244 results found
Ale A, Crepin VF, Collins, et al., 2016, Model of host-pathogen Interaction dynamics links In vivo optical imaging and immune responses, Infection and Immunity, Vol: 85, ISSN: 1098-5522
Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.
Pollard DJ, Young JC, Covarelli V, et al., 2016, The type III secretion system effector SeoC of Salmonella enterica subspecies salamae and arizonae ADP-ribosylates Src and inhibits opsono-phagocytosis, Infection and Immunity, Vol: 84, Pages: 3618-3628, ISSN: 1098-5522
Salmonella spp. utilize type III secretion systems (T3SS) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study we compared a draft genome assembly of S. enterica subsp. salamae strain 3588/07 (S. salamae) against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and S. bongori strain 12419. S. salamae encode the Salmonella pathogenicity island (SPI)-1; SPI-2 and the locus of enterocyte effacement (LEE) T3SSs. Though several key S. Typhimurium effector genes are missing (e.g. avrA, sopB and sseL), S. salamae invades HeLa cells and contain homologues of S. bongori sboK and sboC, which we named seoC. SboC and SeoC are homologues of EspJ from enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), which inhibits Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates we identified EspJ homologues in S. bongori, S. salamae and S. enterica subsp. arizonae (S. arizonae). The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro. Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized bead into Cos-7 cells stably expressing GFP-FcγRIIa. Concurrently, S. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC dependent manner. These results show that S. bongori, S. salamae and S. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and sheds light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.
Crepin VF, Collins JW, Habibzay M, et al., 2016, Citrobacter rodentium mouse model of bacterial infection., Nature Protocols, Vol: 11, Pages: 1851-1876, ISSN: 1754-2189
Infection of mice with Citrobacter rodentium is a robust model to study bacterial pathogenesis, mucosal immunology, the health benefits of probiotics and the role of the microbiota during infection. C. rodentium was first isolated by Barthold from an outbreak of mouse diarrhea in Yale University in 1972 and was 'rediscovered' by Falkow and Schauer in 1993. Since then the use of the model has proliferated, and it is now the gold standard for studying virulence of the closely related human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Here we provide a detailed protocol for various applications of the model, including bacterial growth, site-directed mutagenesis, mouse inoculation (from cultured cells and after cohabitation), monitoring of bacterial colonization, tissue extraction and analysis, immune responses, probiotic treatment and microbiota analysis. The main protocol, from mouse infection to clearance and analysis of tissues and host responses, takes ∼5 weeks to complete.
Furniss RCD, Slater S, Frankel G, et al., 2016, Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signalling axis to prevent recycling endosome maturation during infection, Journal of Molecular Biology, Vol: 428, Pages: 3399-3407, ISSN: 1089-8638
Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate a plethora of host cell processes to establish infection of the gut mucosa. This manipulation is achieved via the injection of bacterial effector proteins into host cells using a Type III secretion system. We have previously reported that the conserved EHEC and EPEC effector EspG disrupts recycling endosome function, reducing cell surface levels of host receptors through accumulation of recycling cargo within the host cell. Here we report that EspG interacts specifically with the small GTPases ARF6 and Rab35 during infection. These interactions target EspG to endosomes and prevent Rab35-mediated recycling of cargo to the host cell surface. Furthermore, we show that EspG has no effect on Rab35-mediated uncoating of newly formed endosomes, and instead leads to the formation of enlarged EspG/TfR/Rab11 positive, EEA1/Clathrin negative stalled recycling structures. Thus, this paper provides a molecular framework to explain how EspG disrupts recycling whilst also reporting the first known simultaneous targeting of ARF6 and Rab35 by a bacterial pathogen.
