127 results found
Richards E, Bouché L, Panico M, et al., 2018, The S-layer protein of a Clostridium difficile SLCT-11 strain displays a complex glycan required for normal cell growth and morphology., Journal of Biological Chemistry, Vol: 293, Pages: 18123-18137, ISSN: 0021-9258
Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficilestrain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using mass spectrometry and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide-linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-β-Gal-peptide, (ii) a repeating pentasaccharide with the sequence -4-β-Rha-4-α-Glc-3-β-Rha-4-(α-Rib-3-)β-Rha-, and (iii) a non-reducing end-terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24 kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile. In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficilesporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.
Vidor CJ, Watts TD, Adams V, et al., 2018, Clostridium sordellii pathogenicity locus plasmid pCS1-1 encodes a novel clostridial conjugation locus, mBio, Vol: 9, ISSN: 2150-7511
A major virulence factor in Clostridium sordellii-mediated infection is the toxin TcsL, which is encoded within a region of the genome called the pathogenicity locus (PaLoc). C. sordellii isolates carry the PaLoc on the pCS1 family of plasmids, of which there are four characterized members. Here, we determined the potential mobility of pCS1 plasmids and characterized a fifth unique pCS1 member. Using a derivative of the pCS1-1 plasmid from strain ATCC 9714 which had been marked with the ermB erythromycin resistance gene, conjugative transfer into a recipient C. sordellii isolate, R28058, was demonstrated. Bioinformatic analysis of pCS1-1 identified a novel conjugation gene cluster defined as the C. sordellii transfer (cst) locus. Interruption of genes within the cst locus resulted in loss of pCS1-1 transfer, which was restored upon complementation in trans. These studies provided clear evidence that genes within the cst locus are essential for the conjugative transfer of pCS1-1. The cst locus is present on all pCS1 subtypes, and homologous loci were identified on toxin-encoding plasmids from Clostridium perfringens and Clostridium botulinum and also carried within genomes of Clostridium difficile isolates, indicating that it is a widespread clostridial conjugation locus. The results of this study have broad implications for the dissemination of toxin genes and, potentially, antibiotic resistance genes among members of a diverse range of clostridial pathogens, providing these microorganisms with a survival advantage within the infected host.
Peltier J, Shaw HA, wren B, et al., 2017, Disparate subcellular location 1 of putative sortase substrates in Clostridium difficile, Scientific Reports, Vol: 7, ISSN: 2045-2322
Clostridium difficile is a gastrointestinal pathogen but how the bacterium colonises this niche is still little understood. Sortase enzymes covalently attach specific bacterial proteins to the peptidoglycan cell wall and are often involved in colonisation by pathogens. Here we show C. difficile proteins CD2537 and CD3392 are functional substrates of sortase SrtB. Through manipulation of the C-terminal regions of these proteins we show the SPKTG motif is essential for covalent attachment to the cell wall. Two additional putative substrates, CD0183 which contains an SPSTG motif, and CD2768 which contains an SPQTG motif, are not cleaved or anchored to the cell wall by sortase. Finally, using an in vivo asymmetric cleavage assay, we show that despite containing a conserved SPKTG motif, in the absence of SrtB these proteins are localised to disparate cellular compartments.
Buckley AM, Jukes C, Candlish D, et al., 2016, Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains, Scientific Reports, Vol: 6, ISSN: 2045-2322
The 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.
Serrano 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: 1365-2958
Engulfment 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.
Fairweather NF, Willing SE, Richards EJ, et al., 2015, Increased toxin expression in a Clostridium difficile mfd mutant, BMC Microbiology, ISSN: 1471-2180
Charlton T, Kovacs-Simon A, Michell S, et al., 2015, Quantitative lipoproteomics in Clostridium difficile reveals a role for lipoproteins in sporulation, Chemistry & Biology, Vol: 22, ISSN: 1074-5521
Bacterial lipoproteins are surface exposed, anchored to the membrane by Sdiacylglyceryl modification of the N-terminal cysteine thiol. They play important roles inmany essential cellular processes and in bacterial pathogenesis. For example,Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinaldisease, however, its lipoproteome remains poorly characterized. Here we describe theapplication of metabolic tagging with alkyne-tagged lipid analogues, in combinationwith quantitative proteomics, to profile protein lipidation across diverse C. difficilestrains and on inactivation of specific components of the lipoprotein biogenesispathway. These studies provide the first comprehensive map of the C. difficilelipoproteome, demonstrate the existence of two active lipoprotein signal peptidasesand provide insights into lipoprotein function, implicating the lipoproteome intransmission of this pathogen.
