The aim of this project was to investigate the possible role of the biofilm matrix as a barrier to drug diffusion in Candida biofilms and in mixed species fungal-bacterial biofilms. The penetration of antifungal agents through single- and mixed-species biofilms containing Candida was investigated using a novel filter disk bioassay. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. Drug penetration was more extensive with C. albicans than with the other species and the rates of diffusion of either drug through biofilms of three strains of C. albicans were similar. In all cases, after 3 to 6h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms under these conditions. It has been reported that the production of extracellular matrix by Candida biofilms growing under static incubation conditions is relatively minimal, but increases dramatically when developing biofilms are subjected to a liquid flow. In this study, Candida biofilms were grown under flow conditions in a modified Robbins device (MRD). Biofilms of C. albicans grown in the MRD produced more matrix material than those grown statically, and were significantly more resistant (P<0.001) to amphotericin B. Biofilms of C. tropicalis synthesized large amounts of matrix material even when grown statically, and such biofilms were completely resistant to both amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis RP62A, when grown statically or in the MRD, were also completely resistant to amphotericin B and fluconazole. Mixed-species biofilms of C. albicans and S. epidermidis M7, on the other hand, were completely drug resistant only when grown under flow conditions. Overall, these findings demonstrate that the matrix can make a significant contribution to drug resistance in Candida biofilms, especially under conditions similar to those found in catheter infections in vivo, and that the composition of the matrix material is an important determinant in resistance.

Cell-substrate interactions are of interest in modern biology. The system of surface bound hydrogels is commonly used as a cell culture surface in the field of cell biomechanics. However, the effect gel geometry (thickness, width and length) has on both the mechanics of the gel and the cells behaviour has usually been ignored. It was discovered that a cell differentially spreads and preferentially accumulates at a certain position with respect to the local variation in thickness along a wedge gel (thickness varied from ~50 to 400 µm). This happened although this range of thickness is supposed to be sufficient to prevent the cells from sensing the underlying rigidity of the supporting glass. The mechanical anisotropy of the gel due to its being bound to the substrate was hypothesised to be the cause of the cell behaviour observed. It was later proven that lateral swelling varies exponentially with thickness. The consequences are the decrease in lateral compression and the lateral dilution of gel network density with increasing gel thickness. Both could cause variations of substrate mechanics. The amount of crosslinker, the geometry of the bound area and type of bathing medium all changed the degree of lateral swelling, and thus are contributing factors influencing the lateral mechanics of the swollen gel. Surface bound square gels (50-2000 μm thick) were found to be stiffer with increasing thickness as measured with an atomic force microscope (AFM). This could be due to a change in osmotic pressure. These indentation based measurements of vertical mechanics might be of little relevance with respect to the cellular response though. This was supported with another set of cell experiments on such samples, where the cell did not respond in accordance with the stiffness as measured by AFM. It was therefore implied that the difference in cell behaviour observed on the substrates of different height might be a result of an interplay between the lateral mechanics and the rate of liquid flow though the gel. The x-, y-aspect ratio was also found to influence cell alignment. Cells tended to align randomly on square (aspect ratio: 1:1), and perpendicular to the direction of the long axis of the gels in high aspect ratio (1:4 – 1:11) gels. This preference could be impaired by inhibition of the interaction between actin and myosin II using blebbistatin treatment. This suggests that actomyosin activity is necessary for such the behaviour. The set of studies stressed the importance of x-, y- and z- macrogeometries of surface bound gels as these factors influence mechanical surface anisotropy. These results could have an implication not only in pure cell biology and cell biomechanics but also in regenerative medicine, physiology, wound healing, embryo development, and oncogenesis, wherever cells are in contact with soft biomaterials or orient themselves with respect to mechanical or other features

