Burkholderia cepacia complex (Bcc) are well known for their ability to produce antifungal agents. However, the majority of past studies have screened only a limited number of isolates, which were not taxonomically well characterized. In addition, while the activity of Bcc bacteria against plant pathogenic fungal species has been characterized, very few studies have examined the interaction between Bcc and saprotrophic wood decay basidiomycete species. The overall aim of this study was to begin to the characterization of the interactions of Bcc and other Burkholderia with the model ascomycete Candida albicans and woodland basidiomycete fungi. Methods. The study made use of a large collection (397 isolates) of taxonomically well identified Burkholderia species. A systematic screening of the antifungal activity of this collection was made against C. albicans and various wood decay basidiomycetes: Bjerkandera adusta, Trametes versicolor, Hypholoma fasciculare, Resinicium bicolor and Phanerochaete velutina. A wide range of media were evaluated for Burkholderia growth, antifungal metabolite production and the ability to support the mycelial growth of basidiomycetes. An agar overlay assay was used to examine the anti-candidal activity of Burkholderia isolates, while a novel overlay assay was developed using homogenised mycelial material for the basidiomycete species. Novel and existing extraction techniques were evaluated for their ability to partially purify active Burkholderia metabolites. The purification and identification of the active Burkholderia antibiotics were performed using both preparative thin layer chromatography (TLC) and liquid chromatography combined with mass spectrometry (LC-MS). A novel TLC-bioautography assay was also developed to reveal the presence of active Burkholderia metabolites in the antimicrobial extracts. The minimum inhibitory concentration (MIC) of the semi-purified Burkholderia antifungals was also determined. The natural diversity of Burkholderia and other bacteria associated with different rhizosphere environments was evaluated by cultivating isolates from local maize crops and examining a collection of Burkholderia isolates obtained from the rainforest in Sabah, Malaysia. The genetic basis for the secretion of antifungal agents by B. ambifaria was examined using transposon mutagenesis and screening for the mutants which had lost their anti-candidal activity. Additional transposon mutants that had lost their anti-bacterial activity and had been obtained in a previous screen were also examined for alterations in antifungal activity. Finally, the Burkholderia genome database and bioinformatic techniques were used to genetically characterize these mutants and other genetic loci that had been previously implicated in antibiotic production. Results. The majority of Burkholderia isolates possessed the same antagonistic activity towards C. albicans as they did towards B. adusta. Of the 397 isolates examined, antifungal activity was the greatest in the following Burkholderia species: B. ambifaria, B. cepacia, B. cenocepacia and B. contaminans. Interestingly, no antifungal activity was observed for B. multivorans (27 isolates) and B. stabilis (18 isolates) under the experimental conditions tested. The type of medium used to study the interaction between Burkholderia and the fungi considerably effected antifungal activity, with the most suitable media for studying antagonistic activity being Sabouraud agar and an acidic minimal salts medium (BSM; pH 6) supplemented with 0.4% glycerol. Of the 397 isolates screened, 47.85% were anticandidal (190 isolates; 89.47% of these were Bcc species and 10.53% were other Burkholderia species), while 48.10% were active against B. adusta (191 isolates; 93.19% of these were Bcc species and 6.81% were other Burkholderia). Extraction using Amberlite XAD16 resin binding followed by elution with methanol was an excellent means to isolate active Burkholderia antifungals. Fractionation of these extracts using silica gel TLC and ethyl-acetate:methanol:water (20:1:0.5) as the elution solvent was optimal for the separation of the majority of the active metabolites. A TLC-bioautography assay was developed in this current study as a preliminary screening technique to detect anti-microbial components of novel Burkholderia extracts. The assay was very useful for detecting anti-candidal and anti-bacterial compounds and was adapted for use for the first time with basidiomycetes. Antifungal Burkholderia isolates produced between 1 and 10 antifungal metabolites. Several antifungal agents were identified as enacyloxin, pyrrolnitrin, bactobolin and quinolines. Novel compounds and novel bactobolin derivatives were also present. For B. ambifaria strain AMMD a number of chromosomal loci were found to be involved in the production of antifungal metabolites. Quorum sensing was essential for the expression of antifungal activity as transposon mutations in the CepI synthase-encoding gene prevented the biosynthesis of all B. ambifaria anti-candidal compounds. B. ambifaria AMMD extracts containing enacyloxin IIa also produced an inhibitory effect at low concentration against E. coli, B. multivorans and A. baumannii. Conclusions. Burkholderia are potentially a very large reservoir of known and novel antimicrobial agents, especially with nearly 50% of isolates screened exhibiting antifungal activity. Antimicrobial compounds such as enacyloxin IIa were discovered for the first time to be produced by Burkholderi and novel extraction and bioassay methods for anti-fungals were developed in this study. Overall, we now have the tools to considerably advance the isolation and characterization of antifungal Burkholderia metabolites.

