Publications by authors named "Gad Frankel"

215 Publications

Type III secretion system effectors form robust and flexible intracellular virulence networks.

Science 2021 03;371(6534)

Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College, London, UK.

Infections with many Gram-negative pathogens, including , , , and , rely on type III secretion system (T3SS) effectors. We hypothesized that while hijacking processes within mammalian cells, the effectors operate as a robust network that can tolerate substantial contractions. This was tested in vivo using the mouse pathogen (encoding 31 effectors). Sequential gene deletions showed that effector essentiality for infection was context dependent and that the network could tolerate 60% contraction while maintaining pathogenicity. Despite inducing very different colonic cytokine profiles (e.g., interleukin-22, interleukin-17, interferon-γ, or granulocyte-macrophage colony-stimulating factor), different networks induced protective immunity. Using data from >100 distinct mutant combinations, we built and trained a machine learning model able to predict colonization outcomes, which were confirmed experimentally. Furthermore, reproducing the human-restricted enteropathogenic effector repertoire in was not sufficient for efficient colonization, which implicates effector networks in host adaptation. These results unveil the extreme robustness of both T3SS effector networks and host responses.
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http://dx.doi.org/10.1126/science.abc9531DOI Listing
March 2021

Overview of the Effect of Citrobacter rodentium Infection on Host Metabolism and the Microbiota.

Methods Mol Biol 2021 ;2291:399-418

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK.

Citrobacter rodentium is a natural enteric mouse pathogen that models human intestinal diseases, such as pathogenic E. coli infections, ulcerative colitis, and colon cancer. Upon reaching the monolayer of intestinal epithelial cells (IECs) lining the gut, a complex web of interactions between the host, the pathogen, and the microbiota ensues. A number of studies revealed surprisingly rapid changes in IEC bioenergetics upon infection, involving a switch from oxidative phosphorylation to aerobic glycolysis, leading to mucosal oxygenation and subsequent changes in microbiota composition. Microbiome studies have revealed a bloom in Enterobacteriaceae during C. rodentium infection in both resistant (i.e., C57BL/6) and susceptible (i.e., C3H/HeN) strains of mice concomitant with a depletion of butyrate-producing Clostridia. The emerging understanding that dysbiosis of cholesterol metabolism is induced by enteric infection further confirms the pivotal role immunometabolism plays in disease outcome. Inversely, the host and microbiota also impact upon the progression of infection, from the susceptibility of the distal colon to C. rodentium colonization to clearance of the pathogen, both via opsonization from the host adaptive immune system and out competition by the resident microbiota. Further complicating this compendium of interactions, C. rodentium exploits microbiota metabolites to fine-tune virulence gene expression and promote colonization. This chapter summarizes the current knowledge of the myriad of pathogen-host-microbiota interactions that occur during the progression of C. rodentium infection in mice and the broader implications of these findings on our understanding of enteric disease.
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http://dx.doi.org/10.1007/978-1-0716-1339-9_20DOI Listing
April 2021

Cryoelectron-microscopy structure of the enteropathogenic type III secretion system EspA filament.

Proc Natl Acad Sci U S A 2021 Jan;118(2)

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom;

Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.
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http://dx.doi.org/10.1073/pnas.2022826118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812819PMC
January 2021

Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

PLoS Biol 2020 12 30;18(12):e3000986. Epub 2020 Dec 30.

Functional Proteomics Group, Chester Beatty Laboratories, The Institute of Cancer Research, London, United Kingdom.

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.
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http://dx.doi.org/10.1371/journal.pbio.3000986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773185PMC
December 2020

Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death.

Cell Microbiol 2020 Dec 23:e13306. Epub 2020 Dec 23.

Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.

Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD-mediated pyroptosis via activation of caspase-1, caspase-4 and caspase-8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island-1 (SPI-1) injectisome, HilA-mediated overexpression of the SPI-1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O-antigen length regulator, which induces the production of very long O-antigen chains. Using a ΔfepE mutant we established that the very long O-antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome-mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar-specific inflammasome modulation, which mirrors the pro- and anti-inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome-mediated detection through the elaboration of very long LPS O-polysaccharides.
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http://dx.doi.org/10.1111/cmi.13306DOI Listing
December 2020

Cryoelectron-Microscopic Structure of the pKpQIL Conjugative Pili from Carbapenem-Resistant Klebsiella pneumoniae.

