• Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles.

      Darpel, Karin E; Langner, Kathrin F A; Nimtz, Manfred; Anthony, Simon J; Brownlie, Joe; Takamatsu, Haru-Hisa; Mellor, Philip S; Mertens, Peter P C; Pirbright Laboratory, Vector-borne Disease Programme, Institute for Animal Health, Woking, United Kingdom. karin.darpel@bbsrc.ac.uk (2011)
      Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.
    • Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD.

      Büttner, Carina R; Sorg, Isabel; Cornelis, Guy R; Heinz, Dirk W; Niemann, Hartmut H; Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2008-01-25)
      Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 A resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely alpha-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.