@article{RaketteDonatOhlsenetal.2012, author = {Rakette, Sonja and Donat, Stefanie and Ohlsen, Knut and Stehle, Thilo}, title = {Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {6}, doi = {10.1371/journal.pone.0039136}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135369}, pages = {e39136}, year = {2012}, abstract = {Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 angstrom resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.}, language = {en} } @phdthesis{Hampe2018, author = {Hampe, Irene Aurelia Ida}, title = {Analysis of the mechanism and the regulation of histatin 5 resistance in \(Candida\) \(albicans\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159634}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Antimycotics such as fluconazole are frequently used to treat C. albicans infections of the oral mucosa. Prolonged treatment of the fungal infection with fluconazole pose a risk to resistance development. C. albicans can adapt to these stressful environmental changes by regulation of gene expression or by producing genetically altered variants that arise in the population. Adapted variants frequently carry activating mutations in zinc cluster transcription factors, which cause the upregulation of their target genes, including genes encoding efflux pumps that confer drug resistance. MDR1, regulated by the zinc cluster transcription factor Mrr1, as well as CDR1 and CDR2, regulated by the zinc cluster transcription factor Tac1, are well-known examples of genes encoding efflux pumps that extrude the antimycotic fluconazole from the fungal cell and thus contribute to the survival of the fungus. In this study, it was investigated if C. albicans can develop resistance to the antimicrobial peptide histatin 5, which serves as the first line of defence in the oral cavity of the human host. Recently, it was shown that C. albicans transports histatin 5 outside of the Candia cell via the efflux pump Flu1. As efflux pumps are often regulated by zinc cluster transcription factors, the Flu1 efflux pump could also be regulated by a zinc cluster transcription factor which could in a hyperactive form upregulate the expression of the efflux pump, resulting in increased export of histatin 5 and consequently in histatin 5 resistance. In order to find a zinc cluster transcription factor that upregulates FLU1 expression, a comprehensive library of C. albicans strains containing artificially activated forms of zinc cluster transcription factors was screened for suitable candidates. The screening was conducted on medium containing mycophenolic acid because mycophenolic acid is also a substrate of Flu1 and a strain expressing a hyperactive zinc cluster transcription factor that upregulates FLU1 expression should exhibit an easily recognisable mycophenolic acid-resistant phenotype. Further, FACS analysis, quantitative real-time RT-PCR analysis, broth microdilution assays as well as histatin 5 assays were conducted to analyse the mechanism and the regulation of histatin 5 resistance. Several zinc cluster transcription factors caused mycophenolic acid resistance and upregulated FLU1 expression. Of those, only hyperactive Mrr1 was able to confer increased histatin 5 resistance. Finding Mrr1 to confer histatin 5 resistance was highly interesting as fluconazole-resistant strains with naturally occurring Mrr1 gain of function mutations exist, which were isolated from HIV-infected patients with oral candidiasis. These Mrr1 gain of function mutations as well as artificially activated Mrr1 cause fluconazole resistance by upregulation of the efflux pump MDR1 and other target genes. In the course of the study, it was found that expression of different naturally occurring MRR1 gain-of-function mutations in the SC5314 wild type background caused increased FLU1 expression and increased histatin 5 resistance. The same was true for fluconazole-resistant clinical isolates with Mrr1 gain of function mutations, which also caused the overexpression of FLU1. Those cells were less efficiently killed by histatin 5 dependent on Mrr1. Surprisingly, FLU1 contributed only little to histatin 5 resistance, rather, overexpression of MDR1 mainly contributed to the Mrr1-mediated histatin 5 resistance, but also additional Mrr1-target genes were involved. These target genes are yet to be uncovered. Moreover, if a link between the yet unknown Mrr1-target genes contributing to fluconazole resistance and increased histatin 5 resistance can be drawn remains to be discovered upon finding of the responsible target genes. Collectively, this study contributes to the understanding of the impact of prolonged antifungal exposure on the interaction between host and fungus. Drug therapy can give rise to resistance evolution resulting in strains that have not only developed resistance to fluconazole but also to an innate host mechanism, which allows adaption to the host niche even in the absence of the drug.