36 results found
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
Jégouzo SAF, Nelson C, Hardwick T, et al., 2020, Mammalian lectin arrays for screening host–microbe interactions, Journal of Biological Chemistry, Vol: 295, Pages: 4541-4555, ISSN: 0021-9258
Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen–receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.
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
Watson J, Sanchez-garrido J, Goddard P, et al., 2019, Shigella sonnei O-Antigen Inhibits Internalization, Vacuole Escape, and Inflammasome Activation, mBio, Vol: 10, Pages: 1-14, ISSN: 2150-7511
Two Shigella species, flexneri and sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle and high-income countries. S. flexneri is a prototypiccytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By contrast little is known about the invasion, vacuole escape and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen, which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-antigen onto its surface compared to other Shigella species.
Torraca V, Kaforou M, Watson J, et al., 2019, Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence, PLoS Pathogens, Vol: 15, Pages: 1-26, ISSN: 1553-7366
Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.
Watson JL, Sanchez-Garrido J, Goddard PJ, et al., 2019, <i>Shigella sonnei</i>O-antigen inhibits internalisation, vacuole escape and inflammasome activation, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>Two<jats:italic>Shigella</jats:italic>species,<jats:italic>flexneri</jats:italic>and<jats:italic>sonnei</jats:italic>, cause approximately 90% of bacterial dysentery worldwide. While<jats:italic>S. flexneri</jats:italic>is the dominant species in low-income countries,<jats:italic>S. sonnei</jats:italic>causes the majority of infections in middle and high-income countries.<jats:italic>S. flexneri</jats:italic>is a prototypic cytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By contrast little is known about the invasion, vacuole escape and induction of pyroptosis during<jats:italic>S. sonnei</jats:italic>infection of macrophages. We demonstrate that<jats:italic>S. sonnei</jats:italic>causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic<jats:italic>S. sonnei</jats:italic>and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen, which in<jats:italic>S. sonnei</jats:italic>is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings suggest that<jats:italic>S. sonnei</jats:italic>has adapted to an extracellular lifestyle by incorporating additional O-antigen into its surface structures compared to other<jats:italic>Shigella</jats:italic>species.</jats:p>
Wong JLC, Romano M, Kerry LE, et al., 2019, OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo, NATURE COMMUNICATIONS, Vol: 10, ISSN: 2041-1723
Torraca V, Kaforou M, Watson J, et al., 2019, <i>Shigella sonnei</i>infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p><jats:italic>Shigella flexneri</jats:italic>is historically regarded as the primary agent of bacillary dysentery, yet the closely-related<jats:italic>Shigella sonnei</jats:italic>is replacing<jats:italic>S. flexneri</jats:italic>, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (<jats:italic>Danio rerio</jats:italic>) model of<jats:italic>Shigella</jats:italic>infection, we discover that<jats:italic>S. sonnei</jats:italic>is more virulent than<jats:italic>S. flexneri in vivo</jats:italic>. Whole animal dual-RNAseq and testing of bacterial mutants suggest that<jats:italic>S. sonnei</jats:italic>virulence depends on its O-antigen oligosaccharide (which is unique among<jats:italic>Shigella</jats:italic>species). We show<jats:italic>in vivo</jats:italic>using zebrafish and<jats:italic>ex vivo</jats:italic>using human neutrophils that<jats:italic>S. sonnei</jats:italic>O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables<jats:italic>S. sonnei</jats:italic>to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of<jats:italic>S. sonnei</jats:italic>and zebrafish survival. Strikingly, larvae primed with a sublethal dose of<jats:italic>S. sonnei</jats:italic>are protected against a secondary lethal dose of<jats:italic>S. sonnei</jats:italic>in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against<jats:italic>S. sonnei</jats:italic>. Collectively, these findings reveal O-antigen as an important therapeutic
Furniss RCD, Low WW, Mavridou DAI, et al., 2018, Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces, Journal of Biological Chemistry, Vol: 293, Pages: 17188-17199, ISSN: 0021-9258
Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing (A/E) lesion on the surface of infected cells causes significant remodelling of the host cell surface, however limited information is available about changes at the protein level. Here we employed "plasma membrane profiling", a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor CD55 exhibited the greatest reduction in cell surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE, and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.
Torres VVL, Heinz E, Stubenrauch CJ, et al., 2018, An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis, MOLECULAR MICROBIOLOGY, Vol: 109, Pages: 584-599, ISSN: 0950-382X
Shenoy AR, Furniss RCD, Goddard PJ, et al., 2018, Modulation of host cell processes by T3SS effectors, Escherichia coli, a Versatile Pathogen, Editors: Frankel, Ron, Publisher: Springer Verlag
Two of the enteric Escherichia coli pathotypes-enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)-have a conserved type 3 secretion system which is essential for virulence. The T3SS is used to translocate between 25 and 50 bacterial proteins directly into the host cytosol where they manipulate a variety of host cell processes to establish a successful infection. In this chapter, we discuss effectors from EPEC/EHEC in the context of the host proteins and processes that they target-the actin cytoskeleton, small guanosine triphosphatases and innate immune signalling pathways that regulate inflammation and cell death. Many of these translocated proteins have been extensively characterised, which has helped obtain insights into the mechanisms of pathogenesis of these bacteria and also understand the host pathways they target in more detail. With increasing knowledge of the positive and negative regulation of host signalling pathways by different effectors, a future challenge is to investigate how the specific effector repertoire of each strain cooperates over the course of an infection.
