Imperial College London

DrVladimirPelicic

Faculty of MedicineDepartment of Medicine

Reader in Molecular Bacteriology & Infection
 
 
 
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Contact

 

+44 (0)20 7594 2080v.pelicic Website

 
 
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Location

 

3.20Flowers buildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

48 results found

Goosens VJ, Busch A, Georgiadou M, Castagnini M, Forest KT, Waksman G, Pelicic Vet al., 2017, Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili., Proc Natl Acad Sci U S A, Vol: 114, Pages: E4978-E4986

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gram-negative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.

JOURNAL ARTICLE

Gurung I, Berry J-L, Hall AMJ, Pelicic Vet al., 2017, Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology., Nucleic Acids Res, Vol: 45

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.

JOURNAL ARTICLE

Berry J-L, Xu Y, Ward PN, Lea SM, Matthews SJ, Pelicic Vet al., 2016, A Comparative Structure/Function Analysis of Two Type IV Pilin DNA Receptors Defines a Novel Mode of DNA Binding., Structure, Vol: 24, Pages: 926-934

DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.

JOURNAL ARTICLE

Gurung I, Spielman I, Davies MR, Lala R, Gaustad P, Biais N, Pelicic Vet al., 2016, Functional analysis of an unusual type IV pilus in the Gram-positive Streptococcus sanguinis., Mol Microbiol, Vol: 99, Pages: 380-392

Type IV pili (Tfp), which have been studied extensively in a few Gram-negative species, are the paradigm of a group of widespread and functionally versatile nano-machines. Here, we performed the most detailed molecular characterisation of Tfp in a Gram-positive bacterium. We demonstrate that the naturally competent Streptococcus sanguinis produces retractable Tfp, which like their Gram-negative counterparts can generate hundreds of piconewton of tensile force and promote intense surface-associated motility. Tfp power 'train-like' directional motion parallel to the long axis of chains of cells, leading to spreading zones around bacteria grown on plates. However, S. sanguinis Tfp are not involved in DNA uptake, which is mediated by a related but distinct nano-machine, and are unusual because they are composed of two pilins in comparable amounts, rather than one as normally seen. Whole genome sequencing identified a locus encoding all the genes involved in Tfp biology in S. sanguinis. A systematic mutational analysis revealed that Tfp biogenesis in S. sanguinis relies on a more basic machinery (only 10 components) than in Gram-negative species and that a small subset of four proteins dispensable for pilus biogenesis are essential for motility. Intriguingly, one of the piliated mutants that does not exhibit spreading retains microscopic motility but moves sideways, which suggests that the corresponding protein controls motion directionality. Besides establishing S. sanguinis as a useful new model for studying Tfp biology, these findings have important implications for our understanding of these widespread filamentous nano-machines.

JOURNAL ARTICLE

Berry J-L, Pelicic V, 2015, Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives., FEMS Microbiol Rev, Vol: 39, Pages: 134-154

Prokaryotes have engineered sophisticated surface nanomachines that have allowed them to colonize Earth and thrive even in extreme environments. Filamentous machineries composed of type IV pilins, which are associated with an amazing array of properties ranging from motility to electric conductance, are arguably the most widespread since distinctive proteins dedicated to their biogenesis are found in most known species of prokaryotes. Several decades of investigations, starting with type IV pili and then a variety of related systems both in bacteria and archaea, have outlined common molecular and structural bases for these nanomachines. Using type IV pili as a paradigm, we will highlight in this review common aspects and key biological differences of this group of filamentous structures.

JOURNAL ARTICLE

Georgiadou M, Pelicic V, 2014, Bacterial Pili, Bacterial Pili, Editors: Barocchi, Telford, Publisher: CABI, ISBN: 9781780642550

This book discusses the synthesis, structure, evolution, function and role in pathogenesis of these complex structures, and their basis for vaccine development and therapeutics for Streptococcus pathogens.

BOOK CHAPTER

Berry J-L, Cehovin A, McDowell MA, Lea SM, Pelicic Vet al., 2013, Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species., PLoS Genet, Vol: 9

Natural transformation is the widespread biological process by which "competent" bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or "transformed". To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence) watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComPsub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche.

