Abstract: Clavibacter michiganensis, subsp. michiganensis is a Gram-positive plant pathogen infecting tomato (Solanum lycopersicum). Despite a considerable economic importance due to significant losses of infected plants and fruits, knowledge about virulence factors of C. michiganensis subsp. michiganensis and host-pathogen interactions on a molecular level are rather limited. In the study presented here, the proteome of culture supernatants from C. michiganensis subsp. michiganensis NCPPB382 was analyzed. In total, 1872 proteins were identified in M9 and 1766 proteins in xylem mimicking medium. Filtration of supernatants before protein precipitation reduced these to 1276 proteins in M9 and 976 proteins in the xylem mimicking medium culture filtrate. The results obtained indicate that C. michiganensis subsp. michiganensis reacts to a sucrose- and glucose-depleted medium similar to the xylem sap by utilizing amino acids and host cell polymers as well as their degradation products, mainly peptides, amino acids and various C5 and C6 sugars. Interestingly, the bacterium expresses the previously described virulence factors Pat-1 and CelA not exclusively after host cell contact in planta but already in M9 minimal and xylem mimicking medium.
Abstract: Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.
Abstract: The growth and productivity of ruminants depends on a complex microbial community found in their fore-stomach (rumen), which is able to breakdown plant polysaccharides and ferment the released sugars. Butyrivibrio proteoclasticus B316T is a Gram-positive polysaccharide-degrading, butyrate-producing bacterium that is present at high numbers in the rumen of animals consuming pasture or grass silage based diets. B316T is one of a small number of rumen fibrolytic microbes capable of efficiently degrading and utilizing xylan, as well as being capable of utilizing arabinose, xylose, pectin and starch. We have therefore carried out a proteomic analysis of B316T to identify intracellular enzymes that are implicated in the metabolism of internalized xylan. Three hundred and ninety four proteins were identified including enzymes that have potential to metabolize assimilated products of extracellular xylan digestion. Identified enzymes included arabinosidases, esterases, an endoxylanase, and β-xylosidase. The presence of intracellular debranching enzymes indicated that some hemicellulosic side-chains may not be removed until oligosaccharides liberated by extracellular digestion have been assimilated by the cells. The results support a model of extracellular digestion of hemicellulose to oligosaccharides that are then transported to the cytoplasm for further digestion by intracellular enzymes.
Abstract: Antimony (SbIII) and miltefosine (MIL) are important drugs for the treatment of Leishmania parasite infections. The mitochondrion is likely to play a central role in SbIII and MIL induced cell death in this parasite. Enriched mitochondrial samples from Leishmania promastigotes selected step by step for in vitro resistance to SbIII and MIL were subjected to differential proteomic analysis. A shared decrease in both mutants in the levels of pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, and isocitrate dehydrogenase was observed, as well as a differential abundance in two calcium-binding proteins and the unique dynamin-1-like protein of the parasite. Both mutants presented a shared increase in the succinyl-CoA:3-ketoacid-coenzyme A transferase and the abundance of numerous hypothetical proteins was also altered in both mutants. In general, the proteomic changes observed in the MIL mutant were less pronounced than in the SbIII mutant, probably due to the early appearance of a mutation in the miltefosine transporter abrogating the need for a strong mitochondrial adaptation. This study is the first analysis of the Leishmania mitochondrial proteome and offers powerful insights into the adaptations to this organelle during SbIII and MIL drug resistance.
Abstract: The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context.
Abstract: We produced a tear proteome of the genome mouse, C57BL/6, that contained 139 different protein identifications: 110 from a two-dimensional (2D) gel with subsequent trypsin digestion, 19 from a one-dimensional (1D) gel with subsequent trypsin digestion and ten from a 1D gel with subsequent Asp-N digestion. We compared this tear proteome with a C57BL/6 mouse saliva proteome produced previously. Sixteen of the 139 tear proteins are shared between the two proteomes, including six proteins that combat microbial growth. Among the 123 other tear proteins, were members of four large protein families that have no counterparts in humans: Androgen-binding proteins (ABPs) with different members expressed in the two proteomes, Exocrine secreted peptides (ESPs) expressed exclusively in the tear proteome, major urinary proteins (MUPs) expressed in one or both proteomes and the mouse-specific Kallikreins (subfamily b KLKs) expressed exclusively in the saliva proteome. All four families have members with suggested roles in mouse communication, which may influence some aspect of reproductive behavior. We discuss this in the context of functional adaptation involving tear and saliva proteins in the secretions of mouse lacrimal and salivary glands, respectively.