A group II intron in a conjugative transposon from the gram-positive bacterium,Clostridium difficile

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.     

A mariner-Based Transposon System for In Vivo Random Mutagenesis of Clostridium difficile

Understanding the molecular basis of Clostridium difficile infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of C. difficile, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (±0.4) × 10⁻⁴ per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene cspBA and an auxotroph with an insertion in the pyrimidine biosynthesis gene pyrB. These results validate our mariner-based transposon system for use in forward genetic studies of C. difficile.Clostridium difficile infection is widely recognized as the leading cause of health care-associated diarrhea in North America and Europe. Infection usually follows antibiotic treatment, which disrupts the native gastrointestinal microflora and thus allows C. difficile to proliferate. The emergence of so-called “epidemic” or “hypervirulent” strains of C. difficile over the last 5 to 10 years has compounded an already serious problem. Classed as BI/NAP1/027, these epidemic strains are believed to cause a more severe disease and lead to increased mortality and relapse rates (11, 20, 24).Understanding the genetic and molecular basis of C. difficile infection will be a crucial step in the development of effective countermeasures. Methods for directed gene inactivation in C. difficile have recently been described (7, 21). This has opened the way for reverse genetic studies, in which the exact role of a specific gene, hypothesized to be important in a given phenotype, can be elucidated experimentally. By way of contrast, forward genetic studies aim to identify the genetic basis of a particular phenotype without making any assumptions about the genes involved. In forward genetic studies, transposons are often used to generate libraries of random insertion mutants. Libraries are then screened to identify mutants that are defective in a particular phenotype. Identification of the gene or genes which have been inactivated by transposon insertion then implicates them as having a role in that particular phenotype. Recently, just such an approach was used to identify a novel toxin-regulatory locus in Clostridium perfringens (29). This study elegantly demonstrated the power of forward genetic studies in bacterial pathogens.A number of transposon mutagenesis systems have been described for Gram-positive bacteria (2, 3, 15, 16, 29, 32). Two different systems have recently been developed for use in C. perfringens (15, 29). Both are in vitro mutagenesis systems which rely on being able to transform the recipient organism. As such, they are not suitable for use in C. difficile because in the laboratory at present, recombinant DNA can be transferred into C. difficile only via conjugation. The conjugative transposons Tn916 and Tn5397 have been studied in C. difficile, but both have been found either to have a strong target site preference or to yield multiple insertions in individual clones (9, 30). Therefore, neither is well suited to generating libraries of random C. difficile mutants.We reasoned that a mariner-based transposon mutagenesis system would be an effective tool for generating libraries of random C. difficile mutants. The mariner-transposable element Himar1 has been shown to insert randomly into the genomes of many bacterial species (3, 6, 16, 17, 32). The cognate Himar1 transposase is the only factor required for transposition, which occurs via a cut-and-paste mechanism (13, 14). The transposon itself is defined by inverted terminal repeats (ITRs) at either end and inserts into a TA target site. This is highly appropriate for an organism with a low-GC content such as C. difficile. In this study, we have developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile. Moreover, we have demonstrated the system in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. This new genetic tool opens the way for forward genetic studies of C. difficile.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

EhRho1, a RhoA-Like GTPase of Entamoeba histolytica, Is Modified by Clostridial Glucosylating Cytotoxins

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[¹⁴C]glucose.     

Antimicrobial Agent-Associated Colitis and Diarrhea

Although antimicrobial agent-associated colitis has been recognized as a clinicopathologic entity for years, the cause of this disease has been determined only recently. Virtually all cases of pseudomembranous colitis and some cases of antimicrobial agent-associated nonspecific colitis or diarrhea have been shown to be caused by a toxin of Clostridium difficile. Methods for cultivating C difficile from feces and for detecting the toxin have been developed. Oral administration of vancomycin has proved to be effective for the treatment of C difficile-induced colitis, although isolated instances of relapse after treatment have been documented.The discovery of C difficile as a human intestinal pathogen has provided an explanation for some, but not all cases of antimicrobial agent-associated diarrhea. The epidemiology, pathogenesis and means of prevention of C difficile toxin-induced diarrhea remain to be determined.     

Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro

Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea. A limited degree of success in controlling C. difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized. The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C. difficile by using cultural and molecular techniques. Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C. difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course. Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity. Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C. difficile suppression. NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C. difficile populations. However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance. In the absence of clindamycin, NDO enhanced colonization resistance against C. difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena.     

Co-culture with potentially probiotic microorganisms antagonises virulence factors of <Emphasis Type="Italic">Clostridium difficile</Emphasis> in vitro

Toxigenic strains of Clostridium difficile were co-cultured with different strains of bifidobacteria and lactobacilli. Spent culture supernatants were tested for biological activity on cultured Vero cells. Co-culture of C. difficile with some potentially probiotic strains lead to a reduction of the biological activity of spent culture supernatants. The observed effects cannot be ascribed either to secreted factors from the probiotic strains or to toxin adsorption by bacterial cells. Immunological assays showed that there was significant diminution of both clostridial toxins (TcdA and TcdB) in spent culture supernatants of co-cultures as compared with pure clostridial cultures. Even though co-cultured clostridial cells showed a slight increase of intracellular toxins, this increase did not completely explains the reduction of toxin concentration in culture supernatants. The evidence suggests that the antagonism could be due to the diminution of the synthesis and/or secretion of both clostridial toxins. Our findings provide new insights into the possible mechanisms involved in the protective effect of probiotics in the context of C. difficile infection.     