Fairweather 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: 1083-351X
Background: 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.
Fairweather 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: 1365-2958
Bacteriophages 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.
Couchman EC, Browne HP, Dunn M, et al., 2015, Clostridium sordellii genome analysis reveals plasmid localized toxin genes encoded within pathogenicity loci, BMC Genomics, Vol: 16, ISSN: 1471-2164
BackgroundClostridium sordellii can cause severe infections in animals and humans, the latter associated with trauma, toxic shock and often-fatal gynaecological infections. Strains can produce two large clostridial cytotoxins (LCCs), TcsL and TcsH, related to those produced by Clostridium difficile, Clostridium novyi and Clostridium perfringens, but the genetic basis of toxin production remains uncharacterised.ResultsPhylogenetic analysis of the genome sequences of 44 strains isolated from human and animal infections in the UK, US and Australia placed the species into four clades. Although all strains originated from animal or clinical disease, only 5 strains contained LCC genes: 4 strains contain tcsL alone and one strain contains tcsL and tcsH. Four toxin-positive strains were found within one clade. Where present, tcsL and tcsH were localised in a pathogenicity locus, similar to but distinct from that present in C. difficile. In contrast to C. difficile, where the LCCs are chromosomally localised, the C. sordellii tcsL and tcsH genes are localised on plasmids. Our data suggest gain and loss of entire toxigenic plasmids in addition to horizontal transfer of the pathogenicity locus. A high quality, annotated sequence of ATCC9714 reveals many putative virulence factors including neuraminidase, phospholipase C and the cholesterol-dependent cytolysin sordellilysin that are highly conserved between all strains studied.ConclusionsGenome analysis of C. sordellii reveals that the LCCs, the major virulence factors, are localised on plasmids. Many strains do not contain the LCC genes; it is probable that in several of these cases the plasmid has been lost upon laboratory subculture. Our data are consistent with LCCs being the primary virulence factors in the majority of infections, but LCC-negative strains may precipitate certain categories of infection. A high quality genome sequence reveals putative virulence factors whose role in virulence can be investigated.Keywords: C
Willing SE, Candela T, Shaw HA, et al., 2015, Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII, MOLECULAR MICROBIOLOGY, Vol: 96, Pages: 596-608, ISSN: 0950-382X
Dembek M, Barquist L, Boinett CJ, et al., 2015, High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile, mBio, Vol: 6, ISSN: 2161-2129
Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen.
Phetcharaburanin J, Hong HA, Colenutt C, et al., 2014, The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile, MOLECULAR MICROBIOLOGY, Vol: 92, Pages: 1025-1038, ISSN: 0950-382X
Fagan RP, Fairweather NF, 2014, Biogenesis and functions of bacterial S-layers, NATURE REVIEWS MICROBIOLOGY, Vol: 12, Pages: 211-222, ISSN: 1740-1526
Tulli L, Marchi S, Petracca R, et al., 2013, CbpA: a novel surface exposed adhesin of Clostridium difficile targeting human collagen, CELLULAR MICROBIOLOGY, Vol: 15, Pages: 1674-1687, ISSN: 1462-5814
Chen K, d'Arc S, Setty N, et al., 2013, In Recurrent C. difficile, the CRP Response to the Primary C. difficile Infection Predicts Whether the Same Strain or a Different Strain will Cause a Second Infection, DIGESTIVE DISEASES AND SCIENCES, Vol: 58, Pages: 1683-1688, ISSN: 0163-2116
Dembek M, Stabler RA, Witney AA, et al., 2013, Transcriptional analysis of temporal gene expression in germinating clostridium difficile 630 endospores, PLOS One, Vol: 8, ISSN: 1932-6203
Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical stress and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth.
