Molecular for Frontiers in Microbiology - myassignmenthelp.com

Question: Discuss about theMolecular Pathogenesisfor Frontiers in Microbiology. Answer: The challenges associated with working with type 3 secretion systems of C. violaceum is that the genes clusters Cpi1, 1a and 2 are quite large (Batista da Silva Neto, 2017). It is a challenge to identify the host targets of the effectors encoded by the type 3 secretion systems. Another challenge is the cross-contamination problems when using cell lines. The opportunities include the use of cell lines, learning the techniques of mutant construction and use of animal infection models. This work will be connected with my personal expertise or interest as I have knowledge about the molecular biology techniques that can be used for plasmid and mutant construction. The challenge will be to identify the proteins phosphoryalated by OspG (Zhou et al., 2013). OspG alters the host immune system by blocking the production of IL8. Mutations of OspG will prevent the blocking of the host immune system, but will not be able to determine the proteins that are phosphoryalated by OspG. The opportunity will be to identify the proteins that were previously not known to be phosphoryalated by OspG. It has very little connection with my expertise as it will involve the use of various bioinformatic softwares to determine the binding of the OspG to its necessary targets. The challenges involved are the presence of a large number of studies on this topic, IpaH mutants have no detectable phenotypes in cellular systems and the use of animal models are a cumbersome process (Wandel et al., 2017). The opportunity will be to identify the amino acid residues of IpaH 9.8 that are involved in the binding and subsequent ubiquitinylation. However, it will help me to gain knowledge about new techniques like RNA interference, confocal microscopy, FACS, among others. There is no information about DrrA function or presence in C. burnetti (Larson et al., 2015). The challenge will be to identify the ortholog of DrrA in C. burnetti, showing the same function as DrrA. The opportunity will be that the information obtained will be the first to be reported in the organism. This will help me to gain knowledge about the bioinformatic tools that can be utilized to identify the ortholog of DrrA. The topic, which I would pick is the Evaluation of the pathogenic potential of the T3SS harbored by Chromobacterium violaceum, because type 3 secretion systems in C. violaceum is demonstrated to be the key virulence determinants of the pathogenic microorganism. They are responsible for the delivery of effector proteins in the eukaryotic host cells. Cpi 1 and 1a has been shown to be required for virulence in mouse and human models but not Cpi2. Tthere are other unknown effector proteins important for its virulence. It is also necessary to determine the host targets of these effector proteins. Hypothesis 1: The Cpi 1 and 1a encodes toxins that are responsible for virulence in host organisms. Structure function study of the effector systems and determination of their corresponding host targets. Hypothesis 2: Many Chromatobacterium species remain associated with plant roots. The Cpi 2 encodes effector proteins that binds to targets in insect larvae or fungal pathogens, thereby suppressing plant diseases. Hypothesis 3: Cpi1 and 1a are very similar to the Salmonella pathogenicity island 1 genes that encodes the type 3 secretion systems. The Cpi1, 1a and 2 gene clusters can complement the Salmonella enterica serovar Typhimurium mutants deficient in the Type 3 secretion systems. Hypothesis 1 can be tackled by carrying out mutations of the genes encoding the effector proteins and determining the phenotypic loss associated with each of the mutations. Protein-protein interaction experiments can be carried out to determine the target proteins of the effectors. For hypothesis 2, the genes of the Cpi2 encoding the effector proteins can be cloned and overexpressed in a C. violaceum strain having mutations in the Cpi1, 1a and 2 to determine its function. For hypothesis 3, the Cpi1, 1a and 2 gene clusters can be cloned in an expression plasmid and introduced into Salmonella enterica serovar Typhimurium mutants deficient in the Type 3 secretion systems and determining whether they can complement the mutants by determining the virulence of the mutant strains. Reference List Batista, J. H., da Silva Neto, J. F. (2017). Chromobacterium violaceum Pathogenicity: Updates and Insights from Genome Sequencing of Novel Chromobacterium Species.Frontiers in Microbiology,8, 2213. Larson, C. L., Beare, P. A., Voth, D. E., Howe, D., Cockrell, D. C., Bastidas, R. J., ... Heinzen, R. A. (2015). Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication.Infection and immunity,83(2), 661-670. Wandel, M. P., Pathe, C., Werner, E. I., Ellison, C. J., Boyle, K. B., von der Malsburg, A., ... Randow, F. (2017). GBPs Inhibit Motility of Shigella flexneri but Are Targeted for Degradation by the Bacterial Ubiquitin Ligase IpaH9. 8.Cell Host Microbe,22(4), 507-518. Zhou, Y., Dong, N., Hu, L., Shao, F. (2013). The Shigella type three secretion system effector OspG directly and specifically binds to host ubiquitin for activation.PloS one,8(2), e57558.