Identification of a Novel Zinc Metalloprotease through a Global Analysis of Clostridium difficile Extracellular Proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.     

Sequencing and analysis of the gene encoding the α-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile

A library of total Clostridium novyi DNA was established and screened for the α-toxin gene (tcnα) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcnα fragments were isolated and subsequently the total sequence of tcnα was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M_r 250 166 and pI 5.9. The N-terminal α-toxin (Tcnα) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it consist of different subunits. Overall the amino acid sequence shows 48% homology between the Tcnα and both toxins A (TcdA) and B (TcdB) of Clostridium difficile. The C-terminal 382 residues of Tcnα constitute a repetitive domain similar to those reported for TcdA and TcdB of C. difficile. The individual repeat motifs of these three toxins consist of oligopeptides some 19–52 amino acids in length, arranged in four to five different groups. Genetic, biochemical and pharmacological data thus confirm that the three toxins belong to one subgroup, designated large clostridial cytotoxins (LCT). Further definition of their structure and detailed molecular action should allow the LCTs to be used tools for the analysis of microfilament assembly and function.     

Clostridium beijerinckii and Clostridium difficile Detoxify Methylglyoxal by a Novel Mechanism Involving Glycerol Dehydrogenase

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87–93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome.     

Carriage of Clostridium difficile by Wild Urban Norway Rats (Rattus norvegicus) and Black Rats (Rattus rattus)

Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 “novel” ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration.     

Isolation of a Degeneration-Resistant Mutant of Clostridium acetobutylicum NCIMB 8052

Unless periodically grown from germinated spores, Clostridium acetobutylicum tends to degenerate (that is, to spontaneously lose the capacity both to produce solvents and to develop into spores). To obtain mutants that are deficient in degeneration, C. acetobutylicum NCIMB 8052 was mated with Enterococcus faecalis BM4110 harboring transposon Tn1545. We developed a degeneration resistance assay based on a secondary effect of degeneration, the production of toxic levels of acetic and butyric acids. Erythromycin-resistant transconjugant clones were tested individually for longevity by repeated and timely subculturing. One long-lived mutant, A10, survived 18 ± 3 transfers (mean ± standard deviation; n = 20) before extinction, while the wild type (parental cells) survived 6.6 ± 1.5 transfers (n = 11). The three-fold difference in longevity is statistically significant. In a batch culture in a rich medium, the wild-type cells degenerated within 24 h after inoculation with 1% of an overnight culture derived from germinated spores. In contrast, A10 cells were able to switch to solventogenesis and to sporulate. In a minimal medium with greater buffering capacity, both cell types produced solvents and spores. Southern blots of EcoRI and HindIII restriction digests of A10 chromosomal DNA (but not parental DNA) showed that only one copy of Tn1545 was inserted into the clostridial chromosome. Our findings are consistent with the hypothesis that there was an alteration at a regulatory locus that was effected by the insertion of the transposon.     

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44⁺ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.     

Changes in the cell wall of Clostridium species following passage in animals

Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice. We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae. Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates. A thick granular wall prior to animal inoculation was shown only in C. perfringens, C. butyricum, and C. difficile. This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K. pneumoniae or B. fragilis.     

Excess Mortality Attributable to Clostridium difficile and Risk Factors for Infection in an Historic Cohort of Hospitalised Patients Followed Up in the United Kingdom Death Register

Methods

We compared time from hospital admission to death in a probability sample of 100 Clostridium difficile infected cases and a probability sample of 98 non-cases admitted to an English teaching hospital between 2005 and 2007 with follow up in the UK national death register using survival analysis.

Results

Clostridium difficile infection was associated with a 50% increased risk of death (Hazard Ratio 1.51 (95% CI: 1.05–2.19 p = 0.03) at between five to eight years in Cox Regression analysis adjusting for age, sex, Charlson comorbidity index, diagnosis of a malignant condition and insertion of a nasogastric tube during admission. Acquisition of Clostridium difficile infection was independently associated with an almost six fold higher odds of being admitted with a diagnosis of infection of any other type (OR 5.79 (2.19, 15.25) p<0.001).

Conclusions

Our results strongly support continued priority being given to improve prevention and treatment of Clostridium difficile infection in the English National Health Service particularly in patients admitted with an infection. Our results may be applicable to other health systems.     

Technical guide for genetic advancement of underdeveloped and intractable Clostridium

In recent years, the genus Clostridium has risen to the forefront of both medical biotechnology and industrial biotechnology owing to its potential in applications as diverse as anticancer therapy and production of commodity chemicals and biofuels. The prevalence of hyper-virulent strains of C. difficile within medical institutions has also led to a global epidemic that demands a more thorough understanding of clostridial genetics, physiology, and pathogenicity. Unfortunately, Clostridium suffers from a lack of sophisticated genetic tools and techniques which has hindered the biotechnological exploitation of this important bacterial genus. This review provides a comprehensive summary of biotechnological progress made in clostridial genetic tool development, while also aiming to serve as a technical guide for the advancement of underdeveloped clostridial strains, including recalcitrant species, novel environmental samples, and non-type strains. Relevant strain engineering techniques, from genome sequencing and establishment of a gene transfer methodology through to deployment of advanced genome editing procedures, are discussed in detail to provide a blueprint for future clostridial strain construction endeavors. It is expected that a more thorough and rounded-out genetic toolkit available for use in the clostridia will bring about the construction of superior bioprocessing strains and a more complete understanding of clostridial genetics, physiology, and pathogenicity.     

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model

BackgroundClostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model.MethodsC57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets.ResultsC. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet.ConclusionsLow protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.