Publications
277 results found
Zhong Q, Roumeliotis T, Kozik Z, et al., 2020, Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells, PLoS Biology, Vol: 18, ISSN: 1544-9173
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.
Slater S, Frankel G, 2020, Advances and challenges in studying type III secretion effectors of attaching and effacing pathogens, Frontiers in Cellular and Infection Microbiology, Vol: 10, Pages: 1-7, ISSN: 2235-2988
Sanchez Garrido J, Slater SL, Clements A, et al., 2020, Vying for the control of inflammasomes: the cytosolic frontier of enteric bacterial pathogen - host interactions, Cellular Microbiology, Vol: 22, Pages: 1-19, ISSN: 1462-5814
Enteric pathogen-host interactions occur at multiple interfaces,includingthe intestinal epitheliumand deeper organsof the immune system. Microbial ligands and activities are detected by host sensorsthat elicit a range of immune responses. Membrane-bound Toll-Like Receptors (TLRs) and cytosolic inflammasomepathways are key signal transducers that trigger production of pro-inflammatory molecules such as cytokines and chemokinesand regulate cell deathin response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tusslebetween bacterial pathogens and the host. Inflammasomes are complexes that activate caspase-1and 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 inflammasomeactivation(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 (T3SS) effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasomepathwaysand their immune functions and discuss how enteric bacterial pathogens interact with them.These studies have not only shed light on the inflammasome-mediated immunity, but also the exciting area of mammalian cytosolic immune
Carson D, Barry R, Eve GD H, et al., 2020, Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease, Cellular Microbiology, Vol: 22, Pages: 1-17, ISSN: 1462-5814
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.
Barry R, Ruano-Gallego D, Radhakrishnan ST, et al., 2020, Faecal neutrophil elastase-antiprotease balance reflects colitis severity, Mucosal Immunology, Vol: 13, Pages: 322-333, ISSN: 1933-0219
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.
Mullineaux-Sanders C, Sanchez-Garrido J, Hopkins EGD, et al., 2019, <i>Citrobacter rodentium</i>-host-microbiota interactions: immunity, bioenergetics and metabolism, NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 701-715, ISSN: 1740-1526
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- Citations: 67
Wong JLC, Romano M, Kerry L, et al., 2019, OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo, Nature Communications, Vol: 10, ISSN: 2041-1723
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 suggest 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.
Ruano-Gallego D, Yara DA, Di Ianni L, et al., 2019, A nanobody targeting the translocated intimin receptor inhibits the attachment of enterohemorrhagic <i>E</i>. <i>coli</i> to human colonic mucosa, PLOS PATHOGENS, Vol: 15, ISSN: 1553-7366
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- Citations: 14
Mylona E, Frankel G, 2019, The S. Typhi effector StoD is an E3 ubiquitin ligase which binds K48- and K63-linked di-ubiquitin, Life Science Alliance, Vol: 2, ISSN: 2575-1077
Salmonella enterica (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 Salmonella Typhi STY1076/t1865 protein, named StoD, a homologue of the enteropathogenic Escherichia coli/enterohemorrhagic E. coli/Citrobacter rodentium 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- (KD = 5.6 ± 1 μM) and K48-linked diubiquitin (KD = 15 ± 4 μM). Accordingly, we report the first S. Typhi–specific T3SS effector. We suggest that StoD recognizes and ubiquitinates pre-ubiquitinated targets, thus subverting intracellular signaling by functioning as an E4 enzyme.
Goddard P, Sanchez Garrido J, Slater S, et al., 2019, Enteropathogenic E. coli stimulates effector-driven rapid caspase-4 activation in human macrophages, Cell Reports, Vol: 27, Pages: 1008-1017.e6, ISSN: 2211-1247
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.
Hopkins E, Roumeliotis TI, Mullineaux-Sanders C, et al., 2019, Intestinal epithelial cells and the microbiome undergo swift reprogramming at the inception of colonic Citrobacter rodentium infection, mBio, Vol: 10, ISSN: 2150-7511
We used the mouse attaching and effacing (A/E) pathogen Citrobacter rodentium, which models the human A/E pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during in vivo infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when C. rodentium 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.