So EC, Schroeder GN, Carson D, et al., 2016, The Rab-binding profiles of bacterial virulence factors during infection, Journal of Biological Chemistry, Vol: 291, Pages: 5832-5843, ISSN: 1083-351X
Legionella pneumophila, the causativeagent of Legionnaire’s disease, uses its typeIV secretion system to translocate over 300effector proteins into host cells. Theseeffectors subvert host cell signalingpathways to ensure bacterial proliferation.Despite their importance for pathogenesis,the roles of most of the effectors are yet tobe characterized. Key to understanding thefunction of effectors is the identification ofhost proteins they bind during infection. Wepreviously developed a novel tandemaffinitypurification (TAP) approach usinghexahistidine and BirA-specificbiotinylation tags for isolating translocatedeffector complexes from infected cellswhose composition were subsequentlydeciphered by mass spectrometry. Here wefurther advanced the workflow for the TAPapproach and determined the infectiondependentinteractomes of the effectorsSidM and LidA, which were previouslyreported to promiscuously bind multiple RabGTPases in vitro. In this study we defined astringent subset of Rab GTPases targeted bySidM and LidA during infection, comprisingof Rab1A, 1B, 6 and 10; in addition, LidAtargets Rab14 and 18. Taken together, thisstudy illustrates the power of this approachto profile the intracellular interactomes ofbacterial effectors during infection
Witcomb LA, Collins JW, McCarthy AJ, et al., 2015, Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia coli K1 Experimental Infection in the Neonatal Rat, Infection and Immunity, Vol: 83, Pages: 4528-4540, ISSN: 0019-9567
Key features of Escherichia coli K1-mediated neonatal sepsis and meningitis, such as a strong age dependency and development along the gut-mesentery-blood-brain course of infection, can be replicated in the newborn rat. We examined temporal and spatial aspects of E. coli K1 infection following initiation of gastrointestinal colonization in 2-day-old (P2) rats after oral administration of E. coli K1 strain A192PP and a virulent bioluminescent derivative, E. coli A192PP-lux2. A combination of bacterial enumeration in the major organs, two-dimensional bioluminescence imaging, and three-dimensional diffuse light imaging tomography with integrated micro-computed tomography indicated multiple sites of colonization within the alimentary canal; these included the tongue, esophagus, and stomach in addition to the small intestine and colon. After invasion of the blood compartment, the bacteria entered the central nervous system, with restricted colonization of the brain, and also invaded the major organs, in line with increases in the severity of symptoms of infection. Both keratinized and nonkeratinized surfaces of esophagi were colonized to a considerably greater extent in susceptible P2 neonates than in corresponding tissues from infection-resistant 9-day-old rat pups; the bacteria appeared to damage and penetrate the nonkeratinized esophageal epithelium of infection-susceptible P2 animals, suggesting the esophagus represents a portal of entry for E. coli K1 into the systemic circulation. Thus, multimodality imaging of experimental systemic infections in real time indicates complex dynamic patterns of colonization and dissemination that provide new insights into the E. coli K1 infection of the neonatal rat.
Schroeder GN, Frankel G, Tate EW, et al., 2015, The Legionella pneumophila effector LpdA is a palmitoylated phospholipase D virulence factor, Infection and Immunity, Vol: 83, Pages: 3989-4002, ISSN: 1098-5522
Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.
So EC, Mattheis C, Tate EW, et al., 2015, Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors, Canadian Journal of Microbiology, Vol: 61, Pages: 617-635, ISSN: 1480-3275
The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.
Frankel GM, Habibzay M, Crepin-Sevenou V, et al., 2015, Tir-induced actin remodeling triggers expression of CXCL1 in enterocytes and neutrophil recruitment during Citrobacter rodentium infection, Infection and Immunity, Vol: 83, Pages: 3342-3354, ISSN: 1098-5522
The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated leading to recruitment of Nck, activation of N-WASP and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382-462 or 478-547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478-547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium expressing Tir_Y451A/Y471A recruited significantly less neutrophils to the colon and triggered less colonic hyperplasia on day 14 post infection, compared to infection with the wild type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection.
Messens W, Bolton D, Frankel G, et al., 2015, Defining pathogenic verocytotoxin-producing Escherichia coli (VTEC) from cases of human infection in the European Union, 2007-2010, EPIDEMIOLOGY AND INFECTION, Vol: 143, Pages: 1652-1661, ISSN: 0950-2688
Hunter PJ, Shaw RK, Berger CN, et al., 2015, Older leaves of lettuce (Lactuca spp.) support higher levels of Salmonella enterica ser. Senftenberg attachment and show greater variation between plant accessions than do younger leaves, FEMS Microbiology Letters, Vol: 362, ISSN: 0378-1097
Salmonella can bind to the leaves of salad crops including lettuce and survive for commercially relevant periods. Previous studies have shown that younger leaves are more susceptible to colonization than older leaves and that colonization levels are dependent on both the bacterial serovar and the lettuce cultivar. In this study, we investigated the ability of two Lactuca sativa cultivars (Saladin and Iceberg) and an accession of wild lettuce (L. serriola) to support attachment of Salmonella enterica serovar Senftenberg, to the first and fifth to sixth true leaves and the associations between cultivar-dependent variation in plant leaf surface characteristics and bacterial attachment. Attachment levels were higher on older leaves than on the younger ones and these differences were associated with leaf vein and stomatal densities, leaf surface hydrophobicity and leaf surface soluble protein concentrations. Vein density and leaf surface hydrophobicity were also associated with cultivar-specific differences in Salmonella attachment, although the latter was only observed in the older leaves and was also associated with level of epicuticular wax.
