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Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis. Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5′ ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases.
ABSTRACT
The highly conserved heterotrimeric protein kinase SNF1 is important for metabolic adaptations in the pathogenic yeast Candida albicans. A key function of SNF1 is to inactivate the repressor protein Mig1 and thereby allow the expression of genes that are required for the utilization of alternative carbon sources when the preferred carbon source, glucose, is absent or becomes limiting. However, how SNF1 controls Mig1 activity in C. albicans has remained elusive. Using a phosphoproteomics approach, we found that Mig1 is phosphorylated at multiple serine residues. Replacement of these serine residues by nonphosphorylatable alanine residues strongly increased the repressor activity of Mig1 in cells lacking a functional SNF1 complex, indicating that additional protein kinases are involved in the regulation of Mig1. Unlike wild-type Mig1, whose levels strongly decreased when the cells were grown on sucrose or glycerol instead of glucose, the levels of a mutant Mig1 protein lacking nine phosphorylation sites remained high under these conditions. Despite the increased protein levels and the absence of multiple phosphorylation sites, cells with a functional SNF1 complex could still sufficiently inhibit the hyperactive Mig1 to enable wild-type growth on alternative carbon sources. In line with this, phosphorylated forms of the mutant Mig1 were still detected in the presence and absence of a functional SNF1, demonstrating that Mig1 contains additional, unidentified phosphorylation sites and that downstream protein kinases are involved in the control of Mig1 activity by SNF1.
IMPORTANCE
The SNF1 protein kinase signaling pathway, which is highly conserved in eukaryotic cells, is important for metabolic adaptations in the pathogenic yeast Candida albicans. However, so far, it has remained elusive how SNF1 controls the activity of one of its main effectors, the repressor protein Mig1 that inhibits the expression of genes required for the utilization of alternative carbon sources when glucose is available. In this study, we have identified multiple phosphorylation sites in Mig1 that contribute to its inactivation. Mutation of these sites strongly increased Mig1 repressor activity in the absence of SNF1, but SNF1 could still sufficiently inhibit the hyperactive Mig1 to enable growth on alternative carbon sources. These findings reveal features of Mig1 that are important for controlling its repressor activity. Furthermore, they demonstrate that both SNF1 and additional protein kinases regulate Mig1 in this pathogenic yeast.
Abstract
Protein kinases are central components of almost all signaling pathways that control cellular activities. In the model organism Saccharomyces cerevisiae, the paralogous protein kinases Ypk1 and Ypk2, which control membrane lipid homeostasis, are essential for viability, and previous studies strongly indicated that this is also the case for their single ortholog Ypk1 in the pathogenic yeast Candida albicans. Here, using FLP-mediated inducible gene deletion, we reveal that C. albicans ypk1Δ mutants are viable but slow-growing, explaining prior failures to obtain null mutants. Phenotypic analyses of the mutants showed that the functions of Ypk1 in regulating sphingolipid biosynthesis and cell membrane lipid asymmetry are conserved, but the consequences of YPK1 deletion are milder than in S. cerevisiae. Mutational studies demonstrated that the highly conserved PDK1 phosphorylation site T548 in its activation loop is essential for Ypk1 function, whereas the TORC2 phosphorylation sites S687 and T705 at the C-terminus are important for Ypk1-dependent resistance to membrane stress. Unexpectedly, Pkh1, the single C. albicans orthologue of Pkh1/Pkh2, which mediate Ypk1 phosphorylation at the PDK1 site in S. cerevisiae, was not required for normal growth of C. albicans under nonstressed conditions, and Ypk1 phosphorylation at T548 was only slightly reduced in pkh1Δ mutants. We found that another protein kinase, Pkh3, whose ortholog in S. cerevisiae cannot substitute Pkh1/2, acts redundantly with Pkh1 to activate Ypk1 in C. albicans. No phenotypic effects were observed in cells lacking Pkh3 alone, but pkh1Δ pkh3Δ double mutants had a severe growth defect and Ypk1 phosphorylation at T548 was completely abolished. These results establish that Ypk1 is not essential for viability in C. albicans and that, despite its generally conserved function, the Ypk1 signaling pathway is rewired in this pathogenic yeast and includes a novel upstream kinase to activate Ypk1 by phosphorylation at the PDK1 site.
Author summary
Protein kinases are key components of cellular signaling pathways, and elucidating the specific roles of individual kinases is important to understand how organisms adapt to changes in their environment. The protein kinase Ypk1 is highly conserved in eukaryotic organisms and crucial for the maintenance of cell membrane homeostasis. It was previously thought that Ypk1 is essential for viability in the pathogenic yeast Candida albicans, as in the model organism Saccharomyces cerevisiae. Here, by using forced, inducible gene deletion, we reveal that C. albicans mutants lacking Ypk1 are viable but have a strong growth defect. The phenotypes of the mutants indicate that the known functions of Ypk1 are conserved in C. albicans, but loss of this kinase has less severe consequences than in S. cerevisiae. We also unravel the puzzling previous observation that C. albicans mutants lacking the Ypk1-activating kinase Pkh1, which is essential in S. cerevisiae, have no obvious growth defects. We show that the protein kinase Pkh3, which has not previously been implicated in the Ypk1 signaling pathway, can substitute Pkh1 and activate Ypk1 in C. albicans. These findings provide novel insights into this conserved signaling pathway and how it is rewired in a human-pathogenic fungus.
Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria.
IMPORTANCE: Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.
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.
Increasing antibacterial drug resistance threatens global health, unfortunately, however, efforts to find novel antibacterial agents have been scaled back by the pharmaceutical industry due to concerns about a poor return on investment. Nevertheless, there is an urgent need to find novel antibacterial compounds to combat antibacterial drug resistance. The synthesis of novel drugs from natural sources is mostly cost-intensive due to those drugs’ complicated structures. Therefore, it is necessary to find novel antibacterials by simple synthesis to become more attractive for industrial production. We succeeded in the discovery of four antibacterial compound (sub)classes accessible in a simple one-pot reaction based on fluorinated benzothiophene-indole hybrids. They have been evaluated against various S. aureus and MRSA strains. Structure- and substituent-dependent activities have been found within the (sub)classes and promising lead compounds have been identified. In addition, bacterial pyruvate kinase was found to be the molecular target of the active compounds. In conclusion, simple one-pot synthesis of benzothiophene-indoles represents a promising strategy for the search of novel antimicrobial compounds.
The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid–liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-β-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-β-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-β-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-β-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2–256 µg/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria.
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.
Azobenzene derivatives with activity against drug‐resistant Candida albicans and Candida auris
(2023)
Increasing resistance against antimycotic drugs challenges anti‐infective therapies today and contributes to the mortality of infections by drug‐resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'‐dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole‐susceptible and fluconazole‐resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.