PhD PROPOSAL FOR THE PASTEUR-PARIS UNIVERSITY INTERNATIONAL PROGRAM

 

Deadline for full application: December 15th, 2013

Interviews: March, 2014

Start of the Ph.D.: October 1st, 2014

 

 

Department: Structural Biology and Chemistry

Title of the PhD project: Tackling the diversity generated by phase variation in N. meningitidis using proteogenomics.

Name of the lab: Structural Mass Spectrometry and Proteomics

Head of the lab: Julia Chamot-Rooke

PhD advisor: Julia Chamot-Rooke

Email address: julia.chamot-rooke@pasteur.fr

Web site address of the lab:

http://www.pasteur.fr/ip/easysite/pasteur/fr/recherche/departements-scie...

 

Doctoral school affiliation and University: B3MI

 

Presentation of the laboratory and its research topics:

 

The overarching objective of the Unit is to develop new Mass Spectrometry-based methodologies for the analysis of proteins and protein complexes involved in infectious disease. The unit focuses particularly on “top-down” approaches, performed on the entire protein, that are particularly useful for the complete characterization of post-translationally modified proteins. Top-down and high throughput bottom-up approaches are also combined to identify and fully characterize sets of proteins involved in host-pathogen interactions.

 

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))

 

The expression of most bacterial genes is controlled at the level of transcription via promoter control mechanisms that permit a graded response. However, an increasing number of bacterial genes are found to exhibit an `all-or-none' control mechanism that adapts the bacterium to more than one environment. One such mechanism is phase variation (PV) defined as the high frequency heritable ON<>OFF switching of protein expression [1]. PV has been described in a large and diverse number of human pathogens such as Salmonella, UPEC, Neisseria spp, etc. It generates diversity in the bacterial population during infection but also during isolation and culture. An important consequence is that PV introduces differences between the population that caused the disease and the one frozen down in the hospital. PV is usually ignored because difficult to evaluate but it can be a major confounding factor in host pathogen interaction studies [2]. In certain pathogens such as Neisseria meningitidis the estimated repertoire of genes submitted to PV is in the order of 40, generating the staggering number of 1010 combination [3].

Different mechanisms can be involved in PV: slipped strand mispairing (which produces mispairing of short repeat sequences during DNA synthesis), site specific recombination including DNA inversion or epigenetic modification by methylation. Because of the importance of this process, efficient and new tools are needed to evaluate it.

The present project aims at using high throughput proteomics to identify which gene is submitted to PV in N. meningitidis. Using proteomics to improve gene annotation is an emerging field called proteogenomics [4]. Proteogenomics has already proven to be a unique tool to annotate bacterial genomes allowing multiple missing genes or incorrect start position to be easily detected. In the past months the team of J. Chamot-Rooke has worked on the development of an optimized protocol allowing a deep proteome coverage of N. meningitidis to be obtained. Preliminary results indicate that using state-of-the-art mass spectrometer and appropriate chromatographic separation conditions, more than 1000 meningococcal proteins can be identified in a single experiment, without effort.

In this project, this shotgun bottom-up approach (based on the analysis of peptides after protein digestion) will be further optimized and combined to a top-down one (analysis of intact proteins) to increase proteome sequence coverage. Diverse sample preparation conditions (including N-terminal labeling) and protein fractionation will also be tested [5,6].

Once fully optimized, the MS approaches will be use to address the expression status of phase variable genes in 100 N. meningitidis variants with different phase variation combinations.

Data analysis, which is crucial in proteogenomics studies, will be undertaken by a team in Strasbourg that has a recognized expertise in the field (P. Collet, O. Lecompte) and with whom the Unit of J. Chamot-Rooke already collaborates.

Once genes submitted to PV have been identified, they will be selected for in vivo experiments. G. Duménil, partner of this project, has recently developed a humanized mouse model of infection that recapitulates the vascular damages that occur during infection [7].

Comparing the state of the phase variable genes before and after passage through the animal model will be highly informative on the genes necessary for survival and proliferation in vivo.

The results of this approach will also help determine the dynamics and the extent of PV that has occurred between the effective disease and culture on rich medium and freezing in the laboratory environment. The approach developed in this project will be applicable to other pathogens for which PV is also an issue.

 

In summary, the objectives and expected results of the project are:

  1. Develop new mass spectrometry-based tools to characterize phase variation
  2. Probe the genes involved in phase variation using N. meningitidis as a model
  3. Use this property to identify genes involved in virulence

 

References:

[1] C. D. Bayliss, Determinants of phase variation rate and the fitness implications of differing rates for bacterial pathogens and commensals. FEMS microbiology reviews 33, 504 (May, 2009).

[2] M. T. Anderson, H. S. Seifert, Phase variation leads to the misidentification of a Neisseria gonorrhoeae virulence gene. PloS one 8, e72183 (2013).

[3] P. Martin et al., Experimentally revised repertoire of putative contingency loci in Neisseria meningitidis strain MC58: evidence for a novel mechanism of phase variation. Molecular microbiology 50, 245 (Oct, 2003).

[4] N. Gupta et al., Comparative proteogenomics: combining mass spectrometry and comparative genomics to analyze multiple genomes. Genome research 18, 1133 (Jul, 2008).

[5] J. A. Christie-Oleza, G. Miotello, J. Armengaud, High-throughput proteogenomics of Ruegeria pomeroyi: seeding a better genomic annotation for the whole marine Roseobacter clade. BMC genomics 13, 73 (2012).

[6] S. Gallien et al., Ortho-proteogenomics: multiple proteomes investigation through orthology and a new MS-based protocol. Genome research 19, 128 (Jan, 2009).

[7] K. Melican, P. Michea Veloso, T. Martin, P. Bruneval, G. Dumenil, Adhesion of Neisseria meningitidis to dermal vessels leads to local vascular damage and purpura in a humanized mouse model. PLoS pathogens 9, e1003139 (Jan, 2013).

 

 

Keywords:

Bacterial pathogenesis, proteomics, virulence, phase variation, Neisseria meningitidis

 

Expected profile of the candidate (optional):

 

Ideally a candidate with a strong background in the following topics will be sought: Proteomics; Bioinformatics, Bacterial pathogenesis. Research will be performed through a strong collaboration with the unit headed by Guillaume Duménil at HEGP.

 

Contact: Julia Chamot-Rooke

Mis à jour le 16/09/2013