Structure 2020 12 10;28(12):1321-1328.e2. Epub 2020 Sep 10.

Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22903, USA. Electronic address:

Conjugative pili are important in mediating bacterial conjugation and horizontal gene transfer. Since plasmid transfer can include antibiotic-resistance genes, conjugation is an important mechanism in the spread of antibiotic resistance. Filamentous bacteriophages have been shown to exist in two different structural classes: those with a 5-fold rotational symmetry and those with a one-start helix with approximately 5 subunits per turn. Structures for the F and the F-like pED208 conjugation pilus have shown that they have 5-fold rotational symmetry. Here, we report the cryoelectron-microscopic structure of conjugative pili from carbapenem-resistant Klebsiella pneumoniae, encoded on the IncFIIK pKpQIL plasmid, at 3.9 Å resolution and show that it has a one-start helix. These results establish that conjugation pili can exist in at least two structural classes, consistent with other results showing that relatively small perturbations are needed to change the helical symmetry of polymers.
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http://dx.doi.org/10.1016/j.str.2020.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710920PMC
December 2020

Advances and Challenges in Studying Type III Secretion Effectors of Attaching and Effacing Pathogens.

Front Cell Infect Microbiol 2020 7;10:337. Epub 2020 Jul 7.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom.

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http://dx.doi.org/10.3389/fcimb.2020.00337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358347PMC
July 2020

Vying for the control of inflammasomes: The cytosolic frontier of enteric bacterial pathogen-host interactions.

Cell Microbiol 2020 04;22(4):e13184

Department of Life Sciences, Imperial College London, London, UK.

Enteric pathogen-host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane-bound toll-like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro-inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase-1 and are regulated by related caspases, such as caspase-11, -4, -5 and -8. Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasome activation (e.g. Listeria monocytogenes, Helicobacter pylori), others (e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome-mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.
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http://dx.doi.org/10.1111/cmi.13184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154749PMC
April 2020

Faecal neutrophil elastase-antiprotease balance reflects colitis severity.

Mucosal Immunol 2020 03 26;13(2):322-333. Epub 2019 Nov 26.

Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.

Given the global burden of diarrheal diseases on healthcare it is surprising how little is known about the drivers of disease severity. Colitis caused by infection and inflammatory bowel disease (IBD) is characterised by neutrophil infiltration into the intestinal mucosa and yet our understanding of neutrophil responses during colitis is incomplete. Using infectious (Citrobacter rodentium) and chemical (dextran sulphate sodium; DSS) murine colitis models, as well as human IBD samples, we find that faecal neutrophil elastase (NE) activity reflects disease severity. During C. rodentium infection intestinal epithelial cells secrete the serine protease inhibitor SerpinA3N to inhibit and mitigate tissue damage caused by extracellular NE. Mice suffering from severe infection produce insufficient SerpinA3N to control excessive NE activity. This activity contributes to colitis severity as infection of these mice with a recombinant C. rodentium strain producing and secreting SerpinA3N reduces tissue damage. Thus, uncontrolled luminal NE activity is involved in severe colitis. Taken together, our findings suggest that NE activity could be a useful faecal biomarker for assessing disease severity as well as therapeutic target for both infectious and chronic inflammatory colitis.
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http://dx.doi.org/10.1038/s41385-019-0235-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039808PMC
March 2020

Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease.

Cell Microbiol 2020 01 30;22(1):e13126. Epub 2019 Oct 30.

Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, London, UK.

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell-based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP-heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.
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http://dx.doi.org/10.1111/cmi.13126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003488PMC
January 2020

Citrobacter rodentium-host-microbiota interactions: immunity, bioenergetics and metabolism.

Nat Rev Microbiol 2019 11 20;17(11):701-715. Epub 2019 Sep 20.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK.

Citrobacter rodentium is an extracellular enteric mouse-specific pathogen used to model infections with human pathogenic Escherichia coli and inflammatory bowel disease. C. rodentium injects type III secretion system effectors into intestinal epithelial cells (IECs) to target inflammatory, metabolic and cell survival pathways and establish infection. While the host responds to infection by activating innate and adaptive immune signalling, required for clearance, the IECs respond by rapidly shifting bioenergetics to aerobic glycolysis, which leads to oxygenation of the epithelium, an instant expansion of mucosal-associated commensal Enterobacteriaceae and a decline of obligate anaerobes. Moreover, infected IECs reprogramme intracellular metabolic pathways, characterized by simultaneous activation of cholesterol biogenesis, import and efflux, leading to increased serum and faecal cholesterol levels. In this Review we summarize recent advances highlighting the intimate relationship between C. rodentium pathogenesis, metabolism and the gut microbiota.
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http://dx.doi.org/10.1038/s41579-019-0252-zDOI Listing
November 2019

OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo.

