@article{SeethalerHertleinHopkeetal.2022, author = {Seethaler, Marius and Hertlein, Tobias and Hopke, Elisa and K{\"o}hling, Paul and Ohlsen, Knut and Lalk, Michael and Hilgeroth, Andreas}, title = {Novel effective fluorinated benzothiophene-indole hybrid antibacterials against S. aureus and MRSA strains}, series = {Pharmaceuticals}, volume = {15}, journal = {Pharmaceuticals}, number = {9}, issn = {1424-8247}, doi = {10.3390/ph15091138}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288253}, year = {2022}, abstract = {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.}, language = {en} } @article{MasotaOhlsenSchollmayeretal.2022, author = {Masota, Nelson E. and Ohlsen, Knut and Schollmayer, Curd and Meinel, Lorenz and Holzgrabe, Ulrike}, title = {Isolation and characterization of galloylglucoses effective against multidrug-resistant strains of Escherichia coli and Klebsiella pneumoniae}, series = {Molecules}, volume = {27}, journal = {Molecules}, number = {15}, issn = {1420-3049}, doi = {10.3390/molecules27155045}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286179}, year = {2022}, abstract = {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.}, language = {en} } @article{UmstaetterWernerZerlinetal.2022, author = {Umst{\"a}tter, Florian and Werner, Julia and Zerlin, Leah and M{\"u}hlberg, Eric and Kleist, Christian and Klika, Karel D. and Hertlein, Tobias and Beijer, Barbro and Domhan, Cornelius and Zimmermann, Stefan and Ohlsen, Knut and Haberkorn, Uwe and Mier, Walter and Uhl, Philipp}, title = {Impact of linker modification and PEGylation of vancomycin conjugates on structure-activity relationships and pharmacokinetics}, series = {Pharmaceuticals}, volume = {15}, journal = {Pharmaceuticals}, number = {2}, issn = {1424-8247}, doi = {10.3390/ph15020159}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-255197}, year = {2022}, abstract = {As multidrug-resistant bacteria represent a concerning burden, experts insist on the need for a dramatic rethinking on antibiotic use and development in order to avoid a post-antibiotic era. New and rapidly developable strategies for antimicrobial substances, in particular substances highly potent against multidrug-resistant bacteria, are urgently required. Some of the treatment options currently available for multidrug-resistant bacteria are considerably limited by side effects and unfavorable pharmacokinetics. The glycopeptide vancomycin is considered an antibiotic of last resort. Its use is challenged by bacterial strains exhibiting various types of resistance. Therefore, in this study, highly active polycationic peptide-vancomycin conjugates with varying linker characteristics or the addition of PEG moieties were synthesized to optimize pharmacokinetics while retaining or even increasing antimicrobial activity in comparison to vancomycin. The antimicrobial activity of the novel conjugates was determined by microdilution assays on susceptible and vancomycin-resistant bacterial strains. VAN1 and VAN2, the most promising linker-modified derivatives, were further characterized in vivo with molecular imaging and biodistribution studies in rodents, showing that the linker moiety influences both antimicrobial activity and pharmacokinetics. Encouragingly, VAN2 was able to undercut the resistance breakpoint in microdilution assays on vanB and vanC vancomycin-resistant enterococci. Out of all PEGylated derivatives, VAN:PEG1 and VAN:PEG3 were able to overcome vanC resistance. Biodistribution studies of the novel derivatives revealed significant changes in pharmacokinetics when compared with vancomycin. In conclusion, linker modification of vancomycin-polycationic peptide conjugates represents a promising strategy for the modulation of pharmacokinetic behavior while providing potent antimicrobial activity.}, language = {en} } @article{HombergerBarquistVogel2022, author = {Homberger, Christina and Barquist, Lars and Vogel, J{\"o}rg}, title = {Ushering in a new era of single-cell transcriptomics in bacteria}, series = {microLife}, volume = {3}, journal = {microLife}, doi = {10.1093/femsml/uqac020}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313292}, year = {2022}, abstract = {Transcriptome analysis of individual cells by single-cell RNA-seq (scRNA-seq) has become routine for eukaryotic tissues, even being applied to whole multicellular organisms. In contrast, developing methods to read the transcriptome of single bacterial cells has proven more challenging, despite a general perception of bacteria as much simpler than eukaryotes. Bacterial cells are harder to lyse, their RNA content is about two orders of magnitude lower than that of eukaryotic cells, and bacterial mRNAs are less stable than their eukaryotic counterparts. Most importantly, bacterial transcripts lack functional