Autophagy is a conserved lysosomal degradation pathway for recycling long-lived proteins and organelles that is thought to be required for life cycle progression and virulence of Leishmania. ATG8 is a ubiquitin like protein that is required for the formation of autophagosomes, and Leishmania uniquely possesses a set of ATG8-like proteins in addition to ATG8, that are distributed in three multi-gene families called ATG8A, ATG8B and ATG8C. The localisation and expression of ATG8A, ATG8B and ATG8C were analysed using GFP fusion proteins and affinity purified antibodies. ATG8A exhibited a dramatic relocalisation to punctate structures under starvation conditions, suggestive of a specific role for ATG8A in starvation induced autophagy. Although ATG8 and ATG8A both participate in a response to starvation, they differed in their sensitivity to the PI(3) kinase inhibitor wortmannin, responded differently to the presence of energy sources, and labelled distinct subsets of vesicles. When the data generated in this thesis was considered together with recent analyses of the functions of the cysteine peptidases ATG4.1 and ATG4.2, evidence for distinct roles of ATG8 and ATG8A emerged. ATG8B and ATG8C localised to single punctate structures close to the flagellar pocket in a small proportion of promastigotes grown under nutrient rich conditions. The distribution of ATG8B and ATG8C labelled structures did not change during differentiation or starvation, suggestive of a role distinct from autophagy. ATG8B labelled structures appeared to be duplicated during cell division, and might be derived from endosomal membranes. ATG8A, ATG8B and ATG8C expression was shown to be developmentally regulated with all expressed at high levels in stationary phase promastigotes. Conjugation of ATG8 to phosphatidylethanolamine (PE) is required for the association of ATG8 with autophagosome membranes, and while ATG8 was shown to be conjugated to a phospholipid, no evidence was obtained to suggest that ATG8 paralogues are modified by a lipid. High molecular weight proteins were detected by western blot with anti-ATG8, ATG8A, ATG8B and ATG8C antibodies, perhaps indicating associations with other proteins in complexes. Two ubiquitin fusion proteins and a putative SNARE were identified in co-immunoprecipitation experiments performed with anti-ATG8B antibodies, although further experiments are required to determine the validity of these interactions. To analyse the role of a predicted presenilin-1 (PS1) homologue in L. major, Δps1 null mutants were generated. These mutants were not defective in their ability to differentiate into infective metacyclic promastigotes, and could establish infections in vivo and in vitro, demonstrating that PS1 is not essential and is not a good target for drug development. Large autophagosomes accumulated in Δps1 mutants suggesting that PS1 might be involved in the regulation of autophagy, although it seemed that the parasites could compensate for this, as autophagy was restored to normal levels in Δps1 mutants that had undergone differentiation into amastigotes. Antibodies were raised against a PS1 peptide that recognised L. major PS1 only when over-expressed, suggesting that endogenous PS1 is expressed at a low level. PS1-HA that was stably integrated into the genome localised to a structure close to the flagellar pocket, although a different localisation was observed when PS1-GFP was over-expressed, and investigation is required to clarify the subcellular localisation. In summary, the regulation of autophagy in L. major has been investigated from two different angles, leading to the characterisation of a unique family of ATG8-like proteins and an aspartic peptidase, presenilin-1.

Since the Gram positive anaerobe Clostridium difficile was first isolated and described, it has emerged as an increasingly important nosocomial pathogen in Europe, North America and elsewhere, and the prime causative agent of antibiotic associated dirrhoea and pseudomembranous colitis in humans. The two large toxins, A and B, are the main virulence factors, proteins that are expressed in the gastrointestinal tract after colonisation by C. difficile. The pathological symptoms mediated by these toxins include disruption of the integrity of the epithelium, fluid loss, intestinal inflammation and tissue destruction. Important as the toxins are to C. difficile pathogenesis, several other proteins are known to contribute to colonisation and other aspects of the disease process remain poorly understood. Immunological studies using antisera from the patients revealed a number of candidates and amongst these, proteins present, or thought to be present, at the bacterial surface contribute to adhesion, motility and other interactions with the human host. The aims of this study were to produce a number of surface proteins from C. difficile as recombinant products and to isolate antibodies against these targets via phage display. The goal was to assess if these antibodies could inhibit the normal function of these targets and to confirm their location in C. difficile. Of 11 clostridial proteins, expression and purification of 3 proved impossible (Cwp84, FbpA and Acd) but 8 others (CspA, GroEL, FliC, FliD, a putative sortase, Cwp66, and its amino and carboxy terminal regions) were used for antibody isolation along with recombinant and native forms of SlpA. Phage display yielded a large panel of specific single chain variable fragments (scFv) antibodies that were expressed, purified and characterised. Reaction between the scFvs and their targets took place in ELISA and Western blotting suggesting the recognition of linear rather than conformational epitopes. The binding of scFvs to SlpA and its components showed strain specificity 3 with good recognition of protein from C. difficile 630 but no reaction towards SlpA from R20291, and 027 ribotype. Binding of scFvs of a range of specificities to extracts from C. difficile M120 indicated that a component of the S layer from this strain might possess immunoglobulin binding activities in the manner of a superantigen. The scFvs against flagellar proteins FliC and FliD were able to inhibit bacterial motility and therefore there would be potential in testing whether other scFvs generated in this study were able to inhibit the biological activity of their targets. Some scFvs were tested in immunofluorescence microscopy. The positive results from these experiments showed that the reagents and the strategy pursued could be used to establish surface exposure of the targets and other components of the bacterial surface. Given the high specificity of the reagents, and in the case of Cwp66, the ability to isolate scFvs against defined regions of the protein, the strategy has the capacity to define the orientation of proteins in the bacterial surface. In contrast, the use of scFvs to locate their targets in electron microscopy using immunogold reagents was unsuccessful. As this approach has been successful in other studies, it deserves further investment of effort. Overall, expression of proteins from C. difficile in an E.coli host was generally successful and phage display provided a rapid, highly efficient method for the isolation of specific immunological reagents. These have the potential to explore the location, orientation and activity of proteins from the pathogen.