Nat Commun 2019 09 2;10(1):3957. Epub 2019 Sep 2.

Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK.

Carbapenem-resistance in Klebsiella pneumoniae (KP) sequence type ST258 is mediated by carbapenemases (e.g. KPC-2) and loss or modification of the major non-selective porins OmpK35 and OmpK36. However, the mechanism underpinning OmpK36-mediated resistance and consequences of these changes on pathogenicity remain unknown. By solving the crystal structure of a clinical ST258 OmpK36 variant we provide direct structural evidence of pore constriction, mediated by a di-amino acid (Gly115-Asp116) insertion into loop 3, restricting diffusion of both nutrients (e.g. lactose) and Carbapenems. In the presence of KPC-2 this results in a 16-fold increase in MIC to Meropenem. Additionally, the Gly-Asp insertion impairs bacterial growth in lactose-containing medium and confers a significant in vivo fitness cost in a murine model of ventilator-associated pneumonia. Our data suggests that the continuous selective pressure imposed by widespread Carbapenem utilisation in hospital settings drives the expansion of KP expressing Gly-Asp insertion mutants, despite an associated fitness cost.
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http://dx.doi.org/10.1038/s41467-019-11756-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718652PMC
September 2019

A nanobody targeting the translocated intimin receptor inhibits the attachment of enterohemorrhagic E. coli to human colonic mucosa.

PLoS Pathog 2019 08 29;15(8):e1008031. Epub 2019 Aug 29.

Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM-Cantoblanco, Madrid, Spain.

Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection.
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http://dx.doi.org/10.1371/journal.ppat.1008031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738647PMC
August 2019

The . Typhi effector StoD is an E3/E4 ubiquitin ligase which binds K48- and K63-linked diubiquitin.

Life Sci Alliance 2019 06 29;2(3). Epub 2019 May 29.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, UK

(e.g., serovars Typhi and Typhimurium) relies on translocation of effectors via type III secretion systems (T3SS). Specialization of typhoidal serovars is thought to be mediated via pseudogenesis. Here, we show that the Typhi STY1076/t1865 protein, named StoD, a homologue of the enteropathogenic /enterohemorrhagic NleG, is a T3SS effector. The StoD C terminus (StoD-C) is a U-box E3 ubiquitin ligase, capable of autoubiquitination in the presence of multiple E2s. The crystal structure of the StoD N terminus (StoD-N) at 2.5 Å resolution revealed a ubiquitin-like fold. In HeLa cells expressing StoD, ubiquitin is redistributed into puncta that colocalize with StoD. Binding assays showed that StoD-N and StoD-C bind the same exposed surface of the β-sheet of ubiquitin, suggesting that StoD could simultaneously interact with two ubiquitin molecules. Consistently, StoD interacted with both K63- (K = 5.6 ± 1 μM) and K48-linked diubiquitin (K = 15 ± 4 μM). Accordingly, we report the first Typhi-specific T3SS effector. We suggest that StoD recognizes and ubiquitinates pre-ubiquitinated targets, thus subverting intracellular signaling by functioning as an E4 enzyme.
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http://dx.doi.org/10.26508/lsa.201800272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545606PMC
June 2019

Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages.

Cell Rep 2019 04;27(4):1008-1017.e6

Department of Medicine, Medical Research Council Centre for Molecular Bacteriology & Infection, Imperial College London, London, UK. Electronic address:

Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.
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http://dx.doi.org/10.1016/j.celrep.2019.03.100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486487PMC
April 2019

Intestinal Epithelial Cells and the Microbiome Undergo Swift Reprogramming at the Inception of Colonic Citrobacter rodentium Infection.

mBio 2019 04 2;10(2). Epub 2019 Apr 2.

Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College, London, United Kingdom

We used the mouse attaching and effacing (A/E) pathogen , which models the human A/E pathogens enteropathogenic and enterohemorrhagic (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when resides in the caecum. In contrast, at 4 DPI, the day of colonic colonization, despite only sporadic adhesion to the apex of the crypt, we observed robust upregulation of cell cycle and DNA repair processes, which were associated with expansion of the crypt Ki67-positive replicative zone, and downregulation of multiple metabolic processes (including the tricarboxylic acid [TCA] cycle and oxidative phosphorylation). Moreover, we observed dramatic depletion of goblet and deep crypt secretory cells and an atypical regulation of cholesterol homeostasis in IECs during early infection, with simultaneous upregulation of cholesterol biogenesis (e.g., 3-hydroxy-3-methylglutaryl-coenzyme A reductase [Hmgcr]), import (e.g., low-density lipoprotein receptor [Ldlr]), and efflux (e.g., AbcA1). We also detected interleukin 22 (IL-22) responses in IECs (e.g., Reg3γ) on the day of colonic colonization, which occurred concomitantly with a bloom of commensal Enterobacteriaceae on the mucosal surface. These results unravel a new paradigm in host-pathogen-microbiome interactions, showing for the first time that sensing a small number of pathogenic bacteria triggers swift intrinsic changes to the IEC composition and function, in tandem with significant changes to the mucosa-associated microbiome, which parallel innate immune responses. The mouse pathogen is a widely used model for colonic infection and has been a major tool in fundamental discoveries in the fields of bacterial pathogenesis and mucosal immunology. Despite extensive studies probing acute infection, our understanding of the early stages preceding the infection climax remains relatively undetailed. To this end, we apply a multiomics approach to resolve temporal changes to the host and microbiome during early infection. Unexpectedly, we found immediate and dramatic responses occurring on the day of colonic infection, both in the host intestinal epithelial cells and in the microbiome. Our study suggests changes in cholesterol and carbon metabolism in epithelial cells are instantly induced upon pathogen detection in the colon, corresponding with a shift to primarily facultative anaerobes constituting the microbiome. This study contributes to our knowledge of disease pathogenesis and mechanisms of barrier regulation, which is required for development of novel therapeutics targeting the intestinal epithelium.
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http://dx.doi.org/10.1128/mBio.00062-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445932PMC
April 2019

The Galleria mellonella Infection Model for Investigating the Molecular Mechanisms of Legionella Virulence.

Methods Mol Biol 2019 ;1921:333-346

Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, UK.

Legionella species evolved virulence factors to exploit protozoa as replicative niches in the environment. Cell culture infection models demonstrated that many of these factors also enable the bacteria to thrive in human macrophages; however, these models do not recapitulate the complex interactions between macrophages, lung epithelial, and additional immune cells, which are crucial to control bacterial infections. Thus, suitable infection models are required to understand which bacterial factors are important to trigger disease. Guinea pigs and, most frequently, mice have been successfully used as mammalian model hosts; however, ethical and economic considerations impede their use in high-throughput screening studies of Legionella isolates or small molecule inhibitors.Here, we describe the larvae of the lepidopteran Galleria mellonella as insect model of Legionella pathogenesis. Larvae can be obtained from commercial suppliers in large numbers, maintained without the need of specialized equipment, and infected by injection. Although lacking the complexity of a mammalian immune system, the larvae mount humoral and cellular immune responses to infection. L. pneumophila strain 130b and other prototype isolates withstand these responses and use the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) type IV secretion system (T4SS ) to inject effectors enabling survival and replication in hemocytes, insect phagocytes, ultimately leading to the death of the larvae. Differences in virulence between L. pneumophila isolates or gene deletion mutants can be analyzed using indicators of larval health and immune induction, such as pigmentation, mobility, histopathology, and survival. Bacterial replication can be measured by plating hemolymph or by immunofluorescence microscopy of isolated circulating hemocytes from infected larvae. Combined, these straightforward experimental readouts make G. mellonella larvae a versatile model host to rapidly assess the virulence of different Legionella isolates and investigate the role of specific virulence factors in overcoming innate host defense mechanisms.
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http://dx.doi.org/10.1007/978-1-4939-9048-1_22DOI Listing
June 2019

Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification.

Methods Mol Biol 2019 ;1921:289-303

Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, UK.

The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.
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http://dx.doi.org/10.1007/978-1-4939-9048-1_19DOI Listing
June 2019

More than 18,000 effectors in the genus genome provide multiple, independent combinations for replication in human cells.

