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Sharma, A. & Puhar, A. (2019). Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri. BIO-PROTOCOL, 9(13), Article ID UNSP e3292.
Open this publication in new window or tab >>Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri
2019 (English)In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 13, article id UNSP e3292Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
BIO-PROTOCOL, 2019
Keywords
Shigella, Intracellular pathogen, Invasion, Type III Secretion System, Intestinal epithelial cells
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-162003 (URN)10.21769/BioProtoc.3292 (DOI)000476647800009 ()
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Sharma, A. & Puhar, A. (2019). Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells. BIO-PROTOCOL, 9(13), Article ID UNSP e3293.
Open this publication in new window or tab >>Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells
2019 (English)In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 13, article id UNSP e3293Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
BIO-PROTOCOL, 2019
Keywords
Intracellular bacteria, Intestinal epithelial cells, Giemsa stain, Shigella, Invasion, Dissemination
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-162002 (URN)10.21769/BioProtoc.3293 (DOI)000476647800010 ()
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Boal, F., Puhar, A., Xuereb, J.-M., Kunduzova, O., Sansonetti, P. J., Payrastre, B. & Tronchére, H. (2016). PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment. Cell reports, 14(4), 750-759
Open this publication in new window or tab >>PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment
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2016 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, no 4, p. 750-759Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Cell Press, 2016
National Category
Natural Sciences Biological Sciences Immunology Microbiology
Research subject
Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-115891 (URN)10.1016/j.celrep.2015.12.079 (DOI)000369104500007 ()26776508 (PubMedID)
Available from: 2016-02-05 Created: 2016-02-05 Last updated: 2019-03-03Bibliographically approved
Bravo, V., Puhar, A., Sansonetti, P., Parsot, C. & Toro, C. S. (2015). Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp. PLoS ONE, 10(3), Article ID e0121785.
Open this publication in new window or tab >>Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, article id e0121785Article in journal (Refereed) Published
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.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-156941 (URN)10.1371/journal.pone.0121785 (DOI)25811616 (PubMedID)
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-03-08Bibliographically approved
Puhar, A. & Sansonetti, P. J. (2014). Dye-uptake Experiment through Connexin Hemichannels. Bio-protocol, 4(17), Article ID e1221.
Open this publication in new window or tab >>Dye-uptake Experiment through Connexin Hemichannels
2014 (English)In: Bio-protocol, ISSN 2331-8325, Vol. 4, no 17, article id e1221Article in journal (Refereed) Published
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.

National Category
Natural Sciences Biological Sciences Cell Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-156956 (URN)10.21769/BioProtoc.1221 (DOI)
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-03-11Bibliographically approved
Puhar, A. & Sansonetti, P. J. (2014). Induction of Connexin-hemichannel Opening. Bio-Protocol, 4(17), Article ID e1220.
Open this publication in new window or tab >>Induction of Connexin-hemichannel Opening
2014 (English)In: Bio-Protocol, ISSN 2331-8325, Vol. 4, no 17, article id e1220Article in journal (Refereed) Published
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)].

National Category
Natural Sciences Biological Sciences Cell Biology Microbiology
Identifiers
urn:nbn:se:umu:diva-156957 (URN)10.21769/BioProtoc.1220 (DOI)
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-03-11Bibliographically approved
Puhar, A. & Sansonetti, P. J. (2014). Type III secretion system. Current Biology, 24(17), R784-R791
Open this publication in new window or tab >>Type III secretion system
2014 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 24, no 17, p. R784-R791Article in journal (Refereed) Published
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.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-156942 (URN)10.1016/j.cub.2014.07.016 (DOI)25202865 (PubMedID)
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-03-08Bibliographically approved
Puhar, A., Tronchère, H., Payrastre, B., Nhieu, G. T. & Sansonetti, P. J. (2013). A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P. Immunity, 39(6), 1121-1131
Open this publication in new window or tab >>A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P
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2013 (English)In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 39, no 6, p. 1121-1131Article in journal (Refereed) Published
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.

National Category
Microbiology Immunology
Identifiers
urn:nbn:se:umu:diva-156943 (URN)10.1016/j.immuni.2013.11.013 (DOI)24332032 (PubMedID)
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-03-08Bibliographically approved
Teo, I., Toms, S. M., Marteyn, B., Barata, T. S., Simpson, P., Johnston, K. A., . . . Shaunak, S. (2012). Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers. EMBO Molecular Medicine, 4(9), 866-881
Open this publication in new window or tab >>Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers
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2012 (English)In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 4, no 9, p. 866-881Article in journal (Refereed) Published
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.

National Category
Immunology in the medical area Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-156944 (URN)10.1002/emmm.201201290 (DOI)22887873 (PubMedID)
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-03-08Bibliographically approved
Tran, S.-L., Guillemet, E., Ngo-Camus, M., Clybouw, C., Puhar, A., Moris, A., . . . Ramarao, N. (2011). Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages. Cellular Microbiology, 13(1), 92-108
Open this publication in new window or tab >>Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages
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2011 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 13, no 1, p. 92-108Article in journal (Refereed) Published
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.

National Category
Microbiology Immunology
Identifiers
urn:nbn:se:umu:diva-156947 (URN)10.1111/j.1462-5822.2010.01522.x (DOI)20731668 (PubMedID)
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-03-08Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0002-9915-002x

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