@article{BauriedlGerovacHeidrichetal.2020, author = {Bauriedl, Saskia and Gerovac, Milan and Heidrich, Nadja and Bischler, Thorsten and Barquist, Lars and Vogel, J{\"o}rg and Schoen, Christoph}, title = {The minimal meningococcal ProQ protein has an intrinsic capacity for structure-based global RNA recognition}, series = {Nature Communications}, volume = {11}, journal = {Nature Communications}, doi = {10.1038/s41467-020-16650-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230040}, year = {2020}, abstract = {FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition. FinO-domain proteins are bacterial RNA-binding proteins with a wide range of target specificities. Here, the authors employ UV CLIP-seq and show that minimal ProQ protein of Neisseria meningitidis binds to various small non-coding RNAs and mRNAs involved in virulence.}, language = {en} } @article{Vogel2020, author = {Vogel, J{\"o}rg}, title = {An RNA biology perspective on species-specific programmable RNA antibiotics}, series = {Molecular Microbiology}, volume = {113}, journal = {Molecular Microbiology}, number = {3}, doi = {10.1111/mmi.14476}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214869}, pages = {550 -- 559}, year = {2020}, abstract = {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.}, language = {en} } @article{MayrRamirezZavalaKruegeretal.2020, author = {Mayr, Eva-Maria and Ram{\´i}rez-Zavala, Bernardo and Kr{\"u}ger, Ines and Morschh{\"a}user, Joachim}, title = {A Zinc Cluster Transcription Factor Contributes to the Intrinsic Fluconazole Resistance of Candida auris}, series = {mSphere}, volume = {5}, journal = {mSphere}, number = {2}, doi = {10.1128/mSphere.00279-20}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229937}, year = {2020}, abstract = {ABSTRACT The recently emerged pathogenic yeast Candida auris is a major concern for human health, because it is easily transmissible, difficult to eradicate from hospitals, and highly drug resistant. Most C. auris isolates are resistant to the widely used antifungal drug fluconazole due to mutations in the target enzyme Erg11 and high activity of efflux pumps, such as Cdr1. In the well-studied, distantly related yeast Candida albicans, overexpression of drug efflux pumps also is a major mechanism of acquired fluconazole resistance and caused by gain-of-function mutations in the zinc cluster transcription factors Mrr1 and Tac1. In this study, we investigated a possible involvement of related transcription factors in efflux pump expression and fluconazole resistance of C. auris. The C. auris genome contains three genes encoding Mrr1 homologs and two genes encoding Tac1 homologs, and we generated deletion mutants lacking these genes in two fluconazole-resistant strains from clade III and clade IV. Deletion of TAC1b decreased the resistance to fluconazole and voriconazole in both strain backgrounds, demonstrating that the encoded transcription factor contributes to azole resistance in C. auris strains from different clades. CDR1 expression was not or only minimally affected in the mutants, indicating that Tac1b can confer increased azole resistance by a CDR1-independent mechanism. IMPORTANCE Candida auris is a recently emerged pathogenic yeast that within a few years after its initial description has spread all over the globe. C. auris is a major concern for human health, because it can cause life-threatening systemic infections, is easily transmissible, and is difficult to eradicate from hospital environments. Furthermore, C. auris is highly drug resistant, especially against the widely used antifungal drug fluconazole. Mutations in the drug target and high activity of efflux pumps are associated with azole resistance, but it is not known how drug resistance genes are regulated in C. auris. We have investigated the potential role of several candidate transcriptional regulators in the intrinsic fluconazole resistance of C. auris and identified a transcription factor that contributes to the high resistance to fluconazole and voriconazole of two C. auris strains from different genetic clades, thereby providing insight into the molecular basis of drug resistance of this medically important yeast."}, language = {en} } @article{AlzheimerSvenssonKoenigetal.2020, author = {Alzheimer, Mona and Svensson, Sarah L. and K{\"o}nig, Fabian and Schweinlin, Matthias and Metzger, Marco and Walles, Heike and Sharma, Cynthia M.}, title = {A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni}, series = {PLoS Pathogens}, volume = {16}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1008304}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229454}, year = {2020}, abstract = {The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.}, language = {en} } @article{SchulteSchweinlinWestermannetal.2020, author = {Schulte, Leon N. and Schweinlin, Matthias and Westermann, Alexander J. and Janga, Harshavardhan and Santos, Sara C. and Appenzeller, Silke and Walles, Heike and Vogel, J{\"o}rg and Metzger, Marco}, title = {An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection}, series = {mBio}, volume = {11, 2020}, journal = {mBio}, number = {1}, doi = {10.1128/mBio.03348-19}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229428}, year = {2020}, abstract = {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.