Glucose homeostasis depends on the ability of insulin to stimulate glucose uptake into both muscle and adipose tissue by promoting the translocation of glucose transporters (GLUT4) from intracellular sites to the plasma membrane (PM). In individuals with Type 2 diabetes the ability of insulin to stimulate glucose transport is impaired. The incidence of Type 2 diabetes is increasing worldwide, highlighting the need to understand the molecular basis of insulin-stimulated glucose uptake. GLUT4 translocation is a specialised example of vesicular trafficking. Within the context of vesicle trafficking, all eukaryotic cells contain a common set of conserved components responsible for the execution of membrane fusion. Central to this machinery are members of the SNARE (soluble NSF attachment protein receptor) family of proteins. The process of SNARE-mediated membrane fusion needs to be tightly regulated and the SNARE proteins are partially responsible for the specificity in communication between eukaryotic subcellular organelles. Other proteins such as the Sec1p/ Munc18 (SM) proteins were shown to be essential for SNARE-mediated membrane fusion. Several methods were used to test the ability of SNARE proteins to drive membrane fusion, and one of the most important methods described to date is the in vitro fusion assay used in this study. The first topic addressed in this thesis was related to the molecular interactions between the regulatory SM protein Munc18c and the SNARE proteins VAMP2 and syntaxin 4. The use of pull-down assays revealed the novel fact that Munc18c interacts not only with the t-SNARE syntaxin 4 but also with the v-SNARE VAMP2 via its SNARE motif. The SM:v-SNARE interaction was disrupted by the presence of syntaxin 4 revealing that these two SNARE proteins compete for binding to Munc18c. Next, the role of Munc18c in membrane fusion driven by four different versions of syntaxin 4 plus SNAP23 and VAMP2 liposomes, was investigated using the well-characterised in vitro fusion assay. Results suggested that Munc18c negatively regulates SNARE- mediated membrane fusion by inhibiting the formation of SNARE complexes. Interestingly, deletion of the first 36 amino acids of syntaxin 4 was not sufficient to suppress Munc18c negative regulation of fusion indicating that this inhibition might involve other interactions apart from the short N-terminal peptide of syntaxin 4. Finally, the role of phosphorylation in SNARE complex formation was assessed using several techniques such as site-directed mutagenesis, pull-down assays and radiolabelling studies. Data obtained revealed that both syntaxin 4 and Munc18c become phosphorylated in vitro by a recombinant cytoplasmic insulin receptor kinase (CIRK). Munc18c phosphorylated by CIRK was unable to bind syntaxin 4 in vitro. Furthermore, phosphomimetic mutations were also introduced on both proteins and pull-down assays indicated that phosphorylated Munc18c is unable to interact with both syntaxin 4 and VAMP2, whereas phosphomimetic mutations in syntaxin 4 did not affect the interaction with its cognate SNARE proteins and Munc18c. These results were very useful to further understand and confirm the importance of phosphorylation in SNARE complex formation. Collectively, these data suggest that Munc18c acts through different modes of interaction with its cognate SNARE proteins, and support a model in which Munc18c negatively regulates SNARE complex formation. However, this regulation might also be dependent on other factors such as phosphorylation upon insulin signalling.

No description available.

In vivo expression technology (IVET) is a promoter-trap strategy deigned to identify genes whose expression in induced in a specific environment, typically that encountered in a host. Signature-tagged mutagenesis (STM) uses comparative hybridisation to isolate mutants unable to survive specified environmental conditions and has been used to identify genes critical for survival in the host. Both methods have been used to identify virulence genes in S. aureus. The main aim of this project was to find any probable new genes of S. aureus that are essential for biofilm formation and infection mouse model by STM. A library of tagged insertion mutants of S. aureus and a series of selected tags in plasmids of S. aureus strain RN6390 were used. Most of the experiments with both the library and selected tags had problems with cross-hybridisation. All the selected tags were therefore sequenced and 33 tags with less than 50% identity were chosen for future experiments. A library of 825 mutants was made with the 33 selected tags. The mutants were arrayed in 25 pools of 33 mutants. Different tests were done to determine that the new library was reliable for a cross-hybridisation free screening. The library was then used in an infection model in mouse and biofilm formation. A total of 12 mutants with significantly reduced signals were sequenced. 7 out of 12 attenuated mutants showed homology to different genes in S. aureus and other bacteria. Tetrahydrodipicolinate succinylase homolog, opp-2F, acetoin utilization AcuC protein, phosphate ABC transporter, dapD, branched-chain-amino-acid transporter, pepF, and flaR genes were identified. This work was the first STM screening in a biofilm system, and the dapD gene was identified in a biofilm for the first time. 2 out of 12 genes had also been identified in previous STM screens. 5 out of 12 attenuated mutants showed homology to some hypothetical proteins. A hypothetical protein of the same locus was identified in two mutants.

