Macrophages are essential for both, to clear pathogens and preserve tissue homeostasis, yet the molecular regulators of this equilibrium remain incompletely defined. Here, we identify SAILR (survival associated immune-regulatory RNA), a primate-specific long noncoding RNA (lncRNA), as a critical modulator of macrophage viability under infection conditions. SAILR is induced during monocyte-to-macrophage differentiation, but rapidly downregulated upon bacterial challenge in a nuclear factor kappa B (NF-κB) dependent manner. In both naïve and immune-activated macrophages, SAILR dampens the expression of adhesion, phagocytosis, and invasion factors, which include SIGLEC1 and MMP7. During infection with Salmonella Typhimurium, depletion of SAILR sensitizes macrophages to apoptosis, resulting in loss of intracellular replication niches and reduced bacterial recovery. Conversely, enforced SAILR expression promotes macrophage survival and increases intracellular pathogen burden. Mechanistically, SAILR interacts with the antiapoptotic adaptor protein 14-3-3β to support macrophage survival. Notably, downregulation of SAILR is mirrored in circulating immune cells from patients with severe COVID-19 and sepsis. Together, our findings position SAILR as a central regulator in linking macrophage survival to host-pathogen interaction and disease pathophysiology.

Single-cell RNA sequencing is increasingly applied to bacterial model species, but a dedicated technique for anaerobic microbiota members of the Bacteroidota phylum has been lacking. Here, working with Bacteroides thetaiotaomicron, we describe experimental steps for transcriptome stabilization, fluorescence-activated cell sorting (FACS) collection, and optimized lysis of single cells from this group of organisms. We detail procedures for reverse transcription of RNAs via the multiple annealing and dC-tailing-based quantitative single-cell RNA sequencing (MATQ-seq) protocol, sensitive Cas9-based depletion of ribosomal sequences, and cDNA library generation.For complete details on the use and execution of this protocol, please refer to Bornet et al.1

Abstract Microbial interactions unfold within environments structured by physical transport and chemical gradients. Yet most mechanistic studies rely on well-mixed systems that mask the reciprocal influences of environmental heterogeneity on metabolism and ecology. Here, we investigate how the physical environment modulates the interaction between the gut commensal Bacteroides thetaiotaomicron and Escherichia coli . In anoxic liquid culture, cell-resolved isotope imaging and genetic perturbations reveal exploitative cross-feeding, where E. coli consumes diffusible sugars released by B . thetaiotaomicron during starch degradation. When exposed to intestinal-like oxygen gradients in microfluidics, the interaction is restructured by spatial organization. The species self-organize into complementary niches: E. coli locally depletes sugars and oxygen, thereby expanding the anoxic niche required by B. thetaiotaomicron . A reactive transport model confirms that this organization arises from coupled feedback between physical transport and metabolic reaction rates. Together, our results reveal how physical structure and chemical gradients convert an exploitative cross-feeding interaction into a dynamic niche-construction process that generates emergent spatial organization and stabilizes coexistence.

Paramount to human health, symbiotic bacteria in the gastrointestinal tract rely on the breakdown of complex polysaccharides to thrive in this sugar-deprived environment. Gut Bacteroides are metabolic generalists and deploy dozens of polysaccharide utilization loci (PULs) to forage diverse dietary and host-derived glycans. The expression of the multi-protein PUL complexes is tightly regulated at the transcriptional level. However, how PULs are orchestrated at translational level in response to the fluctuating levels of their cognate substrates is unknown. Here, we identify the RNA-binding protein RbpB and a family of noncoding RNAs as key players in post-transcriptional PUL regulation. We demonstrate that RbpB interacts with numerous cellular transcripts, including a paralogous noncoding RNA family comprised of 14 members, the FopS (family of paralogous sRNAs). Through a series of in-vitro and in-vivo assays, we reveal that FopS sRNAs repress the translation of SusC-like glycan transporters when substrates are limited-an effect antagonized by RbpB. Ablation of RbpB in Bacteroides thetaiotaomicron compromises colonization in the mouse gut in a diet-dependent manner. Together, this study adds to our understanding of RNA-coordinated metabolic control as an important factor contributing to the in-vivo fitness of predominant microbiota species in dynamic nutrient landscapes.