poly(A) tails, precluding simple adaptation of popular standard eukaryotic scRNA-seq protocols that come with the double advantage of specific mRNA amplification and concomitant depletion of rRNA. However, thanks to very recent breakthroughs in methodology, bacterial scRNA-seq is now feasible. This short review will discuss recently published bacterial scRNA-seq approaches (MATQ-seq, microSPLiT, and PETRI-seq) and a spatial transcriptomics approach based on multiplexed in situ hybridization (par-seqFISH). Together, these novel approaches will not only enable a new understanding of cell-to-cell variation in bacterial gene expression, they also promise a new microbiology by enabling high-resolution profiling of gene activity in complex microbial consortia such as the microbiome or pathogens as they invade, replicate, and persist in host tissue.}, language = {en} } @article{GuptaMinochaThapaetal.2022, author = {Gupta, Shishir K. and Minocha, Rashmi and Thapa, Prithivi Jung and Srivastava, Mugdha and Dandekar, Thomas}, title = {Role of the pangolin in origin of SARS-CoV-2: an evolutionary perspective}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {16}, issn = {1422-0067}, doi = {10.3390/ijms23169115}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285995}, year = {2022}, abstract = {After the recent emergence of SARS-CoV-2 infection, unanswered questions remain related to its evolutionary history, path of transmission or divergence and role of recombination. There is emerging evidence on amino acid substitutions occurring in key residues of the receptor-binding domain of the spike glycoprotein in coronavirus isolates from bat and pangolins. In this article, we summarize our current knowledge on the origin of SARS-CoV-2. We also analyze the host ACE2-interacting residues of the receptor-binding domain of spike glycoprotein in SARS-CoV-2 isolates from bats, and compare it to pangolin SARS-CoV-2 isolates collected from Guangdong province (GD Pangolin-CoV) and Guangxi autonomous regions (GX Pangolin-CoV) of South China. Based on our comparative analysis, we support the view that the Guangdong Pangolins are the intermediate hosts that adapted the SARS-CoV-2 and represented a significant evolutionary link in the path of transmission of SARS-CoV-2 virus. We also discuss the role of intermediate hosts in the origin of Omicron.}, language = {en} } @article{HungDreherDiessneretal.2022, author = {Hung, Sophia and Dreher, Liane and Diessner, Joachim and Schwarz, Stefan and Ohlsen, Knut and Hertlein, Tobias}, title = {MRSA infection in the thigh muscle leads to systemic disease, strong inflammation, and loss of human monocytes in humanized mice}, series = {Frontiers in Immunology}, volume = {13}, journal = {Frontiers in Immunology}, issn = {1664-3224}, doi = {10.3389/fimmu.2022.892053}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-278050}, year = {2022}, abstract = {MRSA (Methicillin-resistant Staphylococcus aureus) is the second-leading cause of deaths by antibiotic-resistant bacteria globally, with more than 100,000 attributable deaths annually. Despite the high urgency to develop a vaccine to control this pathogen, all clinical trials with pre-clinically effective candidates failed so far. The recent development of "humanized" mice might help to edge the pre-clinical evaluation closer to the clinical situation and thus close this gap. We infected humanized NSG mice (huNSG: (NOD)-scid IL2R\(_γ\)\(^{null}\) mice engrafted with human CD34+ hematopoietic stem cells) locally with S. aureus USA300 LAC* lux into the thigh muscle in order to investigate the human immune response to acute and chronic infection. These mice proved not only to be more susceptible to MRSA infection than wild-type or "murinized" mice, but displayed furthermore inferior survival and signs of systemic infection in an otherwise localized infection model. The rate of humanization correlated directly with the severity of disease and survival of the mice. Human and murine cytokine levels in blood and at the primary site of infection were strongly elevated in huNSG mice compared to all control groups. And importantly, differences in human and murine immune cell lineages surfaced during the infection, with human monocyte and B cell numbers in blood and bone marrow being significantly reduced at the later time point of infection. Murine monocytes in contrast behaved conversely by increasing cell numbers. This study demonstrates significant differences in the in vivo behavior of human and murine cells towards S. aureus infection, which might help to sharpen the translational potential of pre-clinical models for future therapeutic approaches.