Deadline for full application: December 15th, 2013
Interviews: March, 2014
Start of the Ph.D.: October 1st, 2014
Title of the PhD project: Flagellar motor recruitment to the peptidoglycan layer and role of the lytic transglycosylases
Name of the lab: BGPB
Head of the lab: Ivo BONECA
PhD advisor: Ivo BONECA
Email address: email@example.com
Web site address of the lab: http://www.pasteur.fr/ip/easysite/pasteur/fr/recherche/departements-scie...
Doctoral school affiliation and University: GC2ID/Paris 5 Descartes
Presentation of the laboratory and its research topics:
The unit Biology and genetics of bacterial cell wall (BGPB) studies the biosynthesis and degradation of peptidoglycan and other bacterial cell wall components, and their recognition by the host immune system using different models such as Helicobacter pylori and Leptospira interrogans.
Description of the project:
The peptidoglycan (PG) is a complex heteropolymer composed of glycan chains and small peptides connecting neighboring glycan chains, leading to a mesh surrounding each single bacterial cell. The PG functions as an exoskeleton that needs to be remodel to accommodate cell growth and division as well as assembly of surface macromolecular appendages such as flagella or secretion apparatus. We have recently shown that PG maturation enzymes are essential for flagellum functionality but not for assembly as initially postulated .
We propose to extend these observations using H. pylori as a model organism to understand the underlying mechanisms between lytic transglycosylase activity and flagellum functionality. The project can be separated in two aspects 1) studying the biochemistry of hpMotB and substrate specificity of binding and 2) how the lytic transglycosylases are recruited to the flagellum to fulfill their maturation role.
1) Biochemistry and substrate specificity of hpMotB: The aim is to study by biochemical means the interaction of hpMotB with different substrates to define the exact structures in the PG that are recognized by hpMotB. Using ITC (IsoTermal Calorimetry), we want to study the interaction of the hpMotB to defined substructures of the peptidoglycan. We will generate enzymatically different substrates from intact PG. We have several recombinant enzymes in the laboratory involved in H. pylori peptidoglycan maturation. By this approach, we can distinguish whether hpMotB discriminates effectively anydro muramic acid residues from fully hydrates ones, and whether the peptides are required for hpMotB PG binding. We will complement these biochemical studies with studies using random mutagenesis of the hpMotB PG binding domain to understand the interaction surface with the different PG substrates. Mutations that affect PG binding will be introduced into H. pylori by site directed mutagenesis and analyzed for their impact of motility and hpMotB localization. The hpMotB localization studies will be done using the GFP-hpMotB fusion already described in the heterologous system of E. coli . In H. pylori, we will use a series of antiserum we have generated recently against hpMotB in mouse, guinea pig and rabbit. Finally, because, we propose that MotB binding to PG and flagellum functionality is universal, we will create chimera between hpMotB, ecMotB and lmMotB. The PG binding domains of the different MotBs should be interchangeable based on our current model.
2) Mechanism of recruitment of lytic transglycosylases to the flagellum: During the course of this work, we observed an intriguing phenotype in H. pylori. H. pylori has only two lytic transglycsylases, Slt and MltD. Surprisingly, the recruitment of either for flagellum functionality depends on the strain genetic background. Studying the impact of the inactivation of the two genes, slt and mltD, in 20 different strains of H. pylori, we observed that some strains only require Slt for motility while others use instead MltD (Roure et al. unpublished data). Two strains require both. We believe that the use of either protein or of both is based on the ability of each strain to recruit either Slt or MltD to the flagellum basal body to locally mature the PG allowing correct hpMotB binding to its substrate. We have generated also antiserums against Slt and MltD from mouse, guinea pig and rabbit. This will allow us to study simultaneously the three proteins in single bacteria in different genetic backgrounds by immunofluorescence microscopy. We will also purify flagella from two model strains of H. pylori, B128 for which MltD is required for motility, and X47 for which Slt is required. We will analyze the protein content of purified flagellum from strains B128 and X47 as well as of the corresponding slt and mltD mutants. Recently, the flagellum protein composition of the closely related epsilon proteobacterium, Campylobacter jejuni has been analyzed by electron cryotomography (ECT) . We will also study by ECT the flagellum motor of B128, X47 and their mutants. For example, because H. pylori has polar flagellum, the motor is visible by ECT which is not the case for flagella that are peritrish such as Salmonella or E. coli . Hence, our non-motile mutants should have a loss of density by ECT corresponding to MotB.
1. Roure S, Bonis M, Chaput C, Ecobichon C, Mattox A, et al. (2012) Peptidoglycan maturation enzymes affect flagellar functionality in bacteria. Mol Microbiol 86: 845-856.
2. Chen S, Beeby M, Murphy GE, Leadbetter JR, Hendrixson DR, et al. (2011) Structural diversity of bacterial flagellar motors. EMBO J 30: 2972-2981.
5 mains Publications of the host laboratory:
peptidoglycan, flagellum, motility, MotB, lytic transglycosylases, Helicobacter pylori
Contact: Ivo G BONECA, firstname.lastname@example.org