}, subject = {Histatin 5}, language = {en} } @article{SunkavalliAguilarSilvaetal.2017, author = {Sunkavalli, Ushasree and Aguilar, Carmen and Silva, Ricardo Jorge and Sharan, Malvika and Cruz, Ana Rita and Tawk, Caroline and Maudet, Claire and Mano, Miguel and Eulalio, Ana}, title = {Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {4}, doi = {10.1371/journal.ppat.1006327}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158204}, pages = {e1006327}, year = {2017}, abstract = {MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.}, language = {en} } @article{SharanFoerstnerEulalioetal.2017, author = {Sharan, Malvika and F{\"o}rstner, Konrad U. and Eulalio, Ana and Vogel, J{\"o}rg}, title = {APRICOT: an integrated computational pipeline for the sequence-based identification and characterization of RNA-binding proteins}, series = {Nucleic Acids Research}, volume = {45}, journal = {Nucleic Acids Research}, number = {11}, doi = {10.1093/nar/gkx137}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157963}, pages = {e96}, year = {2017}, abstract = {RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs) and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches. We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using position-specific scoring matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot.}, language = {en} } @article{JaegerPernitzschRichteretal.2012, author = {J{\"a}ger, Dominik and Pernitzsch, Sandy R. and Richter, Andreas S. and Backofen, Rolf and Sharma, Cynthia M. and Schmitz, Ruth A.}, title = {An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains}, series = {Nucleic Acids Research}, volume = {40}, journal = {Nucleic Acids Research}, number = {21}, doi = {10.1093/nar/gks847}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134972}, pages = {10964-10979}, year = {2012}, abstract = {We report on the characterization and target analysis of the small (s) RNA\(_{162}\) in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5' fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA\(_{162}\) (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA\(_{162}\) is crucial for target interactions. Furthermore, our results indicate that sRNA\(_{162}\)-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 50 end of sRNA\(_{162}\) targets the 5'-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA\(_{162}\) acts as an antisense (as) RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.}, language = {en} } @phdthesis{Oesterreich2017, author = {Oesterreich, Babett}, title = {Preclinical development of an immunotherapy against antibiotic-resistant Staphylococcus aureus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123237}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {The Gram-positive bacterium Staphylococcus aureus is the leading cause of nosocomial infections. In particular, diseases caused by methicillin-resistant S. aureus (MRSA) are associated with higher morbidity, mortality and medical costs due to showing resistance to several classes of established antibiotics and their ability to develop resistance mechanisms against new antibiotics rapidly. Therefore, strategies based on immunotherapy approaches have the potential to close the gap for an efficient treatment of MRSA. In this thesis, a humanized antibody specific for the immunodominant staphylococcal antigen A (IsaA) was generated and thoroughly characterized as potential candidate for an antibody based therapy. A murine monoclonal antibody was selected for humanization based on its binding characteristics and the ability of efficient staphylococcal killing in mouse infection models. The murine antibody was humanized by CDR grafting and mouse and humanized scFv as well as scFv-Fc fragments were constructed for comparative binding studies to analyse the successful humanization. After these studies, the full antibody with the complete Fc region was constructed as isotype IgG1, IgG2 and IgG4, respectively to assess effector functions, including antibody-dependent killing of S. aureus. The biological activity of the humanized antibody designated hUK-66 was analysed in vitro with purified human PMNs and whole blood samples taken from healthy donors and patients at high risk of S. aureus infections, such as those with diabetes, end-stage renal disease, or artery occlusive disease (AOD). Results of the in vitro studies show, that hUK-66 was effective in antibody-dependent killing of S. aureus in blood from both healthy controls and patients vulnerable to S. aureus infections. Moreover, the biological activity of hUK-66 and hUK-66 combined with a humanized anti-alpha-toxin antibody (hUK-tox) was investigated in vivo using a mouse pneumonia model. The in vivo results revealed the therapeutic efficacy of hUK-66 and the antibody combination of hUK-66 and hUK-tox to prevent staphylococcal induced pneumonia in a prophylactic set up. Based on the experimental data, hUK-66 represents a promising candidate for an antibody-based therapy against antibiotic resistant MRSA.