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Journal articleSurana S, Shenoy AR, Krishnan Y, 2015,
Designing DNA nanodevices for compatibility with the immune system of higher organisms
, Nature Nanotechnology, Vol: 10, Pages: 741-747, ISSN: 1748-3395DNA is proving to be a powerful scaffold to construct molecularly precise designer DNA devices. Recent trends reveal their ever-increasing deployment within living systems as delivery devices that not only probe but also program and re-program a cell, or even whole organisms. Given that DNA is highly immunogenic, we outline the molecular, cellular and organismal response pathways that designer nucleic acid nanodevices are likely to elicit in living systems. We address safety issues applicable when such designer DNA nanodevices interact with the immune system. In light of this, we discuss possible molecular programming strategies that could be integrated with such designer nucleic acid scaffolds to either evade or stimulate the host response with a view to optimizing and widening their applications in higher organisms.
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Journal articleMostowy S, Shenoy AR, 2015,
The cytoskeleton in cell-autonomous immunity: structural determinants of host defence
, Nature Reviews Immunology, Vol: 15, Pages: 559-573, ISSN: 1474-1741Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence.
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Journal articleSo EC, Mattheis C, Tate EW, et al., 2015,
Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors
, Canadian Journal of Microbiology, Vol: 61, Pages: 617-635, ISSN: 1480-3275The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.
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Journal articleThompson CC, Griffiths C, Nicod SS, et al., 2015,
The Rsb phosphoregulatory network controls availability of the primary sigma factor in Chlamydia trachomatis and influences the kinetics of growth and development
, PLOS Pathogens, Vol: 11, Pages: 1-22, ISSN: 1553-7366Chlamydia trachomatis is the leading cause of both bacterial sexually transmitted infection and infection-derived blindness world-wide. No vaccine has proven protective to date in humans. C. trachomatis only replicates from inside a host cell, and has evolved to acquire a variety of nutrients directly from its host. However, a typical human immune response will normally limit the availability of a variety of essential nutrients. Thus, it is thought that the success of C. trachomatis as a human pathogen may lie in its ability to survive these immunological stress situations by slowing growth and development until conditions in the cell have improved. This mode of growth is known as persistence and how C. trachomatis senses stress and responds in this manner is an important area of research. Our report characterizes a complete signaling module, the Rsb network, that is capable of controlling the growth rate or infectivity of Chlamydia. By manipulating the levels of different pathway components, we were able to accelerate and restrict the growth and development of this pathogen. Our results suggest a mechanism by which Chlamydia can tailor its growth rate to the conditions within the host cell. The disruption of this pathway could generate a strain incapable of surviving a typical human immune response and would represent an attractive candidate as an attenuated growth vaccine.
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Journal articleFairweather NF, Peltier J, Shaw HA, et al., 2015,
Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage
, Journal of Biological Chemistry, Vol: 290, Pages: 24453-24469, ISSN: 1083-351XBackground: Bacteria use variousmechanisms to anchor their surface proteins,including a sortase enzyme.Results: Covalent anchoring of proteins to thepeptidoglycan in Clostridium difficile and itsregulation by cyclic-di-GMP and proteaseactivity are demonstrated.Conclusion: A novel regulatory mechanism ofcell wall protein anchoring is found.Significance: Elucidating how proteins areanchored may shed light on control of bacterialcolonization in vivo.
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ReportCurry S, Leen EN, Sorgeloos F, et al., 2015,
A conserved interaction between a C-terminal motif in Norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation
, Publisher: bioRxivTranslation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5 ́ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5 ́-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C-terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C- terminal peptide of MNV VPg to inhibit translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
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Journal articleLarrouy-Maumus GJ, Gilleron M, Skovierova H, et al., 2015,
A glycomic approach reveals a new mycobacterial polysaccharide
, Glycobiology, Vol: 25, Pages: 1163-1171, ISSN: 1460-2423Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host–pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate–polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10–50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis.
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Journal articleAl Shammari B, Shiomi T, Tezera L, et al., 2015,
The extracellular matrix regulates granuloma necrosis in tuberculosis
, Journal of Infectious Diseases, Vol: 212, Pages: 463-473, ISSN: 1537-6613A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis–infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction.
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Journal articleMoscoso JA, Schramke H, Zhang Y, et al., 2015,
Binding of cyclic Di-AMP to the staphylococcus aureus sensor kinase KdpD occurs via the universal stress protein domain and downregulates the expression of the Kdp potassium transporter
, Journal of Bacteriology, ISSN: 1098-5530Nucleotide signalling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signalling nucleotide cyclic di-adenosine monophosphate (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signalling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here, we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD and amino acids of a conserved SxS-X20-FTAxY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signalling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the up-regulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus. IMPORTANCE: Staphylococcus aureus is an important human pathogen and major cause of food poisoning in western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signalling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inh
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Journal articleBosi E, Fondi M, Maida I, et al., 2015,
Genome-scale phylogenetic and DNA composition analyses of Antarctic Pseudoalteromonas bacteria reveal inconsistencies in current taxonomic affiliation
, Hydrobiologia, Vol: 761, Pages: 85-95, ISSN: 0018-8158Bacteria belonging to the Pseudoalteromonas genus have important ecological implications in marine environments, playing a role in the control of microbial community as producers of bioactive molecules endowed with antifouling activity and able to antagonize larvae, fungi and bacteria, including important human pathogens. For these reasons, representatives of this genus are very promising for biotechnological and biomedical applications. In this work, we used different genome-scale approaches to infer the taxonomy of 38 Pseudoalteromonas representatives (most of which isolated from Antarctica) and whose complete genome has been sequenced. We show that an accurate re-evaluation of the real taxonomic relationships of Pseudoalteromonas representatives is needed since many inconsistencies with the current taxonomic annotation were observed. Moreover, data obtained with different genome-scale methods are consistent, confirming the reliability of the genomic approaches. On the basis of these data, we propose a re-annotation for some Pseudoalteromonas species. This proposal should be validated in the future by comparing the phenotypes of these strains.
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