74 research outputs found
Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants
10.1371/journal.ppat.1003948PLoS Pathogens103
Genome Sequencing and Analysis of a Type A Clostridium perfringens Isolate from a Case of Bovine Clostridial Abomasitis
Clostridium perfringens is a common inhabitant of the avian and mammalian gastrointestinal tracts and can behave commensally or pathogenically. Some enteric diseases caused by type A C. perfringens, including bovine clostridial abomasitis, remain poorly understood. To investigate the potential basis of virulence in strains causing this disease, we sequenced the genome of a type A C. perfringens isolate (strain F262) from a case of bovine clostridial abomasitis. The ∼3.34 Mbp chromosome of C. perfringens F262 is predicted to contain 3163 protein-coding genes, 76 tRNA genes, and an integrated plasmid sequence, Cfrag (∼18 kb). In addition, sequences of two complete circular plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), and two incomplete plasmid fragments, pF262A (48.5 kb) and pF262B (50.0 kb), were identified. Comparison of the chromosome sequence of C. perfringens F262 to complete C. perfringens chromosomes, plasmids and phages revealed 261 unique genes. No novel toxin genes related to previously described clostridial toxins were identified: 60% of the 261 unique genes were hypothetical proteins. There was a two base pair deletion in virS, a gene reported to encode the main sensor kinase involved in virulence gene activation. Despite this frameshift mutation, C. perfringens F262 expressed perfringolysin O, alpha-toxin and the beta2-toxin, suggesting that another regulation system might contribute to the pathogenicity of this strain. Two complete plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), unique to this strain of C. perfringens were identified
The stb Operon Balances the Requirements for Vegetative Stability and Conjugative Transfer of Plasmid R388
The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed
A role for the tfs3 ICE-encoded type IV secretion system in pro-inflammatory signalling by the Helicobacter pylori Ser/Thr kinase, CtkA
Two distinct type IV secretion systems (T4SSs) can be identified in certain Helicobacter pylori strains, encoded on mobile genetic elements termed tfs3 and tfs4. Although their function remains unknown, both have been implicated in clinical outcomes of H. pylori infection. Here we provide evidence that the Tfs3 T4SS is required for activity of the pro-inflammatory Ser/Thr kinase protein, CtkA, in a gastric epithelial cell infection model. Previously, purified recombinant CtkA protein has been shown to upregulate NF-kappaB signalling and induce TNF-alpha and IL-8 cytokine secretion from cultured macrophages suggesting that it may potentiate the H. pylori-mediated inflammatory response. In this study, we show that CtkA expressed from its native host, H. pylori has a similar capacity for stimulation of a pro-inflammatory response from gastric epithelial cells. CtkA interaction was found to be dependent upon a complement of tfs3 T4SS genes, but independent of the T4SSs encoded by either tfs4 or the cag pathogenicity island. Moreover, the availability of CtkA for host cell interaction was shown to be conditional upon the carboxyl-terminus of CtkA, encoding a putative conserved secretion signal common to other variably encoded Tfs3 proteins. Collectively, our observations indicate a role for the Tfs3 T4SS in CtkA-mediated pro-inflammatory signalling by H. pylori and identify CtkA as a likely Tfs3 T4SS secretion substrate
Anaplasma phagocytophilum Ats-1 Is Imported into Host Cell Mitochondria and Interferes with Apoptosis Induction
Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, infects human neutrophils and inhibits mitochondria-mediated apoptosis. Bacterial factors involved in this process are unknown. In the present study, we screened a genomic DNA library of A. phagocytophilum for effectors of the type IV secretion system by a bacterial two-hybrid system, using A. phagocytophilum VirD4 as bait. A hypothetical protein was identified as a putative effector, hereby named Anaplasma translocated substrate 1 (Ats-1). Using triple immunofluorescence labeling and Western blot analysis of infected cells, including human neutrophils, we determined that Ats-1 is abundantly expressed by A. phagocytophilum, translocated across the inclusion membrane, localized in the host cell mitochondria, and cleaved. Ectopically expressed Ats-1 targeted mitochondria in an N-terminal 17 residue-dependent manner, localized in matrix or at the inner membrane, and was cleaved as native protein, which required residues 55–57. In vitro-translated Ats-1 was imported in a receptor-dependent manner into isolated mitochondria. Ats-1 inhibited etoposide-induced cytochrome c release from mitochondria, PARP cleavage, and apoptosis in mammalian cells, as well as Bax-induced yeast apoptosis. Ats-1(55–57) had significantly reduced anti-apoptotic activity. Bax redistribution was inhibited in both etoposide-induced and Bax-induced apoptosis by Ats-1. Taken together, Ats-1 is the first example of a bacterial protein that traverses five membranes and prevents apoptosis at the mitochondria
Solid lipid nanoparticles of irbesartan: preparation, characterization, optimization and pharmacokinetic studies
Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB
Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening?closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA
Structural studies of T4S systems by electron microscopy
Abstract: Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNA and/or proteins through the membranes of bacteria. Analysis of T4S system architecture is an extremely challenging task taking into account their multi protein organisation and lack of overall global symmetry. Nonetheless the last decade demonstrated an amazing progress achieved by X-ray crystallography and cryo-electron microscopy. In this review we present a structural analysis of this dynamic complex based on recent advances in biochemical, biophysical and structural studies
Identification, structure and mode of action of a new regulator of the Helicobacter pylori HP0525 ATPase
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Not AvailableHeat shock proteins (HSPs) play a pivotal role in cell growth and variability. Since conventional approaches are expensive and voluminous protein sequence information is available in the post-genomic era, development of an automated and accurate computational tool is highly desirable for prediction of HSPs, their families and sub-types. Thus, we propose a computational approach for reliable prediction of all these components in a single framework and with higher accuracy as well. The proposed approach achieved an overall accuracy of ∼84% in predicting HSPs, ∼97% in predicting six different families of HSPs, and ∼94% in predicting four types of DnaJ proteins, with bench mark datasets. The developed approach also achieved higher accuracy as compared to most of the existing approaches. For easy prediction of HSPs by experimental scientists, a user friendly web server ir-HSP is made freely accessible at http://cabgrid.res.in:8080/ir-hsp. The ir-HSP was further evaluated for proteome-wide identification of HSPs by using proteome datasets of eight different species, and ∼50% of the predicted HSPs in each species were found to be annotated with InterPro HSP families/domains. Thus, the developed computational method is expected to supplement the currently available approaches for prediction of HSPs, to the extent of their families and sub-types.Not Availabl
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