JOURNAL ARTICLE

Cehovin A, Simpson PJ, McDowell MA, Brown DR, Noschese R, Pallett M, Brady J, Baldwin GS, Lea SM, Matthews SJ, Pelicic Vet al., 2013, Specific DNA recognition mediated by a type IV pilin., Proc Natl Acad Sci U S A, Vol: 110, Pages: 3065-3070

Natural transformation is a dominant force in bacterial evolution by promoting horizontal gene transfer. This process may have devastating consequences, such as the spread of antibiotic resistance or the emergence of highly virulent clones. However, uptake and recombination of foreign DNA are most often deleterious to competent species. Therefore, model naturally transformable gram-negative bacteria, including the human pathogen Neisseria meningitidis, have evolved means to preferentially take up homotypic DNA containing short and genus-specific sequence motifs. Despite decades of intense investigations, the DNA uptake sequence receptor in Neisseria species has remained elusive. We show here, using a multidisciplinary approach combining biochemistry, molecular genetics, and structural biology, that meningococcal type IV pili bind DNA through the minor pilin ComP via an electropositive stripe that is predicted to be exposed on the filaments surface and that ComP displays an exquisite binding preference for DNA uptake sequence. Our findings illuminate the earliest step in natural transformation, reveal an unconventional mechanism for DNA binding, and suggest that selective DNA uptake is more widespread than previously thought.

JOURNAL ARTICLE

Loh E, Kugelberg E, Tracy A, Zhang Q, Gollan B, Ewles H, Chalmers R, Pelicic V, Tang CMet al., 2013, Temperature triggers immune evasion by Neisseria meningitidis., Nature, Vol: 502, Pages: 237-240

Neisseria meningitidis has several strategies to evade complement-mediated killing, and these contribute to its ability to cause septicaemic disease and meningitis. However, the meningococcus is primarily an obligate commensal of the human nasopharynx, and it is unclear why the bacterium has evolved exquisite mechanisms to avoid host immunity. Here we demonstrate that mechanisms of meningococcal immune evasion and resistance against complement increase in response to an increase in ambient temperature. We have identified three independent RNA thermosensors located in the 5' untranslated regions of genes necessary for capsule biosynthesis, the expression of factor H binding protein, and sialylation of lipopolysaccharide, which are essential for meningococcal resistance against immune killing. Therefore increased temperature (which occurs during inflammation) acts as a 'danger signal' for the meningococcus, enhancing its defence against human immune killing. Infection with viral pathogens, such as influenza, leads to inflammation in the nasopharynx with an increased temperature and recruitment of immune effectors. Thermoregulation of immune defence could offer an adaptive advantage to the meningococcus during co-infection with other pathogens, and promote the emergence of virulence in an otherwise commensal bacterium.

JOURNAL ARTICLE

Georgiadou M, Castagnini M, Karimova G, Ladant D, Pelicic Vet al., 2012, Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly., Mol Microbiol, Vol: 84, Pages: 857-873

The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.

JOURNAL ARTICLE

Nagorska K, Silhan J, Li Y, Pelicic V, Freemont PS, Baldwin GS, Tang CMet al., 2012, A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis., Mol Microbiol, Vol: 83, Pages: 1064-1079

Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterized meningococcal base excision repair enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However, MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence shows that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes.

JOURNAL ARTICLE

Cehovin A, Kroll JS, Pelicic V, 2011, Testing the vaccine potential of PilV, PilX and ComP, minor subunits of Neisseria meningitidis type IV pili., Vaccine, Vol: 29, Pages: 6858-6865

Because meningitis and septicaemia caused by Neisseria meningitidis are major public health problems worldwide, the design of a broadly protective vaccine remains a priority. Type IV pili (Tfp) are surface-exposed filaments playing a key role in pathogenesis in a variety of bacterial species, including N. meningitidis, that have demonstrated vaccine potential. Unfortunately, in the meningococcus, the major pilus subunit PilE usually undergoes extensive antigenic variation and is therefore not suitable as a vaccine component. However, we have recently shown that N. meningitidis Tfp contain low abundance subunits PilX, PilV and ComP, collectively called minor pilins, that are highly conserved and modulate Tfp-linked functions key to pathogenesis. This prompted us to examine the vaccine potential of these proteins by assessing whether sera directed against them have bactericidal properties and/or are able to interfere with Tfp-linked functions. Here we show that minor pilin proteins are recognized by sera of patients convalescent from meningococcal disease and that antibodies directed against some of them can selectively interfere with Tfp-linked functions. This shows that, despite their apparent inability to elicit bactericidal antibodies, minor pilins might have vaccine potential.