Watson J, Jenkins C, Clements A, 2018, Shigella sonnei does not utilize amoebae as a protective host, Applied and Environmental Microbiology, Vol: 84, ISSN: 0099-2240
Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii, bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii, unlike those of Legionella pneumophila, which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered.IMPORTANCE The distribution of Shigella species closely mirrors a country's socioeconomic conditions. With the transition of many populous nations from low- to middle-income countries, S. sonnei infections have emerged as a major public health issue. Understanding why S. sonnei infections are resistant to improvements in living conditions is key to developing methods to reduce exposure to this pathogen. We show that free-living amoebae are not likely to be environmental hosts of S. sonnei, as all Shigella strains tested were phagocytosed and degraded by amoebae. Therefore, alternative scenarios are required to explain the emergence and persistence of S. sonnei infections.
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.
Furniss RCD, Clements A, 2017, Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing Pathogens., Journal of Bacteriology, Vol: 200, ISSN: 0021-9193
Attaching and Effacing (AE) pathogens colonise the gut mucosa using a Type Three Secretion System (T3SS) and a suite of effector proteins. The Locus of Enterocyte Effacement (LEE) is the defining genetic feature of the AE pathogens, encoding the T3SS and the core effector proteins necessary for pathogenesis. Extensive research has revealed a complex regulatory network that senses and responds to a myriad of host and microbiota-derived signals in the infected gut to control transcription of the LEE. These signals include microbiota-liberated sugars and metabolites in the gut lumen, molecular oxygen at the gut epithelium and host hormones. Recent research has revealed that AE pathogens also perceive physical signals, such as attachment to the epithelium, and that the act of effector translocation remodels gene expression in infecting bacteria. In this review we summarise our knowledge to date and present an integrated view of how chemical, geographical and physical cues regulate the virulence program of AE pathogens during infection.
Mavridou DAI, Gonzalez D, Clements A, et al., 2016, The pUltra plasmid series: a robust and flexible tool for fluorescent labeling of Enterobacteria, Plasmid, Vol: 87-88, Pages: 65-71, ISSN: 1095-9890
Fluorescent labeling has been an invaluable tool for the study of living organisms andbacterial species are no exception to this. Here we present and characterize the pUltraplasmids which express constitutively a fluorescent protein gene (GFP, RFP, YFP or CFP)from a strong synthetic promoter and are suitable for the fluorescent labeling of a broad rangeof Enterobacteria. The amount of expressed fluorophore from these genetic constructs issuch, that the contours of the cells can be delineated on the basis of the fluorescent signalonly. In addition, labeling through the pUltra plasmids can be used successfully forfluorescence and confocal microscopy while unambiguous distinction of cells labeled withdifferent colors can be carried out efficiently by microscopy or flow cytometry. We comparethe labeling provided by the pUltra plasmids with that of another plasmid series encodingfluorescent proteins and we show that the pUltra constructs are vastly superior in signalintensity and discrimination power without having any detectable growth rate effects for thebacterial population. We also use the pUltra plasmids to produce mixtures of differentiallylabeled pathogenic Escherichia, Shigella and Salmonella species which we test duringinfection of mammalian cells. We find that even inside the host cell, different strains can bedistinguished effortlessly based on their fluorescence. We, therefore, conclude that the pUltraplasmids are a powerful labeling tool especially useful for complex biological experimentssuch as the visualization of ecosystems of different bacterial species or of enteric pathogensin contact with their hosts.
Furniss RCD, Slater S, Frankel G, et al., 2016, Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signalling axis to prevent recycling endosome maturation during infection, Journal of Molecular Biology, Vol: 428, Pages: 3399-3407, ISSN: 1089-8638
Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate a plethora of host cell processes to establish infection of the gut mucosa. This manipulation is achieved via the injection of bacterial effector proteins into host cells using a Type III secretion system. We have previously reported that the conserved EHEC and EPEC effector EspG disrupts recycling endosome function, reducing cell surface levels of host receptors through accumulation of recycling cargo within the host cell. Here we report that EspG interacts specifically with the small GTPases ARF6 and Rab35 during infection. These interactions target EspG to endosomes and prevent Rab35-mediated recycling of cargo to the host cell surface. Furthermore, we show that EspG has no effect on Rab35-mediated uncoating of newly formed endosomes, and instead leads to the formation of enlarged EspG/TfR/Rab11 positive, EEA1/Clathrin negative stalled recycling structures. Thus, this paper provides a molecular framework to explain how EspG disrupts recycling whilst also reporting the first known simultaneous targeting of ARF6 and Rab35 by a bacterial pathogen.