}, language = {en} } @phdthesis{Leimbach2017, author = {Leimbach, Andreas}, title = {Genomics of pathogenic and commensal \(Escherichia\) \(coli\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154539}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {High-throughput sequencing (HTS) has revolutionized bacterial genomics. Its unparalleled sensitivity has opened the door to analyzing bacterial evolution and population genomics, dispersion of mobile genetic elements (MGEs), and within-host adaptation of pathogens, such as Escherichia coli. One of the defining characteristics of intestinal pathogenic E. coli (IPEC) pathotypes is a specific repertoire of virulence factors (VFs). Many of these IPEC VFs are used as typing markers in public health laboratories to monitor outbreaks and guide treatment options. Instead, extraintestinal pathogenic E. coli (ExPEC) isolates are genotypically diverse and harbor a varied set of VFs -- the majority of which also function as fitness factors (FFs) for gastrointestinal colonization. The aim of this thesis was the genomic characterization of pathogenic and commensal E. coli with respect to their virulence- and antibiotic resistance-associated gene content as well as phylogenetic background. In order to conduct the comparative analyses, I created a database of E. coli VFs, ecoli_VF_collection, with a focus on ExPEC virulence-associated proteins (Leimbach, 2016b). Furthermore, I wrote a suite of scripts and pipelines, bac-genomics-scripts, that are useful for bacterial genomics (Leimbach, 2016a). This compilation includes tools for assembly and annotation as well as comparative genomics analyses, like multi-locus sequence typing (MLST), assignment of Clusters of Orthologous Groups (COG) categories, searching for protein homologs, detection of genomic regions of difference (RODs), and calculating pan-genome-wide association statistics. Using these tools we were able to determine the prevalence of 18 autotransporters (ATs) in a large, phylogenetically heterogeneous strain panel and demonstrate that many AT proteins are not associated with E. coli pathotypes. According to multivariate analyses and statistics the distribution of AT variants is instead significantly dependent on phylogenetic lineages. As a consequence, ATs are not suitable to serve as pathotype markers (Zude et al., 2014). During the German Shiga toxin-producing E. coli (STEC) outbreak in 2011, the largest to date, we were one of the teams capable of analyzing the genomic features of two isolates. Based on MLST and detection of orthologous proteins to known E. coli reference genomes the close phylogenetic relationship and overall genome similarity to enteroaggregative E. coli (EAEC) 55989 was revealed. In particular, we identified VFs of both STEC and EAEC pathotypes, most importantly the prophage-encoded Shiga toxin (Stx) and the pAA-type plasmid harboring aggregative adherence fimbriae. As a result, we could show that the epidemic was caused by an unusual hybrid pathotype of the O104:H4 serotype. Moreover, we detected the basis of the antibiotic multi-resistant phenotype on an extended-spectrum beta-lactamase (ESBL) plasmid through comparisons to reference plasmids. With this information we proposed an evolutionary horizontal gene transfer (HGT) model for the possible emergence of the pathogen (Brzuszkiewicz et al., 2011). Similarly to ExPEC, E. coli isolates of bovine mastitis are genotypically and phenotypically highly diverse and many studies struggled to determine a positive association of putative VFs. Instead the general E. coli pathogen-associated molecular pattern (PAMP), lipopolysaccharide (LPS), is implicated as a deciding factor for intramammary inflammation. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype was proposed presumably encompassing strains more adapted to elicit bovine mastitis with virulence traits differentiating them from commensals. We sequenced eight E. coli isolates from udder serous exudate and six fecal commensals (Leimbach et al., 2016). Two mastitis isolate genomes were closed to a finished-grade quality (Leimbach et al., 2015). The genomic sequence of mastitis-associated E. coli (MAEC) strain 1303 was used to elucidate the biosynthesis gene cluster of its O70 LPS O-antigen. We analyzed the phylogenetic genealogy of our strain panel plus eleven bovine-associated E. coli reference strains and found that commensal or MAEC could not be unambiguously allocated to specific phylogroups within a core genome tree of reference E. coli. A thorough gene content analysis could not identify functional convergence of either commensal or MAEC, instead both have only very few gene families enriched in either pathotype. Most importantly, gene content and ecoli_VF_collection analyses showed that no virulence determinants are significantly associated with MAEC in comparison to bovine fecal commensals, disproving the MPEC hypothesis. The genetic repertoire of bovine-associated E. coli, again, is dominated by phylogenetic background. This is also mostly the case for large virulence-associated E. coli gene cluster previously associated with mastitis. Correspondingly, MAEC are facultative and opportunistic pathogens recruited from the bovine commensal gastrointestinal microbiota (Leimbach et al., 2017). Thus, E. coli mastitis should be prevented rather than treated, as antibiotics and vaccines have not proven effective. Although traditional E. coli pathotypes serve a purpose for diagnostics and treatment, it is clear that the current typing system is an oversimplification of E. coli's genomic plasticity. Whole genome sequencing (WGS) revealed many nuances of pathogenic E. coli, including emerging hybrid or heteropathogenic pathotypes. Diagnostic and public health microbiology need to embrace the future by implementing HTS techniques to target patient care and infection control more efficiently.