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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.
Background: The hypothalamus is an important brain region for the regulation of energy balance. Roux-en-Y gastric bypass (RYGB) surgery and gut hormone-based treatments are known to reduce body weight, but their effects on hypothalamic gene expression and signaling pathways are poorly studied. Methods: Diet-induced obese male Wistar rats were randomized into the following groups: RYGB, sham operation, sham + body weight-matched (BWM) to the RYGB group, osmotic minipump delivering PYY3-36 (0.1 mg/kg/day), liraglutide s.c. (0.4 mg/kg/day), PYY3-36 + liraglutide, and saline. All groups (except BWM) were kept on a free choice of high- and low-fat diets. Four weeks after interventions, hypothalami were collected for RNA sequencing. Results: While rats in the RYGB, BWM, and PYY3-36 + liraglutide groups had comparable reductions in body weight, only RYGB and BWM treatment had a major impact on hypothalamic gene expression. In these groups, hypothalamic leptin receptor expression as well as the JAK–STAT, PI3K-Akt, and AMPK signaling pathways were upregulated. No significant changes could be detected in PYY3-36 + liraglutide-, liraglutide-, and PYY-treated groups. Conclusions: Despite causing similar body weight changes compared to RYGB and BWM, PYY3-36 + liraglutide treatment does not impact hypothalamic gene expression. Whether this striking difference is favorable or unfavorable to metabolic health in the long term requires further investigation.
FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.
Ongoing resistance developments against antibiotics that also affect last-resort antibiotics require novel antibacterial compounds. Strategies to discover such novel structures have been dimerization or hybridization of known antibacterial agents. We found novel antibacterial agents by dimerization of indols and hybridization with carbazoles. They were obtained in a simple one-pot reaction as bisindole tetrahydrocarbazoles. Further oxidation led to bisindole carbazoles with varied substitutions of both the indole and the carbazole scaffold. Both the tetrahydrocarbazoles and the carbazoles have been evaluated in various S. aureus strains, including MRSA strains. Those 5-cyano substituted derivatives showed best activities as determined by MIC values. The tetrahydrocarbazoles partly exceed the activity of the carbazole compounds and thus the activity of the used standard antibiotics. Thus, promising lead compounds could be identified for further studies.
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.
A viral infection involves entry and replication of viral nucleic acid in a host organism, subsequently leading to biochemical and structural alterations in the host cell. In the case of SARS-CoV-2 viral infection, over-activation of the host immune system may lead to lung damage. Albeit the regeneration and fibrotic repair processes being the two protective host responses, prolonged injury may lead to excessive fibrosis, a pathological state that can result in lung collapse. In this review, we discuss regeneration and fibrosis processes in response to SARS-CoV-2 and provide our viewpoint on the triggering of alveolar regeneration in coronavirus disease 2019 (COVID-19) patients.
Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes.
The human gastrointestinal tract is in constant contact with microbial stimuli. Its barriers have to ensure co-existence with the commensal bacteria, while enabling surveillance of intruding pathogens. At the centre of the interaction lies the epithelial layer, which marks the boundaries of the body. It is equipped with a multitude of different innate immune sensors, such as Toll-like receptors, to mount inflammatory responses to microbes. Dysfunction of this intricate system results in inflammation-associated pathologies, such as inflammatory bowel disease. However, the complexity of the cellular interactions, their molecular basis and their development remains poorly understood. In recent years, stem cell-derived organoids have gained increasing attention as promising models for both development and a broad range of pathologies, including infectious diseases. In addition, organoids enable the study of epithelial innate immunity in vitro. In this review, we focus on the gastrointestinal epithelial barrier and its regional organization to discuss innate immune sensing and development.
Serine/threonine kinase PknB and its corresponding phosphatase Stp are important regulators of many cell functions in the pathogen S. aureus. Genome-scale gene expression data of S. aureus strain NewHG (sigB\(^+\)) elucidated their effect on physiological functions. Moreover, metabolic modelling from these data inferred metabolic adaptations. We compared wild-type to deletion strains lacking pknB, stp or both. Ser/Thr phosphorylation of target proteins by PknB switched amino acid catabolism off and gluconeogenesis on to provide the cell with sufficient components. We revealed a significant impact of PknB and Stp on peptidoglycan, nucleotide and aromatic amino acid synthesis, as well as catabolism involving aspartate transaminase. Moreover, pyrimidine synthesis was dramatically impaired by stp deletion but only slightly by functional loss of PknB. In double knockouts, higher activity concerned genes involved in peptidoglycan, purine and aromatic amino acid synthesis from glucose but lower activity of pyrimidine synthesis from glucose compared to the wild type. A second transcriptome dataset from S. aureus NCTC 8325 (sigB\(^−\)) validated the predictions. For this metabolic adaptation, PknB was found to interact with CdaA and the yvcK/glmR regulon. The involved GlmR structure and the GlmS riboswitch were modelled. Furthermore, PknB phosphorylation lowered the expression of many virulence factors, and the study shed light on S. aureus infection processes.