JOURNAL ARTICLE

Szeto TH, Dessen A, Pelicic V, 2011, Structure/function analysis of Neisseria meningitidis PilW, a conserved protein that plays multiple roles in type IV pilus biology., Infect Immun, Vol: 79, Pages: 3028-3035

Type IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple functions and play a key role in pathogenesis in several important human pathogens, including Neisseria meningitidis. Tfp biology remains poorly understood at a molecular level because the roles of the numerous proteins that are involved remain mostly obscure. Guided by the high-resolution crystal structure we recently reported for N. meningitidis PilW, a widely conserved protein essential for Tfp biogenesis, we have performed a structure/function analysis by targeting a series of key residues through site-directed mutagenesis and analyzing the corresponding variants using an array of phenotypic assays. Here we show that PilW's involvement in the functionality of Tfp can be genetically uncoupled from its concurrent role in the assembly/stabilization of the secretin channels through which Tfp emerge on the bacterial surface. These findings suggest that PilW is a multifunctional protein.

JOURNAL ARTICLE

Brown DR, Helaine S, Carbonnelle E, Pelicic Vet al., 2010, Systematic functional analysis reveals that a set of seven genes is involved in fine-tuning of the multiple functions mediated by type IV pili in Neisseria meningitidis., Infect Immun, Vol: 78, Pages: 3053-3063

Type IV pili (Tfp), which mediate multiple phenotypes ranging from adhesion to motility, are one of the most widespread virulence factors in bacteria. However, the molecular mechanisms of Tfp biogenesis and associated functions remain poorly understood. One of the underlying reasons is that the roles played by the numerous genes involved in Tfp biology are unclear because corresponding mutants have been studied on a case-by-case basis, in different species, and using different assays, often generating heterogeneous results. Therefore, we have recently started a systematic functional analysis of the genes involved in Tfp biology in a well-characterized clinical isolate of the human pathogen Neisseria meningitidis. After previously studying 16 genes involved in Tfp biogenesis, here we report the characterization of 7 genes that are dispensable for piliation and potentially involved in Tfp biology. Using a battery of assays, we assessed piliation and each of the Tfp-linked functions in single mutants, double mutants in which filament retraction is abolished by a concurrent mutation in pilT, and strains overexpressing the corresponding proteins. This showed that each of the seven genes actually fine-tunes a Tfp-linked function(s), which brings us one step closer to a global view of Tfp biology in the meningococcus.

JOURNAL ARTICLE

Cehovin A, Winterbotham M, Lucidarme J, Borrow R, Tang CM, Exley RM, Pelicic Vet al., 2010, Sequence conservation of pilus subunits in Neisseria meningitidis., Vaccine, Vol: 28, Pages: 4817-4826

The rapid onset and dramatic consequences of Neisseria meningitidis infections make the design of a broadly protective vaccine a priority for public health. There is an ongoing quest for meningococcal components that are surface exposed, widely conserved and can induce protective antibodies. Type IV pili (Tfp) are filamentous structures with a key role in pathogenesis that extend beyond the surface of the bacteria and have demonstrated vaccine potential. However, extensive antigenic variation of PilE, the major subunit of Tfp, means that they are currently considered to be unsuitable vaccine components. Recently it has been shown that Tfp also contain low abundance pilins ComP, PilV and PilX in addition to PilE. This prompted us to examine the prevalence and sequence diversity of these proteins in a panel of N. meningitidis disease isolates. We found that all minor pilins are highly conserved and the major pilin genes are also highly conserved within the ST-8 and ST-11 clonal complexes. These data have important implications for the re-consideration of pilus subunits as vaccine antigens.

JOURNAL ARTICLE

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

BACKGROUND: Genome sequences, now available for most pathogens, hold promise for the rational design of new therapies. 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. This holds true for Neisseria meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicemia. RESULTS: By determining and manually annotating the complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013) and assembling a library of defined mutants in up to 60% of its non-essential genes, we have created NeMeSys, a biological resource for Neisseria meningitidis systematic functional analysis. To further enhance the versatility of this toolbox, we have manually (re)annotated eight publicly available Neisseria genome sequences and stored all these data in a publicly accessible online database. The potential of NeMeSys for narrowing the gap between sequence and function is illustrated in several ways, notably by performing a functional genomics analysis of the biogenesis of type IV pili, one of the most widespread virulence factors in bacteria, and by identifying through comparative genomics a complete biochemical pathway (for sulfur metabolism) that may potentially be important for nasopharyngeal colonization. CONCLUSIONS: By improving our capacity to understand gene function in an important human pathogen, NeMeSys is expected to contribute to the ongoing efforts aimed at understanding a prokaryotic cell comprehensively and eventually to the design of new therapies.

