270 results found
Wong JLC, Romano M, Kerry LE, et al., 2023, Author Correction: OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo., Nat Commun, Vol: 14
Wong J, David S, Sanchez Garrido J, et al., 2022, Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure, Proceedings of the National Academy of Sciences of USA, Vol: 119, Pages: 1-12, ISSN: 0027-8424
Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.
Chaukimath P, Frankel G, Visweswariah SS, 2022, The metabolic impact of bacterial infection in the gut, FEBS JOURNAL, ISSN: 1742-464X
David S, Wong JLC, Sanchez-Garrido J, et al., 2022, Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae., PLoS Pathogens, Vol: 18, Pages: 1-23, ISSN: 1553-7366
Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.
Low WW, Wong J, Beltran L, et al., 2022, Mating pair stabilization mediates bacterial conjugation species specificity, Nature Microbiology, Vol: 7, Pages: 1016-1027, ISSN: 2058-5276
Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating thespread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane(OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNAtransfer. We show that TraN from the plasmids pKpQIL (Klebsiella pneumoniae), R100-1 (Shigella flexneri) and pSLT (SalmonellaTyphimurium), and the prototypical F plasmid (Escherichia coli) interact with OmpK36, OmpW and OmpA, respectively.Cryo-EM analysis revealed that TraN pKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN intoa monomer of the OmpK36 trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified afourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classifica-tion scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ(OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect thedistribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pairstabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emergingresistance plasmids in high-risk bacterial pathogens.
Sanchez Garrido J, Ruano-Gallego D, Choudhary JS, et al., 2022, The type III secretion system effector network hypothesis, Trends in Microbiology, Vol: 30, Pages: 524-533, 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.
Higginson EE, Nkeze J, Permala-Booth J, et al., 2022, Detection of Salmonella Typhi in Bile by Quantitative Real-Time PCR, MICROBIOLOGY SPECTRUM, Vol: 10, ISSN: 2165-0497
Kreutzberger MAB, Sobe RC, Sauder AB, et al., 2022, Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments., Nature Communications, Vol: 13, Pages: 1-13, ISSN: 2041-1723
Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.
Kreutzberger MA, Sobe R, Sauder AB, et al., 2022, Cryo-EM of bacterial flagellar filaments with screw-like surfaces and outer domain sheaths, Publisher: CELL PRESS, Pages: 131-131, ISSN: 0006-3495
Mullineaux Sanders C, Kozik Z, Sanchez Garrido J, et al., 2022, Citrobacter rodentium infection induces persistent molecular changes and interferon gamma-dependent major histocompatibility complex class II expression in the colonic epithelium, mBio, Vol: 13, Pages: 1-18, ISSN: 2150-7511
Most studies of infections at mucosal surfaces have focused on the acute phase of the disease. Consequently, little is known about the molecular processes that underpin tissue recovery and the long-term consequences postinfection. Here, we conducted temporal deep quantitative proteomic analysis of colonic intestinal epithelial cells (cIECs) from mice infected with the natural mouse pathogen Citrobacter rodentium over time points corresponding to the late steady-state phase (10 days postinfection [DPI]), the clearance phase (13 to 20 DPI), and 4 weeks after the pathogen has been cleared (48 DPI). C. rodentium, which relies on a type III secretion system to infect, is used to model infections with enteropathogenic and enterohemorrhagic Escherichia coli. We observe a strong upregulation of inflammatory signaling and nutritional immunity responses during the clearance phase of the infection. Despite morphological tissue recovery, chromogranin B (ChgB)-positive endocrine cells remained significantly below baseline levels at 48 DPI. In contrast, we observed an increased abundance of proteins involved in antigen processing and presentation 4 weeks after pathogen clearance. In particular, long-term changes were characterized by a persistent interferon gamma (IFN-γ) response and the expression of major histocompatibility complex class II (MHCII) molecules in 60% of the EpCAM+ cIECs, which were not seen in Ifnγ−/− mice. Nonetheless, both wild-type and Ifnγ−/− mice mounted similar systemic and colonic IgG responses to C. rodentium and were equally protected from rechallenge, suggesting that cIEC MHCII is not necessary for protective immunity against C. rodentium.
Sperandio V, Frankel G, 2022, Editorial overview: Host-microbe interactions: friends, foes and frenemies, CURRENT OPINION IN MICROBIOLOGY, Vol: 65, Pages: VIII-X, ISSN: 1369-5274
Zhong Q, Chatterjee S, Choudhary JS, et al., 2021, EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death, MOLECULAR MICROBIOLOGY, Vol: 117, Pages: 480-492, 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.
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: 1-21, ISSN: 0036-8075
Infections with many Gram-negative pathogens, including Escherichia coli, Salmonella, Shigella, and Yersinia, 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 Citrobacter rodentium (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 E. coli effector repertoire in C. rodentium 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.
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, SHIGA TOXIN-PRODUCING E. COLI, Vol: 2291, Pages: 399-418, ISSN: 1064-3745
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.
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