Antisense-Regulation der Genexpression von Streptococcus pyogenes in Virulenz und Therapie

Streptococcus pyogenes ist ein strikt humanpathogenes Bakterium, das weltweit eine hohe Belastung der menschlichen Gesundheit und der Gesundheitssysteme darstellt. In dieser Arbeit lag der Fokus auf der Untersuchung der Antisense-Regulation der Genexpression. Zum einen wurden kleine regulatorische RNAs identifiziert und ihre Bedeutung für die Genregulation in S. pyogenes an zwei Beispielen untersucht. Zum anderen wurde ein Antisense-basierter Therapieansatz mit Carrierpeptid-gekoppelten Peptid-Nukleinsäuren für S. pyogenes etabliert

Analysis of differential DNA damage in the mitochondrial genome employing a semi-long run real-time PCR approach

The maintenance of the mitochondrial genomic integrity is a prerequisite for proper mitochondrial function. Due to the high concentration of reactive oxygen species (ROS) generated by the oxidative phosphorylation pathway, the mitochondrial genome is highly exposed to oxidative stress leading to mitochondrial DNA injury. Accordingly, mitochondrial DNA damage was shown to be associated with ageing as well as with numerous human diseases including neurodegenerative disorders and cancer. To date, several methods have been described to detect damaged mitochondrial DNA, but those techniques are semi-quantitative and often require high amounts of genomic input DNA. We developed a rapid and quantitative method to evaluate the relative levels of damage in mitochondrial DNA by using the real time-PCR amplification of mitochondrial DNA fragments of different lengths. We investigated mitochondrial DNA damage in SH-SY5Y human neuroblastoma cells exposed to hydrogen peroxide or stressed by over-expression of the tyrosinase gene. In the past, there has been speculation about a variable vulnerability to oxidative stress along the mitochondrial genome. Our results indicate the existence of at least one mitochondrial DNA hot spot, namely the D-Loop, being more prone to ROS-derived damage

A Glycine Riboswitch in Streptococcus pyogenes Controls Expression of a Sodium:Alanine Symporter Family Protein Gene

Regulatory RNAs play important roles in the control of bacterial gene expression. In this study, we investigated gene expression regulation by a putative glycine riboswitch located in the 5′-untranslated region of a sodium:alanine symporter family (SAF) protein gene in the group A Streptococcus pyogenes serotype M49 strain 591. Glycine-dependent gene expression mediated by riboswitch activity was studied using a luciferase reporter gene system. Maximal reporter gene expression was observed in the absence of glycine and in the presence of low glycine concentrations. Differences in glycine-dependent gene expression were not based on differential promoter activity. Expression of the SAF protein gene and the downstream putative cation efflux protein gene was investigated in wild-type bacteria by RT-qPCR transcript analyses. During growth in the presence of glycine (≥1 mM), expression of the genes were downregulated. Northern blot analyses revealed premature transcription termination in the presence of high glycine concentrations. Growth in the presence of 0.1 mM glycine led to the production of a full-length transcript. Furthermore, stability of the SAF protein gene transcript was drastically reduced in the presence of glycine. We conclude that the putative glycine riboswitch in S. pyogenes serotype M49 strain 591 represses expression of the SAF protein gene and the downstream putative cation efflux protein gene in the presence of high glycine concentrations. Sequence and secondary structure comparisons indicated that the streptococcal riboswitch belongs to the class of tandem aptamer glycine riboswitches

Non-coding RNA detection methods combined to improve usability, reproducibility and precision

<p>Abstract</p> <p>Background</p> <p>Non-coding RNAs gain more attention as their diverse roles in many cellular processes are discovered. At the same time, the need for efficient computational prediction of ncRNAs increases with the pace of sequencing technology. Existing tools are based on various approaches and techniques, but none of them provides a reliable ncRNA detector yet. Consequently, a natural approach is to combine existing tools. Due to a lack of standard input and output formats combination and comparison of existing tools is difficult. Also, for genomic scans they often need to be incorporated in detection workflows using custom scripts, which decreases transparency and reproducibility.</p> <p>Results</p> <p>We developed a Java-based framework to integrate existing tools and methods for ncRNA detection. This framework enables users to construct transparent detection workflows and to combine and compare different methods efficiently. We demonstrate the effectiveness of combining detection methods in case studies with the small genomes of <it>Escherichia coli</it>, <it>Listeria monocytogenes </it>and <it>Streptococcus pyogenes</it>. With the combined method, we gained 10% to 20% precision for sensitivities from 30% to 80%. Further, we investigated <it>Streptococcus pyogenes </it>for novel ncRNAs. Using multiple methods--integrated by our framework--we determined four highly probable candidates. We verified all four candidates experimentally using RT-PCR.</p> <p>Conclusions</p> <p>We have created an extensible framework for practical, transparent and reproducible combination and comparison of ncRNA detection methods. We have proven the effectiveness of this approach in tests and by guiding experiments to find new ncRNAs. The software is freely available under the GNU General Public License (GPL), version 3 at <url>http://www.sbi.uni-rostock.de/moses</url> along with source code, screen shots, examples and tutorial material.</p

Whole-Genome Sequence of Streptococcus pyogenes Strain 591, Belonging to the Genotype emm49

Patenge N, Rückert C, Bull J, Strey K, Kalinowski J, Kreikemeyer B. Whole-Genome Sequence of Streptococcus pyogenes Strain 591, Belonging to the Genotype emm49. Microbiology Resource Announcements. 2021;10(43): e0081621.Streptococcus pyogenes strain 591 is a clinical isolate belonging to the genotype emm49. It has been intensively studied for its pathogenicity traits. In this study, the complete genome of strain 591 was sequenced. It consists of a chromosome of 1,762,765bp with a G+C content of 38.5%

Assembly of the RAG1/RAG2 Synaptic Complex

Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly

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<p>Regulatory RNAs play important roles in the control of bacterial gene expression. In this study, we investigated gene expression regulation by a putative glycine riboswitch located in the 5′-untranslated region of a sodium:alanine symporter family (SAF) protein gene in the group A Streptococcus pyogenes serotype M49 strain 591. Glycine-dependent gene expression mediated by riboswitch activity was studied using a luciferase reporter gene system. Maximal reporter gene expression was observed in the absence of glycine and in the presence of low glycine concentrations. Differences in glycine-dependent gene expression were not based on differential promoter activity. Expression of the SAF protein gene and the downstream putative cation efflux protein gene was investigated in wild-type bacteria by RT-qPCR transcript analyses. During growth in the presence of glycine (≥1 mM), expression of the genes were downregulated. Northern blot analyses revealed premature transcription termination in the presence of high glycine concentrations. Growth in the presence of 0.1 mM glycine led to the production of a full-length transcript. Furthermore, stability of the SAF protein gene transcript was drastically reduced in the presence of glycine. We conclude that the putative glycine riboswitch in S. pyogenes serotype M49 strain 591 represses expression of the SAF protein gene and the downstream putative cation efflux protein gene in the presence of high glycine concentrations. Sequence and secondary structure comparisons indicated that the streptococcal riboswitch belongs to the class of tandem aptamer glycine riboswitches.</p

Pyrenebutyrate Enhances the Antibacterial Effect of Peptide-Coupled Antisense Peptide Nucleic Acids in <i>Streptococcus pyogenes</i>

Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane