The Mtr (Multiple transferable resistance) transport system in Neisseria gonorrhoeae was found to confer the resistance of gonococci to penicillin and structural diverse hydrophobic agents (HAs), such as drugs, dyes, detergents and host-derived compounds (fatty acids and bile salts), as well as some cationic antimicrobial peptides. The mtr operon encodes an energy-dependant efflux pump system and this MtrC-MtrD-MtrE system is negatively regulated by the transcriptional repressor MtrR. The first part of this thesis presents insights into a structural study of the two membrane components, the inner membrane protein MtrD and the outer membrane protein MtrE. MtrD and MtrE have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Unfortunately, due to the difficulty of membrane protein crystallography, the best resolution of the MtrE crystals was only 8A and of the MtrD crystals was 20Å, so the structures of both MtrE and MtrD haven't been solved yet. However, this preliminary X-ray analysis leads to the possibility of solving these structures in the near future. In the mean time, using pull-down assay, growth curve analyses and ITC( Isothermal Titration Calorimetry), we established that the antiporter MtrD, the adaptor MtrC and the outer membrane protein MtrE form a contiguous complex. Assembly of the pump is constitutive, even in absence of substrate. Without the assistance of MtrC, MtrD was unable to export its substrate, Nafcillin, and the efficiency of this assembly could be enhanced by the presence of the outer membrane protein MtrE. MtrD could interact with MtrE independently in pull-down assays, showing they can form a complex even in the absence of MtrC. This behaviour is consistent with the increased hypersensitivity to Vancomycin of the recombinant strain expressing MtrE-MtrD, which indicated that MtrE could be opened by MtrD in the presence of the substrate. However, the interaction between them was not detected by ITC, suggesting this interaction is weak or energetically unfavourable. The a-helical hairpin domain of MtrC was also over expressed and purified to test for its interaction with MtrE or MlrD. Fascinatingly, the cross-linked hairpin formed a hexamer and AFM (Atomic Force Microscopy) studies revealed it arranged into a cylindrical structure. In addition, ITC studies revealed that the hairpin domain could bind to both MtrE and MtrD, suggesting that MtrC might form a channel, one end of which interacts with MtrD and the other with MtrE. Growth curves also showed that the periplasmic hairpin domain could enhance the transport activity of MtrCDE, but couldn't activate the transport of MlrD. indicating it probably works by stabilizeing the open form of MtrE. The expression of mtrCDE is believed to be under the control of a TetR-type transcriptional regulator repressor, mtrR. MtrR foims a dimer but with presence of dsDNA, it forms a tetramer. Fragments of the antimicrobial polypeptide LL-37 were synthesized and tested by titrating to MtrR by ITC. The C-tenninal of LL-37(29-37) is the part that binds to MtrR and N-terminal (I-II) is not. Interestingly, interaction of MtrR with Penicillin G demonstrated for first time that MtrR might work as a ß-lactamase; with its enzyme activity reduced after mutanting His l05 to Tyr, which was reported to be found naturally in penicillin sensitive N, gonorrhoeae strains.

Bacterial contamination and persistent infection is a common cause of impaired wound healing. Generally, non-healing wounds display similar physiological features with regards to mixed bacterial flora, ischemia and production of exudate. The application of topical, broad spectrum antimicrobial compounds embedded in absorbent dressings has been shown to control bioburden and improve healing. Lyophilised, biopolymeric antimicrobial wafers can offer a contemporary, user-friendly, self-adhesive and effective approach for the management of suppuration and polybacterial contamination in a wide range of non-healing wounds. Cohesive, non-friable, porous, disc shape wafers were successfully produced with sodium alginate (SA) (18.17 ± 0.70 Pa.s), guar gum (GG) (82.21 ± 5.41 Pa.s; 95.87 ± 2.31 Pa), xanthan gum (XG) (2.86 ± 0.12 Pa.s; 23.61 ± 0.68 Pa), karaya gum (KAG) (12.89 ± 0.93 Pa.s) and an original gel consisting of a blend of a synergistic SA-KAG (7.75 ± 0.64 Pa.s; 86.34 ± 5.19 Pa) (1:1 ratio). Clinical concentrations of the broad spectrum, topical, antimicrobial compounds, neomycin sulphate (0.5 % w/v NS), chlorhexidine digluconate (0.5 % v/v CHD), povidone iodine (1.0 % v/v PVP-I) and silver sulfadiazine (1.0 % w/v SS) were mixed with compatible biopolymers and appeared to alter the rheological properties of the biopolymers. Rheological analysis of pre-lyophilised gels was undertaken to quantify the flow properties of the gels. The necessity of producing sterile wafers was investigated by exposing all biopolymer-antimicrobial combinations to 25 and 40 kGy of gamma irradiation. Gamma-rays caused total degradation of GG, KAG, SA and SA-KAG, while XG appeared to withstand irradiation. A novel free standing dissolution raft (FSDR) was designed and used to quantify the CHD released from both gels and wafers. CHD released from wafers ranged from 3.5 ± 0.01 to 17.4 ± 0.39 %. Gels and wafers released CHD in a sustained manner and the release profile of wafers was similar to the respective gels, with the exception of GG. Neither gels nor wafers released 100 % of the incorporated antimicrobial indicating that drug-polymer interactions governed the general performance of antimicrobial wafers, in terms of adhesion, expansion ratio (ER), inhibition ratio (IR), water uptake capacity (WUC) and antimicrobial delivery. Molecular modelling studies undertaken for KAG-antimicrobial complexes demonstrated an unusual ‘Z-shape’ geometry for cationic CHD. The charge and geometry of CHD was plausibly responsible for the antimicrobial’s entrapment within biopolymeric networks. The efficacy of antimicrobial wafers was demonstrated in vitro under simulated conditions of an exuding wound using modified disc diffusion and an original antimicrobial diffusion cell (ADC). All wafers were effective in vitro against common chronic wound pathogens of such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), E. coli and P. aeruginosa. Antimicrobial activity depended on the sensitivity of the microorganisms to a specific antimicrobial compound and the presence of organic material. Data obtained demonstrated that the presence of protein (BSA) in the pseudo-exudate inhibited the antimicrobial activity of CHD and PVP-I, while enhancing the antimicrobial activity of SS and NS against MRSA. The general findings summarised in this thesis conclude that factors such as protein content, electrolyte content and pH of exudate play a key role in the efficacy of self-adhesive, absorbent formulations intended for the topical delivery of antimicrobial compounds to non-healing, infected wounds. Drug-polymer interactions developed between biopolymers and incorporated antimicrobial compounds have a profound effect on the general performance of lyophilised antimicrobial wafers.

The aim of this project was to investigate the role of lectin pathway of complement activation in the innate immune defence against S. pneumoniae infection and polymicrobial septic peritonitis. In this study the only available model of total lectin pathway deficiency was used, a gene targeted mouse line deficient of the lectin pathway effector enzyme MASP-2. MASP-2 deficiency increases the susceptibility of mice to S. pneumoniae infection and MASP-2 deficient mice showed a significantly higher bacterial load in blood and in lung tissues after intra nasal challenge with S. pneumoniae when compared to their wild type littermates. The MASP-2 deficient mice showed also a significantly higher rate of mortality when compared to the control wild type littermates after S. pneumoniae infection. The failure of the MASP-2 deficient mice to clear the infection is due to an impaired C3 deposition on the surface of S. pneumoniae and hence impaired opsonophagocytosis. The delayed inflammatory response of MASP-2 deficient mice may be also another factor that results in their inability to clear the infection. In the CLP model of poly-microbial peritonitis, the MASP-2 deficient mice showed no significantly increased in mortality after CLP when compared to their MASP-2 sufficient littermates. Bacterial clearance however was significantly reduced in peritoneal lavage of MASP-2 deficient mice. Significant differences in the cytokine expression profiles between MASP-2 deficient and MASP-2 sufficient animals was also observed with TNF-α and IL-1β expression being significantly reduced in MASP-2 deficient animals following CLP. We conclude that MASP-2 deficiency compromises bacterial clearance, but limits the inflammatory response to septic peritonitis considerably, thus leading to a relative reduction of the inflammation driven mortality during septic shock. The finding that the deficiency of MASP-2 may lead to a reduced inflammatory response during sepsis in CLP model and to the protection of mice from the lethal effect of a TNF-α driven severe inflammatory response has prompted the idea to generate antibodies against human and murine MASP-2 that could deplete MASP-2 and transiently inhibit lectin pathway functional activity. These antibodies could be used as therapeutic intervention during sepsis and septic shock. In addition, these antibodies may also serve as therapeutic agents to limit lectin pathway mediated ischaemia/reperfusion injury following myocardial infarction and other forms of ischemic diseases.