No description available.

This study aimed to investigate the RBT outcome as a function of the bacterial inoculum during adaptation to degrade the HAs dichloroacetic acid (DCA), trichloroacetic acid (TCA) and 2-monochloropropionic acid (2MCPA). The HAs investigated were ranked, in order of greatest recalcitrance; TCA > 2MCPA > DCA.  DCA degradation was associated with enrichment of a Ralstonia like phylotype and dehII expression.  TCA degradation in replicate RBTs was consistently associated with a Bradyrhizobium like phylotype and dehI gene expression, regardless of the inoculum concentration used, suggesting that TCA imposes a highly selective pressure on the community. TCA degradation was affected by the inoculum concentration, as the rate of degradation was slower at the lowest inoculum concentration used, implying that TCA degradation was accomplished by cometabolism. 2MCPA was associated with various Alpha-, Beta- and Gammaproteobacteria, and the presence and expression of both dehI and dehII genes. Biodegradation of 2MCPA was strongly influenced by inoculum concentration. At lower inoculum concentrations, 2MCPA was commonly associated with a biphasic dechlorination curve, which has not been reported previously.  Biphasic dechlorination curves were attributed to the enrichment of an initial degrading organism, always associated with dehII expression, which was succeeded following 50% dechlorination by a second organism, in association with dehI expression, which were supposed to act sequentially on the L- and D-2MCPA isomers, respectively. The delayed enrichment of a dehI containing organism was attributed to their low environmental abundance, relative to dehII containing organisms. This study highlights the utility of cultivation-independent methods to link more precisely community structure and function during adaptation to degrade xenobiotic compounds.

Multiple sclerosis (MS) is generally considered to be an autoimmune disease; it results in areas of focal demyelination known as plaques, where astrocytes are the main constituent and appear hypertrophic and stellate with increased expression of glial fibrillary acidic protein (GFAP). Although these reactive astrocytes can be beneficial for encapsulating areas of tissue necrosis within the glial scar, the pro-inflammatory cytokines and extracellular molecules they secrete are thought to be inhibitors of remyelination. Conversely, astrocytes can secrete anti-inflammatory cytokines in their less activated state and thereby can be beneficial for remyelination; although the mechanisms involved are not clearly understood. We have developed myelinating cultures of embryonic rat mixed spinal cord cells that are plated on neurosphere-derived astrocyte monolayers, which allow us to manipulate their reactive state and determine any effects of these astrocytes on myelination. To identify any differences in gene expression profiles of several purified astrocyte phenotypes we carried out a microarray analysis using myelinating cultures plated on astrocytes on poly-L-lysine (PLL-astrocytes) or on tenascin C (TnC-astrocytes), untreated or treated with ciliary neurotrophic factor (CNTF) since it was previously shown that CNTF could stimulate myelination in these myelinating cultures and that TnC-astrocytes supported myelination less than PLL-astrocytes. The microarray analysis suggested CCL2, CCL7 and SERPINB2 as potential myelination regulators, which were validated by qRT-PCR but failed to affect axonal ensheathment in myelinating cultures. A metabolomics analysis was also carried out using untreated or CNTF-treated PLL- and TnC-astrocytes. A list of metabolites were associated with different astrocyte phenotypes and highlighted the importance of lipid metabolism, which might be crucial for stimulating myelination. These in vitro astrocyte phenotypes were compared with previously identified in vivo astrocyte reactivity markers, which suggested that PLL- and TnC-astrocytes could reflect in vivo astrocytes to some extent and that TnC-astrocytes might not be as “quiescent” as currently considered. It is possible that TnC-astrocytes suppress myelination by creating an extracellular matrix (ECM) rich environment, while CNTF dissolves the ECM and thus creates a more regenerative environment. Despite similar stimulatory effects of CNTF on mixed neural cells on PLL and TnC, CNTF appears to affect them via different mechanisms showing that distinct reactive astrocytes could react to the same stimulus in different ways as also presented in in vivo studies.

