Deadline for full application: December 15th, 2013
Interviews: March, 2014
Start of the Ph.D.: October 1st, 2014
Title of the PhD project: Molecular mechanism of fiber assembly in the type II secretion system
Name of the lab: Molecular genetics Unit
Head of the lab: Anthony P. Pugsley
PhD advisor: Olivera Francetic
Email address: firstname.lastname@example.org
Web site address of the lab: none
Doctoral school affiliation and University: B3MI, Paris 7
Presentation of the laboratory and its research topics:
The Molecular genetics unit is composed of several teams that study molecular aspects of adaptation to stress (Françoise Norel), signal integration in regulation of cellular processes on model proteins of a STAND superfamily (Evelyne Richet), lipoprotein biogenesis (Nienke Buddelmeijer), membrane protein assembly and insertion (Tony Pugsley), pilus assembly systems and protein secretion (Olivera Francetic). We use Gram-negative bacterial models like Salmonella and Escherichia coli to study these processes. Our major aim is to understand fundamental and mechanistic aspects of complex and dynamic systems, using a wide range of approaches integrating genetic, biochemical, biophysical, structural and bioinformatics analysis.
Description of the project:
(1 page, Arial font size 11 : 600 words in total with at least 50% dedicated specifically to the proposed PhD project(s))
Gram-negative bacteria use different mechanisms to secrete toxins, hydrolases or adhesins and expose them on the cell surface or release them in the surrounding milieu. A unique transport mechanism in the type 2 secretion systems (T2SS) allows secretion of proteins in a native, folded form. This system is closely related to a superfamily of molecular machines in archaea and bacteria that function via a common mechanism that involves assembly of helical fibers embedded in the plasma membrane. In Archaea, these systems build flagella to promote swimming in liquid via a rotational mechanism. In bacteria, besides T2SS, they build dynamic type IV pili (T4P), which promote bacterial crawling on solid surfaces via fiber extension, substrate binding and retraction that pulls the bacterial body forward. T4P are involved in adhesion, biofilm formation and virulence, as well as in DNA uptake and natural transformation. All these systems share many common elements, including an integral membrane protein, a cytoplasmic ATPase and pilins, the fiber building blocks. Although T2SS and T4P are highly medically and economically important, little is known about the mechanism of fiber assembly and the way they promote specific functions of these systems.
In T2SS an essential dynamic element involved in protein transport is a periplasmic fiber, the pseudopilus. To understand the mechanism of pseudopilus assembly and its role in protein secretion, our laboratory uses as a model system the well-characterized T2SS from Klebsiella oxytoca. This system is composed of 15 different proteins that form a dynamic multimeric complex spanning the Gram-negative envelope. This system, functionally reconstituted in Escherichia coli, secretes a single lipoprotein substrate pullulanase (PulA). Under specific culture conditions the Pul T2SS assembles pili on the bacterial surface, composed of the major pilin subunit PulG. The assembly of these pili is essential for the function of the PulA secretion system. Our group has determined the detailed atomic structure of PulG pilus, which is so far the only detailed structure of a fiber from this class (1,2). Based on this structure, and studies of other T2SS components (3,4) we have identified the function of specific steps during fiber assembly. Current studies of fiber dynamics using a combination of molecular modeling and biochemical analysis allowed us to propose an assembly model that involves fiber rotation in the inner membrane (5). The goal of this PhD project is to characterize the central motor complex involved in pseudopilus assembly, composed of a hexameric ATPase PulE, a polytopic membrane protein PulF and two single-span membrane proteins PulL and PulM. Together the role of this complex is to promote and couple PulG pilus assembly to protein transport. Although structural information is available for the soluble domains of these proteins, little is known about their transmembrane segments, stoichiometry and interactions. In this study structural modeling in silico and molecular dynamics simulations in model membranes will be used to provide predictions of specific residue contacts between these proteins. These contacts will be tested by site-directed mutagenesis and functional analysis, in order to provide experimental restraints that would be incorporated into the refined structural model. Bacterial two-hybrid analysis, cross-linking and co-purification will be used to study interactions of these proteins in their native membrane environment. Specific mutations that abolish protein contacts in this complex will be functionally characterized using quantitative assays of PulG pilus assembly and of PulA secretion. The dynamics of pilus assembly will be studied by live fluorescence microscopy and single-molecule analysis to test the rotation model.
Together the results of this study should provide the molecular insight into the mechanism of pilus assembly in T2SS and T4P, the two fundamentally and medically highly relevant systems.
1.Campos M, Nilges M, Cisneros DA and O. Francetic (2010) Detailed structural and assembly model of the type II secretion pilus from sparse data. Proc. Natl. Acad. Sci. USA 107: 13081-86.
2.Campos M, Francetic O, M. Nilges (2011) Modeling pilus structures from sparse data. J. Struct Biol. 173: 436-444.
3. Cisneros, D., PJ Bond, AP Pugsley, M Campos, O. Francetic (2012) Minor pseudopilins self-assembly primes the type II secretion pseudopilus elongation. EMBO J. 31: 1041-1053.
4. Cisneros, DA, Péhau-Arnaudet, G. and O. Francetic (2012) The heterologous assembly of type IV pilin by a type II secretion system reveals the role of minor pilins in assembly initiation. Mol Microbiol 86: 805-818.
5. Campos M, Cisneros DA, Nivaskumar M, Francetic O.The type 2 secretion system - a dynamic fiber assembly nanomachine. (A review) Res Microbiol. 2013 164 (6):545-555.
Keywords: type II secretion system, type IV pili, membrane protein assembly, protein dynamics
Expected profile of the candidate (optional):
The candidate should have good theoretical background in biochemistry and molecular biology. Interest in biophysics and structural biology is a plus.
Contact: Olivera Francetic