259 results found
Zhong Q, Chatterjee S, Choudhary JS, et al., 2021, EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death, MOLECULAR MICROBIOLOGY, ISSN: 0950-382X
Sanchez Garrido J, Alberdi L, Chatterjee S, et al., 2021, Type III secretion system effector subnetworks elicit distinct host immune responses to infection, Current Opinion in Microbiology, Vol: 64, Pages: 19-26, ISSN: 1369-5274
Citrobacter rodentium, a natural mouse pathogen which colonises the colon of immuno-competent mice, provides a robust model for interrogating host-pathogen-microbiota interactions in vivo. This model has been key to providing new insights into local host responses to enteric infection, including changes inintestinal epithelial cell immuno metabolism and mucosal immunity. C. rodent iuminjects 31 bacterial effectors into epithelial cells via a type III secretion system (T3SS). Recently, these effectors were shown to be able to form multiple intracellular subnetworks which can withstand significant contractions whilst maintaining virulence. Here we highlight recent advances in understanding gut mucosal responses to infection and effector biology, as well as potential uses for artificial intelligence (AI) in understanding infectious diseaseand speculate on the role of T3SS effector networks in host adaption.
Sanchez Garrido J, Ruano-Gallego D, Choudhary JS, et al., 2021, The type III secretion system effector network hypothesis, Trends in Microbiology, ISSN: 0966-842X
Type III secretion system (T3SS) effectors are key virulence factors that underpin the infection strategy of many clinically important Gram-negative pathogens, including Salmonella enterica, Shigella spp, enteropathogenic and enterohaemorrhagic Escherichia coli and their murine equivalent, Citrobacter rodentium. The cellular processes or proteins targeted by the effectors can be common to multiple pathogens or pathogen-specific. The main approach to understanding T3SS-mediated pathogenesis has been to determine the contribution of one effector at a time, with the aim to piece together individual functions and unveil infection mechanisms. However, in contrast to this prevailing approach, simultaneous deletion of multiple effectors revealed that they function as an interconnected network in vivo, uncoveringeffector co-dependency and context-dependent effector essentiality. This paradigm shift in T3SS biology is at the heart of this opinion.
Mullineaux Sanders C, Carson D, Hopkins E, et al., 2021, Citrobacter amalonaticus inhibits the growth of Citrobacter rodentium in the gut lumen, mBio, Vol: 5, Pages: 1-19, ISSN: 2150-7511
The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing or succumbing to the infection respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in re-localisation of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumen of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon we term antibiotic-induced bacterial commensalisation (AIBC). AIBC C. rodentium was well-tolerated by the host, which showed little signs of inflammation; passaged AIBC C. rodentium robustly infected naïve C3H/HeN mice suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalisation in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in vitro in a contact-dependant manner, and luminal growth of AIBC C. rodentium in vivo. Overall our data suggest that commensal species can confer colonization resistance against closely-related pathogenic species.
Chatterjee S, Choi A, Frankel G, 2021, A systematic review of Sec24 cargo interactome, Traffic, Vol: 22, Pages: 412-424, ISSN: 1398-9219
Endoplasmic reticulum (ER)-to-Golgi trafficking is an essential and highly conserved cellular process. The coat protein complex-II (COPII) arm of the trafficking machinery incorporates a wide array of cargo proteins into vesicles through direct or indirect interactions with Sec24, the principal subunit of the COPII coat. Approximately one-third of all mammalian proteins rely on the COPII mediated secretory pathway for membrane insertion or secretion. There are four mammalian Sec24 paralogs and three yeast Sec24 paralogs with emerging evidence of paralog-specific cargo interaction motifs. Furthermore, individual paralogs also differ in their affinity for a subset of sorting motifs present on cargo proteins. As with many aspects of protein trafficking, we lack a systematic and thorough understanding of the interaction of Sec24 with cargoes. This systematic review focuses on the current knowledge of cargo binding to both yeast and mammalian Sec24 paralogs and their ER export motifs. The analyses show that Sec24 paralog specificity of cargo (and cargo receptors) range from exclusive paralog dependence or preference to partial redundancy. Wealso discuss how the Sec24 secretion system is hijacked by viral (e.g., VSV-G, Hepatitis B envelope protein) and bacterial (e.g., the enteropathogenic E. coli type III secretion system effector NleA/EspI) pathogens.
Chatterjee S, Lekmeechai S, Constantinou N, et al., 2021, The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1, CELLULAR MICROBIOLOGY, Vol: 23, ISSN: 1462-5814
Chisenga CC, Bosomprah S, Simuyandi M, et al., 2021, Shigella-specific antibodies in the first year of life among Zambian infants: A longitudinal cohort study, PLOS ONE, Vol: 16, ISSN: 1932-6203
Mylona E, Sanchez Garrido J, Nguyen Hoang Thu T, et al., 2021, Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death, Cellular Microbiology, Vol: 23, Pages: 1-14, ISSN: 1462-5814
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.
Ruano-Gallego D, Sanchez-Garrido J, Kozik Z, et al., 2021, Type III secretion system effectors form robust and flexible intracellular virulence networks, SCIENCE, Vol: 371, Pages: 1122-+, ISSN: 0036-8075
Zheng W, Peña A, Ilangovan A, et al., 2021, Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament., Proceedings of the National Academy of Sciences of USA, Vol: 118, ISSN: 0027-8424
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (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.
Hopkins EGD, Frankel G, 2021, Overview of the Effect of Citrobacter rodentium Infection on Host Metabolism and the Microbiota., Methods Mol Biol, Vol: 2291, Pages: 399-418
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.
Zheng W, Pena A, Low WW, et al., 2020, Cryoelectron-Microscopic Structure of the pKpQIL Conjugative Pili from Carbapenem-Resistant Klebsiella pneumoniae, STRUCTURE, Vol: 28, Pages: 1321-+, ISSN: 0969-2126
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, Citrobacter rodentium-host-microbiota interactions: immunity, bioenergetics and metabolism, NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 701-715, ISSN: 1740-1526
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 E. coli to human colonic mucosa, PLOS PATHOGENS, Vol: 15, ISSN: 1553-7366
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 Citrobacter rodentium type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses, PLOS PATHOGENS, Vol: 14, ISSN: 1553-7366
Johnson R, Mylona E, Frankel G, 2018, Typhoidal Salmonella: Distinctive virulence factors and pathogenesis, CELLULAR MICROBIOLOGY, Vol: 20, ISSN: 1462-5814
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