Schreiber F, Kay S, Frankel G, et al., 2015, The Hd, Hj, and Hz66 flagella variants of Salmonella enterica serovar Typhi modify host responses and cellular interactions, SCIENTIFIC REPORTS, Vol: 5, ISSN: 2045-2322
Young JC, Clements A, Lang AE, et al., 2014, The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation, NATURE COMMUNICATIONS, Vol: 5, ISSN: 2041-1723
Satoh-Takayama N, Serafini N, Verrier T, et al., 2014, The Chemokine Receptor CXCR6 Controls the Functional Topography of Interleukin-22 Producing Intestinal Innate Lymphoid Cells, IMMUNITY, Vol: 41, Pages: 776-788, ISSN: 1074-7613
Clements A, Stoneham CA, Furniss RCD, et al., 2014, Enterohaemorrhagic Escherichia coli inhibits recycling endosome function and trafficking of surface receptors, CELLULAR MICROBIOLOGY, Vol: 16, Pages: 1693-1705, ISSN: 1462-5814
Pallett MA, Berger CN, Pearson JS, et al., 2014, The Type III Secretion Effector NleF of Enteropathogenic Escherichia coli Activates NF-kappa B Early during Infection, INFECTION AND IMMUNITY, Vol: 82, Pages: 4878-4888, ISSN: 0019-9567
Collins JW, Chervaux C, Raymond B, et al., 2014, Fermented Dairy Products Modulate Citrobacter rodentium-Induced Colonic Hyperplasia, JOURNAL OF INFECTIOUS DISEASES, Vol: 210, Pages: 1029-1041, ISSN: 0022-1899
Collins JW, Keeney KM, Crepin VE, et al., 2014, Citrobacter rodentium: infection, inflammation and the microbiota, NATURE REVIEWS MICROBIOLOGY, Vol: 12, Pages: 612-623, ISSN: 1740-1526
Stevens MP, Frankel GM, 2014, The Locus of Enterocyte Effacement and Associated Virulence Factors of Enterohemorrhagic Escherichia coli., Microbiol Spectr, Vol: 2
A subset of Shiga toxin-producing Escherichia coli strains, termed enterohemorrhagic E. coli (EHEC), is defined in part by the ability to produce attaching and effacing (A/E) lesions on intestinal epithelia. Such lesions are characterized by intimate bacterial attachment to the apical surface of enterocytes, cytoskeletal rearrangements beneath adherent bacteria, and destruction of proximal microvilli. A/E lesion formation requires the locus of enterocyte effacement (LEE), which encodes a Type III secretion system that injects bacterial proteins into host cells. The translocated proteins, termed effectors, subvert a plethora of cellular pathways to the benefit of the pathogen, for example, by recruiting cytoskeletal proteins, disrupting epithelial barrier integrity, and interfering with the induction of inflammation, phagocytosis, and apoptosis. The LEE and selected effectors play pivotal roles in intestinal persistence and virulence of EHEC, and it is becoming clear that effectors may act in redundant, synergistic, and antagonistic ways during infection. Vaccines that target the function of the Type III secretion system limit colonization of reservoir hosts by EHEC and may thus aid control of zoonotic infections. Here we review the features and functions of the LEE-encoded Type III secretion system and associated effectors of E. coli O157:H7 and other Shiga toxin-producing E. coli strains.
Song-Zhao GX, Srinivasan N, Pott J, et al., 2014, Nlrp3 activation in the intestinal epithelium protects against a mucosal pathogen, MUCOSAL IMMUNOLOGY, Vol: 7, Pages: 763-774, ISSN: 1933-0219
Mousnier A, Schroeder GN, Stoneham CA, et al., 2014, A New Method To Determine In Vivo Interactomes Reveals Binding of the Legionella pneumophila Effector PieE to Multiple Rab GTPases, MBIO, Vol: 5, ISSN: 2150-7511
Wlodarska M, Thaiss CA, Nowarski R, et al., 2014, NLRP6 Inflammasome Orchestrates the Colonic Host-Microbial Interface by Regulating Goblet Cell Mucus Secretion, CELL, Vol: 156, Pages: 1045-1059, ISSN: 0092-8674
Lai Y, Rosenshine I, Leong JM, et al., 2013, Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli, CELLULAR MICROBIOLOGY, Vol: 15, Pages: 1796-1808, ISSN: 1462-5814
Harding CR, Schroeder GN, Collins JW, et al., 2013, Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
Harding CR, Mattheis C, Mousnier A, et al., 2013, LtpD Is a Novel Legionella pneumophila Effector That Binds Phosphatidylinositol 3-Phosphate and Inositol Monophosphatase IMPA1, INFECTION AND IMMUNITY, Vol: 81, Pages: 4261-4270, ISSN: 0019-9567
Pearson JS, Giogha C, Ong SY, et al., 2013, A type III effector antagonizes death receptor signalling during bacterial gut infection, NATURE, Vol: 501, Pages: 247-+, ISSN: 0028-0836
Raymond B, Young JC, Pallett M, et al., 2013, Subversion of trafficking, apoptosis, and innate immunity by type III secretion system effectors, TRENDS IN MICROBIOLOGY, Vol: 21, Pages: 430-439, ISSN: 0966-842X
Collins JW, Meganck JA, Kuo C, et al., 2013, 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
Harding CR, Stoneham CA, Schuelein R, et al., 2013, The Dot/Icm Effector SdhA Is Necessary for Virulence of Legionella pneumophila in Galleria mellonella and A/J Mice, INFECTION AND IMMUNITY, Vol: 81, Pages: 2598-2605, ISSN: 0019-9567
Aurass P, Schlegel M, Metwally O, et al., 2013, The Legionella pneumophila Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 11080-11092
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