Chronic kidney disease is a progressive condition resulting in morbidity and mortality. Lost kidney function can be replaced by transplantation or dialysis (haemodialysis or peritoneal dialysis). Although peritoneal dialysis is simple and cheap, there are two major problems: protein loss and peritonitis. Despite protein supplements and aseptic technique, little is known about the correlation between protein loss and infection. The overall aim of this project was to characterise dialysate protein profiles of patients and investigate factors that might increase infection e.g. catecholamines. Protein profiles of daytime and overnight dwells were investigated using proteomic techniques: the total number of proteins in the dialysates was similar. Sequence analysis showed these were plasma proteins: e.g. albumin, fibrin-beta, IgG, complement C3 and transferrin. Three proteins not reported previously were detected: ceruloplasmin, albumin-myristate-azapropazone complex, and albumin-myristate-tri-iodobenzoate complex. Protein concentrations in overnight dwells were higher than in daytime samples. Differences were also found in other parameters, e.g. pH, glucose, and total iron. Direct measurements of ability of S. epidermidis to grow in peritoneal dialysates showed variation between patients. Addition of iron or catecholamines significantly increased growth, indicating dialysates were iron-limited. Analysis of dialysate exposure on S. epidermidis virulence showed that biofilm formation and haemolytic toxin production were significantly stimulated, but that stimulation varied between patients. One-year follow-up peritoneal dialysates showed that higher protein concentration occurred compared with initial dialysates. This may explain the apparent tendency of the one-year dialysate to sustain greater S.epidermidis growth. A correlation was found between dialysate protein concentration, and degree of growth stimulation/virulence. This suggests that a biomarker for infection risk in these patients could be protein concentration in their dialysate. Suggestions are made as to how this might be implemented into patient care.

Iron acquisition is vital for intestinal colonisation by Campylobacter jejuni. Characterisation of a number of iron uptake systems has occurred recently, allowing advancement in the understanding of the iron sources that C. jejuni utilises and how this occurs; however, the molecular basis of iron uptake from host iron-binding glycoproteins, the transferrins, is not known. The research presented here confirms that C. jejuni can use iron from the transferrins for growth and further characterises this process and the factors involved. Iron uptake from the transferrins requires proximity and appears to be receptor specific. Binding of lactoferrin to the cell surface is iron-responsive. Cj0178, a protein similar to TonB-dependent receptors, is required and the involvement of the enterochelin outer membrane receptor protein CfrA, FeoB, the ferrous iron inner membrane transporter, and the ABC transporter system Cj0175c-Cj0173c was also indicated. Less lactoferrin bound to cells without Cj0178 and complementation of the cj0178 mutation was successful. A role for Cj0178 in the uptake of haem was not demonstrated. Regulation of the genes cj0176c-cj0173c and cj0177-tonB1 was shown to require Fur and promoter activity levels increased under iron-restriction. The presence of two Fur-boxes indicated separate regulation of the operons. The catecholamine stress hormone noradrenaline augmented the growth of C. jejuni in the absence and presence of iron and in the presence of the transferrins, but was non-essential. A model is proposed of how transferrin-bound iron is used by C. jejuni. The process appears to be novel, involving a number of systems, but further work is required to confirm how they interact. The system through which noradrenaline may supply iron was investigated; however the mechanisms involved require further characterisation. The involvement of Cj0178 implies that the uptake of transferrin-derived iron is important for successful colonisation, which is vital for establishing an infection.