Levanova N, Mattheis C, Carson D, et al., 2019, The Legionella effector LtpM is a new type of phosphoinositide-activated glucosyltransferase, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 294, Pages: 2862-2879
Gomez-Valero L, Rusniok C, Carson D, et al., 2019, More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 116, Pages: 2265-2273, ISSN: 0027-8424
Frankel G, Schroeder GN, 2019, The Galleria mellonella Infection Model for Investigating the Molecular Mechanisms of Legionella Virulence., Methods Mol Biol, Vol: 1921, Pages: 333-346
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 v
So EC, Mousnier A, Frankel G, et al., 2019, Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification., Methods Mol Biol, Vol: 1921, Pages: 289-303
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.
Connolly JPR, Slater SL, O'Boyle N, et al., 2018, Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion, Nature Communications, Vol: 9, ISSN: 2041-1723
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.
Berger CN, Crepin VF, Roumeliotis TI, et al., 2018, The <i>Citrobacter rodentium</i> type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses, PLOS PATHOGENS, Vol: 14, ISSN: 1553-7366
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- Citations: 20
Johnson R, Mylona E, Frankel G, 2018, Typhoidal <i>Salmonella</i>: Distinctive virulence factors and pathogenesis, CELLULAR MICROBIOLOGY, Vol: 20, ISSN: 1462-5814
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- Citations: 73
Slater SL, Sagfors AM, Pollard DJ, et al., 2018, The Type III Secretion System of Pathogenic Escherichia coli, Editors: Frankel, Ron, Publisher: SPRINGER INTERNATIONAL PUBLISHING AG, Pages: 51-72
Pollard D, Berger CN, So E, et al., 2018, Broad spectrum regulation of non receptor tyrosine kinases by the bacterial ADP ribosyltransferase EspJ, mBio, Vol: 9, ISSN: 2150-7511
Tyrosine phosphorylation is key for signal transduction fromexogenousstimuli, including the defence against pathogens. Conversely, pathogens cansubvert protein phosphorylation to control hostimmune responsesand facilitateinvasionanddissemination. The bacterial 23effectorsEspJand SeoC areinjected into host cellsthough a type III secretion system by enteropathogenic and enterohaemorrhagic Escherichia coli(EPEC and EHEC), Citrobacter rodentiumand Salmonellaentericawhere they inhibit Src kinase bycoupledamidation andADP-ribosylation. C. rodentium, which is used tomodel EPEC and EHEC infections in human, is a mouse pathogen triggeringcolonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparing the proteomes of intestinal epithelial cells isolated from mice infected with wildtype C.rodentiumor C. rodentiumencoding catalyticallyinactive EspJrevealed that EspJ-induced ADP-ribosylationregulatesmultiple non-receptor tyrosine kinasesin vivo. Investigating the substrate repertoire of EspJ revealed that in HeLa and A549 Src and Csk were significantly targeted; in polarised Caco2 cells EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 EspJ modifiedCsk, 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 which targets mammalian kinases,controls immune responses andthe Src/Csk signalling axis.
Baumann D, Salia H, Greune L, et al., 2018, Multitalented EspB of enteropathogenic <i>Escherichia coli</i> (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells, INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, Vol: 308, Pages: 387-404, ISSN: 1438-4221
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- Citations: 7
Mullineaux-Sanders C, Suez J, Elinav E, et al., 2018, Sieving through gut models of colonization resistance, NATURE MICROBIOLOGY, Vol: 3, Pages: 132-140, ISSN: 2058-5276
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- Citations: 39
Mullineaux-Sanders C, Colins JW, Ruano-Gallego D, et al., 2017, Citrobacter rodentium relies on commensals for colonization of the colonic mucosa, Cell Reports, Vol: 21, Pages: 3381-3389, ISSN: 2211-1247
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.
Johnson R, Ravenhall M, Pickard D, et al., 2017, Comparison of Salmonella enterica serovars Typhi and Typhimurium reveals typhoidal-specific responses to bile, Infection and Immunity, Vol: 86, ISSN: 0019-9567
Salmonella enterica 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 S. Typhi transmission. Despite this, S. Typhi responses to bile have been poorly studied. RNA-Seq of S. Typhi Ty2 and a clinical S. Typhi isolate belonging to the globally dominant H58 lineage (129-0238), as well as S. 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. fad genes, the actP-acs operon, and putative sialic acid uptake and metabolism genes (t1787-t1790) were upregulated in all strains following bile exposure, which may represent adaptation to the small intestine environment. Genes within the Salmonella pathogenicity island 1 (SPI-1), encoding a type IIII secretion system (T3SS), and motility genes were significantly upregulated in both S. Typhi strains in bile, but downregulated in S. Typhimurium. Western blots of the SPI-1 proteins SipC, SipD, SopB and SopE validated the gene expression data. Consistent with this, bile significantly increased S. Typhi HeLa cell invasion whilst S. Typhimurium invasion was significantly repressed. Protein stability assays demonstrated that in S. 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 S. Typhi exhibits a specific response to bile, especially with regards to virulence gene expression, which could impact pathogenesis and transmission.
