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Clostridium difficile is a gram positive, anaerobic bacterium that is the leading cause of antibiotic-associated pseudomembranous colitis worldwide. C. difficile is an extremely infectious bacterium that produces spores that are highly resistant to standard disinfectant agents and can survive on surfaces for long periods of time. Both the resistance of the spores combined with multiple patients with low-immune systems has lead to an increase in hospital-acquired C. difficile infection, which has had a severe economic impact on the healthcare system. Due to the emerging antibiotic resistance problems and the common occurrence of patient relapse using the current drugs of choice, alternative therapeutic avenues are being explored. C.difficile produces two potent exotoxins; Toxin A and Toxin B that are the causative agents of infection. These toxins have multi-modular domain organisations, with each domain playing a role in cytotoxicity. Some of these domains have been characterised structurally using X-ray crystallography. In this thesis, the low resolution SAXS structure of Toxin A will be presented along with the advances made towards determining the X-ray crystallographic structure of the full-length Toxin A. In addition to Toxins A and B, some strains of C. difficile produce a binary toxin, CDT, which is made up of two individually produced components, CDTa and CDTb. The CDTa component is the enzymatically active component, whereas CDTb is the transport component, directly involved in translocating CDTa into target cells. The precise role of CDT in pathogenesis is unclear, however there is evidence that CDT ADP-ribosylates monomeric actin in target cells, but the detailed mechanism by which this reaction takes place is unknown. Here site directed mutagenesis of key residues of the active site of CDTa was performed and the effect of these mutations on the enzyme’s cytotoxicity tested. By separately mutating three active site residues, the cytotoxic effect of CDTa can be completely eradicated, details of which will be discussed in this thesis. Additionally, the progress made towards determining the X-ray crystallographic structure of the transport component, CDTb, will be discussed.

Clostridium difficile has raised significant public concern over the past few decades owing to its emergence as a serious and life-threatening nosocomial pathogen. C. difficile-associated disease is often precipitated due to persistent use of broad-spectrum antibiotics which eventually disrupts the normal colonic micro flora thereby promoting the spread of this toxin- producing organism. There are several classical signs and symptoms associated with C. difficile infection including profuse diarrhoea, abdominal cramps, nausea, and fever. The main objective of this investigation was to employ a systems biology approach in order to assess the response of C. difficile strain 630 when exposed to a clinically relevant heat-stress (4l0C relative to 37°C). This was achieved by carrying out gel-based and gel-free proteomic techniques to characterise the soluble subproteome of this bacterium. Several differentially expressed proteins were hence identified, functionally categorised, and physiochemically characterised which made it possible to comment upon the biochemistry of this organism when exposed to heat-stress conditions. Subsequently, in order to complement our proteomics dataset, whole-genome microarrays were performed to gain a transcriptomic overview of the same heat-stress response of C. difficile. Upon data analysis, it was revealed that in addition to several others, the gene expression and protein abundance levels of classical molecular chaperones such as GroEL and DnaK were significantly modulated. Therefore, genes encoding these chaperones were then targeted to attempt and isolate knockout mutants of C. difficile in order to assess the impact of such a disruption upon the overall physiology of this organism. The results obtained from this systems biology investigation provide a comprehensive overview of the heat-stress response of C. difficile and additionally reveals the importance of certain genes in maintaining cellular homeostasis not only upon exposing the organism to stressful environments, but also under optimal growth conditions.

Cancer is a major cause of mortality and morbidity that is likely to both increase in incidence and persist with humankind indefinitely. Conventional therapies are not effective against all types and stages of cancer, representing a huge present and future unmet medical need. Clostridial Directed Enzyme Prodrug Therapy (CDEPT) is a promising new experimental approach to the treatment of solid tumours, whose hypoxic (low-oxygen) regions make them resistant to conventional therapies. These same hypoxic regions can be exploited in therapies using clostridia; strictly-anaerobic bacteria that can grow in hypoxic tumours, but not elsewhere. In CDEPT, the clostridial cells are engineered to produce an enzyme that converts a subsequently-administered, non-toxic prodrug into a highly-toxic drug only at the tumour site, resulting in highly tumour- specific cytotoxicity; an important shortfall of existing therapies. The prodrug 5- (aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) has excellent properties, and is safe. However, CDEPT progress has been hindered by poor CB 1954-activating enzymes and the unavailability of techniques to chromosomally-localise the enzyme-encoding gene, a safety feature required for clinical use. Here, two novel CB 1954-activating enzymes have been isolated and characterised, one of which has kinetic properties with respect to CB 1954 substantially better than previously-reported enzymes, and may prove to be ideal for CDEPT and related therapies. Both separately, and in collaboration with structural biologists, progress has been made towards understanding, and therefore potentially improving, the interactions between CB 1954 and several enzymes. Finally, a method for the integration of DNA into clostridial chromosomes has been developed, which facilitates the construction of clinical CDEPT strains. This method has profound implications beyond CDEPT, particularly because it allows the construction of isogenic mutants, important research tools that were previously difficult or impossible to construct in major pathogens such as Clostridium difficile and Clostridium botulinum, and industrially-important strains such as Clostridium acetobutylicum.