DNA is replicated during S phase of the S. pombe cell cycle so that each chromosome is composed of two identical sister chromatids. During M phase condensed chromosomes align at the cells equator and sister chromatids are attached to microtubules emanating from opposite spindle poles. At anaphase, sister chromatids are triggered to separate rapidly and completely and move to opposite ends of the dividing cell in the equational pattern of nuclear division forming two identical daughters. During sexual development pre-meiotic replication is followed by homologous recombination and two successive rounds of nuclear division, MI and MIT. During MI sister chromatids remain joined and homologues migrate to opposite spindle poles (reductional division). During MIl, sister chromatids separate as in mitosis, forming four haploid spores enclosed in an ascus. Cohesin is a multi-subunit complex that controls chromosome segregation by providing a physical linkage between sister chromatids. Cohesin is loaded onto newly replicated chromosomes during G liS phase and associates with chromatin both at the centromeres and chromosome arms. When cohesion is dissolved at the onset of anaphase (meiotic anaphase II), sister chromatid separation is triggered. In S. pombe Rad21p is an essential cohesion protein first identified for its role DNA repair, while Rec8p cohesin is essential for meiosis, having additional roles in homologous recombination and spore formation. Both Rad21p and Rec8p occupy critical positions in the mitotic and meiotic cohesin complexes, respectively. The aim of this study was to investigate the mitotic and meiotic functions of Rad21p and Rec8p. To this end their specific patterns of expression were exchanged so that rad21+ was expressed during meiotic development and rec8+ during the mitotic cell cycle. This was achieved by swapping their promoter regions so that each would be ectopically expressed both at physiological levels and at the appropriate time in the cell cycle. Replacing Rad21p with Rec8p during the mitotic cell cycle was found to rescue the lethal effect of a rad21+ deletion with cells having wild type morphology. No S phase delay was detected in these cells though a ~5% increased division time was seen. This rescue was also accompanied with a ~30% loss of viability, and a proportion of cells exhibited the lethal rad21 null phenotype. 111 In the absence of Rec8p cells underwent aberrant meioses with abnormal chromosome segregation, reduced spore viability and impaired ascospore development. When Rec8p was replaced with Rad21p, although aberrant meiotic divisions occurred, 66% of asci contained four DNA nuclei in comparison to 51 % for rec8 null meioses. Furthermore, ascospores developed normally and spore viability was improved by ~30% when compared to rec8 null. No delay in S phase progression was detected in cells lacking Rec8p, while expressing Rad21 p in place of Rec8p also did not alter S phase timing. These data demonstrate that the essential mitotic function of Rad21 p can be partially complemented by the meiotic cohesin Rec8p. Conversely, the role ofRec8p in ascospore development appeared to be fully complemented by the mitotic cohesin Rad21 p. Taken together, the findings of this study indicate that, while S. pombe Rad21p and Rec8p cohesins have specialised mitotic and meiotic roles, the functional overlap between them is greater than previously described. They also suggest a novel role for Rad21 p in septation and/ or ascospore development.

Methionine--lyase (MGL) from Trichomonas vaginalis has been crystallised and the structure solved by molecular replacement at 2.2 A. The enzyme has a similar overall secondary structure arrangement as the search model, cystathionine -lyase from Escherichia coli, but differs in the active site. In addition to the holoenzyme, the structure of the enzyme in complex with the acetylenic suicide inhibitor L-propargylglycine has also been solved. This has revealed the mechanism of inhibition by this compound. It has also provided insights into the catalytic mechanism of the enzyme and allowed the postulation of a scheme for its action. Particularly, this predicts core roles for active site tyrosine and cysteine residues (111 and 113, respectively). To test the hypothesised mechanism of catalysis, seven site-directed mutants were produced. The activities of the mutants were determined and the structure solved for one of the most interesting. The resulting data confirm the mode of action of L-propargylglycine and also reveal a proton relay which activates the important active site cysteine which mediates much of the enzyme activity. This mechanism is substantially different from that proposed for other members of the same family of enzymes and explains the substrate specificity of MGL. The structure also reveals a potential role for the enzyme. The active site cysteine is positioned ideally to form an internal persulphide with a sulphur atom released from the substrate. This implies a possible role for the enzyme in iron-sulphur cluster formation in an analogous fashion to the enzyme, cysteine desulphurase. Cultivation of the parasite in medium supplemented with L-cysteine has revealed reduced expression of the enzyme. This is the first demonstration of part of the de novo synthesis of cysteine in the T. vaginalis.

No description available.