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Xavier Nassif

Publications and source records attributed to Xavier Nassif.

At least 19 recordsLinked to original sources
PubMed2006

Identification of candidates for a subunit vaccine against extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause a large spectrum of infections. The majority of ExPEC strains are closely related to the B2 or the D phylogenetic group. The aim of our study was to develop a protein-based vaccine against these ExPEC strains. To this end, we identified ExPEC-specific genomic regions, using a comparative genome analysis, between the nonpathogenic E. coli strain K-12 MG1655 and ExPEC strains C5 (meningitis isolate) and CFT073 (urinary tract infection isolate). The analysis of these genomic regions allowed the selection of 40 open reading frames, which are conserved among B2/D clinical isolates and encode proteins with putative outer membrane localization. These genes were cloned, and recombinant proteins were purified and assessed as vaccine candidates. After immunization of BALB/c mice, five proteins induced a significant protective immunity against a lethal challenge with a clinical E. coli strain of the B2 group. In passive immunization assays, antigen-specific antibodies afforded protection to naive mice against a lethal challenge. Three of these antigens were related to iron acquisition metabolism, an important virulence factor of the ExPEC, and two corresponded to new, uncharacterized proteins. Due to the large number of genetic differences that exists between commensal and pathogenic strains of E. coli, our results demonstrate that it is possible to identify targets that elicit protective immune responses specific to those strains. The five protective antigens could constitute the basis for a preventive subunit vaccine against diseases caused by ExPEC strains.

Animals↗
PubMed2006

Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.

Animals↗
PubMed2006

Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures.

Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte-endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH(2)-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte-endothelial cell interaction.

Cell Adhesion↗
PubMed2006

Bacteriophages and pathogenicity: more than just providing a toxin?

An increasing number of pathogenicity factors carried by bacteriophages have been discovered. This review considers bacteriophage-bacterium interaction and its relation to disease processes. We discuss the search for new bacteriophage-associated pathogenicity factors, with emphasis on recent advances brought by the use of genomic sequence data and the techniques of genomic epidemiology.

Bacteria↗
PubMed2006

Pilus-mediated adhesion of Neisseria meningitidis is negatively controlled by the pilus-retraction machinery.

The type IV pili (Tfp) of Neisseria meningitidis play an essential role in meningococcal virulence by mediating the initial interaction of bacteria with host cells. Tfp are also subject to retraction, which relies on the PilT protein. Among the other components of the Tfp machinery, PilC1, a pilus-associated protein, is important for Tfp biogenesis and adhesion. Adhesion of N. meningitidis to living epithelial cells was previously shown to rely on the upregulation of the pilC1 gene. On the other hand the lack of induction of pilC1 is believed to be responsible for the low adhesion of N. meningitidis onto fixed dead cells. Surprisingly, a pilT mutant, unable to retract its pili, has been shown to adhere very efficiently onto both living and fixed epithelial cells. To elucidate the mechanisms by which the pilus retraction machinery mediates meningococcal adhesion onto fixed cells, an analysis of gene expression levels in wild-type and pilT meningococci was performed using DNA microarrays. One of the upregulated genes in the pilT strain was pilC1. This result was confirmed using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis. The transcription starting point responsible for the upregulation of pilC1 in a pilT background was shown to be different from those controlling the induction of pilC1 upon contact with living host cells. Subsequent work using a strain hyperproducing PilT confirmed that PilT downregulates the production of PilC1. Furthermore using a pilC1 allele under the control of IPTG, we demonstrated that the upregulation of pilC1 in a pilT background was responsible for the adhesive phenotype onto fixed dead cells. Taken together our results demonstrate that the pilus retraction machinery negatively controlled the adhesiveness of the Tfp via the expression of pilC1.

Adenosine Triphosphatases↗
PubMed2006

A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili.

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.

Bacterial Proteins↗
PubMed2006

Meningococcal adhesion suppresses proapoptotic gene expression and promotes expression of genes supporting early embryonic and cytoprotective signaling of human endothelial cells.

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.

Apoptosis↗
PubMed2005

Invasion of endothelial cells by Neisseria meningitidis requires cortactin recruitment by a phosphoinositide-3-kinase/Rac1 signalling pathway triggered by the lipo-oligosaccharide.

Type-IV-pilus-mediated adhesion of Neisseria meningitidis (also known as meningococcus) to human endothelial cells induces the formation of membrane protrusions leading to bacterial uptake. We have previously shown that these protrusions result from a Rho- and Cdc42-dependent cortical actin polymerization, and from the activation of the ErbB2 tyrosine-kinase receptor and the Src kinase, leading to tyrosine phosphorylation of cortactin. We report here that N. meningitidis mutants expressing a deglycosylated lipo-oligosaccharide are poorly invasive. These mutants show structurally altered actin polymerization. Moreover, although they efficiently recruit and activate ErbB2 and Src, these mutants are defective in the recruitment and phosphorylation of cortactin. We demonstrate that phosphorylated cortactin controls the cortical actin polymerization, which leads to membrane protrusion formation. In addition, we show that cortactin recruitment is dependent on the activation of a phosphoinositide-3-kinase/Rac1-GTPase signalling pathway, which is required for actin polymerization and internalization of N. meningitidis, and is not activated by the mutant strains. Altogether, these results define a new role for the lipo-oligosaccharide in triggering a phosphoinositide-3-kinase/Rac1 signalling required to elicit an efficient uptake of N. meningitidis in non-phagocytic cells.

Actin Cytoskeleton↗
PubMed2005

A chromosomally integrated bacteriophage in invasive meningococci.