JOURNAL ARTICLE

Pelicic V, 2008, Type IV pili: e pluribus unum?, Mol Microbiol, Vol: 68, Pages: 827-837

The widespread role of pili as colonization factors in pathogens has long been recognized in Gram-negative bacteria and more recently in Gram-positive bacteria, making the study of these hair-like filaments a perennial hot topic for research. No other pili are found in as many or as diverse bacteria as type IV pili. This is likely a consequence of their ancient origin and unique ability to promote multiple and strikingly different phenotypes such as attachment to surfaces, aggregation, uptake of DNA during transformation, motility, etc. Two decades of investigations in several model species have shed some light on the structure of these filaments and the molecular basis of some of the properties they confer. Moreover, recent discoveries have led to a better knowledge of the genetic basis and molecular mechanisms of type IV pili biogenesis. This brings us a few steps closer to understanding how these filaments are produced, but leaves us wondering whether (as in the famous motto that inspired the title) out of the many models studied will emerge one unifying mechanism.

JOURNAL ARTICLE

Trindade MB, Job V, Contreras-Martel C, Pelicic V, Dessen Aet al., 2008, Structure of a widely conserved type IV pilus biogenesis factor that affects the stability of secretin multimers., J Mol Biol, Vol: 378, Pages: 1031-1039

Type IV pili (Tfp) are arguably the most widespread pili in bacteria, whose biogenesis requires a complex machinery composed of as many as 18 different proteins. This includes the conserved outer membrane-localized secretin, which forms a pore through which Tfp emerge on the bacterial surface. Although, in most model species studied, secretin oligomerization and functionality requires the action of partner lipoproteins, structural information regarding these molecules is limited. We report the high-resolution crystal structure of PilW, the partner lipoprotein of the type IV pilus secretin PilQ from Neisseria meningitidis, which defines a conserved class of Tfp biogenesis proteins involved in the formation and/or stability of secretin multimers in a wide variety of bacteria. The use of the PilW structure as a blueprint reveals an area of high-level sequence conservation in homologous proteins from different pathogens that could reflect a possible secretin-binding site. These results could be exploited for the development of new broad-spectrum antibacterials interfering with the biogenesis of a widespread virulence factor.

JOURNAL ARTICLE

Arraiano CM, Bamford J, Brüssow H, Carpousis AJ, Pelicic V, Pflüger K, Polard P, Vogel Jet al., 2007, Recent advances in the expression, evolution, and dynamics of prokaryotic genomes., J Bacteriol, Vol: 189, Pages: 6093-6100, ISSN: 0021-9193

JOURNAL ARTICLE

Chamot-Rooke J, Rousseau B, Lanternier F, Mikaty G, Mairey E, Malosse C, Bouchoux G, Pelicic V, Camoin L, Nassif X, Duménil Get al., 2007, Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue., Proc Natl Acad Sci U S A, Vol: 104, Pages: 14783-14788, ISSN: 0027-8424

The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis, the etiological agent of cerebrospinal meningitis, crosses cellular barriers after adhering to host cells through type IV pili. Pilin glycosylation genes (pgl) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido-2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by "top-down" MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pili preparations originating from N. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacetamido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glyceramido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pglBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis.

JOURNAL ARTICLE

Helaine S, Dyer DH, Nassif X, Pelicic V, Forest KTet al., 2007, 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili., Proc Natl Acad Sci U S A, Vol: 104, Pages: 15888-15893, ISSN: 0027-8424

Type IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple virulence-related phenotypes. They are composed mainly of pilin subunits, which are processed before filament assembly by dedicated prepilin peptidases. Other proteins processed by these peptidases, whose molecular nature and mode of action remain enigmatic, play critical roles in Tfp biology. We have performed a detailed structure/function analysis of one such protein, PilX from Neisseria meningitidis, which is crucial for formation of bacterial aggregates and adhesion to human cells. The x-ray crystal structure of PilX reveals the alpha/beta roll fold shared by all pilins, and we show that this protein colocalizes with Tfp. These observations suggest that PilX is a minor, or low abundance, pilin that assembles within the filaments in a similar way to pilin. Deletion of a PilX distinctive structural element, which is predicted to be exposed on the filament surface, abolishes aggregation and adhesion. Our results support a model in which surface-exposed motifs in PilX subunits stabilize bacterial aggregates against the disruptive force of pilus retraction and illustrate how a minor pilus component can enhance the functional properties of pili of rather simple composition and structure.

