Biology subjects

Westermann, A. J.

Publications and source records attributed to Westermann, A. J..

16 recordsLinked to original sources

bioRxiv2026

M-CSF stimulated alveolar macrophages safeguard from invasive aspergillosis

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication in immunocompromised individuals, including recipients of allogeneic hematopoietic cell transplantation (allo-HCT). While systemic neutropenia is traditionally considered the primary risk factor for IPA, we demonstrate that tissue-resident alveolar macrophages (AMs), rather than recruited neutrophils, dictate survival during the critical early window after transplantation. Utilizing an ultra-low dose Aspergillus fumigatus infection model that mimics physiological exposure, we identify alveolar macrophages (AMs) as key players in pulmonary antifungal defense. In immunocompromised mice, AMs conferred protection against lethal invasive aspergillosis by day 6, but not day 4 post-allo-HCT. To enhance AM function at the earlier time point, we tested cytokine-based interventions and show that M-CSF, but not IL-34, which both bind to the CSF-1 receptor, promotes migratory activity, phagolysosomal function and fungal killing in both mouse and human primary tissue-resident AMs. In allo-HCT recipient mice, M-CSF treatment preserved lung tissue integrity, suppressed pro-inflammatory cytokines, and protected mice from lethal invasive aspergillosis. The M-CSF-driven protective effect was abrogated upon AM depletion. Our findings demonstrate a critical role of tissue-resident AMs in pulmonary antifungal immunity and suggest that therapeutic modulation of AM activity via M-CSF may offer a promising strategy to combat severe fungal infections in immunocompromised patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/736478v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@49ba37org.highwire.dtl.DTLVardef@607c4forg.highwire.dtl.DTLVardef@814894org.highwire.dtl.DTLVardef@1c413c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Oxygen gradients reshape cross-feeding through emergent spatial organization of gut commensal bacteria

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.

microbiology↗

bioRxiv2025

An RNA regulates iron homeostasis and host mucus colonization in Bacteroides thetaiotaomicron

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.

microbiology↗

bioRxiv2025

Ablation of polysaccharide breakdown in Bacteroides thetaiotaomicron prevents cross-feeding and growth of Salmonella Typhimurium in the mouse gut

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.

microbiology↗

bioRxiv2025

DNA uptake and twitching motility are controlled by the small RNA Arp through repression of pilin translation in Acinetobacter baumannii

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. Significance StatementAcinetobacter baumannii is a multidrug-resistant WHO #1 priority pathogen that acquires antibiotic resistance genes through natural transformation, a process dependent on type IV pili (T4P). This work reveals Arp, the first native post-transcriptional repressor of natural competence in A. baumannii, uncovering a novel regulatory layer that modulates horizontal gene transfer. The widespread presence of arp in pathogenic Acinetobacter strains suggests that sRNA is an important regulator in those organisms. Furthermore, these findings broaden our understanding of RNA-based regulation in this priority pathogen and open potential avenues for interfering with antibiotic resistance dissemination.

microbiology↗

bioRxiv2025

3'UTR-derived small RNA couples acid resistance to metabolic reprogramming of Salmonella within macrophages

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 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. HIGHLIGHTSO_LISalmonella adiA transcript expresses both acid resistance enzyme and sRNA AdiZ C_LIO_LIAdiZ derived from the adiA 3'UTR is induced under an acidic and anaerobic condition C_LIO_LIAdiZ base-pairs with mRNAs to modulate glucose metabolism and anaerobic respiration C_LIO_LIAdiZ promotes Salmonella survival in acidic and hypoxic macrophage vacuoles C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/658255v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@10aec22org.highwire.dtl.DTLVardef@14b5f2org.highwire.dtl.DTLVardef@1b53c8dorg.highwire.dtl.DTLVardef@19a8540_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Intramacrophage RIL-seq uncovers an RNA antagonist of the Salmonella virulence-associated small RNA PinT

Salmonella virulence chiefly relies upon two major pathogenicity islands, SPI-1 and SPI-2, which enable host cell invasion and intracellular survival, respectively. There has been increasing evidence for post-transcriptional control of SPI gene expression by Hfq-dependent small 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 different virulence factor mRNAs. It has been elusive, however, how PinT activity could be counteracted when virulence gene suppression were to be relieved. To identify putative inhibitors of PinT, we have mapped the RNA interactome of Salmonella recovered from infected macrophages, using an optimized version of the RIL-seq method. Next to offering an unprecedented view of Hfq-mediated RNA interactions during Salmonellas intracellular infection stage, RIL-seq uncovered the 3 end-derived sRNA InvS as a direct negative regulator of PinT. Biochemical and genetic experiments suggest a decoy mechanism whereby InvS lifts the PinT-mediated repression of virulence factors. Additionally, InvS acts as an mRNA repressor of the host cell adhesion protein, MipA, and PinT interaction with InvS relieves mipA repression. Together, our work identifies a unique pair of antagonistic sRNAs in a growing post-transcriptional network of virulence gene regulation.

microbiology↗

bioRxiv2024

A small RNA derived from the 5' end of the IS200 tnpA transcript regulates multiple virulence pathways in Salmonella Typhimurium