Haptoglobin is a positive acute-phase serum protein. It is upregulated during infection and is a valuable marker for many inflammatory-related diseases. Its primary known function is to eliminate haemoglobin from plasma to prevent loss of iron through the kidneys, thereby protecting the kidneys from damage and sequestering iron, a key target for invading bacteria. In this thesis, I show that haptoglobin also interacts with lipoteichoic acid (LTA) of Staphylococcus aureus, an important virulence factor. Bacteria are subsequently eliminated by neutrophil-, monocyte- and macrophage-mediated killing. S. aureus is an extremely common human pathogen associated with various diseases including septic arthritis and toxic-shock syndrome. A current major concern is the emergence of multidrug-resistant strains such as methicillin-resistant S. aureus (MRSA) that cause severe infections in hospitals and in the community. To increase our understanding of the defence mechanisms employed by the host against S. aureus, LTA was used to capture LTA-binding proteins from human serum and these were identified using a proteomics-based strategy. A variety of targets were captured including complement proteins, lipid-transport proteins, coagulation-cascade proteins and acute-phase proteins. Haptoglobin was selected for further study, because its expression is known to be upregulated in response to infection, and it interacts with phagocytic cells to stimulate phagocytosis. ELISA and column chromatography studies confirmed that haptoglobin binds LTA directly as well as S. aureus and suggest that the α-chain of haptoglobin is critical for this interaction. The role of haptoglobin in promoting the killing of S. aureus by neutrophils, monocytes and macrophages was evaluated by comparing in vitro bacterial-killing using serum depleted of haptoglobin. For each cell type haptoglobin was found to be a key mediator in phagocyte-mediated bacterial killing. This work highlights an important new role for haptoglobin in immune defence, and explains its upregulation during infection.

The nasopharynx is covered by mucin. Mucin is a glycoprotein and the carbohydrate moieties are potential fermentable substrates for Streptococcus pneumoniae. Previous in vitro studies showed that S. pneumoniae can grow on mucin. This study was undertaken to investigate how S. pneumoniae degrades the mucin carbohydrates to mono and disaccharides for subsequent fermentation. In silico search of pneumococcal genome identified fourteen putative glycosidases. Pneumococcal mutants of each were made and tested for growth in defined medium with mucin as the only source of carbon. Of these, two genes SPD0065 and SPD0247 were chosen for further study. Consequently two novel glycosidases were described, β-galactosidase (BgaC), encoded by gene SPD0065 and a 6-phospho-β-glucosidase (BglA) encoded by gene SPD0247. The knocked-out mutants SPD0065M and SPD0247M could not grow in Sicard’s defined medium supplemented with mucin and exhibited decreased enzymatic activity when compared to the wild type D39. Since gene SPD0562 had been previously identified as encoding a β-galactosidase (BgaA), the relative contribution of BgaA and BgaC to total β-galactosidase activity was investigated by introducing mutations in these genes individually and together. Mutation in the individual genes resulted in significant decrease in the enzymatic activity but the double mutation did not totally abolished activity. BgaC had specificity for galactose (β1,3)-N-acetylgalactosamine. Furthermore, BgaC released galactose from desialylated fetuin. The expression of SPD0065 and SPD0247 in S. pneumoniae grown in mucin containing medium or harvested from tissues from infected animals was significantly up-regulated compared to growth in glucose containing medium. When the mutants were tested in vivo, it was noted that SPD0065M had attenuated growth in the nasopharynx and SPD0247M in the lungs. In this study was demonstrated that BgaC has a role in the sequential deglycosylation of host glycoproteins and that BglA is involved in the further degradation of mucin-derived sugars.

The Mycobacterium tuberculosis genome codes for 11 pairs of CFP-10/ESAT-6 proteins (Esx family) as well as the apparatus required for secretion of these proteins. The core machinery for the secretion of ESAT-6 and CFP-10 is encoded by their surrounding genes. Recent studies also identified a distant region, the Rv3612c-Rv3616c operon, which is essential for the CFP-10/ESAT-6 secretion. Constructs carrying Rv3613c, Rv3614c, Rv3615c and Rv3616c coding regions were produced and used to express the corresponding proteins. However, only Rv3614c and Rv3615c were expressed using an E. coli-based expression system. Analysis using a range of spectroscopic techniques on the purified proteins revealed that both Rv3615c and Rv3614c contain stable secondary structure, but little if any stable tertiary structure and exist in a molten globule-like state. This suggests the proteins probably undergo folding upon binding with possible functional partners. Yeast-two hybrid studies showed no intermolecular interaction between the proteins encoded by the Rv3616c-Rv3612c operon, perhaps suggesting the formation of a higher order multi-protein complex. Together with CFP-10 and ESAT-6, Rv0287 and Rv0288 are the members of the Esx family which are clearly implicated in M. tuberculosis pathogenesis. The expression vectors carrying Rv0287 and Rv0288 coding regions were constructed and used to express the proteins. Analysis using a range of spectroscopic techniques on the purified proteins showed that Rv0288 contains up to 30 % helical secondary structure, but little if any stable tertiary structure and exists in a molten globule-like state. In contrast, Rv0287 has been found to form an unstructured, random coil polypeptide. The work reported here also shows that Rv0287 and Rv0288 form a tight 1:1 complex which is predominantly helical. Furthermore, the Rv0287-Rv0288 complex was found to be significantly more stable to thermal denaturation than CFP-10-ESAT-6. The high resolution solution structure reported here reveals that both proteins, Rv0287 and Rv0288, adopt an elongated helix-turn-helix hairpin structure in which the proteins lie antiparallel to each other, forming a stable four helix bundle. Comparison of the CFP-10-ESAT-6 and Rv0287-Rv0288 complexes also revealed that the overall backbone fold for the complexes is very similar although they display significantly different surface features.

