< Back to previous page

Project

Calcium Controls the Intrinsic Dynamics of the Type 3 Translocase Export gate to Switch Client Secretion

The type three secretion system (T3SS) is a multiprotein (20-30) transmembrane complex used by Gram-negative organisms to deliver bacterial cytoplasmic proteins to the host cell cytoplasm. The T3SS is a very important virulence mechanism for many pathogenic organisms. This system has been studied across the globe in the last three decades but still some of the aspects of this system are yet to be unveiled.

The main aim of this thesis work was to understand the mechanism of T3SS secretion regulation in enteropathogenic Escherichia coli (EPEC) (3rd chapter). While working in this direction we felt the need of a quick and quantitative in vivo secretion system for monitoring of the T3SS function in EPEC, which has been detailed in the second chapter. The activities of my PhD project in the lab were also extended to the upgrading of an online database for subcellular location of the E. coli K12 proteome along with the T3SS of EPEC and different homologues (4th Chapter).

A robust, quick and quantitative assay is essential for studying the function of T3SS components and the regulation of secretion. For this, we developed an in vivo secretion assay (Chapter 2) by using a T3SS-substrate (SctA) and reporter (PhoA) fusion protein (SctA-PhoA). This helped us to monitor and quantify the in vivo translocator secretion through the injectisome of EPEC in spent growth medium of bacterial cultures. This simplified assay is able to measure the extracellular SctA-PhoA secretion in the spent growth medium of a bacterial culture, in the absence of eukaryotic cells. We also verified the utility of this system to screen for T3SS mutants.

In T3SS of EPEC, the regulation of the second switch (i.e. the shift of secretion from middle to late substrates) not fully understood. Here, we showed that Ca2+ interacts with the C-domain of the export gate, SctV, and it works as a conformational switch. SctW plus high Ca2+ concentrations promote “middle/translocator” and suppress “late/effector” client secretion. Low Ca2+ concentrations or loss of SctW reverses this. SctV is the receptor for the SctW/CesL/SepD heterotrimer. We could detect the evidence of interaction Ca2+ with SctV-C for the first time and thereby could explain the probable mechanism for secretion switching from middle to late substrate in EPEC.

The STEPdb is an online database hosted by the Laboratory of Molecular Bacteriology, Rega Institute. It contains a comprehensive annotation and manual curation for all 4,313 protein entries of strain E. coli K12. Over 70 features like structural folds, predicted secondary structures, dynamics, early foldons, aggregation-prone regions, topological, functional information and cellular concentrations were evaluated. The entries were linked to downloadable PDB structure file or predicted structure available in the AlphaFold database. In addition, we have extended similar information for more than 50 T3SS proteins from EPEC and homologues from other Gram-negative bacterial pathogens. The STEPdb 2.0 is available online at stepdb.eu.

Date:4 Oct 2018 →  12 Jun 2023
Keywords:Bacterial Type 3 Protein Translocation System, In-vitro Reconstitution, Enteropathogenic Escherichia coli (EPEC)
Disciplines:Microbiology, Systems biology, Laboratory medicine, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Immunology
Project type:PhD project