JOURNAL ARTICLE

Carbonnelle E, Helaine S, Nassif X, Pelicic Vet al., 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, Vol: 61, Pages: 1510-1522, ISSN: 0950-382X

Although type IV pili (Tfp) are among the commonest virulence factors in bacteria, their biogenesis by complex machineries of 12-15 proteins, and thereby their function remains poorly understood. Interestingly, some of these proteins were reported to merely antagonize the retraction of the fibres powered by PilT, because piliation could be restored in their absence by a mutation in the pilT gene. The recent identification of the 15 Pil proteins dedicated to Tfp biogenesis in Neisseria meningitidis offered us the unprecedented possibility to define their exact contribution in this process. We therefore systematically introduced a pilT mutation into the corresponding non-piliated mutants and characterized them for the rescue of Tfp and Tfp-mediated virulence phenotypes. We found that in addition to the pilin, the main constituent of Tfp, only six Pil proteins were required for the actual assembly of the fibres, because apparently normal fibres were restored in the remaining mutants. Restored fibres were surface-exposed, except in the pilQ/T mutant in which they were trapped in the periplasm, suggesting that the PilQ secretin was the sole Pil component necessary for their emergence on the surface. Importantly, although in most mutants the restored Tfp were not functional, the pilG/T and pilH/T mutants could form bacterial aggregates and adhere to human cells efficiently, suggesting that Tfp stabilization and functional maturation are two discrete steps. These findings have numerous implications for understanding Tfp biogenesis/function and provide a useful groundwork for the characterization of the precise function of each Pil protein in this process.

JOURNAL ARTICLE

Linhartova I, Basler M, Ichikawa J, Pelicic V, Osicka R, Lory S, Nassif X, Sebo Pet al., 2006, Meningococcal adhesion suppresses proapoptotic gene expression and promotes expression of genes supporting early embryonic and cytoprotective signaling of human endothelial cells., FEMS Microbiol Lett, Vol: 263, Pages: 109-118, ISSN: 0378-1097

Neisseria meningitidis colonizes the human nasopharynx and occasionally causes lethal or damaging septicemia and meningitis. Here, we examined the adherence-mediated signaling of meningococci to human cells by comparing gene expression profiles of human umbilical vein endothelial cells (HUVEC) infected by adherent wild-type, frpC-deficient mutant, or the nonadherent (DeltapilD) N. meningitidis. Pili-mediated adhesion of meningococci resulted in alterations of expression levels of human genes known to regulate apoptosis, cell proliferation, inflammatory response, adhesion and genes for signaling pathway proteins such as TGF-beta/Smad, Wnt/beta-catenin and Notch/Jagged. This reveals that adhering piliated meningocci manipulate host signaling pathways controlling cell proliferation while establishing a commensal relationship.

JOURNAL ARTICLE

Pelicic V, Nassif X, 2006, Impact of genome sequences on mutational analysis of fungal and bacterial pathogens, Pathogenomics: genome analysis of pathogenic microbes, Editors: Hacker, Dobrindt, Publisher: John Wiley & Sons, Pages: 1-568, ISBN: 9783527312658

BOOK CHAPTER

Carbonnelle E, Hélaine S, Prouvensier L, Nassif X, Pelicic Vet al., 2005, Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring after pilus assembly, essential for fibre stability and function., Mol Microbiol, Vol: 55, Pages: 54-64, ISSN: 0950-382X

Type IV pili (Tfp) play a critical role in the pathogenic lifestyle of Neisseria meningitidis and N. gonorrhoeae, notably by facilitating bacterial attachment to human cells, but our understanding of their biogenesis, during which the fibres are assembled in the periplasm, then emerge onto the cell surface and are stabilized, remains fragmentary. We therefore sought to identify the genes required for Tfp formation in N. meningitidis by screening a genome-wide collection of mutants for those that were unable to form aggregates, another phenotype mediated by these organelles. Fifteen proteins, of which only seven were previously characterized, were found to be essential for Tfp biogenesis. One novel component, named PilW, was studied in more detail. We found that PilW is an outer-membrane protein necessary for the stabilization of the fibres but not for their assembly or surface localization, because Tfp could be restored on the surface in a pilW mutant by a mutation in the twitching motility gene pilT. However, Tfp-linked properties, including adherence to human cells, were not restored in a pilW/T mutant, which suggests that PilW is also essential for the functionality of the fibres. Together with the finding that PilW is important for the stability of PilQ multimers, our results extend the current model for Tfp biogenesis by suggesting that a multiprotein machinery in the outer-membrane is involved in the terminal stage of Tfp biogenesis during which growing fibres are not only stabilized, but also become perfectly functional.

