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  • 1.
    Boal, Frédéric
    et al.
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Xuereb, Jean-Marie
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Kunduzova, Oksana
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Sansonetti, Philippe J.
    INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Payrastre, Bernard
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France; .
    Tronchére, Héléne
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment2016Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, nr 4, s. 750-759Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella flexneri, the pathogen responsible for bacillary dysentery, has evolved multiple strategies to control the inflammatory response. Here, we show that Shigella subverts the subcellular trafficking of the intercellular adhesion molecule-1 (ICAM-1), a key molecule in immune cell recruitment, in a mechanism dependent on the injected bacterial enzyme IpgD and its product, the lipid mediator PI5P. Overexpression of IpgD, but not a phosphatase dead mutant, induced the internalization and the degradation of ICAM-1 in intestinal epithelial cells. Remarkably, addition of permeant PI5P reproduced IpgD effects and led to the inhibition of neutrophil recruitment. Finally, these results were confirmed in an in vivo model of Shigella infection where IpgD-dependent ICAM-1 internalization reduced neutrophil adhesion. In conclusion, we describe here an immune evasion mechanism used by the pathogen Shigella to divert the host cell trafficking machinery in order to reduce immune cell recruitment.

  • 2. Bravo, Verónica
    et al.
    Puhar, Andrea
    Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France; INSERM, Paris, France.
    Sansonetti, Philippe
    Parsot, Claude
    Toro, Cecilia S
    Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp2015Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 3, artikel-id e0121785Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events.

  • 3. Dal Molin, Federica
    et al.
    Zornetta, Irene
    Puhar, Andrea
    Dipartimento di Scienze Biomediche and Istituto C.N.R. Neuroscienze, Università di Padova, Viale G. Colombo n. 3, 35121 Padova, Italy.
    Tonello, Fiorella
    Zaccolo, Manuela
    Montecucco, Cesare
    cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin2008Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 376, nr 2, s. 429-433Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.

  • 4. Genisset, Christophe
    et al.
    Puhar, Andrea
    Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Padova, Italy..
    Calore, Federica
    de Bernard, Marina
    Dell'Antone, Paolo
    Montecucco, Cesare
    The concerted action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces swelling of isolated endosomes2007Ingår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 9, nr 6, s. 1481-1490Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori, the bacterium associated to gastroduodenal ulcers and stomach cancers. VacA induces formation of cellular vacuoles that originate from late endosomal compartments. VacA forms an anion-selective channel and its activity has been suggested to increase the osmotic pressure in the lumen of these acidic compartments, driving their swelling to vacuoles. Here, we have tested this proposal on isolated endosomes that allow one to manipulate at will the medium. We have found that VacA enhances the v-ATPase proton pump activity and the acidification of isolated endosomes in a Cl- dependent manner. Other counter-anions such as pyruvate, Br-, I- and SCN- can be transported by VacA with stimulation of the v-ATPase. The VacA action on isolated endosomes is associated with their increase in size. Single amino acid substituted VacA with no channel-forming and vacuolating activity is unable to induce swelling of endosomes. These data provide a direct evidence that the transmembrane VacA channel mediates an influx of anions into endosomes that stimulates the electrogenic v-ATPase proton pump, leading to their osmotic swelling and transformation into vacuoles.

  • 5. Konradt, Christoph
    et al.
    Frigimelica, Elisabetta
    Nothelfer, Katharina
    Puhar, Andrea
    Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 25–28 Rue du Dr Roux, 75724 Paris Cedex 15, France, and INSERM U786, Institut Pasteur, 25–28 Rue du Dr Roux, 75724 Paris Cedex 15, France .
    Salgado-Pabon, Wilmara
    di Bartolo, Vincenzo
    Scott-Algara, Daniel
    Rodrigues, Cristina D
    Sansonetti, Philippe J
    Phalipon, Armelle
    The Shigella flexneri type three secretion system effector IpgD inhibits T cell migration by manipulating host phosphoinositide metabolism2011Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 9, nr 4, s. 263-272Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella, the Gram-negative enteroinvasive bacterium that causes shigellosis, relies on its type III secretion system (TTSS) and injected effectors to modulate host cell functions. However, consequences of the interaction between Shigella and lymphocytes have not been investigated. We show that Shigella invades activated human CD4(+) T lymphocytes. Invasion requires a functional TTSS and results in inhibition of chemokine-induced T cell migration, an effect mediated by the TTSS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Upon IpgD-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), the pool of PIP(2) at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus, likely affecting the formation of the polarized edge required for cell migration. These results reveal a bacterial TTSS effector-mediated strategy to impair T cell function.

