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Sonstige beteiligte Institutionen
A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly
(2011)
P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. Pbodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.
A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria.
The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.
IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.
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.
Post-transcriptional RNA modification methods are in high demand for site-specific RNA labelling and analysis of RNA functions. In vitro-selected ribozymes are attractive tools for RNA research and have the potential to overcome some of the limitations of chemoenzymatic approaches with repurposed methyltransferases. Here we report an alkyltransferase ribozyme that uses a synthetic, stabilized S-adenosylmethionine (SAM) analogue and catalyses the transfer of a propargyl group to a specific adenosine in the target RNA. Almost quantitative conversion was achieved within 1 h under a wide range of reaction conditions in vitro, including physiological magnesium ion concentrations. A genetically encoded version of the SAM analogue-utilizing ribozyme (SAMURI) was expressed in HEK293T cells, and intracellular propargylation of the target adenosine was confirmed by specific fluorescent labelling. SAMURI is a general tool for the site-specific installation of the smallest tag for azide-alkyne click chemistry, which can be further functionalized with fluorophores, affinity tags or other functional probes.
Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown. Here, we take a two-pronged approach to search for RNA-binding effectors, combining biocomputational prediction of RNA-binding domains (RBDs) in a newly assembled comprehensive dataset of bacterial secreted proteins, and experimental screening for RNA binding in mammalian cells. Only a small subset of effectors were predicted to carry an RBD, indicating that if RNA targeting was common, it would likely involve new types of RBDs. Our experimental evaluation of effectors with predicted RBDs further argues for a general paucity of RNA binding activities amongst bacterial effectors. We obtained evidence that PipB2 and Lpg2844, effector proteins of Salmonella and Legionella species, respectively, may harbor novel biochemical activities. Our study presenting the first systematic evaluation of the RNA-targeting potential of bacterial effectors offers a basis for discussion of whether or not host RNA is a prominent target of secreted bacterial proteins.
Amidochelocardin overcomes resistance mechanisms exerted on tetracyclines and natural chelocardin
(2020)
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.
A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.
IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.
An expanded evaluation of protein function prediction methods shows an improvement in accuracy
(2016)
Background
A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging.
Results
We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2.
Conclusions
The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent.
Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and –injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf\(^{-/-}\) ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.
Our body is colonized by a vast array of bacteria the sum of which forms our microbiota. The gut alone harbors >1,000 bacterial species. An understanding of their individual or synergistic contributions to human health and disease demands means to interfere with their functions on the species level. Most of the currently available antibiotics are broad‐spectrum, thus too unspecific for a selective depletion of a single species of interest from the microbiota. Programmable RNA antibiotics in the form of short antisense oligonucleotides (ASOs) promise to achieve precision manipulation of bacterial communities. These ASOs are coupled to small peptides that carry them inside the bacteria to silence mRNAs of essential genes, for example, to target antibiotic‐resistant pathogens as an alternative to standard antibiotics. There is already proof‐of‐principle with diverse bacteria, but many open questions remain with respect to true species specificity, potential off‐targeting, choice of peptides for delivery, bacterial resistance mechanisms and the host response. While there is unlikely a one‐fits‐all solution for all microbiome species, I will discuss how recent progress in bacterial RNA biology may help to accelerate the development of programmable RNA antibiotics for microbiome editing and other applications.
To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/.
RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs) and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches. We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using position-specific scoring matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot.
Inflammation in the brain and gut is a critical component of several neurological diseases, such as Parkinson’s disease (PD). One trigger of the immune system in PD is aggregation of the pre-synaptic protein, α-synuclein (αSyn). Understanding the mechanism of propagation of αSyn aggregates is essential to developing disease-modifying therapeutics. Using a brain-first mouse model of PD, we demonstrate αSyn trafficking from the brain to the ileum of male mice. Immunohistochemistry revealed that the ileal αSyn aggregations are contained within CD11c+ cells. Using single-cell RNA sequencing, we demonstrate that ileal CD11c\(^+\) cells are microglia-like and the same subtype of cells is activated in the brain and ileum of PD mice. Moreover, by utilizing mice expressing the photo-convertible protein, Dendra2, we show that CD11c\(^+\) cells traffic from the brain to the ileum. Together these data provide a mechanism of αSyn trafficking between the brain and gut.
Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dosedependent responses to environmental stimuli may involve functional specialization of seemingly coinduced miRNAs in other cellular circuitries as well.
Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dosedependent responses to environmental stimuli may involve functional specialization of seemingly coinduced miRNAs in other cellular circuitries as well.
Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizospheremimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions.
Background:
The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro-and eukaryotic cells without prior fixation or physical disruption of the interaction.
Results:
Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells.
Conclusions:
Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium.
Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315
(2015)
Background: Burkholderia cenocepacia is a soil-dwelling Gram-negative Betaproteobacterium with an important role as opportunistic pathogen in humans. Infections with B. cenocepacia are very difficult to treat due to their high intrinsic resistance to most antibiotics. Biofilm formation further adds to their antibiotic resistance. B. cenocepacia harbours a large, multi-replicon genome with a high GC-content, the reference genome of strain J2315 includes 7374 annotated genes. This study aims to annotate transcription start sites and identify novel transcripts on a whole genome scale. Methods: RNA extracted from B. cenocepacia J2315 biofilms was analysed by differential RNA-sequencing and the resulting dataset compared to data derived from conventional, global RNA-sequencing. Transcription start sites were annotated and further analysed according to their position relative to annotated genes. Results: Four thousand ten transcription start sites were mapped over the whole B. cenocepacia genome and the primary transcription start site of 2089 genes expressed in B. cenocepacia biofilms were defined. For 64 genes a start codon alternative to the annotated one was proposed. Substantial antisense transcription for 105 genes and two novel protein coding sequences were identified. The distribution of internal transcription start sites can be used to identify genomic islands in B. cenocepacia. A potassium pump strongly induced only under biofilm conditions was found and 15 non-coding small RNAs highly expressed in biofilms were discovered. Conclusions: Mapping transcription start sites across the B. cenocepacia genome added relevant information to the J2315 annotation. Genes and novel regulatory RNAs putatively involved in B. cenocepacia biofilm formation were identified. These findings will help in understanding regulation of B. cenocepacia biofilm formation.
The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.