}, subject = {Escherichia coli}, language = {en} } @article{HickeySridharWestermannetal.2012, author = {Hickey, Scott F. and Sridhar, Malathy and Westermann, Alexander J. and Qin, Qian and Vijayendra, Pooja and Liou, Geoffrey and Hammond, Ming C.}, title = {Transgene regulation in plants by alternative splicing of a suicide exon}, series = {Nucleic Acids Research}, volume = {40}, journal = {Nucleic Acids Research}, number = {10}, doi = {10.1093/nar/gks032}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134724}, pages = {4701-4710}, year = {2012}, abstract = {Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation.}, language = {en} } @article{AfonsoGrunzHoffmeierMuelleretal.2015, author = {Afonso-Grunz, Fabian and Hoffmeier, Klaus and M{\"u}ller, S{\"o}ren and Westermann, Alexander J. and Rotter, Bj{\"o}rn and Vogel, J{\"o}rg and Winter, Peter and Kahl, G{\"u}nter}, title = {Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells}, series = {BMC Genomics}, volume = {16}, journal = {BMC Genomics}, number = {323}, doi = {10.1186/s12864-015-1489-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143230}, year = {2015}, abstract = {Background: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro-and eukaryotic cells without prior fixation or physical disruption of the interaction. Results: Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75\% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells. Conclusions: Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium.}, language = {en} } @article{RodriguezRicoYepesetal.2015, author = {Rodriguez, H{\´e}ctor and Rico, Sergio and Yepes, Ana and Franco-Echevarr{\´i}a, Elsa and Antoraz, Sergio and Santamar{\´i}a, Ram{\´o}n I. and D{\´i}az, Margerita}, title = {The two kinases, AbrC1 and AbrC2, of the atypical two-component system AbrC are needed to regulate antibiotic production and differentiation in Streptomyces coelicolor}, series = {Frontiers in Microbiology}, volume = {6}, journal = {Frontiers in Microbiology}, number = {450}, doi = {10.3389/fmicb.2015.00450}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143048}, year = {2015}, abstract = {Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. gRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites.}, language = {en} } @article{NguyenKraftYuetal.2015, author = {Nguyen, Minh Thu and Kraft, Beatrice and Yu, Wenqi and Demicrioglu, Dogan Doruk and Hertlein, Tobias and Burian, Marc and Schmaler, Mathias and Boller, Klaus and Bekeredjian-Ding, Isabelle and Ohlsen, Knut and Schittek, Birgit and G{\"o}tz, Friedrich}, title = {The vSa\(\alpha\) Specific Lipoprotein Like Cluster (lpl) of S. aureus USA300 Contributes to Immune Stimulation and Invasion in Human Cells}, series = {PLoS Pathogens}, volume = {11}, journal = {PLoS Pathogens}, number = {6}, doi = {10.1371/journal.ppat.1004984}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151856}, pages = {e1004984}, year = {2015}, abstract = {All Staphylococcus aureus genomes contain a genomic island, which is termed vSa\(\alpha\) and characterized by two clusters of tandem repeat sequences, i.e. the exotoxin (set) and 'lipoprotein-like' genes (lpl). Based on their structural similarities the vSa\(\alpha\) islands have been classified as type I to IV. The genomes of highly pathogenic and particularly epidemic S. aureus strains (USA300, N315, Mu50, NCTC8325, Newman, COL, JH1 or JH9) belonging to the clonal complexes CC5 and CC8 bear a type I vSa\(\alpha\) island. Since the contribution of the lpl gene cluster encoded in the vSa\(\alpha\) island to virulence is unclear to date, we deleted the entire lpl gene cluster in S. aureus USA300. The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes. Purified lipoprotein Lpl1 was further shown to elicit a TLR2-dependent response. Furthermore, heterologous expression of the USA300 lpl cluster in other S. aureus strains enhanced their immune stimulatory activity. Most importantly, the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin and the non-invasive S. carnosus expressing the lpl gene cluster became invasive. Additionally, in a murine kidney abscess model the bacterial burden in the kidneys was higher in wild type than in mutant mice. In this infection model the lpl cluster, thus, contributes to virulence. The present report is one of the first studies addressing the role of the vSa\(\alpha\) encoded lpl gene cluster in staphylococcal virulence. The finding that the lpl gene cluster contributes to internalization into non-professional antigen presenting cells such as keratinocytes high-lights the lpl as a new cell surface component that triggers host cell invasion by S. aureus. Increased invasion in murine skin and an increased bacterial burden in a murine kidney abscess model suggest that the lpl gene cluster serves as an important virulence factor.