}, language = {en} } @article{IbrahimOhlsen2022, author = {Ibrahim, Eslam S. and Ohlsen, Knut}, title = {The old yellow enzyme OfrA fosters Staphylococcus aureus survival via affecting thiol-dependent redox homeostasis}, series = {Frontiers in Microbiology}, volume = {13}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2022.888140}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-274381}, year = {2022}, abstract = {Old yellow enzymes (OYEs) are widely found in the bacterial, fungal, and plant kingdoms but absent in humans and have been used as biocatalysts for decades. However, OYEs' physiological function in bacterial stress response and infection situations remained enigmatic. As a pathogen, the Gram-positive bacterium Staphylococcus aureus adapts to numerous stress conditions during pathogenesis. Here, we show that in S. aureus genome, two paralogous genes (ofrA and ofrB) encode for two OYEs. We conducted a bioinformatic analysis and found that ofrA is conserved among all publicly available representative staphylococcal genomes and some Firmicutes. Expression of ofrA is induced by electrophilic, oxidative, and hypochlorite stress in S. aureus. Furthermore, ofrA contributes to S. aureus survival against reactive electrophilic, oxygen, and chlorine species (RES, ROS, and RCS) via thiol-dependent redox homeostasis. At the host-pathogen interface, S. aureusΔofrA has defective survival in macrophages and whole human blood and decreased staphyloxanthin production. Overall, our results shed the light onto a novel stress response strategy in the important human pathogen S. aureus.}, language = {en} } @article{RamirezZavalaKruegerDunkeretal.2022, author = {Ram{\´i}rez-Zavala, Bernardo and Kr{\"u}ger, Ines and Dunker, Christine and Jacobsen, Ilse D. and Morschh{\"a}user, Joachim}, title = {The protein kinase Ire1 has a Hac1-independent essential role in iron uptake and virulence of Candida albicans}, series = {PLoS Pathogens}, volume = {18}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1010283}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300225}, year = {2022}, abstract = {Protein kinases play central roles in virtually all signaling pathways that enable organisms to adapt to their environment. Microbial pathogens must cope with severely restricted iron availability in mammalian hosts to invade and establish themselves within infected tissues. To uncover protein kinase signaling pathways that are involved in the adaptation of the pathogenic yeast Candida albicans to iron limitation, we generated a comprehensive protein kinase deletion mutant library of a wild-type strain. Screening of this library revealed that the protein kinase Ire1, which has a conserved role in the response of eukaryotic cells to endoplasmic reticulum stress, is essential for growth of C. albicans under iron-limiting conditions. Ire1 was not necessary for the activity of the transcription factor Sef1, which regulates the response of the fungus to iron limitation, and Sef1 target genes that are induced by iron depletion were normally upregulated in ire1Δ mutants. Instead, Ire1 was required for proper localization of the high-affinity iron permease Ftr1 to the cell membrane. Intriguingly, iron limitation did not cause increased endoplasmic reticulum stress, and the transcription factor Hac1, which is activated by Ire1-mediated removal of the non-canonical intron in the HAC1 mRNA, was dispensable for Ftr1 localization to the cell membrane and growth under iron-limiting conditions. Nevertheless, expression of a pre-spliced HAC1 copy in ire1Δ mutants restored Ftr1 localization and rescued the growth defects of the mutants. Both ire1Δ and hac1Δ mutants were avirulent in a mouse model of systemic candidiasis, indicating that an appropriate response to endoplasmic reticulum stress is important for the virulence of C. albicans. However, the specific requirement of Ire1 for the functionality of the high-affinity iron permease Ftr1, a well-established virulence factor, even in the absence of endoplasmic reticulum stress uncovers a novel Hac1-independent essential role of Ire1 in iron acquisition and virulence of C. albicans.}, language = {en} } @article{MetznerHerzogHeckeletal.2022, author = {Metzner, Valentin and Herzog, Gloria and Heckel, Tobias and Bischler, Thorsten and Hasinger, Julia and Otto, Christoph and Fassnacht, Martin and Geier, Andreas and Seyfried, Florian and Dischinger, Ulrich}, title = {Liraglutide + PYY\(_{3-36}\) combination therapy mimics effects of Roux-en-Y bypass on early NAFLD whilst lacking-behind in metabolic improvements}, series = {Journal of Clinical Medicine}, volume = {11}, journal = {Journal of Clinical Medicine}, number = {3}, issn = {2077-0383}, doi = {10.3390/jcm11030753}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-255244}, year = {2022}, abstract = {Background: Treatment options for NAFLD are still limited. Bariatric surgery, such as Roux-en-Y