  • 6. Lin, Po-Chi
    et al.
    Puhar, Andrea
    Biochemisches Institut, Universität Zürich, Zurich, Switzerland, Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris Cedex 15, France.
    Steuber, Julia
    NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica2008Ingår i: Archives of Microbiology, ISSN 0302-8933, E-ISSN 1432-072X, Vol. 190, nr 4, s. 471-480Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na+ uptake, indicating that a redox-driven, primary Na+ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 micromol min(-1) mg(-1)) to Na+ translocation (2.0 micromol min(-1) mg(-1)). NADH-driven Na+ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na+ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na+ gradient established by complex I.

  • 7. Lin, Po-Chi
    et al.
    Puhar, Andrea
    Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
    Türk, Karin
    Piligkos, Stergios
    Bill, Eckhard
    Neese, Frank
    Steuber, Julia
    A vertebrate-type ferredoxin domain in the Na+-translocating NADH dehydrogenase from Vibrio cholerae2005Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, nr 24, s. 22560-22563Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. Mössbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys(70), Cys(76), Cys(79), and Cys(111). Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.

  • 8.
    Puhar, Andrea
    et al.
    Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Padova, Italy,.
    Dal Molin, Federica
    Horvath, Stéphanie
    Ladant, Daniel
    Ladants, Daniel
    Montecucco, Cesare
    Anthrax edema toxin modulates PKA- and CREB-dependent signaling in two phases2008Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 3, nr 10, artikel-id e3564Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Anthrax edema toxin (EdTx) is an adenylate cyclase which operates in the perinuclear region of host cells. However, the action of EdTx is poorly understood, especially at molecular level. The ability of EdTx to modulate cAMP-dependent signaling was studied in Jurkat T cells and was compared with that of other cAMP-rising agents: Bordetella pertussis adenylate cyclase toxin, cholera toxin and forskolin.

    METHODOLOGY/PRINCIPAL FINDINGS: EdTx caused a prolonged increase of the intracellular cAMP concentration. This led to nuclear translocation of the cAMP-dependent protein kinase (PKA) catalytic subunit, phosphorylation of cAMP response element binding protein (CREB) and expression of a reporter gene under control of the cAMP response element. Neither p90 ribosomal S6 kinase nor mitogen- and stress-activated kinase, which mediate CREB phosphorylation during T cell activation, were involved. The duration of phospho-CREB binding to chromatin correlated with the spatio-temporal rise of cAMP levels. Strikingly, EdTx pre-treated T cells were unresponsive to other stimuli involving CREB phosphorylation such as addition of forskolin or T cell receptor cross-linking.

    CONCLUSIONS/SIGNIFICANCE: We concluded that, in a first intoxication phase, EdTx induces PKA-dependent signaling, which culminates in CREB phosphorylation and activation of gene transcription. Subsequently CREB phosphorylation is impaired and therefore T cells are not able to respond to cues involving CREB. The present data functionally link the perinuclear localization of EdTx to its intoxication mechanism, indicating that this is a specific feature of its intoxication mechanism.