Proc Natl Acad Sci U S A 2019 02 18;116(6):2265-2273. Epub 2019 Jan 18.

Institut Pasteur, Biologie des Bactéries Intracellulaires, 75724 Paris, France;

The genus comprises 65 species, among which is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.
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http://dx.doi.org/10.1073/pnas.1808016116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369783PMC
February 2019

The effector LtpM is a new type of phosphoinositide-activated glucosyltransferase.

J Biol Chem 2019 02 20;294(8):2862-2879. Epub 2018 Dec 20.

the MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom, and

causes Legionnaires' disease, a severe form of pneumonia. translocates more than 300 effectors into host cells via its Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) type IV secretion system to enable its replication in target cells. Here, we studied the effector LtpM, which is encoded in a recombination hot spot in Paris. We show that a C-terminal phosphoinositol 3-phosphate (PI3P)-binding domain, also found in otherwise unrelated effectors, targets LtpM to the -containing vacuole and to early and late endosomes. LtpM expression in yeast caused cytotoxicity. Sequence comparison and structural homology modeling of the N-terminal domain of LtpM uncovered a remote similarity to the glycosyltransferase (GT) toxin PaTox from the bacterium ; however, instead of the canonical DxD motif of GT-A type glycosyltransferases, essential for enzyme activity and divalent cation coordination, we found that a DxN motif is present in LtpM. Using UDP-glucose as sugar donor, we show that purified LtpM nevertheless exhibits glucohydrolase and autoglucosylation activity and demonstrate that PI3P binding activates LtpM's glucosyltransferase activity toward protein substrates. Substitution of the aspartate or the asparagine in the DxN motif abolished the activity of LtpM. Moreover, whereas all glycosyltransferase toxins and effectors identified so far depend on the presence of divalent cations, LtpM is active in their absence. Proteins containing LtpM-like GT domains are encoded in the genomes of other isolates and species, suggesting that LtpM is the first member of a novel family of glycosyltransferase effectors employed to subvert hosts.
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http://dx.doi.org/10.1074/jbc.RA118.005952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393602PMC
February 2019

Author Correction: Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

Nat Commun 2018 11 29;9(1):5148. Epub 2018 Nov 29.

Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK.

The original version of this Article contained an error in the spelling of the author David Ruano-Gallego, which was incorrectly given as David R. Gallego. This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-07612-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265335PMC
November 2018

The Citrobacter rodentium type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses.

PLoS Pathog 2018 10 26;14(10):e1007406. Epub 2018 Oct 26.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom.

Infection with Citrobacter rodentium triggers robust tissue damage repair responses, manifested by secretion of IL-22, in the absence of which mice succumbed to the infection. Of the main hallmarks of C. rodentium infection are colonic crypt hyperplasia (CCH) and dysbiosis. In order to colonize the host and compete with the gut microbiota, C. rodentium employs a type III secretion system (T3SS) that injects effectors into colonic intestinal epithelial cells (IECs). Once injected, the effectors subvert processes involved in innate immune responses, cellular metabolism and oxygenation of the mucosa. Importantly, the identity of the effector/s triggering the tissue repair response is/are unknown. Here we report that the effector EspO ,an orthologue of OspE found in Shigella spp, affects proliferation of IECs 8 and 14 days post C. rodentium infection as well as secretion of IL-22 from colonic explants. While we observed no differences in the recruitment of group 3 innate lymphoid cells (ILC3s) and T cells, which are the main sources of IL-22 at the early and late stages of C. rodentium infection respectively, infection with ΔespO was characterized by diminished recruitment of sub-mucosal neutrophils, which coincided with lower abundance of Mmp9 and chemokines (e.g. S100a8/9) in IECs. Moreover, mice infected with ΔespO triggered significantly lesser nutritional immunity (e.g. calprotectin, Lcn2) and expression of antimicrobial peptides (Reg3β, Reg3γ) compared to mice infected with WT C. rodentium. This overlapped with a decrease in STAT3 phosphorylation in IECs. Importantly, while the reduced CCH and abundance of antimicrobial proteins during ΔespO infection did not affect C. rodentium colonization or the composition of commensal Proteobacteria, they had a subtle consequence on Firmicutes subpopulations. EspO is the first bacterial virulence factor that affects neutrophil recruitment and secretion of IL-22, as well as expression of antimicrobial and nutritional immunity proteins in IECs.
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http://dx.doi.org/10.1371/journal.ppat.1007406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221368PMC
October 2018

Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion.