}, language = {en} } @article{DingemansMonsieursYuetal.2016, author = {Dingemans, Josef and Monsieurs, Pieter and Yu, Sung-Huan and Crabb{\´e}, Aur{\´e}lie and F{\"o}rstner, Konrad U. and Malfroot, Anne and Cornelis, Pierre and Van Houdt, Rob}, title = {Effect of Shear Stress on Pseudomonas aeruginosa Isolated from the Cystic Fibrosis Lung}, series = {mBio}, volume = {7}, journal = {mBio}, number = {4}, doi = {10.1128/mBio.00813-16}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165821}, pages = {e00813-16}, year = {2016}, abstract = {Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle.}, language = {en} } @article{AshrafYasrebiHertleinetal.2017, author = {Ashraf, Kerolos and Yasrebi, Kaveh and Hertlein, Tobias and Ohlsen, Knut and Lalk, Michael and Hilgeroth, Andreas}, title = {Novel effective small-molecule antibacterials against \(Enterococcus\) strains}, series = {Molecules}, volume = {22}, journal = {Molecules}, number = {12}, doi = {10.3390/molecules22122193}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172628}, year = {2017}, abstract = {\(Enterococcus\) species cause increasing numbers of infections in hospitals. They contribute to the increasing mortality rates, mostly in patients with comorbidities, who suffer from severe diseases. \(Enterococcus\) resistances against most antibiotics have been described, including novel antibiotics. Therefore, there is an ongoing demand for novel types of antibiotics that may overcome bacterial resistances. We discovered a novel class of antibiotics resulting from a simple one-pot reaction of indole and \(o\)-phthaldialdehyde. Differently substituted indolyl benzocarbazoles were yielded. Both the indole substitution and the positioning at the molecular scaffold influence the antibacterial activity towards the various strains of \(Enterococcus\) species with the highest relevance to nosocomial infections. Structure-activity relationships are discussed, and the first lead compounds were identified as also being effective in the case of a vancomycin resistance.}, language = {en} } @article{HassanVasquezGuoLiangetal.2017, author = {Hassan, Musa A. and Vasquez, Juan J. and Guo-Liang, Chew and Meissner, Markus and Siegel, T. Nicolai}, title = {Comparative ribosome profiling uncovers a dominant role for translational control in \(Toxoplasma\) \(gondii\)}, series = {BMC Genomics}, volume = {18}, journal = {BMC Genomics}, doi = {10.1186/s12864-017-4362-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172376}, year = {2017}, abstract = {Background The lytic cycle of the protozoan parasite \(Toxoplasma\) \(gondii\), which involves a brief sojourn in the extracellular space, is characterized by defined transcriptional profiles. For an obligate intracellular parasite that is shielded from the cytosolic host immune factors by a parasitophorous vacuole, the brief entry into the extracellular space is likely to exert enormous stress. Due to its role in cellular stress response, we hypothesize that translational control plays an important role in regulating gene expression in \(Toxoplasma\) during the lytic cycle. Unlike transcriptional profiles, insights into genome-wide translational profiles of \(Toxoplasma\) \(gondii\) are lacking. Methods We have performed genome-wide ribosome profiling, coupled with high throughput RNA sequencing, in intracellular and extracellular \(Toxoplasma\) \(gondii\) parasites to investigate translational control during the lytic cycle. Results Although differences in transcript abundance were mostly mirrored at the translational level, we observed significant differences in the abundance of ribosome footprints between the two parasite stages. Furthermore, our data suggest that mRNA translation in the parasite is potentially regulated by mRNA secondary structure and upstream open reading frames. Conclusion We show that most of the \(Toxoplasma\) genes that are dysregulated during the lytic cycle are translationally regulated.}, language = {en} } @article{DugarSvenssonBischleretal.2016, author = {Dugar, Gaurav and Svensson, Sarah L. and Bischler, Thorsten and Waldchen, Sina and Reinhardt, Richard and Sauer, Markus and Sharma, Cynthia M.}, title = {The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni}, series = {Nature Communications}, volume = {7}, journal = {Nature Communications}, doi = {10.1038/ncomms11667}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173201}, year = {2016}, abstract = {The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.}, language = {en} } @article{SelleHertleinOesterreichetal.2016, author = {Selle, Martina and Hertlein, Tobias and Oesterreich, Babett and Klemm, Theresa and Kloppot, Peggy and M{\"u}ller, Elke and Ehricht, Ralf and Stentzel, Sebastian and Br{\"o}ker, Barbara M. and Engelmann, Susanne and Ohlsen, Knut}, title = {Global antibody response to Staphylococcus aureus live-cell vaccination}, series = {Scientific Reports}, volume = {6}, journal = {Scientific Reports}, doi = {10.1038/srep24754}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181245}, year = {2016}, abstract = {The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration.