• Collection and analysis of salivary proteins from the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae).

      Langner, Kathrin F A; Darpel, Karin E; Denison, Eric; Drolet, Barbara S; Leibold, Wolfgang; Mellor, Philip S; Mertens, Peter P C; Nimtz, Manfred; Greiser-Wilke, Irene; USDA-ARS, Arthropod-Borne Animal Diseases Research Laboratory, 1000 E. University Ave., Laramie, WY 82071, USA. klangner@uwyo.edu (2007-03)
      Salivary proteins of hematophagous Culicoides spp. are thought to play an important role in pathogen transmission and skin hypersensitivity. Analysis of these proteins, however, has been problematic due to the difficulty in obtaining adequate amounts of secreted Culicoides saliva. In the current study, a collection method for midge saliva was developed. Over a 3-d period, 3- to 5-d-old male and female Culicoides nubeculosus Meigen (Diptera: Ceratopogonidae) were repeatedly placed onto the collection system and allowed to deposit saliva into a filter. Salivary products were eluted from the filters and evaluated by gel electrophoresis and mass spectrometry as well as by intradermal testing and determination of clotting time. Gel electrophoresis revealed approximately 55 protein spots displaying relative molecular masses from 5 to 67 kDa and isoelectric points ranging from 4.5 to 9.8. The majority of molecular species analyzed by mass spectrometry showed high convergence with salivary proteins recently obtained from a cDNA library of Culicoides sonorensis Wirth & Jones, including proteins involved in sugarmeal digestion, defense, and coagulation inhibition as well as members of the D7 family and unclassified salivary proteins. In addition, the proteome analysis revealed a number of peptides that were related to proteins from insect species other than Culicoides. Intradermal injection of the saliva in human skin produced edema, vasodilatation, and pruritus. The anticoagulant activity of the saliva was demonstrated by significantly prolonged clotting times for human platelets. The potential role of the identified salivary proteins in the transmission of pathogens and the induction of allergies is discussed.
    • Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.

      Baumgärtner, Maja; Kärst, Uwe; Gerstel, Birgit; Loessner, Martin; Wehland, Jürgen; Jänsch, Lothar; Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), D-38124 Braunschweig, Germany. (2007-01)
      Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.
    • The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis.

      Dahl, Jan-Ulrik; Radon, Christin; Bühning, Martin; Nimtz, Manfred; Leichert, Lars I; Denis, Yann; Jourlin-Castelli, Cécile; Iobbi-Nivol, Chantal; Méjean, Vincent; Leimkühler, Silke; et al. (2013-02-22)
      The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.