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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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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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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 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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Journal articleFairweather NF, Sekulovic1 O, Bedoya MO, et al., 2015,
The Clostridium difficile Cell Wall Protein CwpV Confers Phase-Variable Phage Resistance
, Molecular Microbiology, Vol: 98, Pages: 329-342, ISSN: 1365-2958Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell-surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase-variable, is classified into five types, each differing in their repeat-containing C-terminal domain. When expressed constitutively from a plasmid or the chromosome of locked ‘ON’ cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10−7 for phages ϕCD38-2, ϕCD111 and ϕCD146. Protection against the myophages ϕMMP01 and ϕCD52 was weaker, with EOP values between 9.0 × 10−3 and 1.1 × 10−1. The C-terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host-encoded and phase-variable antiphage system in C. difficile.
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Conference paperPolo LM, Grundy G, Rulten S, et al., 2015,
New structural insights into PARP3 function
, 40th Congress of the Federation-of-European-Biochemical-Societies (FEBS) - The Biochemical Basis of Life, Publisher: WILEY-BLACKWELL, Pages: 397-397, ISSN: 1742-464X -
Journal articleWheat WH, Dhouib R, Angala SK, et al., 2015,
The presence of a galactosamine substituent on the arabinogalactan of Mycobacterium tuberculosis abrogates full maturation of human peripheral blood monocyte-derived dendritic cells and increases secretion of IL-10
, TUBERCULOSIS, Vol: 95, Pages: 476-489, ISSN: 1472-9792- Author Web Link
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Journal articleLarrouy-Maumus GJ, 2015,
Cholesterol acquisition by Mycobacterium tuberculosis
, Virulence, ISSN: 2150-5608
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