Bacterial small proteins impact diverse physiological processes, however, technical challenges posed by small size hampered their systematic identification and biochemical characterization. In our quest to uncover small proteins relevant for Salmonella pathogenicity, we previously identified YjiS, a 54 amino acid protein, which is strongly induced during this pathogen's intracellular infection stage. Here, we set out to further characterize the role of YjiS. Cell culture infection assays with Salmonella mutants lacking or overexpressing YjiS suggested this small protein to delay bacterial escape from macrophages. Mutant scanning of the protein's conserved, arginine-rich DUF1127 domain excluded a major effect of single amino acid substitutions on the infection phenotype. A comparative dual RNA-seq assay uncovered the molecular footprint of YjiS in the macrophage response to infection, with host effects related to oxidative stress and the cell cortex. Bacterial cell fractionation experiments demonstrated YjiS to associate with the inner membrane and proteins interacting with YjiS in pull-down experiments were enriched for inner membrane processes. Among the YjiS interactors was the two-component system SsrA/B, the master transcriptional activator of intracellular virulence genes and a suppressor of flagellar genes. Indeed, in the absence of YjiS, we observed elevated expression of motility genes and an increased number of flagella per bacterium. Together, our study points to a role for Salmonella YjiS as a membrane-associated timer of pathogen dissemination.

Microbiomes are complex communities of microorganisms that are essential for biochemical processes on Earth and for the health of humans, animals and plants. Many environmental and host-associated microbiomes are dominated by anaerobic microbes, some of which cannot tolerate oxygen. Anaerobic microbial communities have been extensively studied over the last 20 years using molecular techniques, especially next-generation sequencing. However, there is a renewed interest in microbial cultivation because isolates provide the basis for understanding the taxonomic and functional units of biodiversity, elucidating novel biochemical pathways and the mechanisms underlying microbe-microbe and microbe-host interactions and opening new avenues for biotechnological and clinical applications. In this Perspective, we present areas of research and applications that will benefit from advancement in anaerobic microbial cultivation. We highlight key technical and infrastructural hurdles associated with the development and deployment of sophisticated cultivation workflows. Improving the performance of cultivation techniques will set new trends in functional microbiome research in the coming years.

Salmonella virulence chiefly relies upon two major pathogenicity islands, SPI-1 and SPI-2, which enable host cell invasion and intracellular survival, respectively. Growing evidence suggests post-transcriptional control of SPI gene expression by Hfq-dependent small regulatory RNAs (sRNAs) such as PinT. This 80-nucleotide sRNA is highly expressed after Salmonella enters host cells and modulates the transition from the SPI-1 to SPI-2 program by targeting the mRNAs of different virulence factors. However, it remains unclear how PinT activity can be counteracted when the suppression of virulence genes needs to be relieved. Here, we mapped the RNA interactome of Salmonella recovered from infected macrophages, using an optimized version of RIL-seq. In addition to offering an unprecedented view of Hfq-mediated RNA interactions during Salmonella's intracellular infection stage, RIL-seq uncovered the previously described 3' end-derived sRNA InvS as a direct negative regulator of PinT. Biochemical and genetic experiments suggest a decoy mechanism by which InvS lifts PinT-mediated target repression. Moreover, InvS acts as an mRNA repressor of the adhesion protein MipA and PinT interaction with InvS relieves mipA repression. Together, our work identifies a pair of antagonistic sRNAs in a growing post-transcriptional network of virulence gene regulation.

Pathogens invading the intestine compete for nutrients with the resident microbiota. However, there is evidence that commensal members of the gut also provide nutritional resources to enteropathogens and thus promote their outgrowth. In this study, we investigated metabolic cross-feeding mechanisms between the abundant gut commensal Bacteroides thetaiotaomicron and the model enteropathogen Salmonella enterica serovar Typhimurium. We discovered that the processing of various dietary and host-derived glycans by B. thetaiotaomicron liberated building blocks available to Salmonella and identified a range of cross-fed metabolites. Interfering with polysaccharide degradation in B. thetaiotaomicron by genetic manipulation of specific polysaccharide utilization loci (PUL) inhibited pathogenic cross-feeding, both in vitro and in a gnotobiotic mouse model. Our findings highlight the complex metabolic commensal-pathogen interaction in the intestine and propose the disruption of polysaccharide breakdown as a potential microbiota-centric strategy to intervene in intestinal infections. ### Competing Interest Statement The authors have declared no competing interest.