  • 9.
    Puhar, Andrea
    et al.
    Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, I-35121 Padua, Italy.
    Johnson, E A
    Rossetto, O
    Montecucco, C
    Comparison of the pH-induced conformational change of different clostridial neurotoxins2004Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 319, nr 1, s. 66-71Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Clostridial neurotoxins are internalized inside acidic compartments, wherefrom the catalytic chain translocates across the membrane into the cytosol in a low pH-driven process, reaching its proteolytic substrates. The pH range in which the structural rearrangement of clostridial neurotoxins takes place was determined by 8-anilinonaphthalene-1-sulfonate and tryptophan fluorescence measurements. Half conformational change was attained at pH 4.55, 4.50, 4.40, 4.60, 4.40, and 4.40 for tetanus neurotoxin and botulinum neurotoxin serotypes /A, /B, /C, /E, and /F, respectively. This similarity indicates the key residues for the conformation transition are strongly conserved. Acidic liposomes support the conformational rearrangement shifting the effect versus higher pH values, whereas zwitterionic liposomes do not. The disulfide bridge linking the light and the heavy chains together needs to be oxidized to allow toxin membrane insertion, indicating that in vivo its reduction follows exposure to the cytosol after penetration of the endosomal membrane.

  • 10.
    Puhar, Andrea
    et al.
    Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, viale G. Colombo 3, I-35121 Padua, Italy.
    Montecucco, Cesare
    Where and how do anthrax toxins exit endosomes to intoxicate host cells?2007Ingår i: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 15, nr 11, s. 477-482Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of Bacillus anthracis virulence factors in its pathogenesis has been subjected to intense investigation with the aim of finding novel preventive and therapeutic protocols. Toxins that are endocytosed and act in the cytosol of host cells have a central role in B. anthracis infection. Understanding of anthrax toxin cell entry has increased during the past few years and a composite picture is emerging. Nevertheless, unanswered and controversial questions remain, particularly concerning the site and mode of anthrax toxin cell entry, the role of anthrax toxin receptors in the process and the possible involvement of cytosolic chaperones, which might affect entry efficiency. Here, the current model of anthrax toxin cell entry, an alternative model and experimental approaches for clarifying unanswered questions will be discussed.

  • 11.
    Puhar, Andrea
    et al.
    Inserm U786 and Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France.
    Sansonetti, Philippe J.
    Microbiologie et Maladies Infectieuses, Collège de France, Paris, France.
    Dye-uptake Experiment through Connexin Hemichannels2014Ingår i: Bio-protocol, ISSN 2331-8325, Vol. 4, nr 17, artikel-id e1221Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    [Abstract] Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes and immune cells. Hemichannels allow the passage of molecules of up to 1-2 kDa along the concentration gradient. When surface-exposed, hemichannels mediate the exchange of molecules between the cytosol and the extracellular space. Hemichannels are closed by default, but several cues inducing their opening have been described, e.g. a drop in the extracellular Ca2+ concentration (Evans et al., 2006) or infection with enteric pathogens (Puhar et al., 2013; Tran Van Nhieu et al., 2003). Hemichannel opening can be measured by electrophysiology, by quantifying the release of a hemichannel-permeable molecule into the extracellular medium or by quantifying the uptake of a hemichannel-permeable, plasma membrane-impermeant molecule. As the extent of uptake of a molecule is proportional to its concentration, exposure time, temperature (these parameters are controlled) and, importantly, to the number of active hemichannels on the cell surface, uptake assays are routinely used to assess hemichannel opening. This protocol for the uptake of the fluorescent dye ethidium bromide was used with Hela cells that were stably transfected with Cx26 or Cx43 (Paemeleire et al., 2000). Nevertheless, it could likely be used with other Cx-expressing cell types.

