EVOGENE Seminar: New Master students Evogene April2018

Henrik Kvalheim Eriksen (Linke group): Colicins - a future tool in preventing bacterial diseases?

Sai Priya Sharma Kandanur (Leo group): Role of Secondary Structure Elements in the Translocator Domain of the Inverse Autotranporter Protein Intimin

Verena Mertes (Butenko group): A Phosphoproteomics approach to unravel the IDA Signalling Pathway

Henrik Kvalheim Eriksen:  Colicins - a future tool in preventing bacterial diseases? Abstract: Due to rise in multi-resistant microbes, the search and  the need for an alternative to antibiotics is acute. In this search, a specific type of bacterial proteins has come into focus - colicins. Could the investigation and research of these proteins lead to a much needed answer? Colicins are antimicrobial proteins originally discovered to be produced by, and active against, certain strains of Escherichia coli and closely related bacteria in the Enterobacteroaceae family. Much is known about different types of colicins and their mode of action - however, one particular member of the colicin family distinguishes itself from the others. Namely the Colicin Js. My task is to help further characterize and understand the properties and mechanisms of this protein. 

Sai Priya Sharma Kandanur: Role of Secondary Structure Elements in the Translocator Domain of the Inverse Autotranporter Protein Intimin. Abstract: Gram negative bacteria have proteins called autotransporters that transport part of their polypeptide chain to the bacterial cell surface through a transmembrane β-barrel domain that resides in the outer membrane of the cell. This transported part of the protein is called the passenger domain. Intimin, a prototype of large family of adhesins in Gram negative bacteria, is with the prototype of an autotransporter pathway called the type Ve secretion system.  In classical (type Va) autotransporter secretion systems, the β-barrel domain is C-terminal to the extracellular passenger domain. In type Ve secretion system of Intimin, the passenger domain is C-terminal to the β-barrel domain, giving rise to the name inverse autotransport as the domain order is reversed compared to type Va autotransporters.During studies on the inverse autotransport process of Intimin, an epitope tag was inserted into the N-terminus of the passenger domain resulting in a stalled secretion mutant. Further studies on the stalled mutant of Intimin showed that the β-barrel was folded and the passenger domain is translocated to the extracellular space by a hairpin mechanism. Based on these and other studies, it was suggested that the β-barrel of intimin, was involved in passenger translocation.In this project, I will perform a mutagenesis study on the β-barrel domain of Intimin to investigate the molecular details of hairpin formation and passenger secretion. The aim of the project is To study, in detail, the role of three regions in the β-barrel in then formation of the hairpin and passenger secretion. To this end, I will make point mutations and small deletions in the β-barrel domain. Formation of the hairpin will be investigated using the stalled secretion variant mentioned above; the hairpin can be detected by exposure of a hemagglutinin tag at the cell surface. Using this variant, hairpin formation and passenger domain secretion can be uncoupled. Secretion mutants will be studied by exposure of the C-terminus of Intimin. All point mutations will be made in both Intimin variants to see the effect on both hairpin formation and passenger domain secretion. All mutations will be rationally designed based on the available crystal structure of the Intimin β-barrel. The three regions are:

-          The linker connecting the the β-barrel domain to the passenger domain and interacting residues facing the lumen of the β-barrel.

-          The α-helical turn on the periplasmic side of the β-barrel.

-          The small β-sheet formed between the linker and two loops of the β-barrel on the extracellular surface of the β-barrel.

For all mutations, appropriate control experiments will be performed to make sure the protein is still expressed and inserted into the membrane. The results obtained from this study will shed light on the detailed molecular mechanisms of the inverse autotransport process.The techniques used in this study will include cloning and site-directed mutagenesis, detection methods (Western blot, immunofluorescence microscopy), biochemical methods (outer membrane isolation, urea extractions) and basic structural biology.

Verena Mertes: A Phosphoproteomics approach to unravel the IDA Signalling Pathway

 

Published Mar. 12, 2018 1:49 PM - Last modified Apr. 5, 2018 2:31 PM