Nat Commun 2018 10 10;9(1):4187. Epub 2018 Oct 10.

Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence.
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http://dx.doi.org/10.1038/s41467-018-06701-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180029PMC
October 2018

The Type III Secretion System of Pathogenic Escherichia coli.

Curr Top Microbiol Immunol 2018;416:51-72

Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK.

Infection with enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), enteroinvasive E. coli (EIEC) and Shigella relies on the elaboration of a type III secretion system (T3SS). Few strains also encode a second T3SS, named ETT2. Through the integration of coordinated intracellular and extracellular cues, the modular T3SS is assembled within the bacterial cell wall, as well as the plasma membrane of the host cell. As such, the T3SS serves as a conduit, allowing the chaperone-regulated translocation of effector proteins directly into the host cytosol to subvert eukaryotic cell processes. Recent technological advances revealed high structural resolution of the T3SS apparatus and how it could be exploited to treat enteric disease. This chapter summarises the current knowledge of the structure and function of the E. coli T3SSs.
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http://dx.doi.org/10.1007/82_2018_116DOI Listing
June 2019

Typhoidal Salmonella: Distinctive virulence factors and pathogenesis.

Cell Microbiol 2018 09 9;20(9):e12939. Epub 2018 Aug 9.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK.

Although nontyphoidal Salmonella (NTS; including Salmonella Typhimurium) mainly cause gastroenteritis, typhoidal serovars (Salmonella Typhi and Salmonella Paratyphi A) cause typhoid fever, the treatment of which is threatened by increasing drug resistance. Our understanding of S. Typhi infection in human remains poorly understood, likely due to the host restriction of typhoidal strains and the subsequent popularity of the S. Typhimurium mouse typhoid model. However, translating findings with S. Typhimurium across to S. Typhi has some limitations. Notably, S. Typhi has specific virulence factors, including typhoid toxin and Vi antigen, involved in symptom development and immune evasion, respectively. In addition to unique virulence factors, both typhoidal and NTS rely on two pathogenicity-island encoded type III secretion systems (T3SS), the SPI-1 and SPI-2 T3SS, for invasion and intracellular replication. Marked differences have been observed in terms of T3SS regulation in response to bile, oxygen, and fever-like temperatures. Moreover, approximately half of effectors found in S. Typhimurium are either absent or pseudogenes in S. Typhi, with most of the remaining exhibiting sequence variation. Typhoidal-specific T3SS effectors have also been described. This review discusses what is known about the pathogenesis of typhoidal Salmonella with emphasis on unique behaviours and key differences when compared with S. Typhimurium.
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http://dx.doi.org/10.1111/cmi.12939DOI Listing
September 2018

Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ.

mBio 2018 04 10;9(2). Epub 2018 Apr 10.

Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College, London United Kingdom, London, United Kingdom

Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic (EPEC and EHEC, respectively), , and , where they inhibit Src kinase by coupled amidation and ADP-ribosylation. , which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type or encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis. Enteropathogenic and enterohemorrhagic (EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide. is a mouse pathogen used to model EPEC and EHEC pathogenesis Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type or an catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response .
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http://dx.doi.org/10.1128/mBio.00170-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893879PMC
April 2018

Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells.

Int J Med Microbiol 2018 Apr 12;308(3):387-404. Epub 2018 Mar 12.

Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung - ZMBE, Westfälische Wilhelms-Universität Münster, Münster, Germany. Electronic address:

