@article{BarquistMayhoCumminsetal.2016, author = {Barquist, Lars and Mayho, Matthew and Cummins, Carla and Cain, Amy K. and Boinett, Christine J. and Page, Andrew J. and Langridge, Gemma C. and Quail, Michael A. and Keane, Jacqueline A. and Parkhill, Julian}, title = {The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries}, series = {Bioinformatics}, volume = {32}, journal = {Bioinformatics}, number = {7}, doi = {10.1093/bioinformatics/btw022}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189667}, pages = {1109-1111}, year = {2016}, abstract = {Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology.}, language = {en} } @article{YuVogelFoerstner2018, author = {Yu, Sung-Huan and Vogel, J{\"o}rg and F{\"o}rstner, Konrad U.}, title = {ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes}, series = {GigaScience}, volume = {7}, journal = {GigaScience}, doi = {10.1093/gigascience/giy096}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178942}, year = {2018}, abstract = {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/.}, language = {en} } @phdthesis{Ponath2023, author = {Ponath, Falk Fred Finn}, title = {Investigating the molecular biology of \(Fusobacterium\) \(nucleatum\)}, doi = {10.25972/OPUS-30351}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303516}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {The anaerobe Fusobacterium nucleatum (F. nucleatum) is an important member of the oral microbiome but can also colonize different tissues of the human body. In particular, its association with multiple human cancers has drawn much attention. This association has prompted growing interest into the interaction of F. nucleatum with cancer, with studies focusing primarily on the host cells. At the same time, F. nucleatum itself remains poorly understood, which includes its transcriptomic architecture but also gene regulation such as global stress responses that typically enable survival of bacteria in new environments. An important aspect of such regulatory networks is the post-transcriptional regulation, which is entirely unknown in F. nucleatum. This paucity extents to any knowledge on small regulatory RNAs (sRNAs), despite their important role as post-transcriptional regulators of the bacterial physiology. Investigating the above stated aspects is further complicated by the fact that F. nucleatum is phylogenetically distant from all other bacteria, displays very limited genetic tractability and lacks genetic tools for dissecting gene function. This leaves many open questions on basic gene regulation in F. nucleatum, such as if the bacterium combines transcriptional and post-transcriptional regulation in its adaptation to a changing environment. To begin answering this question, this works elucidated the transcriptomic landscape of F. nucleatum by performing differential RNA-seq (dRNA-seq). Conducted for five representative strains of all F. nucleatum subspecies and the closely related F. periodonticum, the analysis globally uncovered transcriptional start sites (TSS), 5'untranslated regions (UTRs) and improved the existing annotation. Importantly, the dRNA-seq analysis also identified a conserved suite of sRNAs specific to Fusobacterium. The development of five genetic tools enabled further investigations of gene functions in F. nucleatum. These include vectors that enable the expression of different fluorescent proteins, inducible gene expression and scarless gene deletion in addition to transcriptional and translational reporter systems. These tools enabled the dissection of a Sigma E response and uncovered several commonalities with its counterpart in the phylogenetically distant Proteobacteria. The similarities include the upregulation of genes involved in membrane homeostasis but also a Simga E-dependent regulatory sRNA. Surprisingly, oxygen was found to activated Sigma E in F. nucleatum contrasting the typical role of the factor in envelope stress. The non-coding Sigma E-dependent sRNA, named FoxI, was shown to repress the translation of several envelope proteins which represented yet another parallel to the envelope stress response in Proteobacteria. Overall, this work sheds light on the RNA landscape of the cancer-associated bacterium leading to the discovery of a conserved global stress response consisting of a coding and a non-coding arm. The development of new genetic tools not only aided the latter discovery but also provides the means for further dissecting the molecular and infection biology of this enigmatic bacterium.}, language = {en} } @phdthesis{Venturini2021, author = {Venturini, Elisa}, title = {Small proteins in \(Salmonella\): an updated annotation and a global analysis to find new regulators of virulence}, doi = {10.25972/OPUS-24702}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-247029}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Small proteins, often defined as shorter than 50 amino acids, have been implicated in fundamental cellular processes. Despite this, they have been largely understudied throughout all domains of life, since their size often makes their identification and characterization challenging. This work addressed the knowledge gap surrounding small proteins with a focus on the model bacterial pathogen Salmonella Typhimurium. In a first step, new small proteins were identified with a combination of computational and experimental approaches. Infection-relevant datasets were then investigated with the updated Salmonella annotation to prioritize promising candidates involved in virulence. To implement the annotation of new small proteins, predictions from the algorithm sPepFinder were merged with those derived from Ribo-seq. These were added to the Salmonella annotation and used to (re)analyse different datasets. Information regarding expression during infection (dual RNA-seq) and requirement for virulence (TraDIS) was collected for each given coding sequence. In parallel, Grad-seq data were mined to identify small proteins engaged in intermolecular interactions. The combination of dual RNA-seq and TraDIS lead to the identification of small proteins with features of virulence factors, namely high intracellular induction and a virulence phenotype upon transposon insertion. As a proof of principle of the power of this approach in highlighting high confidence candidates, two small proteins were characterized in the context of Salmonella infection. MgrB, a known regulator of the PhoPQ two-component system, was shown to be