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Functional genome analysis in a genetically intractable system: an interdisciplinary proof-of-concept study

English title Functional genome analysis in a genetically intractable system: an interdisciplinary proof-of-concept study
Applicant Viollier Patrick
Number 141837
Funding scheme Sinergia
Research institution Dépt Microbiologie et Médecine Moléculaire Faculté de Médecine Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Experimental Microbiology
Start/End 01.09.2012 - 30.11.2015
Approved amount 1'200'000.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Medical Microbiology

Keywords (8)

genetic intractibility; Waddlia chondrophila; functional genome analysis; differentiation; Chlamydiae; small molecule screen; E. coli; pharmacogenetics

Lay Summary (English)

Lead
Lay summary

After >50 years of molecular biology research, the genetic tractability of a few model systems has established itself as the principal pillar of functional genome analyses. As the analysis of many sequenced genomes is still hampered by their genetic intractability, new alternative and broadly applicable procedures are needed to permit functional in vivo studies, particularly in pathogens whose biology is poorly understood. Pharmacological methods present an appealing alternative to inactivate (or enhance the activity of) a client protein in vivo. We will test a novel, interdisciplinary and broadly applicable pharmacogenetic strategy to conduct functional genome analyses in the genetically intractable bacterium Waddlia chondrophila, a strict intracellular pathogen and member of the phylum Chlamydiae. Like the etiological agent of trachoma (Chlamydia trachomatis,) the most prevalent sexually transmitted bacterial disease worldwide that causes blindness, infertility and extra-uterine pregnancy, W. chondrophila features the typical chlamydial differentiation cycle -  from an infectious cell type to a replicative one and back - whose genetic basis is unknown.

As W. chondrophila is much easier to cultivate in the laboratory than C. trachomatis,  our interdisciplinary team of three distinct university research units (led by two PhDs and a clinician MD-PhD) in the Arc Lémanique will explore the function of 10 W. chondrophila regulatory genes using a simple three-step strategy.  First, we will design a functional readout (reporter assay) by heterologous expression of W. chondrophila target protein(s) in the genetically tractable surrogate host Escherichia coli (UNIGE: CMU). Next, we will screen small molecule libraries for compounds that inactivate/enhance the target protein in the E. coli reporter and determine their cytotoxicity on human/amoeba cells (UNIGE: PHARM). Finally, we will use these compounds to inactivate/enhance the native target proteins in W. chondrophila and other Chlamydiae within human/amoeba cells and infer their function by evaluating the consequences of administering the compounds to Chlamydia (UNIL: CHUV).

By targeting regulatory proteins in. W. chondrophila that are conserved among the Chlamydiae, we anticipate a) defining the genetic basis of the mysterious chlamydial differentiation program and b) illuminating the underlying molecular mechanisms, while also c) discovering new potential antibiotics that could be used to treat chlamydial (or other bacterial) infections.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Collaboration

Group / person Country
Types of collaboration
UNIGE: PHARM: Dr. Philippe Christen Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
UNIL: Electron Microscopy Platform Dr. Humbel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
UNIGE:PHARM: Dr. Karl Perron Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Swiss Pharma Science Day 2015 Poster Plant Extract Screening to Discover New Compounds against Chlamydiales 19.08.2015 Bern, Switzerland De Barsy Marie; Greub Gilbert; Walter Chantal; Cuendet Muriel; Viollier Patrick; Frandi Antonio;
International congress on Rickettsia and other intracellular bacteria Talk given at a conference Functional characterization of the Waddlia transcription factor Euo 12.06.2015 Lausanne, Switzerland De Barsy Marie;
Host-Pathogen Interactions symposium Poster Metabolomic and molecular biology approach to understand the development of Waddlia chondrophila as a genetically intractable organism model system 21.05.2015 Villars sur Ollon, Switzerland De Barsy Marie; Walter Chantal; Viollier Patrick; Greub Gilbert; Frandi Antonio; Cuendet Muriel;
Host-Pathogen Interactions symposium Talk given at a conference Functional genome analysis in a genetically intractable system: an interdisciplinary proof-of-concept study 21.05.2015 Villars sur Ollon, Switzerland Greub Gilbert; Walter Chantal; Cuendet Muriel; De Barsy Marie; Martin Nicolas; Viollier Patrick; Frandi Antonio;
BacNet15 Talk given at a conference Comprehensive identification of regulatory network interactions in Chlamydiae 12.05.2015 sant feliu, Spain Viollier Patrick;
Chlamydia Basic Research Society Poster Functional characterizarion of the Waddlia transcription factor Euo 31.03.2015 new orleans, United States of America De Barsy Marie;
Chlamydia Basic Research Society Talk given at a conference Comprehensive identification of regulatory network interactions in Chlamydiae 30.03.2015 new orleans, United States of America Frandi Antonio;
Swiss Pharma Science Day 2014 Poster New approaches for functional genome analysis in a genetically intractable system organism (Waddlia chondrophila) 20.08.2014 Bern, Switzerland Viollier Patrick; Cuendet Muriel; Frandi Antonio; Greub Gilbert; De Barsy Marie; Walter Chantal;