Clostridium difficile is the major underlying cause of antibiotic-associated diarrhoea and poses a risk for healthcare systems worldwide. Endospores produced during sporulation are widely regarded to be the infectious agent of C. difficile associated diarrhoea. These spores are able to withstand a variety of antimicrobial agents and industrial cleaning products and are therefore able to reside on surfaces in healthcare settings for prolonged periods of lime. In order to cause disease in susceptible individuals, spores need to abjure dormancy and return to vegetative cell growth through germination. Sporulation and germination have been studied extensively in Bacillus spp. Knowledge about the sporulation and germination pathways in C. difficile, however, remains incomplete. Here, forward and reverse genetics methods were employed to analyse sporulation and germination phenotypes of C. dfficile. Using forward genetics, 19 mutants with potential sporulation and/or germination phenotypes were isolated, three of which were completely deficient for sporulation. In an attempt to explore the use of transposon suicide vectors, a protocol for the successful transformation of C. difficile was developed. A reverse genetic mutant in the germination specific lytic transglycosylase Slee created by ClosTron mutagenesis was used to study spore germination in vivo. This study is the first report of the use of a germination mutant in vivo. The sporulation characteristics of 52 clinical C. difficile isolates have been analysed indicating that a variation in the rate of sporulation is not associated with molecular type. The germination characteristics of 37 clinical C. difficile isolates were examined, indicating that different isolates exhibit varying germination characteristics in response to bile salts.

Clostridium acetobutylicum is a Gram-type positive, strictly fennentative, endospore-forming bacterium. It gains energy by converting sugars into organic acids during the first part of its life cycle and into solvents during the second part. This work investigated whether RNPP-type quorum sensing is involved in the regulation of key features such as solventogenesis and sporulation. It identified eight putative RNPP-type Quorum ,S,ensing ,S,ystems (qssA to qssH) in the genome of C. acetobutylicul11, each consisting of a putative Quorum ,S,ensing Regulator (qsr) and Quorum ,S,ensing E.eptide (qsp). ClosTron inactivation of six regulators negatively affected solventogenesis, morphological development or sporulation, whereas inactivation of qsrB had a positive effect. Synthetic QspB-derived pep tides could relieve the repression of solvent formation in a qsrB-overexpressing strain, suggesting that QsrB and QspB fonn a functional quorum sensing system. Two orphan Qsr-type regulators, qsrl and qsrJ, were identified. These regulators are encoded without qsp homologues in a gene cluster that is conserved among the genus Clostridium sensu stricto with qsrl preceding qsrJ. qsrl and qsrJ knockout mutants resembled spoOA mutants as total solvent formation was reduced to below 15 mM and sporulation or granulose accumulation did not occur. The phenotypic changes were con finned at transcriptional level by an RNA-Seq analysis, which also revealed that over 600 genes were significantly differentially regulated, including genes encoding secreted proteins and the Agr-type quorum sensing system. Co-culture and cross streak experiments provided further evidence that QsrI controls the Agrtype and possibly other quorum sensing systems. Key outcomes of this work are the evidence that RNPP-type quorum sensing extends to anaerobic FiJ'micutes and that solvent formation in a Clostridium sp. is controlled by quorum sensing. The discovery of the regulators QrsI and QsrJ was of major importance, showing that other master regulators ex ist beside SpoOA.

Clostridium difficile is a leading cause of hospital-acquired diarrhoea, responsible for over 30% of cases of antibiotic-associated colitis, nearly all cases of pseudomembranous colitis and costs the NHS over œ200 million per year. This bacterium is able to persist in the hospital environment to cause recurrent infection by the formation of stable spores, refractile to current decontamination procedures. A more comprehensive understanding of the sporulation signal transduction pathway is essential for the design of a decontamination regime effective in removing the spores from the nosocomial environment and the logical design of novel antimicrobial agents. This project aimed to elucidate the mechanism of sporulation initiation . regulation and the role of sporulation-associated proteins in other C. difficile virulence processes, such as toxin production and colonisation. Analysis of sporulation in response to various hospital cleaning agents showed that the combination of a neutral detergent (such as Hospec) with EDTA is a more effective cleaning agent than the chlorine-based agents currently used, as the combination product is uniquely able to both kill vegetative cells and inhibit spore formation. A variety of molecular approaches were used to elucidate information regarding the C. difficile sporulation initiation pathway and the relationship between sporulation and toxin production. Three putative C. difficile sporulation-associated sensor histidine kinases (CD1A, CD2A and CD3B) were identified and shown to be independently involved in sporulation initiation. Furthermore, CD3B has been shown to directly phosphorylate the master response regulator SpoOA, strongly suggesting that this pathway is a two-component system, as opposed to the extended phosphore lay pathway found in B. subtilis. Previous studies on bacteria capable of both toxin production and endospore formation have described links between the two processes. Data presented here indicates SpoOA has a role in indirectly regulating C. difficile toxin A and B production, as the protein is capable of specifically binding promoter regions of the toxin regulatory genes tcdC and tcdD. Inoculation of a triple-stage continuous-culture chemostat that modelled the human gut with C. difficile spoDA- mutant provided further evidence that SpoDA has a key role in both colonisation a!1d toxin production. Overall, this work adds to the growing body of evidence that SpaDA is a master global regulator and has a crucial role in the pathogenicity of C. difficile, making it an excellent target for future novel antimicrobial therapies.

No description available.

No description available.