essential for the infection of epithelial cells and macrophages, possibly via its stabilizing effect on flagella or by interacting with other sensor kinases of twocomponent systems. YjiS, so far uncharacterized in Salmonella, had an opposite role in infection, with its deletion rendering Salmonella hypervirulent. The mechanism underlying this, though still obscure, likely relies on the interaction with inner-membrane proteins. Overall, this work provides a global description of Salmonella small proteins in the context of infection with a combinatorial approach that expedites the identification of interesting candidates. Different high-throughput datasets available for a broad range of organisms can be analysed in a similar manner with a focus on small proteins. This will lead to the identification of key factors in the regulation of various processes, thus for example providing targets for the treatment of bacterial infections or, in the case of commensal bacteria, for the modulation of the microbiota composition.}, subject = {Salmonella Typhimurium}, language = {en} } @article{StelznerBoynyHertleinetal.2021, author = {Stelzner, Kathrin and Boyny, Aziza and Hertlein, Tobias and Sroka, Aneta and Moldovan, Adriana and Paprotka, Kerstin and Kessie, David and Mehling, Helene and Potempa, Jan and Ohlsen, Knut and Fraunholz, Martin J. and Rudel, Thomas}, title = {Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells}, series = {PLoS Pathogens}, volume = {17}, journal = {PLoS Pathogens}, number = {9}, doi = {10.1371/journal.ppat.1009874}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-263908}, year = {2021}, abstract = {Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.}, language = {en} } @article{GerovaWickeChiharaetal.2021, author = {Gerova, Milan and Wicke, Laura and Chihara, Kotaro and Schneider, Cornelius and Lavigne, Rob and Vogel, J{\"o}rg}, title = {A grad-seq view of RNA and protein complexes in Pseudomonas aeruginosa under standard and bacteriophage predation conditions}, series = {mbio}, volume = {12}, journal = {mbio}, number = {1}, doi = {10.1128/mBio.03454-20}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259054}, pages = {e03454-20}, year = {2021}, abstract = {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.}, language = {en} } @article{BalasubramanianSkafHolzgrabeetal.2018, author = {Balasubramanian, Srikkanth and Skaf, Joseph and Holzgrabe, Ulrike and Bharti, Richa and F{\"o}rstner, Konrad U. and Ziebuhr, Wilma and Humeida, Ute H. and Abdelmohsen, Usama R. and Oelschlaeger, Tobias A.}, title = {A new bioactive compound from the marine sponge-derived Streptomyces sp. SBT348 inhibits staphylococcal growth and biofilm formation}, series = {Frontiers in Microbiology}, volume = {9}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2018.01473}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221408}, year = {2018}, abstract = {Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients carrying surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms leads to refractory and persistent device-related infections (DRIs). Staphylococci organized in biofilms are more tolerant to antibiotics and immune responses, and thus are difficult-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm-based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal, and silicone surfaces. A bioassay-guided fractionation was performed to isolate the active compound (SKC3) from the crude SBT348 extract. Our results demonstrated that SKC3 effectively inhibits the growth (MIC: 31.25 \(\mu\)g/ml) and biofilm formation (sub-MIC range: 1.95-<31.25 \(\mu\)g/ml) of S. epidermidis RP62A in vitro. Chemical characterization of SKC3 by heat and enzyme treatments, and mass spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258.3 Da). Cytotoxicity profiling of SKC3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria mellonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of SKC3 treated S. epidermidis RP62A has further unmasked its negative effect on central metabolism such as carbon flux as well as, amino acid, lipid, and energy metabolism. Taken together, these findings suggest a potential of SKC3 as a putative drug to prevent staphylococcal DRIs.}, language = {en} } @article{KrausBrinkSiegel2019, author = {Kraus, Amelie J. and Brink, Benedikt G. and Siegel, T. Nicolai}, title = {Efficient and specific oligo-based depletion of rRNA}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-48692-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224829}, year = {2019}, abstract = {In most organisms, ribosomal RNA (rRNA) contributes to >85\% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5\% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.}, language = {en} } @article{BurySoundararajanBhartietal.2018, author = {Bury, Susanne and Soundararajan, Manonmani and Bharti, Richa and von B{\"u}nau, Rudolf and F{\"o}rstner, Konrad U. and Oelschlaeger, Tobias A.}, title = {The probiotic escherichia coli strain Nissle 1917 combats lambdoid bacteriophages stx and lambda}, series = {Frontiers in Microbiology}, volume = {9}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2018.00929}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221960}, year = {2018}, abstract = {Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coil strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype 0104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coll strains which got infected by stx2-encoding lambdoid phages turning the E. coil into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN toward not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.}, language = {en} } @article{AllertFoersterSvenssonetal.2018, author = {Allert, Stefanie and F{\"o}rster, Toni M. and Svensson, Carl-Magnus and Richardson, Jonathan P. and Pawlik, Tony and Hebecker, Betty and Rudolphi, Sven and Juraschitz, Marc and Schaller, Martin and Blagojevic, Mariana and Morschh{\"a}user, Joachim and Figge, Marc Thilo and Jacobsen, Ilse D. and Naglik, Julian R. and Kasper, Lydia and Mogavero, Selene and Hube, Bernhard}, title = {\(Candida\) \(albicans\)-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers}, series = {mBio}, volume = {9}, journal = {mBio}, number = {3}, doi = {10.1128/mBio.00915-18}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221084}, pages = {1-20}, year = {2018}, abstract = {Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans, usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.}, language = {en} }