The contact system proteases play disparate roles in streptococcal sepsis

Sepsis causes an activation of the human contact system, an inflammatory response mechanism against foreign surfaces, proteins and pathogens. The serine proteases of the contact system, factor XII and plasma kallikrein, are decreased in plasma of septic patients, which was previously associated with an unfavorable outcome. However, the precise mechanisms and roles of contact system factors in bacterial sepsis are poorly understood. We, therefore, studied the physiological relevance of factor XII and plasma kallikrein in a mouse model of experimental sepsis. We show that decreased plasma kallikrein concentration in septic mice is a result of reduced mRNA expression plasma prekallikrein gene, indicating that plasma kallikrein belong to negative acute phase proteins. Investigations regarding the pathophysiological function of contact system proteases during sepsis revealed different roles for factor XII and plasma kallikrein. In vitro, factor XII decelerated bacteria induced fibrinolysis, whereas plasma kallikrein supported it. Remarkably, depletion of plasma kallikrein (but not factor XII) by treatment with antisense-oligonucleotides, dampens bacterial dissemination and growth in multiple organs in the mouse sepsis model. These findings identify plasma kallikrein as a novel host pathogenicity factor in Streptococcus pyogenes sepsis

The Cyanobacterial Hepatotoxin Microcystin Binds to Proteins and Increases the Fitness of Microcystis under Oxidative Stress Conditions

Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites

Membrane Protein Identification in Rodent Brain Tissue Samples and Acute Brain Slices

Acute brain slices are a sample format for electrophysiology, disease modeling, and organotypic cultures. Proteome analyses based on mass spectrometric measurements are seldom used on acute slices, although they offer high-content protein analyses and explorative approaches. In neuroscience, membrane proteins are of special interest for proteome-based analysis as they are necessary for metabolic, electrical, and signaling functions, including myelin maintenance and regeneration. A previously published protocol for the enrichment of plasma membrane proteins based on aqueous two-phase polymer systems followed by mass spectrometric protein identification was adjusted to the small sample size of single acute murine slices from newborn animals and the reproducibility of the results was analyzed. For this, plasma membrane proteins of 12 acute slice samples from six animals were enriched and analyzed by liquid chromatography-mass spectrometry. A total of 1161 proteins were identified, of which 369 were assigned to membranes. Protein abundances showed high reproducibility between samples. The plasma membrane protein separation protocol can be applied to single acute slices despite the low sample size and offers a high yield of identifiable proteins. This is not only the prerequisite for proteome analysis of organotypic slice cultures but also allows for the analysis of small-sized isolated brain regions at the proteome level

Profiling stage-dependent changes of protein expression in Caenorhabditis elegans by mass spectrometric proteome analysis leads to the identification of stage-specific marker proteins

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Mitochondrial Protein Lipoylation Does Not Exclusively Depend on the mtKAS Pathway of de Novo Fatty Acid Synthesis in Arabidopsis1[W][OA]

The photorespiratory Arabidopsis (Arabidopsis thaliana) mutant gld1 (now designated mtkas-1) is deficient in glycine decarboxylase (GDC) activity, but the exact nature of the genetic defect was not known. We have identified the mtkas-1 locus as gene At2g04540, which encodes β-ketoacyl-[acyl carrier protein (ACP)] synthase (mtKAS), a key enzyme of the mitochondrial fatty acid synthetic system. One of its major products, octanoyl-ACP, is regarded as essential for the intramitochondrial lipoylation of several proteins including the H-protein subunit of GDC and the dihydrolipoamide acyltransferase (E2) subunits of two other essential multienzyme complexes, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. This view is in conflict with the fact that the mtkas-1 mutant and two allelic T-DNA knockout mutants grow well under nonphotorespiratory conditions. Although on a very low level, the mutants show residual lipoylation of H protein, indicating that the mutation does not lead to a full functional knockout of GDC. Lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase E2 subunits is distinctly less reduced than that of H protein in leaves and remains unaffected from the mtKAS knockout in roots. These data suggest that mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of lipoate biosynthesis in leaves and may occur independently of this pathway in roots

Proteomic Comparison of Two Invasive Polychaete Species and Their Naturally Occurring F₁-hybrids

The mud worm genus <i>Marenzelleria</i> is highly invasive and is therefore studied intensively. In recently invaded habitats, sympatric populations of the sibling species <i>Marenzelleria viridis</i> and <i>Marenzelleria neglecta</i> are found. In these secondary contact zones, hybridization occurs frequently, revealing incomplete reproductive isolation between these recently diverged species. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometric methods were applied for a comparative analysis of these species and their F₁-hybrids. Nineteen proteins were identified by cross-species identification strategies. A low degree of interindividual variability within either species allowed characterizing qualitative species-specific differences in 2-DE spot patterns as well as in peptide maps. Species-specific peptides were found in tryptic digests of various proteins, such as glyceraldehyde-3-phosphate dehydrogenase, troponin C, gelsolin-like protein, and peroxiredoxin-1. F₁-hybrids of <i>M. viridis</i> and <i>M. neglecta</i> showed additivity of protein spot patterns, and the presence of both parental traits was confirmed by mass spectrometric data. This study is one of few dealing with global protein expression in polychaetes and is the first proteomic description of natural F₁-hybrids in polychaetes. It furthermore indicates the feasibility of proteomic methods for analyses of speciation in <i>Marenzelleria</i> siblings as well as of hybridization events in secondary contact zones in general