New Microbicidal Functions of Tracheal Glands: Defective Anti-Infectious Response to Pseudomonas aeruginosa in Cystic Fibrosis

Tracheal glands (TG) may play a specific role in the pathogenesis of cystic fibrosis (CF), a disease due to mutations in the cftr gene and characterized by airway inflammation and Pseudomonas aeruginosa infection. We compared the gene expression of wild-type TG cells and TG cells with the cftr ΔF508 mutation (CF-TG cells) using microarrays covering the whole human genome. In the absence of infection, CF-TG cells constitutively exhibited an inflammatory signature, including genes that encode molecules such as IL-1α, IL-β, IL-32, TNFSF14, LIF, CXCL1 and PLAU. In response to P. aeruginosa, genes associated with IFN-γ response to infection (CXCL10, IL-24, IFNγR2) and other mediators of anti-infectious responses (CSF2, MMP1, MMP3, TLR2, S100 calcium-binding proteins A) were markedly up-regulated in wild-type TG cells. This microbicidal signature was silent in CF-TG cells. The deficiency of genes associated with IFN-γ response was accompanied by the defective membrane expression of IFNγR2 and altered response of CF-TG cells to exogenous IFN-γ. In addition, CF-TG cells were unable to secrete CXCL10, IL-24 and S100A8/S100A9 in response to P. aeruginosa. The differences between wild-type TG and CF-TG cells were due to the cftr mutation since gene expression was similar in wild-type TG cells and CF-TG cells transfected with a plasmid containing a functional cftr gene. Finally, we reported an altered sphingolipid metabolism in CF-TG cells, which may account for their inflammatory signature. This first comprehensive analysis of gene expression in TG cells proposes a protective role of wild-type TG against airborne pathogens and reveals an original program in which anti-infectious response was deficient in TG cells with a cftr mutation. This defective response may explain why host response does not contribute to protection against P. aeruginosa in CF

Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity

Des systèmes impliqués dans la formation du biofilm chez Pseudomonias aeruginosa (biologie et régulation des Pili FLP)

Les infections à Pseudomonas aeruginosa chez l homme représentent un problème de santé publique important car elles sont souvent graves et peu de solutions thérapeutiques sont disponibles face aux souches multi-résistantes isolées de façon extrêmement fréquente. La multi-résistance de cette bactérie et sa persistance est connue maintenant pour être associée à sa capacité à vivre en communauté in vivo comme c est le cas par exemple dans les voies aériennes des patients atteints de mucoviscidose. Dans ce contexte, P. aeruginosa s établit sous forme de microcolonies dont le regroupement va résulter dans la formation d un biofilm structuré, auquel différents acteurs moléculaires participent de façon séquentielle et synergique ou antagoniste. Parmi ceux-ci, une machinerie d assemblage de pili de type IVb codée par 12 gènes formant le locus tad, assemble des appendices formés par l oligomérisation de sous-unités pilines Flp, et confère à la bactérie la capacité à former des agrégats et à initier un biofilm. Mon travail de thèse a permis d identifier les conditions d expression chromosomique de la sous-unité Flp qui se révèlent être optimales en phase tardive de croissance et en conditions aérobies. La présence dans le locus d un couple de gènes codant pour un système à deux composants (TCS) classique, le TCS PprA-B suggérait qu il pouvait avoir un rôle dans l expression du locus tad. Le régulateur de réponse PprB contrôle l expression des 5 unités transcriptionnelles du locus tad. TadF correspond à l'unique piline mineure du système, et n est pas essentielle à la formation du pilus. La protéine RcpC, qui n est présente qu au sein des machineries Tad, semble impliquée dans la modification post-traductionnelle de la sous-unité Flp et module le phénotype d adhérence des bactéries aux cellules épithéliales bronchiques. Le pilus Flp n est qu un des acteurs moléculaires de l adhérence et de la formation du biofilm. Le second axe de ma thèse a été la mise au point d une puce dédiée à l étude de la transcription de ces gènes ou adhésome . Elle permettra d avoir une vue globale de l expression de ces systèmes au cours des différentes étapes du développement du biofilm et de l infection ex vivo comme chez les patients atteints de mucoviscidose.Human P. aeruginosa infections have become a serious threat to public health because they are often serious and very difficult to treat due to the emergence of strains resistant to all known antibiotics. Multi-resistance and persistence of this bacterium is known to be associated with its capacity to live within a sedentary community lifestyle in vivo, called biofilm, as it is the case in the airways of cystic fibrosis patients. In the biofilm, bacteria are associated in microcolonies which grow within an autoproduced matrix and lead to a highly structured community. A large number of macromolecular systems are expressed in a sequential manner and probably act in synergy or antagonism. Among them, the Tad machine encoded by the tad locus constituted of 12 genes, assembles Flp type IVb pili at the surface of the bacteria and helps bacteria to aggregate and to initiate biofilm formation. During my PhD work, we identified the conditions of chromosomal production of the Flp pilin subunit, which is highly produced in the very late stationary growth phase and under aerobic conditions. The identification of two genes encoding a classical two-component system within the tad locus, PprAB, suggested that it could play a role in the control of tad genes. The response regulator, PprB, positively controls the expression of the five transcriptional units forming this tad locus. TadF is the unique minor pilin of the Tad system but no implication in Flp pilus biology could be unravelled. The RcpC protein, which is unique to Tad machines, controls an Flp pilin post-translational modification but not its assembly into pilus. The RcpC-dependent Flp pilin modification which could be a glycosylation, affects the efficiency of the Flp-host receptor interaction. The type IVb Flp pilus is not the unique macromolecular system involved in the adhesion and P. aeruginosa biofilm structuring. A second axis of my PhD period was devoted to the development of a dedicated chip (ADH chip) with the aim to obtain a global view of the expression of these systems during the formation of a biofilm in vitro and ex vivo in a large number of clinical strains isolated during the infection in the lungs of cystic fibrosis patientsAIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Les systèmes "chaperone-usher" CupB et CupC chez pseudomonas aeruginosa (assemblage et fonctions, caractérisation du "P-usher", une protéine bi-fonctionnelle impliquée dans l'assemblage de fimbriae et la sécrétion d'une protéine TpsA)

