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Project

Development of proteomics workflows and study of the mechanism for secretion through T3SS in Enteropathogenic E.coli (EPEC)

Bacteria have evolved sophisticated nano-machines that allow them to perform specialized functions. Among these are protein secretion systems making feasible the protein translocation from the cell cytoplasm to the extracellular environment. One such nanomachine is the Type III protein secretion system (T3SS), a ~ 3-4 MDa structure composed by more than 20 proteins. T3SS penetrates both membranes of Gram-negative bacteria and extends a needle-like structure facing outwards of the cell surface. Protein secretion through T3SS is highly regulated and the type of secretory proteins can be classified into three sequential steps: secretion of early substrates, translocators and effectors. This hierarchical order of events is essential for proper assembly and function of T3SS nanomachine. Through the assembled needle structure, toxins translocate directly to the cytoplasm of the host cell, bypassing the extracellular space.

The large number of interacting proteins, the localization of the system in two membrane bilayers and a tight hierarchical regulation of the secretion process of different classes of substrates, introduce a significant technical challenge in the elucidation of the molecular mechanism of T3SS function. We followed a multidisciplinary approach, combining methods such as molecular genetics, structural biology, cellular biology, biophysics, in vitro reconstitution and mass spectrometry, exploring at a molecular level 1) how secretory proteins are targeted to the T3SS, 2) which T3SS protein(s) act as secretory protein receptors and 3) how is the process of hierarchical switching between substrates regulated.

We dissect the secretion process into two steps: protein targeting to the membrane and protein secretion and we focus only in the protein targeting step. We start by studying one step of the secretion process using the model pair of chaperone/secretory protein complex CesAB/EspA. We show that the C-tail of CesAB acts as a membrane targeted signal that guides the loaded with the substrate chaperone to the membrane. We identified the gate-keeper protein SepL as the major interacting partner for CesAB/EspA complex on the membrane and we mapped possible interaction sites. We localize SepL to the periphery of the translocase EscV and identify their interaction sites. Also, we provide clues for substrate specificity regulation from the gate-keeper proteins SepD and SepL showing that while both gate-keeper proteins SepL and SepD are essential for EspA translocation, deletion of SepD does not affect SepL localization to the membrane and CesAB/EspA binding affinity to the T3SS injectisomes. We show that the gate-keeper proteins regulate the binding affinity of different class of translocator (CesAB/EspA) or effector protein complexes (CesT/Tir) to the translocase EscV, with a not completely defined mechanism.

The detailed structural and functional characterization of T3SS regulation and secretion can have a broad range of applications. Acquired knowledge can be used for the development of T3SS specific assays for drug discovery studies as well as for the targeted engineering of the system for protein delivery, making T3SS an interesting system for biomedical and biotechnological research.

Date:19 Feb 2015 →  24 Nov 2017
Keywords:Microbiology, Proteomics, Type III secretion system
Disciplines:Microbiology, Systems biology, Laboratory medicine, Immunology, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering
Project type:PhD project