Cerebrospinal meningitis is a feared disease that can cause the death of a previously healthy individual within hours. Paradoxically, the causative agent, Neisseria meningitidis, is a common inhabitant of the human nasopharynx, and as such, may be considered a normal, commensal organism. Only in a small proportion of colonized people do the bacteria invade the bloodstream, from where they can cross the blood-brain barrier to cause meningitis. Furthermore, most meningococcal disease is caused by bacteria belonging to only a few of the phylogenetic groups among the large number that constitute the population structure of this genetically variable organism. However, the genetic basis for the differences in pathogenic potential remains elusive. By performing whole genome comparisons of a large collection of meningococcal isolates of defined pathogenic potential we brought to light a meningococcal prophage present in disease-causing bacteria. The phage, of the filamentous family, excises from the chromosome and is secreted from the bacteria via the type IV pilin secretin. Therefore, this element, by spreading among the population, may promote the development of new epidemic clones of N. meningitidis that are capable of breaking the normal commensal relationship with humans and causing invasive disease.

Base Sequence↗
PubMed2005

Human and bovine milk oligosaccharides inhibit Neisseria meningitidis pili attachment in vitro.

Milk oligosaccharides have been shown to interfere with adhesion of many pathogens to host mucosal surfaces. Characterization of the adhesion mechanisms of the bacteria to host cell surface is needed to develop novel functional food, infant formulas, and anti-infective drugs. Adhesion of Neisseria meningitidis, a human specific pathogen causing meningitis and septicemia, is not completely understood but is mediated by type IV pili. Here, we developed a microtiter well pili binding assay to investigate the binding activities of N. meningitidis isolated type IV pili to different glycoproteins. Pili binding activities to bovine thyroglobulin and human salivary agglutinin but not to chicken ovalbumin were present. Inhibition of these binding activities was demonstrated by fractionated human or bovine milk oligosaccharides. The binding of neisserial pili to bovine thyroglobulin was most effective and was clearly inhibited by human milk neutral or bovine milk acidic oligosaccharides.

Animals↗
PubMed2005

Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring after pilus assembly, essential for fibre stability and function.

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.

Cells, Cultured↗
PubMed2005

PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells.

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.

Bacterial Adhesion↗
PubMed2005

Contact with host cells induces a DNA repair system in pathogenic Neisseriae.

DNA repair systems play a major role in maintaining the integrity of bacterial genomes. Neisseria meningitidis, a human pathogen capable of colonizing the human nasopharynx, possesses numerous DNA repair genes but lacks inducible DNA repair systems such as the SOS response, present in most bacteria species. We recently identified a set of genes upregulated by contact with host cells. An open reading frame having high homology with the small subunit of Escherichia coli exonuclease VII (xseB) belongs to this regulon. The increased sensitivity of a mutant in this coding sequence to UV irradiation, alkylating agent and nalidixic acid demonstrates the participation of this gene in meningococcal DNA repair. In addition, the upregulation of the transcription of this open reading frame upon interaction of N. meningitidis with host cells increased not only the bacterial ability to repair its DNA but also the rate of phase variation by frameshifting. Together these data demonstrate that N. meningitidis possesses an inducible DNA repair system that might be used by the bacteria to adapt to its niches when it is colonizing a new host.

Bacterial Proteins↗
PubMed2004

Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins.

Pathogenic Neisseria express type IV pili (tfp), which have been shown to play a central role in the interactions of bacteria with their environment. The regulation of piliation thus constitutes a central element in bacterial life cycle. The PilC proteins are outer membrane-associated proteins that have a key role in tfp biogenesis since PilC-null mutants appear defective for fibre expression. Moreover, tfp are also subjected to retraction, which is under the control of the PilT nucleotide-binding protein. In this work, we bring evidence that fibre retraction involves the translocation of pilin subunits to the cytoplasmic membrane. Furthermore, by engineering meningococcal strains that harbour inducible pilC genes, and with the use of meningococcus-cell interaction as a model for the sequential observation of fibre expression and retraction, we show that the PilC proteins regulate PilT-mediated fibre retraction.

Adenosine Triphosphatases↗
PubMed2004

Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili.

Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109-11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37 degrees C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA+ phenotype even in the presence of a functional periplasmic DsbA.

Amino Acid Sequence↗
PubMed2004

The siderophore receptor IroN, but not the high-pathogenicity island or the hemin receptor ChuA, contributes to the bacteremic step of Escherichia coli neonatal meningitis.

Using a neonatal rat meningitis model, we examined the involvement of three iron uptake systems, namely, the high-pathogenicity island, the hemin receptor ChuA, and the siderophore receptor IroN, in the pathogenesis of Escherichia coli neonatal meningitis. Only IroN appeared to play a major role during the bacteremic step of the disease.

Animals↗
PubMed2003

Molecular analysis and experimental virulence of French and North American Escherichia coli neonatal meningitis isolates: identification of a new virulent clone.

Phylogenetic relationships, virulence factors, alone and in specific combinations, and virulence in a rat meningitis model were examined among 132 isolates of Escherichia coli neonatal meningitis from France and North America. Isolates belonging to phylogenetic groups A (n=11), D (n=20), and B2 (n=99) had similar high prevalence rates of the siderophores aerobactin and yersiniabactin and the K1 capsule (>/=70%) yet induced different level of experimental bacteremia. Ectochromosomal DNA-like domains involved in blood-brain barrier passage (PAI III(536) [sfa/foc and iroN; 34%]; GimA [ibeA and ptnC; 38%]; PAI II(J96) [hly, cnf1, and hra; 10%]) were restricted to B2 isolates. Among group B2 isolates, representatives of the O45:K1 clonal group (n=30), which lacked these domains, were as able as the archetypal O18:K1 strain C5 to cause meningitis. Molecular epidemiology combined with experimental virulence assays demonstrate that known virulence factors are insufficient to fully explain the pathophysiology of ECNM and to allow for rational search for new virulence factors.

Animals↗