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1.
Nat Commun ; 13(1): 970, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35217634

RESUMO

Many bacteria and archaea possess a two-dimensional protein array, or S-layer, that covers the cell surface and plays crucial roles in cell physiology. Here, we report the crystal structure of SlpA, the main S-layer protein of the bacterial pathogen Clostridioides difficile, and use electron microscopy to study S-layer organisation and assembly. The SlpA crystal lattice mimics S-layer assembly in the cell, through tiling of triangular prisms above the cell wall, interlocked by distinct ridges facing the environment. Strikingly, the array is very compact, with pores of only ~10 Å in diameter, compared to other S-layers (30-100 Å). The surface-exposed flexible ridges are partially dispensable for overall structure and assembly, although a mutant lacking this region becomes susceptible to lysozyme, an important molecule in host defence. Thus, our work gives insights into S-layer organisation and provides a basis for development of C. difficile-specific therapeutics.


Assuntos
Clostridioides difficile , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Clostridioides difficile/genética
2.
Sci Rep ; 11(1): 3244, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547340

RESUMO

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Clostridioides difficile/fisiologia , GMP Cíclico/análogos & derivados , DNA Bacteriano/metabolismo , Biofilmes/crescimento & desenvolvimento , Clostridioides difficile/ultraestrutura , Infecções por Clostridium/microbiologia , GMP Cíclico/metabolismo , Humanos
3.
J Biol Chem ; 293(47): 18123-18137, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30275012

RESUMO

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. difficile strain 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 MS 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-ß-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-ß-Rha-4-α-Glc-3-ß-Rha-4-(α-Rib-3-)ß-Rha-; and (iii) a nonreducing 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. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.


Assuntos
Clostridioides difficile/metabolismo , Glicoproteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Clostridioides difficile/genética , Clostridioides difficile/crescimento & desenvolvimento , Glicosilação , Espectrometria de Massas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Peso Molecular , Polissacarídeos/química , Conformação Proteica , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo
4.
mBio ; 9(1)2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29339424

RESUMO

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.IMPORTANCEC. sordellii is a bacterial pathogen that causes severe infections in humans and animals, with high mortality rates. While the pathogenesis of C. sordellii infections is not well understood, it is known that the toxin TcsL is an important virulence factor. Here, we have shown the ability of a plasmid carrying the tcsL gene to undergo conjugative transfer between distantly related strains of C. sordellii, which has far-reaching implications for the ability of C. sordellii to acquire the capacity to cause disease. Plasmids that carry tcsL encode a previously uncharacterized conjugation locus, and individual genes within this locus were shown to be required for conjugative transfer. Furthermore, homologues on toxin plasmids from other clostridial species were identified, indicating that this region represents a novel clostridial conjugation locus. The results of this study have broad implications for the dissemination of virulence genes among members of a diverse range of clostridial pathogens.


Assuntos
Clostridium sordellii/genética , Conjugação Genética , Transferência Genética Horizontal , Loci Gênicos , Plasmídeos , Biologia Computacional , Genes Bacterianos , Família Multigênica
5.
Sci Rep ; 7(1): 9204, 2017 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-28835650

RESUMO

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.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Clostridioides difficile/metabolismo , Cisteína Endopeptidases/metabolismo , Motivos de Aminoácidos , Aminoaciltransferases/química , Proteínas de Bactérias/química , Parede Celular/metabolismo , Cisteína Endopeptidases/química , Ligação Proteica , Transporte Proteico , Proteólise , Especificidade por Substrato
6.
Sci Rep ; 6: 23463, 2016 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-26996606

RESUMO

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.


Assuntos
Técnicas Biossensoriais/métodos , Clostridioides difficile/genética , Imagem Molecular/métodos , Imagem Óptica/métodos , Biossíntese de Proteínas , Proteínas de Bactérias/biossíntese , Clostridioides difficile/metabolismo , Proteínas do Citoesqueleto/biossíntese , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Transporte Proteico
7.
Mol Microbiol ; 100(1): 204-28, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26690930

RESUMO

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 SpoIIQ(H120S) interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQ(H120S) , binds Zn(2+) , and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQ(H120S) 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.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridioides difficile/fisiologia , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência
8.
BMC Microbiol ; 15: 280, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26679502

RESUMO

BACKGROUND: The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY. RESULTS: During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations. CONCLUSIONS: This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Enterotoxinas/genética , Mutagênese Insercional/genética , Fatores de Transcrição/genética , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/genética , Chlorocebus aethiops , Clostridioides difficile/metabolismo , Clostridioides difficile/fisiologia , Enterotoxinas/metabolismo , Enterotoxinas/toxicidade , Regulação Bacteriana da Expressão Gênica , Células HT29 , Humanos , Mutagênese Insercional/imunologia , Transcrição Gênica , Regulação para Cima , Células Vero
9.
Chem Biol ; 22(11): 1562-1573, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26584780

RESUMO

Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.