}, language = {en} } @article{MichauxHansenJennichesetal.2020, author = {Michaux, Charlotte and Hansen, Elisabeth E. and Jenniches, Laura and Gerovac, Milan and Barquist, Lars and Vogel, J{\"o}rg}, title = {Single-Nucleotide RNA Maps for the Two Major Nosocomial Pathogens Enterococcus faecalis and Enterococcus faecium}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {10}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2020.600325}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217947}, year = {2020}, abstract = {Enterococcus faecalis and faecium are two major representative clinical strains of the Enterococcus genus and are sadly notorious to be part of the top agents responsible for nosocomial infections. Despite their critical implication in worldwide public healthcare, essential and available resources such as deep transcriptome annotations remain poor, which also limits our understanding of post-transcriptional control small regulatory RNA (sRNA) functions in these bacteria. Here, using the dRNA-seq technique in combination with ANNOgesic analysis, we successfully mapped and annotated transcription start sites (TSS) of both E. faecalis V583 and E. faecium AUS0004 at single nucleotide resolution. Analyzing bacteria in late exponential phase, we capture ~40\% (E. faecalis) and 43\% (E. faecium) of the annotated protein-coding genes, determine 5′ and 3′ UTR (untranslated region) length, and detect instances of leaderless mRNAs. The transcriptome maps revealed sRNA candidates in both bacteria, some found in previous studies and new ones. Expression of candidate sRNAs is being confirmed under biologically relevant environmental conditions. This comprehensive global TSS mapping atlas provides a valuable resource for RNA biology and gene expression analysis in the Enterococci. It can be accessed online at www.helmholtz-hiri.de/en/datasets/enterococcus through an instance of the genomic viewer JBrowse.}, language = {en} } @article{EneLohseVladuetal.2016, author = {Ene, Iuliana V. and Lohse, Matthew B. and Vladu, Adrian V. and Morschh{\"a}user, Joachim and Johnson, Alexander D. and Bennett, Richard J.}, title = {Phenotypic Profiling Reveals that Candida albicans Opaque Cells Represent a Metabolically Specialized Cell State Compared to Default White Cells}, series = {mBio}, volume = {7}, journal = {mBio}, number = {6}, doi = {10.1128/mBio.01269-16}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165818}, pages = {e01269-16}, year = {2016}, abstract = {The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state.}, language = {en} } @article{WestermannBarquistVogel2017, author = {Westermann, Alexander J. and Barquist, Lars and Vogel, J{\"o}rg}, title = {Resolving host-pathogen interactions by dual RNA-seq}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1006033}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171921}, year = {2017}, abstract = {The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables "dual RNA-seq" studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.}, language = {en} } @article{BalasubramanianOthmanKampiketal.2017, author = {Balasubramanian, Srikkanth and Othman, Eman M. and Kampik, Daniel and Stopper, Helga and Hentschel, Ute and Ziebuhr, Wilma and Oelschlaeger, Tobias A. and Abdelmohsen, Usama R.}, title = {Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation}, series = {Frontiers in Microbiology}, volume = {8}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2017.00236}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171844}, year = {2017}, abstract = {Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections.}, language = {en} } @article{MietrachSchlosserGeibel2019, author = {Mietrach, Nicole and Schlosser, Andreas and Geibel, Sebastian}, title = {An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization}, series = {Acta Crystallographica Section F}, volume = {75}, journal = {Acta Crystallographica Section F}, number = {12}, doi = {10.1107/S2053230X1901495X}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213681}, pages = {725-730}, year = {2019}, abstract = {The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16\% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 {\AA} resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 {\AA}, α = β = γ = 90°.}, language = {en} } @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{BlaettnerDasPaprotkaetal.2016, author = {Bl{\"a}ttner, Sebastian and Das, Sudip and Paprotka, Kerstin and Eilers, Ursula and Krischke, Markus and Kretschmer, Dorothee and Remmele, Christian W. and Dittrich, Marcus and M{\"u}ller, Tobias and Schuelein-Voelk, Christina and Hertlein, Tobias and Mueller, Martin J. and Huettel, Bruno and Reinhardt, Richard and Ohlsen, Knut and Rudel, Thomas and Fraunholz, Martin J.}, title = {Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes}, series = {PLoS Pathogens}, volume = {12}, journal = {PLoS Pathogens}, number = {9}, doi = {10.1371/journal.ppat.1005857}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180380}, year = {2016}, abstract = {Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.