Dr Vladimir Pelicic 

Our research consists of two linked streams.

1. Functional profiling of the Neisseria meningitidis genome

By revealing complete repertoires of genes, genome sequences provide the key to a better, and eventually global, understanding of the biology of living organisms. It is widely accepted that this will have important consequences on human health and economy by leading to the rational design of novel therapies against pathogens infecting mankind, livestock or crops. However, biological resources for genome-scale identification of gene function (notably genes involved in pathogenesis and/or genes essential for cell viability), which are necessary to achieve this goal, are often sorely lacking.

As shown in Saccharomyces cerevisiae, the model organism for genomics, the most valuable toolbox for determining gene function on a genome scale is a comprehensive archived collection of mutants constructed by a systematic targeted mutagenesis. In bacteria, archived collections of mutants containing mutations in all non-essential genes have so far been constructed only in two species (Escherichia coli and Acinetobacter baylyi), none of which a pathogen. The first goal of our research is to create such a resource in N. meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicaemia, and to use it to perform an exhaustive functional profiling of this species genome.

We have thus created NeMeSys a biological resource for Neisseria meningitidis systematic functional analysis. We have determined and manually annotated the complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013) and assembled a library of defined mutants in ~60% of its non-essential genes. The design of an archived collections of mutants containing mutations in each non-essential gene is currently under way. To further enhance the versatility of this toolbox, we have manually (re)annotated 8 publicly available Neisseria genome sequences and stored all these data in a publicly accessible online database (http://www.genoscope.cns.fr/agc/nemesys/).

2. Systematic analysis of the biology of a universal bacterial virulence factor: the type IV pilus.

Bacterial attachment to surfaces, including to host cells in pathogenic species, is often mediated by hair-like appendages known as pili. Among these organelles, type IV pili (Tfp) are the most widespread. They might be present in as many as 150 different species, both Gram-negative and Gram-positive, spanning most bacterial phyla. An  explanation to this might be that unlike other types of pili, Tfp are multi-functional machineries that in addition to being adhesive organelles are involved in a variety of other functions: a form of locomotion known as twitching motility, the formation of bacterial aggregates, competence for DNA transformation, etc.

Despite this virtual ubiquity and their key role in virulence in important human pathogens (enteropathogenic Escherichia coli, N. meningitidis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Vibrio cholerae, etc.), the molecular mechanisms underlying Tfp biogenesis (how pili are assembled and exported) and the multiple properties mediated by these organelles remain poorly understood. Providing some answers to these questions is the main goal of our research.

We have therefore started a systematic and global analysis of Tfp biology in what appears to be a perfect model organism: the 8013 clinical isolate of N. meningitidis that is heavily piliated, presents all the phenotypes classically linked with Tfp and has been used to design the NeMeSys biological resource (see above). Using NeMeSys, we have identified 16 genes "essential" for Tfp biogenesis (mutants are non-piliated) and 7 that are "accessory" (mutants are piliated) but modulate Tfp-mediated functions. Exhaustive phenotypic characterization of the corresponding mutants has outlined the first global picture in a single genetic background of the importance of most, if not all, the genes involved in Tfp biology.

Next, we started elucidating the function of the corresponding proteins using a multi-disciplinary approach combining molecular genetics, biochemistry and structural biology. This long-term effort, which is well under way, has already led to significant results both for the "essential" and "accessory" Pil proteins as can be seen in the two examples below.

By using a specific genetic approach, we could define at which step of Tfp biogenesis each of the 15 Pil proteins essential for piliation is involved, which led us to propose a four-step model for pilus biogenesis. Perhaps surprisingly, this revealed that as much as 8 Pil proteins are not involved in pilus assembly per se since Tfp can be expressed in their absence when pilus retraction is abolished. Therefore, the machinery assembling these organelles is apparently simpler than anticipated because it consists, in addition to the pilin PilE, in a set of only 6 proteins (PilD, PilF, PilM, PilN, PilO and PilP).

By combining genetics, biochemistry and structural biology, we showed that PilX, which is dispensable for Tfp biogenesis, is crucial for the formation of bacterial aggregates and attachment to human cells. We found that this protein, which co-localizes with Tfp and represents 3% of the pilus proteins, has a 3D structural fold shared by all pilins (see picture). Altogether, this suggests that PilX is a minor, or low abundance, pilin that assembles within the filaments in a similar way to the major pilus component PilE. Strikingly, deletion of a PilX distinctive structural element, which is predicted to be exposed on the filament surface, is sufficient to abolish aggregation and adhesion. This led us to propose a model in which surface-exposed motifs in PilX subunits stabilize bacterial aggregates against the disruptive force of pilus retraction. This study illustrates how a minor pilus component can enhance the functional properties of filaments of rather simple composition and structure.

Selected recent publications

1. Carbonnelle, E., S. Helaine, X. Nassif and V. Pelicic. 2006. A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili. Mol. Microbiol. 61: 1510-1522.

2. Helaine, S., D. H. Dyer, X. Nassif, V. Pelicic and K. T. Forest. 2007. 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili. Proc. Natl. Acad. Sci. USA. 104: 15888-15893.

3. V. Pelicic. 2008. Type IV pili: e pluribus unum? Mol. Microbiol. 68: 827-837.

4. Rusniok, C., D. Vallenet, S. Floquet, H. Ewles, C. Mouzé-Soulama, D. Brown, A. Lajus, C. Buchrieser, C. Médigue, P. Glaser and V. Pelicic. 2009. NeMeSys: a resource for narrowing the gap between sequence and function in the human pathogen Neisseria meningitidis. Genome Biol. 10: R110.

5. Brown, D., S. Helaine, E. Carbonnelle and V. Pelicic. 2010. Systematic functional analysis reveals that a set of 7 genes is involved in fine-tuning of the multiple functions mediated by type IV pili in Neisseria meningitidis. Infect. Immun. 78 : 3053-3063.

Funding

Our research is/has been generously supported by Agence Nationale de la Recherche (ANR), Biotechnology and Biological Sciences Research Council (BBSRC), European Union (FP7), Meningitis Research Foundation, Royal Society and Wellcome Trust.