JOURNAL ARTICLE

Hélaine S, Carbonnelle E, Prouvensier L, Beretti J-L, Nassif X, Pelicic Vet al., 2005, PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells., Mol Microbiol, Vol: 55, Pages: 65-77, ISSN: 0950-382X

The attachment of pathogenic Neisseria species to human cells, in which type IV pili (Tfp) play a key but incompletely defined role, depends on the ability of these bacteria to establish contacts with the target cells but also interbacterial interactions. In an effort to improve our understanding of the molecular mechanisms of N. meningitidis adherence to human cells, we screened a collection of defined mutants for those presenting reduced attachment to a human cell line. Besides underscoring the central role of Tfp in this process, this analysis led to the identification of mutants interrupted in a novel gene termed pilX, that displayed an adherence as impaired as that of a non-piliated mutant but quantitatively and qualitatively unaltered fibres. Moreover, the pilX gene, which encodes a pilin-like protein that copurifies with Tfp fibres, was also found to be essential for bacterial aggregation. We provide here several piece of evidence suggesting that PilX has intrinsic aggregative but no adhesive properties and that the reduced numbers of adherent bacteria seen with a pilX mutant result from the absence of interbacterial interactions. These data extend the current model for Tfp-facilitated adherence of N. meningitidis to human cells by suggesting that the pili lead to an increase in net initial adherence primarily by mediating a cooperation between the bacteria, which is supported by the finding that a major effect on initial adherence could be observed in a wild-type (WT) genetic background after a mechanical removal of the bacterial aggregates.

JOURNAL ARTICLE

Forman S, Linhartova I, Osicka R, Nassif X, Sebo P, Pelicic Vet al., 2003, Neisseria meningitidis RTX proteins are not required for virulence in infant rats., Infect Immun, Vol: 71, Pages: 2253-2257, ISSN: 0019-9567

RTX cytotoxins play an important role in virulence of numerous gram-negative pathogens. Unexpectedly, however, we show here that the RTX proteins of Neisseria meningitidis are dispensable for virulence in the infant rat model of infection.

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Geoffroy M-C, Floquet S, Métais A, Nassif X, Pelicic Vet al., 2003, Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis., Genome Res, Vol: 13, Pages: 391-398, ISSN: 1088-9051

The biologic role of a majority of the Neisseria meningitidis 2100 predicted coding regions is still to be assigned or experimentally confirmed. Determining the phenotypic effect of gene disruption being a fundamental approach to understanding gene function, we used high-density signature-tagged transposon mutagenesis, followed by a large-scale sequencing of the transposon insertion sites, to construct a genome-wide collection of mutants. The sequencing results for the first half of the 4548 mutants composing the library suggested that we have mutations in 80%-90% of N. meningitidis nonessential genes. This was confirmed by a whole-genome identification of the genes required for resistance to complement-mediated lysis, a key to meningococcal virulence. We show that all the genes we identified, including four previously uncharacterized, were important for the synthesis of the polysialic acid capsule or the lipooligosaccharide (LOS), suggesting that these are likely to be the only meningococcal attributes necessary for serum resistance. Our work provides a valuable and lasting resource that may lead to a global map of gene function in N. meningitidis.

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Raynaud C, Guilhot C, Rauzier J, Bordat Y, Pelicic V, Manganelli R, Smith I, Gicquel B, Jackson Met al., 2002, Phospholipases C are involved in the virulence of Mycobacterium tuberculosis., Mol Microbiol, Vol: 45, Pages: 203-217, ISSN: 0950-382X

Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild-type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the Delta plcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT-PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.

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Boechat N, Bouchonnet F, Bonay M, Grodet A, Pelicic V, Gicquel B, Hance AJet al., 2001, Culture at high density improves the ability of human macrophages to control mycobacterial growth., J Immunol, Vol: 166, Pages: 6203-6211, ISSN: 0022-1767

The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 x 10(4) cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.

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