PDP2019-08

Life and death of Salmonella-containing vacuoles

Supervisors

Kheya Sengupta / CINaM / sengupta@cinam.univ-mrs.fr

Stephane Meresse / CIML / meresse@ciml.univ-mrs.fr

Abstract

The capacity to replicate inside host cells is critical for Salmonella virulence and relies upon the establishment and the stability of a replication niche, called the Salmonella-containing vacuole (SCV). Enclosed within the SCV, the bacterium secretes effector proteins which reprogram the infected cell and the membrane dynamics of SCVs. An important feature of the SCV is the generation of Salmonella-induced filaments (Sif), which are lipid nano-tubes emanating from the SCV in a microtubule/kinesin dependent manner. Sifs may persist on host cell fission, connecting the daughter cells, and probably play a role in systemic virulence. The aim here is to develop a minimal in vitro mimic of the SCV that reconstitutes and analyses Salmonella tubulation and fission. We will define condition allowing the encapsulation of a single bacterium in a liposome, and the translocation of effector proteins across its membrane. This unique minimal system will be used to dissect the biochemical role of the effectors, as well as the biophysical role of the membrane.

Keywords

Giant unilamellar vesicles (GUVs), Salmonella containing vacuole, minimal system, membrane physics.

Objectives

The aim is to dissect the bio-chemistry and biophysics of SCVs. Two specific areas of focus will be:

1. Tubule-formation: Explore regulatory mechanisms for recruitment and activation of kinesin-1 in SCV
2. Fission: Explore the mechanism of the concomitant splitting of SCV during Salmonella cell division

The major technical goal is to enclose a single bacterium in a single liposome to mimic a SCV, using customized liposomal systems; and to develop novel tools to quantify, analyze and model liposomal shape changes.

Proposed approach

The project will build on on-going experiments in Meresse lab (pioneer in SCV [1,2]), using giant unilamellar vesicles (GUVs) from which tubes are pulled using microtubules and kinesins. Means to enclose a single Salmonella in such GUVs will be developed using fluidics-based methods [3]. Salmonella will be grown in conditions allowing the expression of the pore-inducing type III secretion system. We will define conditions to allow bacterial encapsulation, pore formation at the GUV membrane, and secretion of bacterial effectors. Proteins and nutrients will be fed by small vesicle fusion on the GUV (used already in Sengupta lab in a different context [4] ). Outside medium and lipid composition will be tailored, transmembrane proteins incorporated, and tension controlled/measured. Protein binding and membrane shape will be monitored by advanced microscopy and quantified using customized software (Sengupta lab., expert in GUV based biophysics [4,5,6]). New computational analysis and modelling of the dynamic shape changes of the GUVs will help decipher the role of membrane physics.

International collaborations: Dr. Chaitanya Athale, Pune, India, for expertise in microtubules and related motor proteins and Pr. Ana Smith, Erlangen, Germany, for theoretical modelling of the membrane.

Interdisciplinarity

The project brings together Kheya Sengupta, a physicist (attached to the physics doctoral school), who has long worked with GUVs as test cells and Stephane Meresse, a biologist (attached to the biology doctoral school), who made pioneering discoveries on SCV, with the goal of harnessing the power of model membranes to decipher strategies that bacterial pathogens have developed to hijack hosts. Similar approaches, that combine expertise in model membranes and molecular/cell biology have brought tremendous insight into membrane trafficking (for example the recent work on the role of BAR domains and ESCRT proteins in Institute Curie). We hope to shed light on aspects of SCV using a similar combination. The insights should be applicable to other pathogenic vacuoles. The success of the above-mentioned project is based on a synergistic use of the competences of the two laboratories and the common will of the two P.I. to develop this project.

Expected profile

The candidate should have a PhD degree in biology, chemistry, physics or a related field. (S)he should have already done extensive experimental work and be motivated to work on an interdisciplinary subject. Preference will be given to candidates with experience in soft condensed matter, soft matter chemistry, biophysics, or micro/cell biology; interdisciplinary training will also be an asset.

References

(1) E. Boucrot, T. Henry, J. P. Borg, J. P. Gorvel, S. Meresse. Science 308, 1174 –1178 (2005).
(2) N. Schroeder, L. J. Mota, S. Meresse. Trends Microbiol. 19:268 –277 (2011).
(3) C.E.S. Guedes, J.G.B. Lima, E. Helfer, P.S.T. Veras, A. Viallat. PloS ONE 10 8 e0134925 (2015).
(4) C. Monzel, D. Schmidt, … A.-S. Smith, K. Sengupta and R. Merkel. Nat. Comm. 6, 8162 (2015).
(5) S. F. Fenz, K. Sengupta, Integr. Biol., 4, 982–995 (2012).
(6) S. F. Fenz, T. Bihr, D. Schmidt, R. Merkel, U. Seifert, K. Sengupta, and A-S. Smith. Nature Physics, 13, 906–913 (2017).

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