The insertion sequence IS200 is widely distributed in Eubacteria. Despite its prevalence, IS200 does not appear to be mobile and as such is considered an ancestral component of bacterial genomes. Previous work in Salmonella enterica revealed that the IS200 tnpA transcript is processed to form a small, highly structured RNA (5'tnpA) that participates in the posttranscriptional control of invF expression, encoding a key transcription factor in this enteropathogen's invasion regulon. To further examine the scope of 5'tnpA transcript integration into Salmonella gene expression networks, we performed comparative RNA-seq, revealing the differential expression of over 200 genes in a Salmonella SL1344 5'tnpA disruption strain. This includes the genes for the master regulators of both invasion and flagellar regulons (HilD and FlhDC, respectively), plus genes involved in cysteine biosynthesis and an operon (phsABC) encoding a thiosulfate reductase complex. These expression changes were accompanied by an 80-fold increase in Salmonella invasion of HeLa cells. Follow-up experimentation suggested an additional direct target of 5'tnpA to be the small RNA PinT, which has previously been shown to be a negative regulator of invasion genes through its inhibitory action on key transcription factors governing the Salmonella pathogenicity island 1 regulon. This study provides a powerful new example of bacterial transposon domestication that is based not on the production/use of a regulatory protein or regulatory DNA sequences, but on the function of a transposon-derived small RNA.

Acinetobacter baumannii is a major opportunistic pathogen capable of natural transformation, a process driven by type IV pili (T4P) that facilitates horizontal gene transfer and accelerates the spread of antimicrobial resistance. While the transcriptional regulation of T4P is increasingly understood, post-transcriptional mechanisms controlling pilus assembly remain unexplored. Here, we identify and characterise a small RNA, Arp (Acinetobacter repressor of pilin), as a post-transcriptional repressor of T4P-mediated functions in A. baumannii. In a previous Hi-GRIL-seq experiment, we detected specific ligation events between Arp and the ribosome binding site of the pilA mRNA, encoding the major pilin subunit PilA. In-line probing and translational reporter assays revealed that Arp represses pilA translation by sequestering the Shine-Dalgarno sequence and the first 17 codons of the mRNA. Overexpression of Arp significantly impairs DNA uptake and twitching motility, two hallmark T4P-dependent phenotypes. Together, our findings identify a native A. baumannii sRNA that modulates natural competence by targeting pilin synthesis, revealing a new regulatory layer that could be exploited to disrupt horizontal gene transfer in multidrug-resistant strains.

Human cytomegalovirus (HCMV) is a master of immune evasion and a potent modulator of the human immune system. The best-characterized mechanism employed by HCMV to suppress host immunity is the production of a viral interleukin-10 homolog (CMVIL-10). While CMVIL-10 is known to suppress immune responses and promote viral persistence, its capacity to promote increased susceptibility to co-infecting pathogens like Aspergillus fumigatus remains unknown. Therefore, we studied the impact of wild-type (WT) HCMV (strain TB40), a CMVIL-10-deficient HCMV mutant (ΔUL111A), and recombinant CMVIL-10 on the immune activity of monocyte-derived dendritic cells (moDCs) during co-infection with A. fumigatus. Using a combination of transcriptomic and phenotypic readouts, our data revealed a strong and time-dependent immuno-paralytic effect of HCMV by suppressing pathogen recognition pathways, cytokine production, DC maturation, and expression of genes that are essential for host defense and tissue repair. Although infection with ΔUL111A lacking CMVIL-10 led to stronger expression of type I interferons, IFN-γ-inducible chemokines, and proinflammatory cytokines than WT infection, interference with antifungal immune defense and fungal clearance during co-infection was largely similar between both strains. The limited effect of CMVIL-10 on antifungal immune defense persisted even after prolonged pre-exposure of DCs to the recombinant virokine. In summary, although CMVIL-10 contributes to shaping an anti-inflammatory environment, HCMV's suppression of antifungal immunity appears to be multifactorial, with CMVIL-10 alone playing a rather subtle role in altering DC responses to A. fumigatus during viral-fungal co-infection.IMPORTANCEHuman cytomegalovirus (HCMV) is a highly prevalent herpesvirus that establishes lifelong latency and frequently reactivates in immunocompromised individuals, including hematopoietic stem cell transplant recipients. Reactivation not only causes direct disease but also increases the risk of secondary infections, such as invasive pulmonary aspergillosis caused by Aspergillus fumigatus. Specifically, studies estimated that about 6%-25% of critically ill HCMV-positive patients develop HCMV-associated pulmonary aspergillosis. However, the mechanisms by which HCMV creates a permissive environment for fungal superinfection remain poorly understood. HCMV encodes a viral homolog of interleukin-10 (CMVIL-10), which mimics host IL-10 and elicits potent immunomodulatory activity. Here, we show that CMVIL-10 dampens specific anti-viral responses, DC activation, and cytokine signaling. However, HCMV-mediated impairment of fungal control in co-infection settings occurred largely independent of CMVIL-10 expression. These findings suggest that HCMV undermines antifungal defenses through multifactorial mechanisms beyond CMVIL-10, highlighting the need for targeted strategies to restore immune function in high-risk patients.

