Lysine Phoshoglycerylation Is Widespread in Bacteria and Overlaps with Acylation.

Phosphoglycerylation is a non-enzymatic protein modification in which a phosphoglyceryl moiety is covalently bound to the ε-amino group of lysine. It is enriched in glycolytic enzymes from humans and mice and is thought to provide a feedback mechanism for regulating glycolytic flux. We report the first proteomic analysis of this post-translational modification in bacteria by profiling phosphoglyceryl-lysine during the growth of in different culture media.

Dynamic Protein Phosphorylation in during Growth, Stationary Phase, and Starvation.

Phosphorylation of proteins at serine, threonine, and tyrosine residues plays an important role in physiological processes of bacteria, such as cell cycle, metabolism, virulence, dormancy, and stationary phase functions. Little is known about the targets and dynamics of protein phosphorylation in , which possesses a single known transmembrane serine/threonine kinase belonging to the class of PASTA kinases. A proteomics and phosphoproteomics workflow was performed with serotype M49 under different growth conditions, stationary phase, and starvation.

Synthetic mRNA delivered to human cells leads to expression of Cpl-1 bacteriophage-endolysin with activity against .

Endolysins are bacteriophage-encoded hydrolases that show high antibacterial activity and a narrow substrate spectrum. We hypothesize that an mRNA-based approach to endolysin therapy can overcome some challenges of conventional endolysin therapy, namely organ targeting and bioavailability. We show that synthetic mRNA applied to three human cell lines (HEK293T, A549, HepG2 cells) leads to expression and cytosolic accumulation of the Cpl-1 endolysin with activity against Addition of a human lysozyme signal peptide sequence translocates the Cpl-1 to the endoplasmic reticulum leading to secretion (hlySP-sCpl-1).

Pyrenebutyrate Enhances the Antibacterial Effect of Peptide-Coupled Antisense Peptide Nucleic Acids in .

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.

Antimicrobial Activity of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pneumoniae.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for multiple other infectious diseases, such as meningitis and otitis media, in children. Resistance to penicillins, macrolides, and fluoroquinolones is increasing and, since the introduction of pneumococcal conjugate vaccines (PCVs), vaccine serotypes have been replaced by non-vaccine serotypes. Antisense peptide nucleic acids (PNAs) have been shown to reduce the growth of several pathogenic bacteria in various infection models.

Validation of Suitable Carrier Molecules and Target Genes for Antisense Therapy Using Peptide-Coupled Peptide Nucleic Acids (PNAs) in Streptococci.

Antisense peptide nucleic acids (PNAs) targeting genes involved in metabolism or virulence are a possible means to treat infections or to investigate pathogenic bacteria. Potential targets include essential genes, virulence factor genes, or antibiotic resistance genes. For efficient cellular uptake, PNAs can be coupled to cell-penetrating peptides (CPPs).

Volatile scents of influenza A and S. pyogenes (co-)infected cells.

Influenza A is a serious pathogen itself, but often leads to dangerous co-infections in combination with bacterial species such as Streptococcus pyogenes. In comparison to classical biochemical methods, analysis of volatile organic compounds (VOCs) in headspace above cultures can enable destruction free monitoring of metabolic processes in vitro. Thus, volatile biomarkers emitted from biological cell cultures and pathogens could serve for monitoring of infection processes in vitro.

Influence of Different Cell-Penetrating Peptides on the Antimicrobial Efficiency of PNAs in Streptococcus pyogenes.

Streptococcus pyogenes is an exclusively human pathogen causing a wide range of clinical manifestations from mild superficial infections to severe, life-threatening, invasive diseases. S. pyogenes is consistently susceptible toward penicillin, but therapeutic failure of penicillin treatment has been reported frequently.

A Glycine Riboswitch in 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 serotype M49 strain 591. Glycine-dependent gene expression mediated by riboswitch activity was studied using a luciferase reporter gene system.

The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes.

Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced.

Quantification of DNA Damage and Repair in Mitochondrial, Nuclear, and Bacterial Genomes by Real-Time PCR.

DNA damage caused by genotoxic insults is often used as an indicator of specific diseases, environmental challenges, and metabolic processes. To date, various different methods have been described to detect damaged DNA. Many techniques need high amounts of DNA for the analysis and/or require the exact determination of DNA template concentration.

Genome-wide analyses of small non-coding RNAs in streptococci.

Streptococci represent a diverse group of Gram-positive bacteria, which colonize a wide range of hosts among animals and humans. Streptococcal species occur as commensal as well as pathogenic organisms. Many of the pathogenic species can cause severe, invasive infections in their hosts leading to a high morbidity and mortality.

Inhibition of Growth and Gene Expression by PNA-peptide Conjugates in Streptococcus pyogenes.

While Streptococcus pyogenes is consistently susceptible toward penicillin, therapeutic failure of penicillin treatment has been reported repeatedly and a considerable number of patients exhibit allergic reactions to this substance. At the same time, streptococcal resistance to alternative antibiotics, e.g.

Common regulators of virulence in streptococci.

Streptococcal species are a diverse group of bacteria which can be found in animals and humans. Their interactions with host organisms can vary from commensal to pathogenic. Many of the pathogenic species are causative agents of severe, invasive infections in their hosts, accounting for a high burden of morbidity and mortality, associated with high economic costs in industry and health care.

Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays.

Background: Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action.

Streptococcus pyogenes M49 plasminogen/plasmin binding facilitates keratinocyte invasion via integrin-integrin-linked kinase (ILK) pathways and protects from macrophage killing.

The entry into epithelial cells and the prevention of primary immune responses are a prerequisite for a successful colonization and subsequent infection of the human host by Streptococcus pyogenes (group A streptococci, GAS). Here, we demonstrate that interaction of GAS with plasminogen promotes an integrin-mediated internalization of the bacteria into keratinocytes, which is independent from the serine protease activity of potentially generated plasmin. α(1)β(1)- and α(5)β(1)-integrins were identified as the major keratinocyte receptors involved in this process.

Insights into Streptococcus pyogenes pathogenesis from transcriptome studies.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a major human pathogen, causing diseases ranging from mild superficial infections of the skin and pharyngeal mucosal membrane, up to severe systemic and invasive diseases and autoimmune sequelae. The capability of GAS to cause this wide variety of infections is due to the expression of a large set of virulence factors, their concerted transcriptional regulation, and bacterial adaptation mechanisms to various host niches, which we are now beginning to understand on a molecular level. The addition of -omics technologies for GAS pathogenesis investigation, on top of traditional molecular methods, led to fast progress in understanding GAS pathogenesis mechanisms.

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

Background: 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.

Leucine-rich repeat kinase 2 induces alpha-synuclein expression via the extracellular signal-regulated kinase pathway.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes alpha-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD.

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.

Parkin protects mitochondrial genome integrity and supports mitochondrial DNA repair.

Mutations in the parkin gene are the most common cause of recessive familial Parkinson disease (PD). Parkin has been initially characterized as an ubiquitin E3 ligase, but the pathological relevance of this activity remains uncertain. Recently, an impressive amount of evidence has accumulated that parkin is involved in the maintenance of mitochondrial function and biogenesis.

Parkin protects against tyrosinase-mediated dopamine neurotoxicity by suppressing stress-activated protein kinase pathways.

Parkinson's disease (PD) motor symptoms are caused by degeneration of nigrostriatal dopaminergic (DAergic) neurons. The most common causes of hereditary PD are mutations in the PARKIN gene. The ubiquitin ligase parkin has been shown to mediate neuroprotection in cell culture and in vivo, but the molecular mechanisms are not well understood.