gastric bypass (RYGB), has been shown to improve metabolic and histologic markers of NAFLD. Glucagon-like-peptide-1 (GLP-1) analogues lead to improvements in phase 2 clinical trials. We directly compared the effects of RYGB with a treatment using liraglutide and/or peptide tyrosine tyrosine 3-36 (PYY\(_{3-36}\)) in a rat model for early NAFLD. Methods: Obese male Wistar rats (high-fat diet (HFD)-induced) were randomized into the following treatment groups: RYGB, sham-operation (sham), liraglutide (0.4 mg/kg/day), PYY\(_{3-36}\) (0.1 mg/kg/day), liraglutide+PYY\(_{3-36}\), and saline. After an observation period of 4 weeks, liver samples were histologically evaluated, ELISAs and RNA sequencing + RT-qPCRs were performed. Results: RYGB and liraglutide+PYY\(_{3-36}\) induced a similar body weight loss and, compared to sham/saline, marked histological improvements with significantly less steatosis. However, only RYGB induced significant metabolic improvements (e.g., adiponectin/leptin ratio 18.8 ± 11.8 vs. 2.4 ± 1.2 in liraglutide+PYY\(_{3-36}\)- or 1.4 ± 0.9 in sham-treated rats). Furthermore, RNA sequencing revealed a high number of differentially regulated genes in RYGB treated animals only. Conclusions: The combination therapy of liraglutide+PYY\(_{3-36}\) partly mimics the positive effects of RYGB on weight reduction and on hepatic steatosis, while its effects on metabolic function lack behind RYGB.}, language = {en} } @phdthesis{Wencker2022, author = {Wencker, Freya Dorothea Ruth}, title = {The methionine biosynthesis operon in \(Staphylococcus\) \(aureus\): Role of concerted RNA decay in transcript stability and T-box riboswitch turnover}, doi = {10.25972/OPUS-20712}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207124}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Methionine is the first amino acid of every newly synthesised protein. In combination with its role as precursor for the vital methyl-group donor S-adenosylmethionine, methionine is essential for every living cell. The opportunistic human pathogen Staphylococcus aureus is capable of synthesising methionine de novo, when it becomes scarce in the environment. All genes required for the de novo biosynthesis are encoded by the metICFE-mdh operon, except for metX. Expression is controlled by a hierarchical network with a methionyl-tRNA-specific T-box riboswitch (MET-TBRS) as centrepiece, that is also referred to as met leader (RNA). T-box riboswitches (TBRS) are regulatory RNA elements located in the 5'-untranslated region (5'-UTR) of genes. The effector molecule of T-box riboswitches is uncharged cognate tRNA. The prevailing mechanism of action is premature termination of transcription of the nascent RNA in the absence of the effector (i.e. uncharged cognate tRNA) due to formation of a hairpin structure, the Terminator stem. In presence of the effector, a transient stabilisation of the alternative structure, the Antiterminator, enables transcription of the downstream genes ('read-through'). Albeit, after the read-through the thermodynamically more stable Terminator eventually forms. The Terminator and the Antiterminator are two mutually exclusive structures. Previous work of the research group showed that in staphylococci the MET-TBRS ensures strictly methionine-dependent control of met operon expression. Uncharged methionyl-tRNA that activates the system is only present in sufficient amounts under methionine-deprived conditions. In contrast to other bacterial TBRS, the staphylococcal MET-TBRS has some characteristic features regarding its length and predicted secondary structure whose relevance for the function are yet unkown. Aim of the present thesis was to experimentally determine the structure of the met leader RNA and to investigate the stability of the met operon-specific transcripts in the context of methionine biosynthesis control. Furthermore, the yet unknown function of the mdh gene within the met operon was to be determined. In the context of this thesis, the secondary structure of the met leader was determined employing in-line probing. The structural analysis revealed the presence of almost all highly conserved T-box riboswitch structural characteristics. Furthermore, three additional stems, absent in all T-box riboswitches analysed to date, could be identified. Particularly remarkable is the above average length of the Terminator stem which renders it a potential target of the double-strand-specific endoribonuclease III (RNase III). The RNase III-dependent cleavage of the met leader could be experimentally verified by the use of suitable mutants. Moreover, the exact cleavage site within the Terminator was determined. The unusual immediate separation of the met leader from the met operon mRNA via the RNase III cleavage within the Terminator stem