Permpoonpattana P, Phetcharaburanin J, Mikelsone A, et al., 2013, Functional Characterization of Clostridium difficile Spore Coat Proteins, JOURNAL OF BACTERIOLOGY, Vol: 195, Pages: 1492-1503, ISSN: 0021-9193
Deakin LJ, Clare S, Fagan RP, et al., 2012, The Clostridium difficile spo0A Gene Is a Persistence and Transmission Factor, INFECTION AND IMMUNITY, Vol: 80, Pages: 2704-2711, ISSN: 0019-9567
Tierney R, Nakai T, Parkins CJ, et al., 2012, A single-dose cytomegalovirus-based vaccine encoding tetanus toxin fragment C induces sustained levels of protective tetanus toxin antibodies in mice, VACCINE, Vol: 30, Pages: 3047-3052, ISSN: 0264-410X
Dang THT, Fagan RP, Fairweather NF, et al., 2012, Novel inhibitors of surface layer processing in Clostridium difficile, Bioorganic & Medicinal Chemistry, Vol: 20, Pages: 614-621, ISSN: 1464-3391
Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-pathogen interactions. The key structural components of the S-layer are two proteins derived from a polypeptide precursor, SlpA, via proteolytic cleavage by the protease Cwp84. Here, we report the design, synthesis and in vivo characterization of a panel of protease inhibitors and activity-based probes (ABPs) designed to target S-layer processing in live C. difficile cells. Inhibitors based on substrate-mimetic peptides bearing a C-terminal Michael acceptor warhead were found to be promising candidates for further development.
Dembek M, Reynolds CB, Fairweather NF, 2012, Clostridium difficile Cell Wall Protein CwpV Undergoes Enzyme-independent Intramolecular Autoproteolysis, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 1538-1544
Fagan RP, Fairweather NF, 2011, Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 286, Pages: 27483-27493
Fagan RP, Janoir C, Collignon A, et al., 2011, A proposed nomenclature for cell wall proteins of Clostridium difficile, JOURNAL OF MEDICAL MICROBIOLOGY, Vol: 60, Pages: 1225-1228, ISSN: 0022-2615
de la Riva L, Willing SE, Tate EW, et al., 2011, Roles of Cysteine Proteases Cwp84 and Cwp13 in Biogenesis of the Cell Wall of Clostridium difficile, JOURNAL OF BACTERIOLOGY, Vol: 193, Pages: 3276-3285, ISSN: 0021-9193
Permpoonpattana P, Hong HA, Phetcharaburanin J, et al., 2011, Immunization with Bacillus Spores Expressing Toxin A Peptide Repeats Protects against Infection with Clostridium difficile Strains Producing Toxins A and B, INFECTION AND IMMUNITY, Vol: 79, Pages: 2295-2302, ISSN: 0019-9567
Merrigan M, Venugopal A, Roxas JL, et al., 2011, Clostridium difficile Strains of Human and Veterinary Origin Attach Efficiently to Human Intestinal Epithelial Cells via the Major Surface-Layer Protein Slpa, Conference on Digestive Disease Week 2011, Publisher: W B SAUNDERS CO-ELSEVIER INC, Pages: S636-S636, ISSN: 0016-5085
Reynolds CB, Emerson JE, de la Riva L, et al., 2011, The Clostridium difficile Cell Wall Protein CwpV is Antigenically Variable between Strains, but Exhibits Conserved Aggregation-Promoting Function, PLOS PATHOGENS, Vol: 7, ISSN: 1553-7366
Robinson KS, Mousnier A, Hemrajani C, et al., 2010, The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B, MICROBIOLOGY-SGM, Vol: 156, Pages: 1815-1823, ISSN: 1350-0872
Scott N, Qazi O, Wright MJ, et al., 2010, Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding, MOLECULAR IMMUNOLOGY, Vol: 47, Pages: 1931-1941, ISSN: 0161-5890
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