Listeria monocytogenes causes a potentially deadly disease of man and is a major source of contamination in food industry. The mechanism of survival and persistence of L. monocytogenes in the environment is not fully known. The present study investigates the possible role of Acanthamoebae in the survival and persistence of L. monocytogenes in the environment. This was achieved through experiments that brings together the two organisms in a co-culture and then examined ability of bacteria to survive in the presence of amoeba, inside amoeba trophozoites and in their cysts. The effects of intracellular survival on L. monocytogenes’ morphology, ability to form biofilms and respond to biocides inside and outside the cysts were also examined. In summary, L. monocytogenes Scott A was found to survive and grow in Acanthamoeba over 72 h. In addition, exposure of bacteria to manganese enhanced intracellular growth and survival of L. monocytogenes within Acanthamoeba. While L. monocytogenes Scott A survived and replicated in A. castellanii, it barely survived in A. polyphaga and never survived in A. culbertsoni. None of the other strains of L. monocytogenes tested were able to survive in Acanthamoeba. Autophagy, which was previously shown to aid survival of L. monocytogenes in macrophages, was also found contribute to survival within Acanthamoeba. In addition to surviving within A. castellanii trophozoites, L. monocytogenes Scott A also survived encystment of the host amoeba. L. monocytogenes sequestered in cysts were protected from high level of chlorine that is lethal to free bacteria. In addition, L. monocytogenes recovered from cysts were predominantly filamentous and demonstrated enhanced ability to form biofilm and also exhibited increased resistance to a disinfectant and some antibiotics that are normally used in treatment of listerial infections. The observations suggest that A.castellanii could potentially contribute to the survival, dissemination, and persistence of bacteria in the environment.

Ten sub-fragments (N-, P-, C-, NS-, NP-, NPC-, S-, SP-, SPC- and PC-domains) of recombinant S. mansoni calreticulin (SM-CRT), a highly immunogenic antigen, were expressed and purified. A cDNA library of adult S. mansoni was used as a template to amplify the coding sequences of these fragments which were expressed in bacterial expression system using the vector pRSETB. SM-CRT sub-fragments (except those with N-terminal domain) were shown to bind calcium using “Stains-all” stain or interact with 45Ca2. With the exception of the P-domain, SM-CRT sub-fragments bound and inhibited C1q-dependent haemolysis in vitro. In S. mansoni infected mice, specific antibodies against the carboxy-terminal part of SM-CRT appeared 46 days p.i., while antibodies against the N-terminal part of SM-CRT were detectable at 59 days p.i.. Cercarial Transformation Fluid (CTF) and Solube Egg Antigens (SEA) specific antibodies were detectable at day 12 p.i.. Use for diagnosis of S. mansoni in humans, CTF and SEA showed 89.7% sensitivity, while the PC-domain of SM-CRT showed sensitivity of 71.1% (the highest SM-CRT sub-fragment sensitivity). When testing the cross-reactivity with sera drawn from patients with other diseases, SM-CRT N-domain showed the lowest degree of cross-reactivity. Analysing non-endemic controls, a specificity >95% was shown for all of the antigens used. When using endemic controls, SM-CRT N-domain revealed the highest degree of specificity (94.7%), while SEA revealed 26.3% specificity and CTF showed 68.4% specificity. BALB/c mice immunised with recombinant SM-CRT achieved a 49.9% (P >0.05) reduction in Schistosoma adult worm numbers (on exposure to S. mansoni cercariae), comparing to the non-immunised mice. The eggs numbers in the liver decreased by 41.8% in the immunised mouse group with a non-significant statistical difference. The immunised mice responded to SM-CRT immunisation by producing specific IgG1 and IgG2a, reflecting a Th1/Th2 with a predominance of Th2 immune response profile.