Associated projects

Number Title Start Funding scheme
162603 Cell division mechanism and regulation of the Chlamydia-related pathogen Waddlia chondrophila 01.01.2016 Project funding (Div. I-III)
143660 Regulatory interplay of cell cycle transcription factors in compartmentalized Caulobacter cells 01.10.2012 Project funding (Div. I-III)
124843 Virulence mechanisms of Parachlamydia acanthamoebae: characterization of its Type Three Secretion System 01.04.2009 Project funding (Div. I-III)
116445 Interactions of chlamydia-like organisms with macrophages and their role as agents of pneumonia 01.04.2007 Project funding (Div. I-III)

Abstract

Title: Functional genome analysis in a genetically intractable system:an interdisciplinary proof-of-concept study (Applicant: P. Viollier)1. Summary: Next-generation sequencing led to an abundance of information for many biological secrets that remain to be uncovered. The current challenge is to exploit this wealth of information by advancing beyond the nucleotide level to a functional genome. After >50 years of experimentation, the genetic tractability of a biological system has proven as the most powerful tool for the functional genome analyses. However, the analysis of the overwhelming majority of the sequenced genomes is still hampered by the genetic tractability. Therefore, new alternative and broadly applicable procedures are needed to permit such functional in vivo studies, particularly for pathogens whose biology is still poorly understood.When targeted mutagenesis is not possible, pharmacological methods are an ideal alternative to inactivate (or enhance) the activity of a protein. Accordingly, we propose a novel, interdisciplinary and broadly applicable pharmacogenetic strategy to conduct functional studies in genetically intractable organisms such as Waddlia chondrophila, a strict intracellular bacterial pathogen from the phylum Chlamydiae that differentiates from an infectious cell type to a replicative one and back. In a concerted and integrated effort, three distinct university research units (led by two PhDs and a clinician MD-PhD) in the Arc Lémanique will rely on three successive and interdependent experimental pillars (P1-3) for the study:P1) design a functional readout (reporter assay) by heterologous expression of W. chondrophila target protein(s) in a genetically tractable surrogate host (done at UNIGE: CMU);P2) screen small molecule libraries for compounds that inactivate/enhance the protein targets with the surrogate reporter and determine their cytotoxicity on human/amoeba cells (UNIGE: PHARM); and P3) use the compounds to inactivate/enhance target proteins of W. chondrophila and other Chlamydiae within human/amoeba cells to infer their function in the natural context (UNIL: CHUV). A critical precondition for the general feasibility of our strategy is to focus on a class of target proteins that can be easily built into a simple functional reporter system in a surrogate (genetically tractable) host such as E. coli. Because of this and our long-term goal of deciphering the regulatory landscape of the W. chondrophila genome, we will specifically rely on a transcription interference assay in E. coli using W. chondrophila transcription factors (TFs) that repress expression of a reporter by binding to their DNA target/operator sites [identified by chromatin-immunoprecipitation/deep-sequencing (ChIP-SEQ), bioinformatics and biochemistry] engineered into a synthetic promoter of the reporter gene (P1). After (P2) screening chemical libraries with this reporter and assessing the cytotoxicity of candidate compounds, we will use them (P3) to pharmacologically target the TFs in W. chondrophila and other Chlamydiae.By focusing our ChIP-SEQ experiments on the 10 W. chondrophila TFs that represent the core set of TFs present in other members of the phylum Chlamydiae, our studies will also unearth the landscape of a regulatory (pan-)genome at a hitherto unprecedented scale and ultimately extract: a) the logic of the core transcriptional circuitry of Chlamydiae, b) the unknown transcriptional regulator(s) orchestrating the chlamydial differentiation program and c) new potential antibiotics to treat chlamydial (or other) infections.
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