Isogenic bacteria can be phenotypically diverse. This heterogeneity is evident in the Bacteroidota, a predominant phylum of the human gut microbiota. These bacteria adopt diverse morphologies, yet the molecular basis of their morphological heterogeneity is poorly understood. Here, we systematically characterize the variation in cellular morphology of Bacteroides thetaiotaomicron cells during laboratory growth and after isolation from different host niches. We develop a sensitive transcriptomics approach and apply it to B. thetaiotaomicron sorted into sub-populations of varying cell sizes. Differential expression analysis indicates metabolic specialization associated with morphology. Transcriptomic data also reveal morphological marker genes, whose size-dependent expression is validated through fluorescence in situ hybridization. Morphological characterization of deletion and overexpression mutants reveals that specific marker genes causally contribute to B. thetaiotaomicron cell-size determination. Since phenotypic heterogeneity is a common feature of microbial consortia, this study serves as a blueprint for understanding the role of bacterial genes in morphological variation.

Acid resistance is crucial for enterobacteria to withstand host acidic environments during infection, including the gastrointestinal tract and macrophage phagosomes. A key acid resistance mechanism of the facultative intracellular pathogen Salmonella is the expression of the arginine decarboxylase AdiA. While AdiA confers acid resistance via an H+-consuming reaction, we discover that the 3'-untranslated region (UTR) of adiA mRNA is processed by RNase E into a regulatory small RNA, AdiZ. Through RNA-RNA interactome profiling and transcriptomic analysis, followed by in vitro structural probing and in vivo validations, we demonstrate that AdiZ directly base-pairs with and negatively regulates ptsG, pykF, and dmsA mRNAs involved in glucose uptake, glycolysis, and anaerobic respiration, respectively. Intriguingly, AdiZ is induced and facilitates Salmonella survival within macrophages, where acidic and hypoxic stresses prevail. Thus, simultaneous expression of AdiA and AdiZ from a single mRNA ties arginine-dependent acid resistance to metabolic reprogramming of Salmonella in the host intracellular niches.

Studying the molecular basis of intestinal infections caused by enteric pathogens at the tissue level is challenging, because most human intestinal infection models have limitations, and results obtained from animals may not reflect the human situation. Infections with Salmonella enterica serovar Typhimurium (STm) have different outcomes between organisms. 3D tissue modeling of primary human material provides alternatives to animal experimentation, but epithelial co-culture with immune cells remains difficult. Macrophages, for instance, contribute to the immunocompetence of native tissue, yet their incorporation into human epithelial tissue models is challenging. A 3D immunocompetent tissue model of the human small intestine based on decellularized submucosa enriched with monocyte-derived macrophages (MDM) is established. The multicellular model recapitulated in vivo-like cellular diversity, especially the induction of GP2 positive microfold (M) cells. Infection studies with STm reveal that the pathogen physically interacts with these M-like cells. MDMs show trans-epithelial migration and phagocytosed STm within the model and the levels of inflammatory cytokines are induced upon STm infection. Infected epithelial cells are shed into the supernatant, potentially reflecting an intracellular reservoir of invasion-primed STm. Together, the 3D model of the human intestinal epithelium bears potential as an alternative to animals to identify human-specific processes underlying enteric bacterial infections.

Symbiotic bacteria in the human intestinal microbiota provide many pivotal functions to human health and occupy distinct biogeographic niches within the gut. Yet the molecular basis underlying niche-specific colonization remains poorly defined. To address this, we conducted a time-resolved dual RNA-seq experiment to simultaneously monitor the transcriptional co-adaptations of human commensal Bacteroides thetaiotaomicron and human gut epithelial cells in an anaerobe-epithelium co-culture system. Comparative transcriptomic analysis of mucus-associated versus supernatant Bacteroides populations unveiled small RNAs (sRNAs) that are differentially regulated between spatially segregated subpopulations. Among these, we identified IroR as a key sRNA that facilitates B. thetaiotaomicron adaptation to the mucus-rich, iron-limiting niche, partly by modulating expression of bacterial capsule genes. This work provides new insights into the spatiotemporal dynamics of gut colonization and underscores a previously underappreciated role for bacterial sRNAs in shaping mutualistic interactions between the human microbiota and the gut epithelium.

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.

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.

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.

Streptococcus agalactiae (Group B Streptococcus) strain COH1 is a representative strain of serotype III, multi-locus sequence type 17, which is disproportionately associated with neonatal meningitis. Here we report the transcriptome of COH1 when interacting with human brain endothelial cells compared with COH1 alone.

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.