Enteropathogenic Escherichia coli (EPEC) subvert host cell signaling pathways by injecting effector proteins via a Type 3 Secretion System (T3SS). The T3SS-dependent EspB protein is a multi-functional effector protein, which contributes to adherence and translocator pore formation and after injection exhibits several intracellular activities. In addition, EspB is also secreted into the environment. Effects of secreted EspB have not been reported thus far. As a surrogate for secreted EspB we employed recombinant EspB (rEspB) derived from the prototype EPEC strain E2348/69 and investigated the interactions of the purified protein with different human epithelial and immune cells including monocytic THP-1 cells, macrophages, dendritic cells, U-937, epithelial T84, Caco-2, and HeLa cells. To assess whether these proteins might exert a cytotoxic effect we monitored the release of lactate dehydrogenase (LDH) as well as propidium iodide (PI) uptake. For comparison, we also investigated several homologs of EspB such as IpaD of Shigella, and SipC, SipD, SseB, and SseD of Salmonella as purified recombinant proteins. Interestingly, cytotoxicity was only observed in THP-1 cells and macrophages, whereas epithelial cells remained unaffected. Cell fractionation and immune fluorescence experiments showed that rEspB enters cells autonomously, which suggests that EspB might qualify as a novel cell-penetrating effector protein (CPE). Using specific organelle tracers and inhibitors of signaling pathways we found that rEspB destroys the mitochondrial membrane potential - an indication of programmed cell death induction in THP-1 cells. Here we show that EspB not only constitutes an essential part of the T3SS-nanomachine and contributes to the arsenal of injected effector proteins but, furthermore, that secreted (recombinant) EspB autonomously enters host cells and selectively induces cell death in immune cells.
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http://dx.doi.org/10.1016/j.ijmm.2018.03.005DOI Listing
April 2018

Sieving through gut models of colonization resistance.

Nat Microbiol 2018 02 22;3(2):132-140. Epub 2018 Jan 22.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, UK.

The development of innovative high-throughput genomics and metabolomics technologies has considerably expanded our understanding of the commensal microorganisms residing within the human body, collectively termed the microbiota. In recent years, the microbiota has been reported to have important roles in multiple aspects of human health, pathology and host-pathogen interactions. One function of commensals that has attracted particular interest is their role in protection against pathogens and pathobionts, a concept known as colonization resistance. However, pathogens are also able to sense and exploit the microbiota during infection. Therefore, obtaining a holistic understanding of colonization resistance mechanisms is essential for the development of microbiome-based and microbiome-targeting therapies for humans and animals. Achieving this is dependent on utilizing physiologically relevant animal models. In this Perspective, we discuss the colonization resistance functions of the gut microbiota and sieve through the advantages and limitations of murine models commonly used to study such mechanisms within the context of enteric bacterial infection.
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http://dx.doi.org/10.1038/s41564-017-0095-1DOI Listing
February 2018

Citrobacter rodentium Relies on Commensals for Colonization of the Colonic Mucosa.

Cell Rep 2017 Dec;21(12):3381-3389

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, UK. Electronic address:

We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course.
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http://dx.doi.org/10.1016/j.celrep.2017.11.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746604PMC
December 2017

Comparison of Salmonella enterica Serovars Typhi and Typhimurium Reveals Typhoidal Serovar-Specific Responses to Bile.

Infect Immun 2018 03 20;86(3). Epub 2018 Feb 20.

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom

serovars Typhi and Typhimurium cause typhoid fever and gastroenteritis, respectively. A unique feature of typhoid infection is asymptomatic carriage within the gallbladder, which is linked with Typhi transmission. Despite this, Typhi responses to bile have been poorly studied. Transcriptome sequencing (RNA-Seq) of Typhi Ty2 and a clinical Typhi isolate belonging to the globally dominant H58 lineage (strain 129-0238), as well as Typhimurium 14028, revealed that 249, 389, and 453 genes, respectively, were differentially expressed in the presence of 3% bile compared to control cultures lacking bile. genes, the operon, and putative sialic acid uptake and metabolism genes (t1787 to t1790) were upregulated in all strains following bile exposure, which may represent adaptation to the small intestine environment. Genes within the pathogenicity island 1 (SPI-1), those encoding a type IIII secretion system (T3SS), and motility genes were significantly upregulated in both Typhi strains in bile but downregulated in Typhimurium. Western blots of the SPI-1 proteins SipC, SipD, SopB, and SopE validated the gene expression data. Consistent with this, bile significantly increased Typhi HeLa cell invasion, while Typhimurium invasion was significantly repressed. Protein stability assays demonstrated that in Typhi the half-life of HilD, the dominant regulator of SPI-1, is three times longer in the presence of bile; this increase in stability was independent of the acetyltransferase Pat. Overall, we found that Typhi exhibits a specific response to bile, especially with regard to virulence gene expression, which could impact pathogenesis and transmission.
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http://dx.doi.org/10.1128/IAI.00490-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820949PMC
March 2018