1. Functional profiling of the Neisseria meningitidis genome
By revealing complete repertoires of genes, genome sequences have provided 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, which are necessary to achieve this goal, are often sorely lacking.
As shown in S. cerevisiae, the model organism for genomics, the most valuable genomic toolbox for determining gene function is a comprehensive archived collection of mutants constructed by systematic targeted mutagenesis. In bacteria, archived collections of mutants containing mutations in each non-essential gene have so far been constructed only in a few species. 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 for large-scale functional profiling of its genome.
We have therefore designed NeMeSys a biological resource for Neisseria meningitidis systematic functional analysis (2). This toolbox consists of three modules. The first module is the manually annotated complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013). In addition, we have manually (re)annotated eight publicly available Neisseria genome sequences, which constitutes the second module of NeMeSys. Finally, the last module is an archived exhaustive library of mutants with mutations in 1,591 non-essential genes, showing that the minimal meningococcal genome consists of 388 genes. This collection of mutants is currently being prepared for distribution and will soon be available upon request. All these data are stored in a freely accessible online database (NeMeSys).
2. Global analysis of the biology of a universal bacterial virulence factor: the type IV pilus (Tfp)
Bacterial attachment to surfaces, including to host cells in pathogenic species, is often mediated by hair-like appendages known as pili. Among these organelles, Tfp (see picture) are the most widespread. Tfp are the paradigm of a class of nano-machines called type IV filamentous (Tff) nano-machines, which are found in more than 1,800 different species spanning almost all phyla of Bacteria and Archaea (6).
Tfp are multi-functional nano-machines that in addition to being adhesive organelles are also involved in a form of locomotion known as twitching motility, the formation of bacterial aggregates, competence for DNA transformation etc. Despite their wide distribution and key role in virulence in important human pathogens (enteropathogenic E. coli, N. gonorrhoeae, N. meningitidis, V. cholerae etc.), the molecular mechanisms underlying Tfp biogenesis (how pili are assembled) and the multiple properties mediated by these organelles remain poorly understood. Providing some answers to these fundamental questions is the main goal of our research. We have therefore started a systematic and global analysis of Tfp biology in two very distant bacterial species: N. meningitidis and the Gram-positive opportunistic pathogen Streptococcus sanguinis, which causes infective endocarditis.
N. meningitidis Tfp
Our historical model is 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 15 genes essential for Tfp biogenesis (mutants are non-piliated) and seven that are accessory (mutants are piliated but affected for Tfp-mediated functions). An exhaustive phenotypic characterisation of the corresponding mutants has outlined the first global picture of the role of most, if not all, the genes involved in Tfp biology in a single genetic background.
We are currently elucidating the function of the corresponding proteins using a multi-disciplinary approach combining molecular genetics, biochemistry and structural biology. This ongoing effort has already led to significant results both for the essential and accessory Pil proteins.
By using a genetic approach, we have defined a four-step model for Tfp biogenesis and determine at which step each of the proteins essential for piliation is involved. Perhaps surprisingly, this revealed that as many as eight 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 Tfp assembly machinery is apparently simpler than anticipated because it consists, in addition to the pilin PilE, of a set of only seven proteins (PilD, PilF, PilG, PilM, PilN, PilO and PilP). A large-scale study of the interactions between these proteins outlined a complex protein interaction network (2) and has notably improved our understanding of the topology of the sub-complex involved in pilus assembly. This has provided a useful blueprint for a recently published study (9) in which we used a synthetic biology approach to reconstitute, in a non-native heterologous host, a minimal machinery capable of building Tfp. We showed that the above eight synthetic genes are sufficient to promote Tfp assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterised biochemically. These results contribute to a better mechanistic understanding of the filament assembly.
Other studies by our group have illustrated how minor pilus components can enhance the functional properties of filaments of rather simple composition and structure. For example, we have shown that PilX, which is dispensable for Tfp biogenesis, is crucial for the formation of bacterial aggregates and attachment to human cells. This protein, which co-localises with Tfp, has the 3D structural fold shared by all pilins (see picture). PilX is thus a minor, or low abundance, pilin that assembles within the filaments in a similar way to the major pilus subunit PilE. Strikingly, deletion of a distinctive structural element in PilX, which is predicted to be exposed on the surface of the filaments, was found to be sufficient to abolish aggregation and adhesion. This led us to propose a model in which surface-exposed motifs in PilX subunits stabilise bacterial aggregates against the disruptive force of pilus retraction (1).