Larrouy-Maumus GJ, Abigail Clements, Alain Filloux, et al., 2015, Direct detection of lipid A on intact Gram-negative bacteria byMALDI-TOF mass spectrometry, Journal of Microbiological Methods, Vol: 120, Pages: 68-71, ISSN: 1872-8359
The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry.
Young JC, Clements A, Lang AE, et al., 2014, The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation, Nature Communications, Vol: 5, ISSN: 2041-1723
The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck–WIP–N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.
Clements A, Stoneham CA, Furniss RCD, et al., 2014, Enterohaemorrhagic Escherichia coli inhibits recycling endosome function and trafficking of surface receptors, CELLULAR MICROBIOLOGY, Vol: 16, Pages: 1693-1705, ISSN: 1462-5814
Raymond B, Young JC, Pallett M, et al., 2013, Subversion of trafficking, apoptosis, and innate immunity by type III secretion system effectors, TRENDS IN MICROBIOLOGY, Vol: 21, Pages: 430-439, ISSN: 0966-842X
Velkov T, Soon RL, Chong PL, et al., 2013, Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A, INNATE IMMUNITY, Vol: 19, Pages: 265-277, ISSN: 1753-4259
Clements A, Berger CN, Lomma M, et al., 2013, Type 3 secretion effectors, Escherichia coliPathotypes and Principles of Pathogenesis, Editors: Donnenberg, ISBN: 978-0-12-397048-0
Wong ARC, Clements A, Raymond B, et al., 2012, The Interplay between the Escherichia coli Rho Guanine Nucleotide Exchange Factor Effectors and the Mammalian RhoGEF Inhibitor EspH, mBio, Vol: 3, ISSN: 2161-2129
Rho GTPases are important regulators of many cellular processes. Subversion of Rho GTPases is a common infection strategy employed by many important human pathogens. Enteropathogenic Escherichia coli and enterohemorrhagic Escherichia coli (EPEC and EHEC) translocate the effector EspH, which inactivates mammalian Rho guanine exchange factors (GEFs), as well as Map, EspT, and EspM2, which, by mimicking mammalian RhoGEFs, activate Rho GTPases. In this study we found that EspH induces focal adhesion disassembly, triggers cell detachment, activates caspase-3, and induces cytotoxicity. EspH-induced cell detachment and caspase-3 activation can be offset by EspT, EspM2, and the Salmonella Cdc42/Rac1 GEF effector SopE, which remain active in the presence of EspH. EPEC and EHEC therefore use a novel strategy of controlling Rho GTPase activity by translocating one effector to inactivate mammalian RhoGEFs, replacing them with bacterial RhoGEFs. This study also expands the functional range of bacterial RhoGEFs to include cell adhesion and survival.IMPORTANCE Many human pathogens use a type III secretion system to translocate effectors that can functionally be divided into signaling, disabling, and countervirulence effectors. Among the signaling effectors are those that activate Rho GTPases, which play a central role in coordinating actin dynamics. However, many pathogens also translocate effectors with antagonistic or counteractive functions. For example, Salmonella translocates SopE and SptP, which sequentially turn Rac1 and Cdc42 on and off. In this paper, we show that enteropathogenic E. coli translocates EspH, which inactivates mammalian RhoGEFs and triggers cytotoxicity and at the same time translocates the bacterial RhoGEFs EspM2 and EspT, which are insensitive to EspH, and so neutralizes EspH-induced focal adhesion disassembly, cell detachment, and caspase-3 activation. Our data point to an intriguing infection strategy in which EPEC and EHEC override cellular Rho GTPase
Clements A, Young JC, Constantinou N, et al., 2012, Infection strategies of enteric pathogenic Escherichia coli, GUT MICROBES, Vol: 3, Pages: 71-87, ISSN: 1949-0976
Clements A, Smollett K, Lee SF, et al., 2011, EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function, CELLULAR MICROBIOLOGY, Vol: 13, Pages: 1429-1439, ISSN: 1462-5814
Wilksch JJ, Yang J, Clements A, et al., 2011, MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression, PLOS Pathogens, Vol: 7, ISSN: 1553-7366
Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with
Clements A, Bursac D, Gatsos X, et al., 2009, The reducible complexity of a mitochondrial molecular machine, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 106, Pages: 15791-15795, ISSN: 0027-8424
Clements A, Gaboriaud F, Duval JFL, et al., 2008, The major surface-associated saccharides of Klebsiella pneumoniae contribute to host cell association, PLOS One, Vol: 3, ISSN: 1932-6203
Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces.
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