  • 12.
    Puhar, Andrea
    et al.
    Inserm U786 and Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France.
    Sansonetti, Philippe J.
    Microbiologie et Maladies Infectieuses, Collège de France, Paris, France.
    Induction of Connexin-hemichannel Opening2014Ingår i: Bio-Protocol, ISSN 2331-8325, Vol. 4, nr 17, artikel-id e1220Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    [Abstract] Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes and immune cells. Hemichannels allow the passage of molecules of up to 1-2 kDa along the concentration gradient. When surface-exposed, hemichannels mediate the exchange of molecules between the cytosol and the extracellular space. Hemichannels are closed by default, but several cues inducing their opening have been described, e.g. a drop in the extracellular Ca2+ concentration (Evans et al., 2006) or infection with enteric pathogens (Puhar et al., 2013; Tran Van Nhieu et al., 2003). This protocol was used with epithelial cells, in particular with polarized and non-polarized intestinal epithelial TC7 cells and with Hela cells that were stably transfected with Cx26 or Cx43 (Paemeleire et al., 2000). Nevertheless, it could likely be used with other Cx-expressing cell types. Whether hemichannels are open can be determined by electrophysiology or by measuring the release into the extracellular medium of a hemichannel permeable molecule (for example, ATP) or the uptake of a hemichannel-permeable, plasma membrane-impermeant molecule [for example, the fluorescent dye ethidium bromide-see associated protocol “Dye-uptake Experiment through Connexin Hemichannels” (Puhar and Sansonetti, 2014)].

  • 13.
    Puhar, Andrea
    et al.
    Inserm U768 and Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, 75724 Paris Cedex 15, France..
    Sansonetti, Philippe J
    Inserm U768 and Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, 75724 Paris Cedex 15, France.
    Type III secretion system2014Ingår i: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 24, nr 17, s. R784-R791Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The type III secretion system (T3SS) is a membrane-embedded nanomachine found in several Gram-negative bacteria. Upon contact between bacteria and host cells, the syringe-like T3SS (Figure 1) transfers proteins termed effectors from the bacterial cytosol to the cytoplasm or the plasma membrane of a single target cell. This is a major difference from secretion systems that merely release molecules into the extracellular milieu, where they act on potentially distant target cells expressing the relevant surface receptors. The syringe architecture is conserved at the structural and functional level and supports injection into a great variety of hosts and tissues. However, the pool of effectors is species specific and determines the outcome of the interaction, via modulation of target-cell function.

  • 14.
    Puhar, Andrea
    et al.
    Inserm U786, Unité de Pathogénie Microbienne Moléculaire, 75724 Paris Cedex 15, France; Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, 75724 Paris Cedex 15, France.
    Tronchère, Hélène
    Payrastre, Bernard
    Nhieu, Guy Tran Van
    Sansonetti, Philippe J
    A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P2013Ingår i: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 39, nr 6, s. 1121-1131Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Upon infection with Shigella flexneri, epithelial cells release ATP through connexin hemichannels. However, the pathophysiological consequence and the regulation of this process are unclear. Here we showed that in intestinal epithelial cell ATP release was an early alert response to infection with enteric pathogens that eventually promoted inflammation of the gut. Shigella evolved to escape this inflammatory reaction by its type III secretion effector IpgD, which blocked hemichannels via the production of the lipid PtdIns5P. Infection with an ipgD mutant resulted in rapid hemichannel-dependent accumulation of extracellular ATP in vitro and in vivo, which preceded the onset of inflammation. At later stages of infection, ipgD-deficient Shigella caused strong intestinal inflammation owing to extracellular ATP. We therefore describe a new paradigm of host-pathogen interaction based on endogenous danger signaling and identify extracellular ATP as key regulator of mucosal inflammation during infection. Our data provide new angles of attack for the development of anti-inflammatory molecules.

  • 15.
    Sharma, Atin
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri2019Ingår i: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, nr 13, artikel-id UNSP e3292Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella flexneri is an intracellular bacterial pathogen that gains access to the gut epithelium using a specialized Type III Secretion System (T3SS). Various determinants mediating this invasive infection have been experimentally verified using the classical gentamicin protection assay presented here. In this assay epithelial cell lines are infected by bacteria in vitro and the extracellular bacteria are killed by gentamicin. The internalized bacteria, which are protected from the bactericidal action of gentamicin, are recovered by lysing the epithelial cells and enumerated by determining the colonies formed on solid medium. Various techniques based on light microscopy, such as immunofluorescence and bacteria expressing fluorescent proteins, are also used for studying intracellular bacteria. However, these techniques are not only labor intensive and require sophisticated equipment, but mostly are also not quantitative. Despite being an easy quantitative method to study invasiveness of bacteria, the gentamicin protection assay cannot distinguish between the survival and multiplication of the internalized bacteria over longer incubation periods. To alleviate the complications created by multiplication and dissemination of internalized bacteria, complementary assays like plaque formation assays are required. This protocol presents an easy and cost-effective method to determine the invasiveness and the capacity to establish an infection of Shigella under different conditions.

