Genetically Engineered Frameshifted YopN-TyeA Chimeras Influence Type III Secretion System Function in Yersinia pseudotuberculosis
| Autor: | Åke Forsberg, Ummehan Avican, Ayad A. A. Amer, Salah I. Farag, Tiago R. D. Costa, Matthew S. Francis |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
SHINE-DALGARNO SEQUENCE
Mutant Chimeric Proteins translocation Yersinia pseudotuberculosis Infections hierarchy Yersinia Type three secretion system BACTERIAL PATHOGENS Mice Yersinia pseudotuberculosis EFFECTOR PROTEINS GENE-EXPRESSION ribosome slippage Multidisciplinary Host cell cytosol biology Virulence Intracellular Signaling Peptides and Proteins Biochemistry and Molecular Biology SUBSTRATE-SPECIFICITY Multidisciplinary Sciences Host-Pathogen Interactions INNER-ROD Medicine Science & Technology - Other Topics Research Article Bacterial Outer Membrane Proteins General Science & Technology Science Molecular Sequence Data Mutagenesis (molecular biology technique) ENTEROPATHOGENIC ESCHERICHIA-COLI Microbiology Microbiology in the medical area Bacterial genetics InvE family Open Reading Frames Bacterial Proteins MD Multidisciplinary Mikrobiologi inom det medicinska området Animals Humans Secretion Amino Acid Sequence TARGET-CELL CONTACT 3-DIMENSIONAL STRUCTURE SHIGELLA-FLEXNERI Science & Technology Base Sequence Membrane Proteins Gene Expression Regulation Bacterial biology.organism_classification secretion control virulence Mikrobiologi Open reading frame Mutagenesis Site-Directed Protein Multimerization Carrier Proteins Biokemi och molekylärbiologi |
| Zdroj: | PLoS ONE, Vol 8, Iss 10, p e77767 (2013) PLoS ONE |
| Popis: | Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis . To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis , we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica , which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro . Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro . In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia -host cell contact. |
| Databáze: | OpenAIRE |
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