This study was designed to study the effects of ethyl acetate extracts from A. wilkesiana (9EA) and D. grandiflora (75EA-L and 75EA-B) and the respective bioactive fractions from these plants on methicillin resistance Staphylococcus aureus (MRSA ATCC 43300). A bioassay-guided isolation was used for fractionation of the crude extracts by combinations of liquid chromatography methods. The minimum inhibitory concentration (MIC) of crude extracts and fractions ranged between 12 to 0.75 mg/ml for MRSA and 6 to 0.75 mg/ml for methicillin sensitive S. aureus (MSSA ATCC 11630). The MIC values of beta-lactam antibiotics against MRSA strain (i.e. MIC of ampicillin = 50 µg/ml) used in this study were higher compared to MSSA (MIC of ampicillin = 6.25 µg/ml). The crude extracts and selected fractions were evaluated for synergistic activity with ampicillin. The kinetic growth curve experiment illustrated that combination of ampicillin and 9EA or 75EA-L or the fractions derived from these extracts (9EA-FC, 9EA-FD, FC-B, and 75EA-L) suppressed MRSA growth markedly. Results of fractional inhibitory concentration (FIC) index interpretation indicated synergism present in combination treatments of ampicillin and the plant test agents (FIC index < 0.05). Two fractions, FC-B and 75EA-L-F10 were identified to reduce MIC of ampicillin from 50 µg/ml to 1.56 µg/ml and 0.78 µg/ml respectively. These fractions were found to inhibit PBP2a production either alone or in combination with ampicillin in Western blot assay which offered a plausible explanation for restoration of ampicillin’s activity in combination treatment. The same fractions were investigated in MRSA biofilm study. Results showed that FC-B or 75EA-L-F10 alone inhibited MRSA biofilm production (~70-80% inhibition). Findings from microtiter attachment assay suggested that these fractions prevent cell-surface attachment (more than 90% inhibition) which is the initial step in biofilm formation. Whereas the PBP2a latex agglutination showed occurrence of low level of PBP2a in MRSA biofilm treated with FC-B or 75EA-L-F10 implicating possible disruption of cell-cell interactions required for microcolonies development. Ampicillin on the other hand has an inferior activity in preventing cell-surface attachment (37.8% inhibition) although it managed to inhibit MRSA biofilm production by 84.5%. A high performance liquid chromatography (HPLC) and phytochemical analysis showed the studied extracts and fractions are complex mixtures of plant metabolites belonging to the class of tannins, saponins, alkaloids, flavonoids, sterols/steroids, and glycosides. The resistance modifying properties and the anti-biofilm action found in this study are attributed to presence of these phytochemicals. Therefore, we propose that metabolites occurring in A. wilkesiana and D. grandiflora may be good candidates for development of new treatment for MRSA or as an adjuvant for the current antibiotics.

Plants have evolved complex and highly regulated stress response mechanisms which elicit major transcriptional reprogramming to mitigate stress. Examination and network inference modelling of high resolution temporal transcriptomic datasets across a range of biotic and abiotic stresses, identified the AtNF-Y transcription factor (TF) family as important regulators of stress responses based on their differential expression patterns and high connectivity in gene regulatory networks (GRNs). In plants, each subunit of the heterotrimeric NF-Y complex is encoded by a multigene family; such extensive combinatorial diversity likely evolved to facilitate transcriptional fine-tuning. A comprehensive investigation into the formation of protein-protein interactions amongst all family members, revealed a widespread ability of AtNF-YB and AtNF-YC subunits to heterodimerise, and also identified interactions between AtNF-YA subunits and a subset of AtNF-YCs expressed during stress conditions. Detailed functional analysis of loss- and gain-of-function mutants in five AtNF-Ys identified partially overlapping roles for AtNF-YA2, AtNF-YA4 and AtNF-YA7 in jasmonic acid(JA)-/ abscisic acid (ABA)-mediated signalling, and enabled the generation of local GRNs centred around each AtNF-Y. A promoter fragment from the JA-biosynthetic gene LIPOXYGENASE3, bound five AtNF-YC subunits in yeast one-hybrid assays, implicating AtNF-Y TF complexes in the regulation of this gene and hence, JA biosynthesis. Stable lines of epitope-tagged translational fusions were generated for one of these subunits, AtNF-YC2, and co-immunoprecipitation of the tagged protein complex in vivo successfully identified AtNF-YB2 as an interacting protein. Investigation of the LIPOXYGENASE3 promoter architecture revealed a requirement for two discrete cis-elements for effective AtNF-Y binding, suggesting that cooperative interactions between NF-Y complexes, and conceivably other TFs, are an important mechanism in their transcriptional regulation, with NF-Ys potentially functioning as pioneer TFs.

