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Journal articleBuckley AM, Jukes C, Candlish D, et al., 2016,
Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains
, Scientific Reports, Vol: 6, ISSN: 2045-2322The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.
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Journal articleDomingues L, Ismail A, Charro N, et al., 2016,
The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells
, Cellular Microbiology, Vol: 18, Pages: 949-969, ISSN: 1462-5822Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.
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Journal articleSo EC, Schroeder GN, Carson D, et al., 2016,
The Rab-binding profiles of bacterial virulence factors during infection
, Journal of Biological Chemistry, Vol: 291, Pages: 5832-5843, ISSN: 1083-351XLegionella pneumophila, the causativeagent of Legionnaire’s disease, uses its typeIV secretion system to translocate over 300effector proteins into host cells. Theseeffectors subvert host cell signalingpathways to ensure bacterial proliferation.Despite their importance for pathogenesis,the roles of most of the effectors are yet tobe characterized. Key to understanding thefunction of effectors is the identification ofhost proteins they bind during infection. Wepreviously developed a novel tandemaffinitypurification (TAP) approach usinghexahistidine and BirA-specificbiotinylation tags for isolating translocatedeffector complexes from infected cellswhose composition were subsequentlydeciphered by mass spectrometry. Here wefurther advanced the workflow for the TAPapproach and determined the infectiondependentinteractomes of the effectorsSidM and LidA, which were previouslyreported to promiscuously bind multiple RabGTPases in vitro. In this study we defined astringent subset of Rab GTPases targeted bySidM and LidA during infection, comprisingof Rab1A, 1B, 6 and 10; in addition, LidAtargets Rab14 and 18. Taken together, thisstudy illustrates the power of this approachto profile the intracellular interactomes ofbacterial effectors during infection
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Journal articleCorrigan RM, Bellows LE, Wood A, et al., 2016,
ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria
, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E1710-E1719, ISSN: 1091-6490The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases—RsgA, RbgA, Era, HflX, and ObgE—as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.
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Journal articleFilloux A, Whitfield C, 2016,
Editorial: The many wonders of the bacterial cell surface
, FEMS MICROBIOLOGY REVIEWS, Vol: 40, Pages: 161-163, ISSN: 0168-6445- Author Web Link
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Journal articleSerrano M, Crawshaw AD, Dembek M, et al., 2016,
The SpoIIQ-SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis
, Molecular Microbiology, Vol: 100, Pages: 204-228, ISSN: 1365-2958Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc-binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQH120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQH120S, binds Zn2+, and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQH120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post-engulfment, forespore and mother cell-specific gene expression, suggesting a channel-like function. Both engulfment and a channel-like function may be ancestral functions of SpoIIQ-SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms.
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Journal articleFisher RA, Cheverton AM, Helaine S, 2016,
Analysis of Macrophage-Induced Salmonella Persisters.
, Methods in Molecular Biology, Vol: 1333, Pages: 177-187, ISSN: 1940-6029A small subpopulation of non-replicating, multidrug-tolerant bacteria is present within clonal populations of many bacterial species. Known as persisters, these bacteria are probably the cause of relapsing infections such as typhoid fever. Formation of non-growing Salmonella persisters is stimulated by macrophage phagocytosis. This chapter outlines methods to identify and study persisters resulting from interactions between bacterial pathogens and their hosts. We use their antibiotic tolerance for isolation and enumeration and developed a method to study the heterogeneity of growth within clonal populations through single-cell analysis.
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Journal articleTaylor J, Taylor G, Hare S, et al., 2016,
Structures of the DfsB protein family suggest a cationic, helical sibling-lethal factor peptide
, Journal of Molecular Biology, Vol: 428, Pages: 554-560, ISSN: 1089-8638Bacteria have developed a variety of mechanisms for survivingharsh environmental conditions, nutrient stress and overpopulation.Paenibacillus dendritiformis produces a lethal protein (Slf) that is ableto induce cell death in neighboring colonies and a phenotypic switch inmore distant ones. Slf is derived from the secreted precursor protein,DfsB, after proteolytic processing. Here, we present new crystalstructures of DfsB homologues from a variety of bacterial species and asurprising version present in the yeast Saccharomyces cerevisiae.Adopting a four-helix bundle decorated with a further three short heliceswithin intervening loops, DfsB belongs to a non-enzymatic class of theDinB fold. The structure suggests that the biologically-active Slffragment may possess a C-terminal helix rich in basic and aromaticresidues that suggest a functional mechanism akin to that for cationicantimicrobial peptides.
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Journal articleKlionsky DJ, Abdelmohsen K, Abe A, et al., 2016,
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
, Autophagy, Vol: 12, Pages: 1-215, ISSN: 1554-8635 -
Journal articleLeen EN, Sorgeloos F, Correia S, et al., 2016,
A conserved interaction between a C-terminal motif in norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation
, PLOS Pathogens, Vol: 12, ISSN: 1553-7366Translation 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 in vitro 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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