Dans l environnement, les bactéries vivent le plus souvent attachées à une surface, en un mode de vie communautaire pluricellulaire, le biofilm. Pseudomonas aeruginosa est un pathogène humain opportuniste à Gram négatif, responsable d infections nosocomiales et d infections respiratoires chez les patients atteints de mucoviscidose. La vie communautaire chez cette bactérie est un moyen de persistance chez l hôte qu il colonise et constitue un mécanisme passif ou actif de résistance aux molécules antimicrobiennes. L établissement de P. aeruginosa sous forme communautaire est le fait de machineries macromoléculaires, dont la voie chaperone-usher (CU). Mon travail de thèse a consisté à caractériser les systèmes CU, CupB et CupC de P. aeruginosa. Ces deux systèmes sont fonctionnels et assemblent des fimbriae de façon spécifique au travers de leur protéine de membrane externe, le usher . Le cluster cupB code pour deux chaperones périplasmiques CupB2 et CupB4 dont nous montrons qu elles adressent respectivement la piline majeure CupB1 et l adhésine CupB6 à la protéine usher CupB3. Le cluster cupB code également pour un substrat des systèmes TPS, CupB5 dont la sécrétion est assurée par le P-usher , résultant de la fusion d un domaine POTRA et d un domaine usher. Le P-usher est une protéine bi-fonctionnelle capable d assembler un fimbriae CU et de sécréter un substrat TPS. Le cluster cupB de P. aeruginosa aurait subi au cours de l évolution un réarrangement génétique aboutissant à la disparition du gène tpsB et à la création du gène hybride cupB3 codant pour le P-usher , hypothèse confirmée par la sécrétion de la protéine TPS de P. aeruginosa par la protéine de membrane externe TpsB de P. fluorescens présente dans le cluster cupB de cette bactérie.L étude de ces fimbriae CupB et CupC a montré leur implication dans la formation du biofilm et la réponse de la cellule hôte, plus particulièrement la protéine CupB5 qui joue un rôle dans la phagocytose.In the environment, bacteria live mostly attached to a surface in a sedentary and multicellular community called biofilm. Pseudomonas aeruginosa is a human opportunistic Gram negative pathogen, responsible for nosocomial infections and respiratory infections of cystic fibrosis patients. This bacterial lifestyle represents a strategy to persist in colonized hosts and constitutes a passive or active mechanism of resistance towards antimicrobial molecules. The establishment of P. aeruginosa in biofilm is due to macromolecular machines of the chaperone-usher (CU) family. My thesis work consisted in the characterisation of P. aeruginosa CupB and CupC systems of the CU family. These two systems are functional and specifically assemble fimbriae through their own outer membrane protein, the usher. The cupB cluster encodes two periplasmic chaperones CupB2 and CupB4, which are respectively responsible for the capture in the périplasme and targeting of the major pilin CupB1 and the adhesin CupB6, respectively, to the CupB3 usher protein. The cupB cluster encodes a TpsA substrate of the two partner secretion system (TPS), CupB5, which secretion occurs through the P-usher . The P-usher is therefore a bi-functional protein capable of both the assembly of CupB fimbriae and the secretion of CupB5 protein. The P-usher results from the fusion of a POTRA domain and of an usher domain, highlighting a genetic rearrangement in the P. aeruginosa cupB gene cluster. This is strongly supported by the restoration of the P. aeruginosa TPS substrate (CupB5) secretion, in the absence of the P-usher , by the introduction of the heterologous TpsB outer membrane protein from the P. fluorescens cupB cluster. The study of the fimbriae CupB and CupC fimbriae showed their implication in biofilm formation and host cell response, particularly of the CupB5 protein which plays a role in phagocytosis.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

ETUDE DES MECANISMES CELLULAIRES ET MOLECULAIRES IMPLIQUES DANS LES INTERACTIONS STAPHYLOCOCCUS AUREUS - CELLULES EPITHELIALES RESPIRATOIRES HUMAINES (DOCTORAT (GENIE BIOLOGIQUE))