Assuntos
Clostridioides difficile/fisiologia , Lipoproteínas/metabolismo , Proteoma/análise , Proteômica , Alcinos/química , Biocatálise , Cromatografia Líquida de Alta Pressão , Lipoproteínas/química , Ácido Mirístico/química , Esporos Bacterianos/metabolismo , Espectrometria de Massas em Tandem
10.
J Biol Chem ; 290(40): 24453-69, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26283789

RESUMO

In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro, and seven potential substrates have been identified. Here, we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem. We show that CD2831 protein levels are elevated in the presence of high intracellular cyclic diGMP (c-diGMP) concentrations, in agreement with the control of CD2831 expression by a c-diGMP-dependent type II riboswitch. Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells. This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch. These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Clostridioides difficile/metabolismo , GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Metaloproteases/metabolismo , Peptídeo Hidrolases/metabolismo , Adesinas Bacterianas/metabolismo , Motivos de Aminoácidos , Aminoaciltransferases/metabolismo , Membrana Celular/metabolismo , GMP Cíclico/química , Cisteína Endopeptidases/metabolismo , Perfilação da Expressão Gênica , Microscopia de Fluorescência , Mutação , Oligonucleotídeos/metabolismo , Peptidoglicano/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de Massas em Tandem , Fatores de Virulência/metabolismo
11.
Mol Microbiol ; 98(2): 329-42, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26179020

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/crescimento & desenvolvimento , Parede Celular/química , Clostridioides difficile/metabolismo , Clostridioides difficile/virologia , Animais , Proteínas de Bactérias/genética , Bacteriófagos/patogenicidade , Bacteriófagos/fisiologia , Parede Celular/metabolismo , Clostridioides difficile/química , Clostridioides difficile/genética , DNA Viral/genética , Humanos , Análise de Sequência de DNA
12.
BMC Genomics ; 16: 392, 2015 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-25981746

RESUMO

BACKGROUND: Clostridium 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. RESULTS: Phylogenetic 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. CONCLUSIONS: Genome 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.


Assuntos
Toxinas Bacterianas/genética , Clostridium sordellii/genética , Clostridium sordellii/patogenicidade , Genoma Bacteriano/genética , Plasmídeos/metabolismo , Fatores de Virulência/genética , Mapeamento Cromossômico , Clostridium sordellii/classificação , Transferência Genética Horizontal , Loci Gênicos/genética , Neuraminidase/genética , Filogenia , Plasmídeos/genética , Análise de Sequência de DNA , Fosfolipases Tipo C/genética
13.
Mol Microbiol ; 96(3): 596-608, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25649385

RESUMO

Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria.


Assuntos
Motivos de Aminoácidos , Parede Celular/metabolismo , Clostridioides difficile/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos Bacterianos/metabolismo , Técnicas de Silenciamento de Genes , Ligação Proteica , Sequências Repetitivas de Aminoácidos
14.
mBio ; 6(2): e02383, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25714712

RESUMO

UNLABELLED: 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. IMPORTANCE: Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.


Assuntos
Clostridioides difficile/crescimento & desenvolvimento , Genes Bacterianos , Genes Essenciais , Testes Genéticos/métodos , Ensaios de Triagem em Larga Escala , Mutagênese Insercional/métodos , Esporos Bacterianos/crescimento & desenvolvimento , Clostridioides difficile/genética , Elementos de DNA Transponíveis , Humanos , Esporos Bacterianos/genética
15.
J Am Chem Soc ; 136(22): 7869-72, 2014 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-24846811

RESUMO

Hydrogenation of esters is vital to the chemical industry for the production of alcohols, especially fatty alcohols that find broad applications in consumer products. Current technologies for ester hydrogenation rely on either heterogeneous catalysts operating under extreme temperatures and pressures or homogeneous catalysts containing precious metals such as ruthenium and osmium. Here, we report the hydrogenation of esters under relatively mild conditions by employing an iron-based catalyst bearing a PNP-pincer ligand. This catalytic system is also effective for the conversion of coconut oil derived fatty acid methyl esters to detergent alcohols without adding any solvent.

