPR Interference-Based Functional Small RNA Genomics.

Small RNAs (sRNAs) are versatile regulators universally present in species across the prokaryotic kingdom, yet their functional characterization remains a major bottleneck. Gene inactivation through random transposon insertion has proven extremely valuable in discovering hidden gene functions. However, this approach is biased toward long genes and usually results in the underrepresentation of sRNA mutants. In contrast, CRISPR interference (CRISPRi) harnesses guide RNAs to recruit cleavage-deficient Cas nucleases to specific DNA loci. The ensuing steric hindrance inhibits RNA polymerase assembly at-or migration along-predefined genes, allowing for targeted knockdown screens without major length bias. In this chapter, we provide a detailed protocol for CRISPRi-based functional screening of bacterial sRNAs. Using the abundant microbiota species Bacteroides thetaiotaomicron as a model, we describe the design and generation of a guide library targeting the full intergenic sRNA repertoire of this organism and its application to identify sRNA knockdown-associated fitness effects. Our protocol is generic and thus suitable for the systematic assessment of sRNA-associated phenotypes in a wide range of bacterial species and experimental conditions. We expect CRISPRi-based functional genomics to boost sRNA research in understudied bacterial taxa, for instance, members of the gut microbiota.

A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection.

Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.

An integrated transcriptomics-functional genomics approach reveals a small RNA that modulates Bacteroides thetaiotaomicron sensitivity to tetracyclines.

Gene expression plasticity allows bacteria to adapt to diverse environments, tie their metabolism to available nutrients, and cope with stress. This is particularly relevant in a niche as dynamic and hostile as the human intestinal tract, yet transcriptional networks remain largely unknown in gut Bacteroides spp. Here, we map transcriptional units and profile their expression levels in Bacteroides thetaiotaomicron over a suite of 15 defined experimental conditions that are relevant in vivo , such as variation of temperature, pH, and oxygen tension, exposure to antibiotic stress, and growth on simple carbohydrates or on host mucin-derived glycans. Thereby, we infer stress- and carbon source-specific transcriptional regulons, including conditional expression of capsular polysaccharides and polysaccharide utilization loci, and expand the annotation of small regulatory RNAs (sRNAs) in this organism. Integrating this comprehensive expression atlas with transposon mutant fitness data, we identify conditionally important sRNAs. One example is MasB, whose inactivation led to increased bacterial tolerance of tetracyclines. Using MS2 affinity purification coupled with RNA sequencing, we predict targets of this sRNA and discuss their potential role in the context of the MasB-associated phenotype. Together, this transcriptomic compendium in combination with functional sRNA genomics-publicly available through a new iteration of the 'Theta-Base' web browser (www.helmholtz-hiri.de/en/datasets/bacteroides-v2)-constitutes a valuable resource for the microbiome and sRNA research communities alike.

RNA recording in single bacterial cells using reprogrammed tracrRNAs.

Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages.

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB-Irg1-itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32-BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB's role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo.

The ambivalent role of Bacteroides in enteric infections.

Bacteroides spp. are increasingly used as model gut commensals in cocolonization studies with enteropathogens. The collective findings imply common themes of colonization resistance but also pathogen crossfeeding. We discuss how cutting-edge transcriptomics may help to disentangle the molecular basis of the divergent roles of Bacteroides in either protecting against or promoting infection.

Comparative genomics provides structural and functional insights into Bacteroides RNA biology.

Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.

SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo.

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.

Cross-species RNA-seq for deciphering host-microbe interactions.

The human body is constantly exposed to microorganisms, which entails manifold interactions between human cells and diverse commensal or pathogenic bacteria. The cellular states of the interacting cells are decisive for the outcome of these encounters such as whether bacterial virulence programmes and host defence or tolerance mechanisms are induced. This Review summarizes how next-generation RNA sequencing (RNA-seq) has become a primary technology to study host-microbe interactions with high resolution, improving our understanding of the physiological consequences and the mechanisms at play. We illustrate how the discriminatory power and sensitivity of RNA-seq helps to dissect increasingly complex cellular interactions in time and space down to the single-cell level. We also outline how future transcriptomics may answer currently open questions in host-microbe interactions and inform treatment schemes for microbial disorders.

MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT.

A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.

Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.

High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections.

Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.

Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen.

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes 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 to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated 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.

A high-resolution transcriptome map identifies small RNA regulation of metabolism in the gut microbe Bacteroides thetaiotaomicron.

Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called 'Theta-Base' ( www.helmholtz-hiri.de/en/datasets/bacteroides ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes.

Improved bacterial RNA-seq by Cas9-based depletion of ribosomal RNA reads.

A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.