REIMS-BU Santé (514542104) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Méthodes bioinformatiques d'analyses de données génomiques, transcriptomiques et cliniques pour l'étude des mécanismes de résistances aux drogues et leurs relations avec la formation de biofilms

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Assembly of Fimbrial Structures in Pseudomonas aeruginosa: Functionality and Specificity of Chaperone-Usher Machineries

Fimbrial or nonfimbrial adhesins assembled by the bacterial chaperone-usher pathway have been demonstrated to play a key role in pathogenesis. Such an assembly mechanism has been exemplified in uropathogenic Escherichia coli strains with the Pap and the Fim systems. In Pseudomonas aeruginosa, three gene clusters (cupA, cupB, and cupC) encoding chaperone-usher pathway components have been identified in the genome sequence of the PAO1 strain. The Cup systems differ from the Pap or Fim systems, since they obviously lack numbers of genes encoding fimbrial subunits. Nevertheless, the CupA system has been demonstrated to be involved in biofilm formation on solid surfaces, whereas the role of the CupB and CupC systems in biofilm formation could not be clearly elucidated. Moreover, these gene clusters were described as poorly expressed under standard laboratory conditions. The cupB and cupC clusters are directly under the control of a two-component regulatory system designated RocA1/S1/R. In this study, we revealed that Roc1-dependent induction of the cupB and cupC genes resulted in a high level of biofilm formation, with CupB and CupC acting with synergy in clustering bacteria for microcolony formation. Very importantly, this phenotype was associated with the assembly of cell surface fimbriae visualized by electron microscopy. Finally, we observed that the CupB and CupC systems are specialized in the assembly of their own fimbrial subunits and are not exchangeable

FppA, a Novel Pseudomonas aeruginosa Prepilin Peptidase Involved in Assembly of Type IVb Pili

Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly

Functional Characterization of Pseudomonas Contact Dependent Growth Inhibition (CDI) Systems

International audienceContact-dependent inhibition (CDI) toxins, delivered into the cytoplasm of target bacterial cells, confer to host strain a significant competitive advantage. Upon cell contact, the toxic C-terminal region of surface-exposed CdiA protein (CdiA-CT) inhibits the growth of CDI- bacteria. CDI+ cells express a specific immunity protein, CdiI, which protects from autoinhibition by blocking the activity of cognate CdiA-CT. CdiA-CT are separated from the rest of the protein by conserved peptide motifs falling into two distinct classes, the "E. coli"- and "Burkholderia-type". CDI systems have been described in numerous species except in Pseudomonadaceae. In this study, we identified functional toxin/immunity genes linked to CDI systems in the Pseudomonas genus, which extend beyond the conventional CDI classes by the variability of the peptide motif that delimits the polymorphic CdiA-CT domain. Using P. aeruginosa PAO1 as a model, we identified the translational repressor RsmA as a negative regulator of CDI systems. Our data further suggest that under conditions of expression, P. aeruginosa CDI systems are implicated in adhesion and biofilm formation and provide an advantage in competition assays. All together our data imply that CDI systems could play an important role in niche adaptation of Pseudomonadaceae

SciN Is an Outer Membrane Lipoprotein Required for Type VI Secretion in Enteroaggregative Escherichia coli▿

Enteroaggregative Escherichia coli (EAEC) is a pathogen implicated in several infant diarrhea or diarrheal outbreaks in areas of endemicity. Although multiple genes involved in EAEC pathogenesis have been identified, the overall mechanism of virulence is not well understood. Recently, a novel secretion system, called type VI secretion (T6S) system (T6SS), has been identified in EAEC and most animal or plant gram-negative pathogens. T6SSs are multicomponent cell envelope machines responsible for the secretion of at least two putative substrates, Hcp and VgrG. In EAEC, two copies of T6S gene clusters, called sci-1 and sci-2, are present on the pheU pathogenicity island. In this study, we focused our work on the sci-1 gene cluster. The Sci-1 apparatus is probably composed of all, or a subset of, the 21 gene products encoded on the cluster. Among these subunits, some are shared by all T6SSs identified to date, including a ClpV-type AAA+ ATPase (SciG) and an IcmF (SciS) and an IcmH (SciP) homologue, as well as a putative lipoprotein (SciN). In this study, we demonstrate that sciN is a critical gene necessary for T6S-dependent secretion of the Hcp-like SciD protein and for biofilm formation. We further show that SciN is a lipoprotein, as shown by the inhibition of its processing by globomycin and in vivo labeling with [3H]palmitic acid. SciN is tethered to the outer membrane and exposed in the periplasm. Sequestration of SciN at the inner membrane by targeting the +2 residue responsible for lipoprotein localization (Gly2Asp) fails to complement an sciN mutant for SciD secretion and biofilm formation. Together, these results support a model in which SciN is an outer membrane lipoprotein exposed in the periplasm and essential for the Sci-1 apparatus function