Portaliou AG, Tsolis KC, Loos MS, et al., 2017, Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic <i>Escherichia coli</i>, EMBO JOURNAL, Vol: 36, Pages: 3517-3531, ISSN: 0261-4189
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- Citations: 43
Cepeda-Molero M, Berger CN, Walsham ADS, et al., 2017, Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors, PLoS Pathogens, Vol: 13, ISSN: 1553-7366
Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces.
Berger C, Crepin V, Roumeliotis TI, et al., 2017, Citrobacter rodentium subverts ATP flux 1 and cholesterol homeostasis in 2 intestinal epithelial cell in vivo, Cell Metabolism, Vol: 26, Pages: 738-752.e6, ISSN: 1550-4131
The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.
Berger CN, 2017, The Enterohemorrhagic Escherichia coli Effector EspW Triggers Actin Remodeling in a Rac1-Dependent Manner, Infection and Immunity, Vol: 85, ISSN: 1098-5522
Enterohemorrhagic Escherichia coli (EHEC) is a diarrheagenic pathogen that colonizes the gut mucosa and induces attaching-and-effacing lesions. EHEC employs a type III secretion system (T3SS) to translocate 50 effector proteins that hijack and manipulate host cell signaling pathways, which allow bacterial colonization and subversion of immune responses and disease progression. The aim of this study was to characterize the T3SS effector EspW. We found espW in the sequenced O157:H7 and non-O157 EHEC strains as well as in Shigella boydii. Furthermore, a truncated version of EspW, containing the first 206 residues, is present in EPEC strains belonging to serotype O55:H7. Screening a collection of clinical EPEC isolates revealed that espW is present in 52% of the tested strains. We report that EspW modulates actin dynamics in a Rac1-dependent manner. Ectopic expression of EspW results in formation of unique membrane protrusions. Infection of Swiss cells with an EHEC espW deletion mutant induces a cell shrinkage phenotype that could be rescued by Rac1 activation via expression of the bacterial guanine nucleotide exchange factor, EspT. Furthermore, using a yeast two-hybrid screen, we identified the motor protein Kif15 as a potential interacting partner of EspW. Kif15 and EspW colocalized in cotransfected cells, while ectopically expressed Kif15 localized to the actin pedestals following EHEC infection. The data suggest that Kif15 recruits EspW to the site of bacterial attachment, which in turn activates Rac1, resulting in modifications of the actin cytoskeleton that are essential to maintain cell shape during infection.
Witcomb LA, Czupryna J, Francis KP, et al., 2017, Non-invasive three-dimensional imaging of Escherichia coli K1 infection using diffuse light imaging tomography combined with micro-computed tomography, Methods, Vol: 127, Pages: 62-68, ISSN: 1046-2023
In contrast to two-dimensional bioluminescence imaging, three dimensional diffuse light imaging tomography with integrated micro-computed tomography (DLIT-μCT) has the potential to realise spatial variations in infection patterns when imaging experimental animals dosed with derivatives of virulent bacteria carrying bioluminescent reporter genes such as the lux operon from the bacterium Photorhabdus luminescens. The method provides an opportunity to precisely localise the bacterial infection sites within the animal and enables the generation of four-dimensional movies of the infection cycle. Here, we describe the use of the PerkinElmer IVIS SpectrumCT in vivo imaging system to investigate progression of lethal systemic infection in neonatal rats following colonisation of the gastrointestinal tract with the neonatal pathogen Escherichia coli K1. We confirm previous observations that these bacteria stably colonize the colon and small intestine following feeding of the infectious dose from a micropipette; invading bacteria migrate across the gut epithelium into the blood circulation and establish foci of infection in major organs, including the brain. DLIT-μCT revealed novel multiple sites of colonisation within the alimentary canal, including the tongue, oesophagus and stomach, with penetration of the non-keratinised oesophageal epithelial surface, providing strong evidence of a further major site for bacterial dissemination. We highlight technical issues associated with imaging of infections in new born rat pups and show that the whole-body and organ bioburden correlates with disease severity.
Pallett MA, Crepin VF, Serafini N, et al., 2017, Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells, Mucosal Immunology, Vol: 10, Pages: 602-612, ISSN: 1935-3456
The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.
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