induces the rapid degradation of the met leader RNA and, most likely, that of the 5'-region of the met mRNA. The met mRNA is degraded from its 5'-end by the exoribonuclease RNase J. The stability of the met mRNA was found to vary over the length of the transcript with an instable 5'-end (metI and metC) and a longer half-life towards the 3'-end (metE and mdh). The varying transcript stability is reflected by differences in the available cellular protein levels. The obtained data suggest that programmed mRNA degradation is another level of regulation in the complex network of staphylococcal de novo methionine biosynthesis control. In addition, the MET-TBRS was studied with regard to a future use as a drug target for novel antimicrobial agents. To this end, effects of a dysregulated methionine biosynthesis on bacterial growth and survival were investigated in met leader mutants that either caused permanent transcription of the met operon ('ON') or prevented operon transcription ('OFF'), irrespective of the methionine status in the cell. Methionine deprivation turned out to be a strong selection pressure, as 'OFF' mutants acquired adaptive mutations within the met leader to restore met operon expression that subsequently re-enabled growth. The second part of the thesis was dedicated to the characterisation of the Mdh protein that is encoded by the last gene of the met operon and whose function is unknown yet. At first, co-transcription and -expression with the met operon could be demonstrated. Next, the Mdh protein was overexpressed and purified and the crystal structure of Mdh was solved to high resolution by the Kisker research group (Rudolf-Virchow-Zentrum W{\"u}rzburg). Analysis of the structure revealed the amino acid residues crucial for catalytic activity, and zinc was identified as a co-factor of Mdh. Also, Mdh was shown to exist as a dimer. However, identification of the Mdh substrate was, in the context of this thesis, (still) unsuccessful. Nevertheless, interactions of Mdh with enzymes of the met operon could be demonstrated by employing the bacterial two-hybrid system. This fact and the high conservation of mdh/Mdh on nucleotide and amino acid level among numerous staphylococcal species suggests an important role of Mdh within the methionine metabolism that should be a worthwhile subject of future research.}, subject = {Staphylococcus aureus}, language = {en} } @phdthesis{Matera2022, author = {Matera, Gianluca}, title = {Global mapping of RNA-RNA interactions in \(Salmonella\) via RIL-seq}, doi = {10.25972/OPUS-26877}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268776}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {RNA represents one of the most abundant macromolecules in both eukaryotic and prokaryotic cells. Since the discovery that RNA could play important gene regulatory functions in the physiology of a cell, small regulatory RNAs (sRNAs) have been at the center of molecular biology studies. Functional sRNAs can be independently transcribed or derived from processing of mRNAs and other non-coding regions and they often associate with RNA-binding proteins (RBPs). Ever since the two major bacterial RBPs, Hfq and ProQ, were identified, the way we approach the identification and characterization of sRNAs has drastically changed. Initially, a single sRNA was annotated and its function studied with the use of low-throughput biochemical techniques. However, the development of RNA-seq techniques over the last decades allowed for a broader identification of sRNAs and their functions. The process of studying a sRNA mainly focuses on the characterization of its interacting RNA partner(s) and the consequences of this binding. By using RNA interaction by ligation and sequencing (RIL-seq), the present thesis aimed at a high-throughput mapping of the Hfq-mediated RNA-RNA network in the major human pathogen Salmonella enterica. RIL-seq was at first performed in early stationary phase growing bacteria, which enabled the identification of ~1,800 unique interactions. In- depth analysis of such complex network was performed with the aid of a newly implemented RIL-seq browser. The interactome revealed known and new interactions involving sRNAs and genes part of the envelope regulon. A deeper investigation led to the identification of a new RNA sponge of the MicF sRNA, namely OppX, involved in establishing a cross-talk between the permeability at the outer membrane and the transport capacity at the periplasm and the inner membrane. Additionally, RIL-seq was applied to Salmonella enterica grown in SPI-2 medium, a condition that mimicks the intracellular lifestyle of this pathogen, and finally extended to in vivo conditions during macrophage infection. Collectively, the results obtained in the present thesis helped unveiling the complexity of such RNA networks. This work set the basis for the discovery of new mechanisms of RNA-based regulation, for the identification of a new physiological role of RNA sponges and finally provided the first resource of RNA interactions during infection conditions in a major human pathogen.