}, language = {en} } @article{EspinaPaganLopezetal.2015, author = {Espina, Laura and Pag{\´a}n, Rafael and L{\´o}pez, Daniel and Garc{\´i}a-Gonzalo, Diego}, title = {Individual Constituents from Essential Oils Inhibit Biofilm Mass Production by Multi-Drug Resistant Staphylococcus aureus}, series = {Molecules}, volume = {20}, journal = {Molecules}, doi = {10.3390/molecules200611357}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151845}, pages = {11357 -- 11372}, year = {2015}, abstract = {Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings.}, language = {en} } @unpublished{Bartfeld2016, author = {Bartfeld, Sina}, title = {Modeling infectious diseases and host-microbe interactions in gastrointestinal organoids}, series = {Developmental Biology}, journal = {Developmental Biology}, issn = {0012-1606}, doi = {http://dx.doi.org/10.1016/j.ydbio.2016.09.014}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-138788}, year = {2016}, abstract = {Advances in stem cell research have allowed the development of 3-dimensional (3D) primary cell cultures termed organoid cultures, as they closely mimic the in vivo organization of different cell lineages. Bridging the gap between 2-dimensional (2D) monotypic cancer cell lines and whole organisms, organoids are now widely applied to model development and disease. Organoids hold immense promise for addressing novel questions in host-microbe interactions, infectious diseases and the resulting inflammatory conditions. Researchers have started to use organoids for modeling infection with pathogens, such as Helicobacter pylori or Salmonella enteritica, gut- microbiota interactions and inflammatory bowel disease. Future studies will broaden the spectrum of microbes used and continue to establish organoids as a standard model for human host-microbial interactions. Moreover, they will increasingly exploit the unique advantages of organoids, for example to address patient-specific responses to microbes.}, language = {en} } @phdthesis{Sharan2017, author = {Sharan, Malvika}, title = {Bio-computational identification and characterization of RNA-binding proteins in bacteria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153573}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {RNA-binding proteins (RBPs) have been extensively studied in eukaryotes, where they post-transcriptionally regulate many cellular events including RNA transport, translation, and stability. Experimental techniques, such as cross-linking and co-purification followed by either mass spectrometry or RNA sequencing has enabled the identification and characterization of RBPs, their conserved RNA-binding domains (RBDs), and the regulatory roles of these proteins on a genome-wide scale. These developments in quantitative, high-resolution, and high-throughput screening techniques have greatly expanded our understanding of RBPs in human and yeast cells. In contrast, our knowledge of number and potential diversity of RBPs in bacteria is comparatively poor, in part due to the technical challenges associated with existing global screening approaches developed in eukaryotes. Genome- and proteome-wide screening approaches performed in silico may circumvent these technical issues to obtain a broad picture of the RNA interactome of bacteria and identify strong RBP candidates for more detailed experimental study. Here, I report APRICOT ("Analyzing Protein RNA Interaction by Combined Output Technique"), a computational pipeline for the sequence-based identification and characterization of candidate RNA-binding proteins encoded in the genomes of all domains of life using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences of an input proteome using position-specific scoring matrices and hidden Markov models of all conserved domains available in the databases and then statistically score them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them according to functionally relevant structural properties. APRICOT performed better than other existing tools for the sequence-based prediction on the known RBP data sets. The applications and adaptability of the software was demonstrated on several large bacterial RBP data sets including the complete proteome of Salmonella Typhimurium strain SL1344. APRICOT reported 1068 Salmonella proteins as RBP candidates, which were subsequently categorized using the RBDs that have been reported in both eukaryotic and bacterial proteins. A set of 131 strong RBP candidates was selected for experimental confirmation and characterization of RNA-binding activity using RNA co-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) experiments. Based on the relative abundance of transcripts across the RIP-Seq libraries, a catalogue of enriched genes was established for each candidate, which shows the RNA-binding potential of 90\% of these proteins. Furthermore, the direct targets of few of these putative RBPs were validated by means of cross-linking and co-immunoprecipitation (CLIP) experiments. This thesis presents the computational pipeline APRICOT for the global screening of protein primary sequences for potential RBPs in bacteria using RBD information from all kingdoms of life. Furthermore, it provides the first bio-computational resource of putative RBPs in Salmonella, which could now be further studied for their biological and regulatory roles. The command line tool and its documentation are available at https://malvikasharan.github.io/APRICOT/.