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Question: Discuss about the Analysis And Proposal confidence prediction. Answer: Article analysis The title of the article is A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome and Trigger Inflammation Summary Burkholderia cenocepacia is a Gram-negative environmental pathogen that causes opportunistic infections in the lungs of patients with cystic fibrosis (Juhas et al., 2012). The type VI secretion system of B. cenocepacia is required for its pathogenesis (Rosales-Reyes et al., 2012). This secretion system inactivates the Rho type GTPases, thereby causing downregulation of the phagocytic activity of macrophages, blocks assesmbly of the NADPH oxidase onto the vacuole containing the B. cenocepacia and also causes disruption of the actin cytoskeleton of macrophages (Rosales?Reyes et al., 2012; Toesca, French Miller, 2014). It also causes activation of the secretion of interlekins (IL)-1/18, macrophage pyroptosis and caspase 1 inflammasome (Gavrilin et al., 2012). This study identifies a non VgrG type VI secretion system effector molecule responsible for disruption of the actin cytoskeleton of macrophages. This Type VI secretion system effector is termed as TecA. This TecA effector causes d eamidation of of the Rho-GTPases. The target amino acid of TecA is asparagine. Deamidation of the asparagines 41 causes inactivation of the GTPases and eventual disruption of the actin cytoskeleton of host macrophages. Deamidation of RhoA by TecA causes activation of the Pyrin inflammasome, which in turn causes inflammation in the lungs. Thus, this study characterizes TecA and identifies its role as a non-VgrG type VI secretion system effector and carries out modification of the eukaryotic host, thereby causing host cell death and inflammation. Knowledge gap addressed and the main thesis of the article Very few reports are available regarding the characterization of Type VI secretion system effectors belonging to the non-VgrG family (Jiang et al., 2014; Miyata et al., 2013). Moreover, the physiological functions of the few non-VgrG family Type VI secretion system effectors reported were not identified (Valvano et al., 2015). Moreover, no reports were previously present that dealt with Type VI secretion system effectors of B. cenocepacia that can carry out disruption of the actin cytoskeletons of macrophages, thereby altering their physiology. The main thesis of the paper is to identify and characterize the mechanism of action of the Type VI secretion system effector TecA in B. cenocepacia pathogenesis. Hypothesis and Experimental methods The hypothesis for the first figure is the identification of the tecA gene encoding a non-VgrG family Type VI secretion system effector that can carry out alterations in host targets. The first hypothesis deals with the genetic mapping of tecA, which was found to be located in the chromosome 2 of B. cenocepacia K56-2 strain. B. cenocepacia tecA mutants when infected into macrophages produced a bead like appearance phenotype, which got abolished when tecA was expressed in trans through a plasmid. Thus, it was found that mutation of TecA affected its ability to carry out actin rearrangements. The hypothesis for the second figure identifies the target of TecA. It was found that TecA causes RhoA deamidation at the asparagines residue located at position 41. Mass spectrometry helped to identify the amino acid that is modified in RhoA. The hypothesis for figure 3 is that TecA is highly essential for deamidation of Rho and when mutated cannot carry out deamidation. Complementation of the te cA mutant with TecA expressed from a plasmid helped to induce RhoA modifiucation and mass spectrometry helped to show that asparagines at position 41 of RhoA gets converted to aspartic acid by TecA. Moreover, Rac1 was found to be modified at position 39, which is homologous to RhoA that undergoes modification at position 41. The hypothesis of the fourth figure is that TecA is a deamidases that modifies Rho type GTPases. BLAST analysis was carried out to determine the other TecA orthologs. Homologs were also identified. A conserved Cys-His-Asp sequence were identified, where the cysteine acts a s a nucleophile. Moreover, in silico modeling were also carried out. The hypothesis for the fifth figure is that RhoA deamidation activates the Pyrin inflammasomes. Mutation of tecA was unable to cause caspase 1 activation, interleukin secretion and pyroptosis. However, mutation of the residues of the catalytic triad resulted in loss of function of TecA. The hypothesis for the last figure is t hat TecA causes lung inflammation and when recognized by Pyrin, it gives protection from severe lung infection in mice. Immunoblotting and ELISA experiments were carried out. The results were highly compelling as these helped to identify the mechanism of action of TecA and also the targets that are modified by TecA in eukaryotic hosts. Conclusion The article is highly compelling as it for the first time helps to demonstrate the effects of a non-VgrG family type VI secretion system effector TecA in the pathogenesis of B. cenocepacia. This study carries out the experiments in mice models, but future experiments are needed to determine the effect of TecA in plant and insect models. Moreover, it is also necessary to determine whether TecA from B. cenocepacia can function in other pathogens using the Type VI secretion system. It is necessary to determine the position of TecA where the substrate RhoA binds. Research Proposal The title of my research proposal is Identification of the binding site of RhoA in the deamidase TecA in B. cenocepacia. The substrate binding site of TecA is not known, so my research objective would be to determine the binding site for RhoA in TecA. Hypothesis Hypothesis 1 The deamidase TecA has a specific binding site for its substrate RhoA. RhoA binding domains have previously been identified in other species, hence it is necessary to determine the binding site of RhoA in the deamidase TecA (Shang et al., 2012). This can be carried out by application of yeast 2 hybrid screens (Stynen et al., 2012). It will help to determine the binding site of RhoA in TecA. Hypothesis 2 The binding sequence for RhoA in TecA of B. cenocepacia shows significant homology with the other identified RhoA binding proteins. Sequence homology needs to be determined by carrying out sequence analysis using bioinformatic tools like BLAST, CLUSTALW, among others. This will help to determine the binding site sequence of TecA. Hypothesis 3 TecA carries out deamidation of RhoA in plant and insect models. To address this hypothesis, mutation of TecA will be carried out, followed by introduction of the B. cenocepacia tecA mutants in plant and insect cell lines. The ability of the mutants can be determined by its ability to carry out actin disruption in the host macrophages. For this phase contrast microscopy can be carried out. This will help to determine whether TecA has the same mechanism of action in other eukaryotic host models. Hypothesis 4 Overexpression of TecA from B. cenocepacia can complement the phenotypes of the tecA mutants in other pathogenic microorganisms. To address this hypothesis, tecA mutant of other pathogenic microorganisms can be complemented with B. cenocepacia TecA. TecA from B. cenocepacia can be cloned in an expression plasmid and overexpressed in trans in the tecA mutants in other species. Phase contrast microscopy can be carried out to determine the effects of overexpression of B. cenocepacia TecA on the stability of macrophages. This experiment will help to determine whether TecA can function in heterologous hosts as well. Bibliography Gavrilin, M. A., Abdelaziz, D. H., Mostafa, M., Abdulrahman, B. A., Grandhi, J., Akhter, A., ... Amer, A. O. (2012). Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia.The Journal of Immunology,188(7), 3469-3477. Jiang, F., Waterfield, N. R., Yang, J., Yang, G., Jin, Q. (2014). A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.Cell host microbe,15(5), 600-610. Juhas, M., Stark, M., von Mering, C., Lumjiaktase, P., Crook, D. W., Valvano, M. A., Eberl, L. (2012). High confidence prediction of essential genes in Burkholderia cenocepacia.PloS one,7(6), e40064. Miyata, S. T., Unterweger, D., Rudko, S. P., Pukatzki, S. (2013). 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