Recently, we have shown that another minor pilin, ComP, which is dispensable for Tfp biogenesis but key for DNA transformation, is at the basis of the well-known preferential uptake by pathogenic Neisseria species of their own DNA, which contains short and genus-specific DUS (DNA uptake sequence) watermarks (4). This helps meningococci protect themselves from uncontrolled transformation by foreign DNA and preserve species structure. We found that meningococcal Tfp bind DNA through ComP, which displays an exquisite binding preference for DUS. ComP is thus the DUS receptor that has remained elusive for decades, and the first pilin with DNA-binding propensity. We have shown that while peripheral bases in the DUS are less important, inner bases are essential for interaction with ComP and transformation (5). Strikingly, naturally occuring DUS variants in the genomes of human Neisseria commensals differing from canonical DUS by only one or two bases were found to be dramatically impaired for transformation of N. meningitidis (5). These DUS variants are preferentially bound by their cognate ComP variants, confirming that a similar mechanism is used by all Neisseriaceae species to promote transformation by their own DNA. The recently determined 3D structures of two ComP orthologs (see picture) helped us defines a new mode of DNA-binding adapted for exported DNA receptors (8). These findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant and widespread mechanism for modulating horizontal gene transfer between competent species sharing the same niche.
S. sanguinis Tfp
Recently, we established S. sanguinis as a useful new model for studying Tfp biology. We performed the most detailed molecular characterisation of Tfp in a Gram-positive bacterium (7), demonstrating that the naturally competent S. sanguinis produces retractable Tfp, which like their Gram-negative counterparts can generate hundreds of piconewton of tensile force and promote twitching motility. Although S. sanguinis Tfp and are unusual because they are composed of two pilins in comparable amounts (rather than one as normally seen), they are assembled by a machinery similar but simpler than in Gram-negative species since only ten components are required. We are currently using this new model to answer some questions that are difficult to tackle in a Gram-negative piliated species.
1. 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.
2. 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.
3. Georgiadou, M., M. Castagnini, G. Karimova, D. Ladant and V. Pelicic. 2012. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a sub-complex involved in pilus assembly. Mol. Microbiol. 84: 857-873.
4. Cehovin, A., P. J. Simpson, M. A. McDowell, D. R. Brown, R. Noschese, M. Pallett, J. Brady, G. S. Baldwin, S. M. Lea, S. J. Matthews and V. Pelicic. 2013. Specific DNA recognition mediated by a type IV pilin. Proc. Natl. Acad. Sci. USA. 110: 3065-3070.
5. Berry, J. L., A. Cehovin, M. A. McDowell, S. M. Lea and V. Pelicic. 2013. Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species. PLoS Genet. 9: e1004014.
6. Berry, J. L., and V. Pelicic. 2015. Exceptionally widespread nano-machines composed of type IV pilins: the prokaryotic Swiss Army knives. FEMS Microbiol. Rev. 39: 134-154.
7. Gurung, I., I. Spielman, M. R. Davies, R. Lala, P. Gaustad, N. Biais and V. Pelicic. 2016. Functional analysis of an unusual type IV pilus in the Gram-positive Streptococcus sanguinis. Mol. Microbiol. 99: 380-392.
8. Berry, J. L., Y. Xu, P. N. Ward, S. M. Lea, S. J. Matthews and V. Pelicic. 2016. A comparative structure/function analysis of two type IV pilin DNA receptors defines a novel mode of DNA-binding. Structure. 24: 926-934.
9. Goosens, V. J., A. Busch, M. Georgiadou, M. Castagnini, K. T. Forest, G. Waksman and V. Pelicic. 2017. Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili. Proc. Natl. Acad. Sci. USA. 114: E4978-E4986.
Dr Vladimir Pelicic
Dr Jamie Berry
Our research is/has been generously supported by the Agence Nationale de la Recherche (ANR), Biotechnology and Biological Sciences Research Council (BBSRC), European Union (FP7), Medical Research Council (MRC), Meningitis Research Foundation, Royal Society and Wellcome Trust.
Research Student Supervision
Brown,DR, PhD Imperial College London
Carbonnelle,E, PhD University Paris Descartes (France)
Ewles,HA, PhD Imperial College London
Georgiadou,M, PhD Imperial College London
Gurung,I, PhD Imperial College London
Helaine,S, PhD University Paris Descartes (France)