}, language = {en} } @article{MasicHurdayalNieuwenhuizenetal.2012, author = {Masic, Anita and Hurdayal, Ramona and Nieuwenhuizen, Natalie E. and Brombacher, Frank and Moll, Heidrun}, title = {Dendritic Cell-Mediated Vaccination Relies on Interleukin-4 Receptor Signaling to Avoid Tissue Damage after Leishmania major Infection of BALB/c Mice}, series = {PLoS Neglected Tropical Diseases}, volume = {6}, journal = {PLoS Neglected Tropical Diseases}, number = {7}, doi = {10.1371/journal.pntd.0001721}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133869}, year = {2012}, abstract = {Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor \(\alpha\) (IL-4R \(\alpha\))-deficient (CD11c\(^{cre}\)IL-4R \(\alpha^{-/lox}\) BALB/c mice were given either wt or IL-4R \(\alpha\)-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2x10\(^5\) stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4R alpha-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4R alpha-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c\(^{cre}\)IL-4R \(\alpha^{-/lox}\) mice immunized with CpG ODN-exposed LmAg-loaded IL-4R \(\alpha\)-deficient DC, indicating the influence of IL-4R \(\alpha\)-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4R \(\alpha\) signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.}, language = {en} } @phdthesis{HagmanngebKischkies2016, author = {Hagmann [geb. Kischkies], Laura Violetta}, title = {Stringent response regulation and its impact on ex vivo survival in the commensal pathogen \(Neisseria\) \(meningitidis\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144352}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments.}, subject = {Neisseria meningitidis}, language = {en} } @article{SchmidtkeFindeissSharmaetal.2011, author = {Schmidtke, Cornelius and Findeiß, Sven and Sharma, Cynthia M. and Kuhfuss, Juliane and Hoffmann, Steve and Vogel, J{\"o}rg and Stadler, Peter F. and Bonas, Ulla}, title = {Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions}, series = {Nucleic Acids Research}, volume = {40}, journal = {Nucleic Acids Research}, number = {5}, doi = {10.1093/nar/gkr904}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131781}, pages = {2020 -- 2031}, year = {2011}, abstract = {The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14\% of all mRNAs are leaderless and 13\% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.}, language = {en} } @article{PilsKoppPetersonetal.2012, author = {Pils, Stefan and Kopp, Kathrin and Peterson, Lisa and Tascon, Julia Delgado and Nyffenegger-Jann, Naja J. and Hauck, Christof R.}, title = {The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {3}, doi = {10.1371/journal.pone.0032808}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131747}, pages = {e32808}, year = {2012}, abstract = {Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.}, language = {en} } @article{RamachandranShearerJacobetal.2012, author = {Ramachandran, Vinoy K. and Shearer, Neil and Jacob, Jobin J. and Sharma, Cynthia M. and Thompson, Arthur}, title = {The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression}, series = {BMC Genomics}, volume = {13}, journal = {BMC Genomics}, number = {25}, doi = {10.1186/1471-2164-13-25}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130625}, year = {2012}, abstract = {Background: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (STEX), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wildtype S. Typhimurium and a ppGpp null strain under growth conditions which model STEX. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium STEX primary transcriptome than previously recognised. Results: Here we report the precise mapping of transcriptional start sites (TSSs) for 78\% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34\% and 20\% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment. Conclusions: The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research.}, language = {en} } @article{WangChenMinevetal.2012, author = {Wang, Huiqiang and Chen, Nanhai G. and Minev, Boris R. and Szalay, Aladar A.}, title = {Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells}, series = {Journal of Translational Medicine}, volume = {10}, journal = {Journal of Translational Medicine}, number = {167}, doi = {10.1186/1479-5876-10-167}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130019}, year = {2012}, abstract = {Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells. Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.}, language = {en} }