Shiga toxin and the Escherichia coli Shiga-like toxins (SLTs) are type 2 ribosome inactivating proteins (RIPs), exhibiting a specific RNA N-glycosidase activity comparable to that of the plant toxin ricin (Endo et al. 1988). Not only do they possess the same enzymatic activity as a plant toxin, but on examination at the molecular level, they may be seen to be classic bipartite toxins. The present study examines in detail the RNA N-glycosidase activity of Shiga-like toxin I (SLT I) ie, the ribosomes susceptibility to the toxin, of a range the exact of different eukaryotic site of SLT I-catalysed depurination of yeast ribosomal RNA and the kinetics of such depurination. The cytotoxic effect of SLT I on Vero cells is also examined. In addition to its characterisation and subsequent comparison to ricin, an attempt has been made to correlate certain structure - function aspects of SLT I. By structural comparison with other bacterial toxins it has been proposed that proteolytic cleavage with thin an arginine rich, trypsin sensitive, disulphide-bonded loop structure is essential for expression of cytotoxicity. Examination of the enzymatic activity and cytotoxicity of a 'protease insensitive' mutant SLT I A subunit (SLT lA) generated in the present study has led to the conclusion tha t proteolytic processing wi thin this disulphide-bonded loop is not essential, but that processing may occur at alternative sites within SLT lA to compensate for loss cleavage at the proposed target sites.

Rotaviruses are a ubiquitous group of viruses responsible for causing acute gastroenteritis in the young of humans and animals. This thesis concerns the production characterization and use of monoclonal antibodies to tissue culture adapted calf rotavirus (UK-Compton strain). Monoclonal antibodies were produced, which as a result of immunoprecipitation and Transblotting experiments were determined to be directed towards VP6, the subgroup protein of calf rotavirus. Varying the immunogen and fusion protocol proved insufficient since only antibodies directed at this extremely immunogenic protein were produced. As a result of competitive binding assays, this subgroup protein was determined to consist of at least eight different epitopes. Two of these epitopes belonged to a group-specific, non-neutralizing region, whilst four epitopes belonged to a group-specific, neutralizing region. The remaining two epitopes belonged to a type-specific, neutralizing region. None of the antibodies had greater than 75% neutralization. An ELISA was developed to use four of these monoclonal antibodies to detect and subgroup rotavirus antigens using human faecal samples collected from children in Lima, Peru between the period 1983-84, during clinical trials of the RIT 4237 rotavirus vaccine. Out of a total of 61 samples, 62% were typed as belonging to subgroup 1. This was similar to a figure obtained for subgrouping human samples in the Midlands during the same period. This reflected a change in subgroup antigenicity. During 1978-82 strains belonging to subgroup 2 had been the most prevalent. The ELISA technique developed proved to be quick, cheap, efficient and reliable, thus enabling a large number of samples to be screened simultaneously.

Shiga toxin (ST) and the Escherichia coli Shiga-like toxins (SLTs) are type 11 ribosome inactivating proteins (RIPs). All members of this group exhibit specific RNA N-glycosidase activity, the prototype being the plant toxin ricin. ST and the SL Ts are bipartite toxins composed of a catalytic A subunit and a pentamer of cell binding B subunits. These toxins show overall structural similarities to ricin, which is also a bipartite toxin with a catalytic A chain and a single cell binding B chain. The A chains of ST and SL T 1 show homology to ricin A chain, particularly in the active site region, and appear identical in their enzymatic mechanism. The respective B chains however are structurally very different and interact with quite different cellular components. In this study, the role of intracellular proteolytic activation of SLT 1 is addressed using a molecular biological approach. The biological characteristics of several mutant SL Ts has been investigated both in vitro, by addition of exogenous protease, and in vivo by comparing the relative cytotoxicities of mutant and wild type proteins in Vero cells. The intracellular processing of these mutant toxins has also been examined. In parallel, the biological properties of a ricin A chain SL T 1 chimeric protein has been investigated. The ultimate aim of this study was to extend our knowledge of the proteolytic processing requirements of SL T 1 and it has led to the conclusion that proteolytic removal ofthe A2 portion of SL T 1 is not an essential prerequisite for intoxication of Vero cells with SLT 1.

No description available.