}, subject = {Small RNA}, language = {en} } @article{SvenssonSharma2022, author = {Svensson, Sarah L. and Sharma, Cynthia M.}, title = {Small RNAs that target G-rich sequences are generated by diverse biogenesis pathways in Epsilonproteobacteria}, series = {Molecular Microbiology}, volume = {117}, journal = {Molecular Microbiology}, doi = {10.1111/mmi.14850}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259602}, pages = {215-233}, year = {2022}, abstract = {Bacterial small RNAs (sRNAs) are widespread post-transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence-based approaches, and similar functionalities can arise by convergent evolution. Here, we found that the virulence-associated CJnc190 sRNA of the foodborne pathogen Campylobacter jejuni resembles the RepG sRNA from the gastric pathogen Helicobacter pylori. However, while both sRNAs bind G-rich sites in their target mRNAs using a C/U-rich loop, they largely differ in their biogenesis. RepG is transcribed from a stand-alone gene and does not require processing, whereas CJnc190 is transcribed from two promoters as precursors that are processed by RNase III and also has a cis-encoded antagonist, CJnc180. By comparing CJnc190 homologs in diverse Campylobacter species, we show that RNase III-dependent processing of CJnc190 appears to be a conserved feature even outside of C. jejuni. We also demonstrate the CJnc180 antisense partner is expressed in C. coli, yet here might be derived from the 3'UTR (untranslated region) of an upstream flagella-related gene. Our analysis of G-tract targeting sRNAs in Epsilonproteobacteria demonstrates that similar sRNAs can have markedly different biogenesis pathways.}, language = {en} } @article{PrezzaRyanMaedleretal.2022, author = {Prezza, Gianluca and Ryan, Daniel and M{\"a}dler, Gohar and Reichardt, Sarah and Barquist, Lars and Westermann, Alexander J.}, title = {Comparative genomics provides structural and functional insights into Bacteroides RNA biology}, series = {Molecular Microbiology}, volume = {117}, journal = {Molecular Microbiology}, number = {1}, doi = {10.1111/mmi.14793}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259594}, pages = {67-85}, year = {2022}, abstract = {Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.}, language = {en} } @phdthesis{ReuterWeissenberger2022, author = {Reuter-Weissenberger, Philipp}, title = {The role of a fungal-specific transcription regulator on vacuolar biology and host interaction in \(Candida\) \(albicans\)}, doi = {10.25972/OPUS-25928}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259287}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Microorganisms that colonize the human body face large fluctuations in their surroundings. Therefore, those microbes developed sophisticated mechanisms that allow them to adapt their cell biology and maintain cellular homeostasis. One organelle vital to preserve cell physiology is the vacuole. The vacuole exhibits a wide range of functions and is able to adjust itself in response to both external and internal stimuli. Moreover, it plays an important role in host interaction and virulence in fungi such as Candida albicans. Despite this connection, only a few regulatory proteins have been described to modulate vacuolar biology in fungal pathogens. Furthermore, whether such regulation alters fungus-host interplay remains largely unknown. This thesis focuses on the characterization of ZCF8, a fungus-specific transcription regulator in the human-associated yeast C. albicans. To this end, I combined genome-wide protein-DNA interaction assays and gene expression analysis that identified genes regulated by Zcf8p. Fluorescence microscopy uncovered that several top targets of Zcf8p localize to the fungal vacuole. Moreover, deletion and overexpression of ZCF8 resulted in alterations in vacuolar morphology and in luminal pH and rendered the fungus resistant or susceptible to a vacuole-disturbing drug. Finally, in vitro adherence assays showed that Zcf8p modulates the attachment of C. albicans to human epithelial cells in a vacuole-dependent manner. Given those findings, I posit that the previously uncharacterized transcription regulator Zcf8p modulates fungal attachment to epithelial cells in a manner that depends on the status of the fungal vacuole. Furthermore, the results highlight that vacuolar physiology is a substantial factor influencing the physical interaction between Candida cells and mammalian mucosal surfaces.}, subject = {Vakuole}, language = {en} }