}, language = {en} } @article{FroehlichHanekePapenfortetal.2016, author = {Fr{\"o}hlich, Kathrin S. and Haneke, Katharina and Papenfort, Kai and Vogel, J{\"o}rg}, title = {The target spectrum of SdsR small RNA in Salmonella}, series = {Nucleic Acids Research}, volume = {44}, journal = {Nucleic Acids Research}, number = {21}, doi = {10.1093/nar/gkw632}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175365}, pages = {10406-10422}, year = {2016}, abstract = {Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these 'core sRNAs' of largely unknown function is the abundant ∼100-nucleotide SdsR sRNA which is transcribed by the general stress σ-factor, σ\(^{S}\) and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present in almost all enterobacterial genomes, suggesting that additional, conserved targets of this sRNA must exist. Here, we have combined SdsR pulse-expression with whole genome transcriptomics to discover 20 previously unknown candidate targets of SdsR which include mRNAs coding for physiologically important regulators such as the carbon utilization regulator, CRP, the nucleoid-associated chaperone, StpA and the antibiotic resistance transporter, TolC. Processing of SdsR by RNase E results in two cellular SdsR variants with distinct target spectra. While the overall physiological role of this orphan core sRNA remains to be fully understood, the new SdsR targets present valuable leads to determine sRNA functions in resting bacteria.}, language = {en} } @phdthesis{Hoer2020, author = {H{\"o}r, Jens}, title = {Discovery of RNA/protein complexes by Grad-seq}, doi = {10.25972/OPUS-21181}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211811}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Complex formation between macromolecules constitutes the foundation of most cellular processes. Most known complexes are made up of two or more proteins interacting in order to build a functional entity and therefore enabling activities which the single proteins could otherwise not fulfill. With the increasing knowledge about noncoding RNAs (ncRNAs) it has become evident that, similar to proteins, many of them also need to form a complex to be functional. This functionalization is usually executed by specific or global RNA-binding proteins (RBPs) that are specialized binders of a certain class of ncRNAs. For instance, the enterobacterial global RBPs Hfq and ProQ together bind >80 \% of the known small regulatory RNAs (sRNAs), a class of ncRNAs involved in post-transcriptional regulation of gene expression. However, identification of RNA-protein interactions so far was performed individually by employing low-throughput biochemical methods and thereby hindered the discovery of such interactions, especially in less studied organisms such as Gram-positive bacteria. Using gradient profiling by sequencing (Grad-seq), the present thesis aimed to establish high-throughput, global RNA/protein complexome resources for Escherichia coli and Streptococcus pneumoniae in order to provide a new way to investigate RNA-protein as well as protein-protein interactions in these two important model organisms. In E. coli, Grad-seq revealed the sedimentation profiles of 4,095 (∼85 \% of total) transcripts and 2,145 (∼49 \% of total) proteins and with that reproduced its major ribonucleoprotein particles. Detailed analysis of the in-gradient distribution of the RNA and protein content uncovered two functionally unknown molecules—the ncRNA RyeG and the small protein YggL—to be ribosomeassociated. Characterization of RyeG revealed it to encode for a 48 aa long, toxic protein that drastically increases lag times when overexpressed. YggL was shown to be bound by the 50S subunit of the 70S ribosome, possibly indicating involvement of YggL in ribosome biogenesis or translation of specific mRNAs. S. pneumoniae Grad-seq detected 2,240 (∼88 \% of total) transcripts and 1,301 (∼62 \% of total) proteins, whose gradient migration patterns were successfully reconstructed, and thereby represents the first RNA/protein complexome resource of a Gram-positive organism. The dataset readily verified many conserved major complexes for the first time in S. pneumoniae and led to the discovery of a specific interaction between the 3'!5' exonuclease Cbf1 and the competence-regulating ciadependent sRNAs (csRNAs). Unexpectedly, trimming of the csRNAs by Cbf1 stabilized the former, thereby promoting their inhibitory function. cbf1 was further shown to be part of the late competence genes and as such to act as a negative regulator of competence.}, subject = {Multiproteinkomplex}, language = {en} } @article{BarYosefGildorRamirezZavalaetal.2018, author = {Bar-Yosef, Hagit and Gildor, Tsvia and Ram{\´i}rez-Zavala, Bernardo and Schmauch, Christian and Weissman, Ziva and Pinsky, Mariel and Naddaf, Rawi and Morschh{\"a}user, Joachim and Arkowitz, Robert A. and Kornitzer, Daniel}, title = {A global analysis of kinase function in Candida albicans hyphal morphogenesis reveals a role for the endocytosis regulator Akl1}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {8}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2018.00017}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197204}, year = {2018}, abstract = {The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.}, language = {en} }