16.
Mol Microbiol ; 92(5): 1025-38, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24720767

RESUMO

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild-type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID50 ) of spores was significantly higher (2-logs) in the bclA1(-) mutant compared to the isogenic wild-type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild-type spores. bclA1(-) spores germinated faster than wild-type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre-spore germination) stages of infection. We also show that the ID50 was higher in mice infected with R20291, a 'hypervirulent' 027 strain, that carries a truncated BclA1 protein.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/metabolismo , Esporos Bacterianos/patogenicidade , Animais , Clostridioides difficile/metabolismo , Cricetinae , Regulação Bacteriana da Expressão Gênica , Camundongos , Esporos Bacterianos/metabolismo
17.
Nat Rev Microbiol ; 12(3): 211-22, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24509785

RESUMO

The outer surface of many archaea and bacteria is coated with a proteinaceous surface layer (known as an S-layer), which is formed by the self-assembly of monomeric proteins into a regularly spaced, two-dimensional array. Bacteria possess dedicated pathways for the secretion and anchoring of the S-layer to the cell wall, and some Gram-positive species have large S-layer-associated gene families. S-layers have important roles in growth and survival, and their many functions include the maintenance of cell integrity, enzyme display and, in pathogens and commensals, interaction with the host and its immune system. In this Review, we discuss our current knowledge of S-layer and related proteins, including their structures, mechanisms of secretion and anchoring and their diverse functions.


Assuntos
Bactérias/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Parede Celular/metabolismo , Variação Genética , Humanos , Glicoproteínas de Membrana/genética , Modelos Biológicos , Família Multigênica/fisiologia , Estrutura Terciária de Proteína
18.
PLoS One ; 8(5): e64011, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23691138

RESUMO

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.


Assuntos
Clostridioides difficile/genética , Perfilação da Expressão Gênica , Esporos Bacterianos , Transcrição Gênica , Clostridioides difficile/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/genética
19.
Cell Microbiol ; 15(10): 1674-87, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23517059

RESUMO

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudomembranous colitis. While the role of toxins in pathogenesis has been extensively described, the contribution of surface determinants to intestinal colonization is still poorly understood. We focused our study on a novel member of the MSCRAMM family, named CbpA (Collagen binding protein A), for its adhesive properties towards collagen. We demonstrate that CbpA, which carries an LPXTG-like cell wall anchoring domain, is expressed on the bacterial surface of C. difficile and that the recombinant protein binds at high affinity to collagens I and V (apparent Kd in the order of 10(-9 ) M). These findings were validated by confocal microscopy studies showing the colocalization of the protein with type I and V collagen fibres produced by human fibroblasts and mouse intestinal tissues. However, the collagen binding activity of the wild-type C. difficile 630 strain was indistinguishable to the cbpA knock-out strain. To overcome this apparent clostridial adherence redundancy, we engineered a Lactococcus lactis strain for the heterologous expression of CbpA. When exposed on the surface of L. lactis, CbpA significantly enhances the ability of the bacterium to interact with collagen and to adhere to ECM-producing cells. The binding activity of L. lactis-CbpA strain was prevented by an antiserum raised against CbpA, demonstrating the specificity of the interaction. These results suggest that CbpA is a newsurface-exposed adhesin contributing to the C. difficile interaction with the host.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Clostridioides difficile/fisiologia , Colágeno/metabolismo , Interações Hospedeiro-Patógeno , Animais , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Cinética , Lactococcus lactis/genética , Lactococcus lactis/fisiologia , Camundongos , Microscopia Confocal , Ligação Proteica
20.
Dig Dis Sci ; 58(6): 1683-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23306854

RESUMO

BACKGROUND: Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and is associated with an increase in morbidity and mortality. There is a wide variance in disease severity with some patients suffering a single, self-limiting episode of diarrhoea while others suffer more intractable problems with recurrent attacks or toxic dilatation. Numerous different C. difficile ribotypes exist, some of which are considered hypervirulent. The magnitude of toxin production alone is not sufficient to explain the varying virulence of these ribotypes, suggesting the involvement of other mechanisms. METHODS: To test the same patient's response to infection with different C. difficile ribotypes, we reviewed 45 patients who suffered two episodes of C. difficile infection and determined by ribotyping and MLVA whether the second episode was due to the same strain or a different strain. RESULTS: Patients harbouring a different strain had significantly higher C-reactive protein (CRP) responses on the first assessed infection (143 mg/L ± 20 vs. 55 ± 9.63, p = 0.0001) and a significantly lower CRP on reinfection (p = 0.048). Same strain patients had a non-significant increase in CRP response on second infection. CONCLUSIONS: This suggests that the inflammatory response to C. difficile is determined by an interaction between host immunobiology, previous exposure and C. difficile strain.


Assuntos
Proteína C-Reativa/metabolismo , Clostridioides difficile/classificação , Enterocolite Pseudomembranosa/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Clostridioides difficile/genética , Clostridioides difficile/isolamento & purificação , Estudos de Coortes , DNA Bacteriano/análise , Enterocolite Pseudomembranosa/sangue , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites , Reação em Cadeia da Polimerase , Recidiva , Estudos Retrospectivos , Ribotipagem
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