  • 16.
    Sharma, Atin
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells2019Ingår i: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, nr 13, artikel-id UNSP e3293Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella flexneri invades the epithelial cells lining the gut lumen and replicates intracellularly. The specialized Type III Secretion System (T3SS) and its effector proteins, encoded on a large virulence plasmid, assist the bacterium to gain access to the cytosol. Thereafter Shigella disseminates to neighboring cells in an epithelial layer without further extracellular steps. Host cell lysis occurs when these bacteria have extensively replicated in the target cell cytosol. Here we describe a simple method to qualitatively as well as quantitatively study the capacity of Shigella to invade and disseminate within an epithelium by assessing the number and size of plaques representing the dead cells in a monolayer of TC7 cells. This classical protocol follows a simple approach of infecting the monolayers of epithelial cell lines with Shigella and visualizing the dead cells as plaques formed against a stained background.

  • 17. Teo, Ian
    et al.
    Toms, Steve M.
    Marteyn, Benoit
    Barata, Teresa S.
    Simpson, Peter
    Johnston, Karen A.
    Schnupf, Pamela
    Puhar, Andrea
    Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire & Unité INSERM 786, Paris, France.
    Bell, Tracey
    Tang, Chris
    Zloh, Mire
    Matthews, Steve
    Rendle, Phillip M.
    Sansonetti, Philippe J.
    Shaunak, Sunil
    Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers2012Ingår i: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 4, nr 9, s. 866-881Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in β-defensin, IL-10, interferon-β, transforming growth factor-β, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases.

  • 18. Tran, Seav-Ly
    et al.
    Guillemet, Elisabeth
    Ngo-Camus, Maud
    Clybouw, Cyril
    Puhar, Andrea
    Unité PMM, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France..
    Moris, Arnaud
    Gohar, Michel
    Lereclus, Didier
    Ramarao, Nalini
    Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages2011Ingår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 13, nr 1, s. 92-108Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

  • 19. Tran, Seav-Ly
    et al.
    Puhar, Andrea
    Unité PMM, INSERM U786, Institut Pasteur, Paris, France, Universite de la Mediterranee, France.
    Ngo-Camus, Maud
    Ramarao, Nalini
    Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus2011Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, nr 9, artikel-id e22876Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.

  • 20. Türk, Karin
    et al.
    Puhar, Andrea
    Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
    Neese, Frank
    Bill, Eckhard
    Fritz, Günter
    Steuber, Julia
    NADH oxidation by the Na+-translocating NADH: quinone oxidoreductase from Vibrio cholerae2004Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 20, s. 21349-21355Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae is a six subunit enzyme containing four flavins and a single motif for the binding of a Fe-S cluster on its NqrF subunit. This study reports the production of a soluble variant of NqrF (NqrF') and its individual flavin and Fe-S-carrying domains using V. cholerae or Escherichia coli as expression hosts. NqrF' and the flavin domain each contain 1 mol of FAD/mol of enzyme and exhibit high NADH oxidation activity (20,000 micromol min(-1) mg(-1)). EPR, visible absorption, and circular dichroism spectroscopy indicate that the Fe-S cluster in NqrF' and its Fe-S domain is related to 2Fe ferredoxins of the vertebrate-type. The addition of NADH to NqrF' results in the formation of a neutral flavosemiquinone and a partial reduction of the Fe-S cluster. The NqrF subunit harbors the active site of NADH oxidation and acts as a converter between the hydride donor NADH and subsequent one-electron reaction steps in the Na(+)-translocating NADH:quinone oxidoreductase complex. The observed electron transfer NADH --> FAD --> [2Fe-2S] in NqrF requires positioning of the FAD and the Fe-S cluster in close proximity in accordance with a structural model of the subunit.

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