The insertion sequence IS200 is widely distributed in Eubacteria. Despite its wide-ranging occurrence, 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 (5tnpA) that participates in the post-transcriptional control of invF expression, encoding a key transcription factor in this enteropathogens invasion regulon. To further examine the scope of 5tnpA transcript integration into Salmonella gene expression networks, we performed comparative RNA-seq revealing the differential expression of over 200 genes in a Salmonella SL1344 5tnpA 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 (cysteine regulon) 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. Likewise, a phsABC disruption strain was associated with an increased invasion specifically under anaerobic growth conditions. Based on these findings, we propose that early induction of invasion and motility regulons in the absence of 5tnpA causes a metabolic stress resulting in cysteine limitation and activation of CysB, which turns down expression of the phsABC operon to increase the flux of thiosulfate in the media towards cysteine production. Taken together, this study provides a powerful new example of bacterial transposon domestication that is based not on the production of a regulatory protein, but of a transposon-derived small RNA.

molecular biology↗

bioRxiv2023

The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gut Bacteroides

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. Ablation of RbpB in Bacteroides thetaiotaomicron displays compromised colonization in the mouse gut in a host diet-dependent manner. Current dogma holds that individual PULs are regulated by dedicated transcriptional regulators. We demonstrate that RbpB acts as a global RNA binder that directly interacts with several hundred cellular transcripts. This includes a paralogous noncoding RNA family comprised of 14 members, the FopS (family of paralogous sRNAs) cluster. Through a series of in-vitro and in-vivo assays, we reveal that FopS sRNAs repress the translation of a SusC-like glycan transporter when substrates are limited--an effect antagonized by RbpB. Together, this study implicates RNA-coordinated metabolic control as an important, yet previously overlooked, factor contributing to the in-vivo fitness of predominant microbiota species in dynamic nutrient landscapes.

microbiology↗

bioRxiv2023

Global analysis of the RNA-RNA interactome in Acinetobacter baumannii AB5075 uncovers a small regulatory RNA repressing the virulence-related outer membrane protein CarO

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to functionally characterise environmental adaptation, antibiotic resistance and pathogenicity of this organism and their genetic regulators to inform intervention strategies. Critical to rapid adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners in A. baumannii, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. We found that 40 sRNA candidates were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii and that sRNAs act as direct regulators of mRNA molecules through antisense base-pairing. In-depth characterisation uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including that of the outer membrane protein CarO and the siderophore receptor BfnH. We show that Aar initiates base-pairing with these mRNA molecules using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in response to multiple stress stimuli suggesting a role in fine-tuning translation of the Aar-target molecules in A. baumannii under hostile conditions. Together, our study provides mechanistic insights into sRNA-mediated gene expression control in A. baumannii and represents a valuable resource for future RNA-centric research endeavours in this ESKAPE pathogen.

microbiology↗

bioRxiv2023

A CRISPR-based genetic screen in Bacteriodes thetaiotamicron reveals a small RNA modulator of bile susceptibility

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 previously uncharacterized sRNA BatR to increase susceptibility to bile salts, likely 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 discovering hidden functions of bacterial sRNAs.

microbiology↗

bioRxiv2023

Improved RNA stability estimation through Bayesian modeling reveals most bacterial transcripts have sub-minute 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 post-transcriptional 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 minute, 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 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 new insights into post-transcriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression. Significance StatementTogether with transcription and translation, RNA decay is one of the major processes governing protein production. Here, we have developed a new statistical approach that corrects for confounding effects when estimating RNA decay rates from RNA-seq in bacteria. Our more accurate decay rate estimates indicate that Salmonella transcripts have half-lives about three times shorter than previously thought. This approach allowed us to measure the effects of RNA-binding proteins (RBPs) on decay rates, identifying large cohorts of transcripts with changes in stability following RBP deletion and conditions where post-transcriptional regulation affects survival. Our method should lead to a reevaluation of RNA stability estimates across diverse bacteria and new insights into the role of RBPs in shaping the transcriptome.

microbiology↗

bioRxiv2023

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.

molecular biology↗

bioRxiv2020

MAPS integrates overlooked 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 preventing 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.

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↗

bioRxiv2018

MetaMap: An atlas of metatranscriptomic reads in human disease-related RNA-seq data

BackgroundWith the advent of the age of big data in bioinformatics, large volumes of data and high performance computing power enable researchers to perform re-analyses of publicly available datasets at an unprecedented scale. Ever more studies imply the microbiome in both normal human physiology and a wide range of diseases. RNA sequencing technology (RNA-seq) is commonly used to infer global eukaryotic gene expression patterns under defined conditions, including human disease-related contexts, but its generic nature also enables the detection of microbial and viral transcripts.\n\nFindingsWe developed a bioinformatic pipeline to screen existing human RNA-seq datasets for the presence of microbial and viral reads by re-inspecting the non-human-mapping read fraction. We validated this approach by recapitulating outcomes from 6 independent controlled infection experiments of cell line models and comparison with an alternative metatranscriptomic mapping strategy. We then applied the pipeline to close to 150 terabytes of publicly available raw RNA-seq data from >17,000 samples from >400 studies relevant to human disease using state-of-the-art high performance computing systems. The resulting data of this large-scale re-analysis are made available in the presented MetaMap resource.\n\nConclusionsOur results demonstrate that common human RNA-seq data, including those archived in public repositories, might contain valuable information to correlate microbial and viral detection patterns with diverse diseases. The presented MetaMap database thus provides a rich resource for hypothesis generation towards the role of the microbiome in human disease.

bioinformatics↗