HIV-1 hijacks cellular trafficking pathways in order to transport newly synthesised viral subunits, such as the structural polyprotein Gag, to virion assembly sites. HIV-1 Gag has been reported to interact with clathrin adaptor proteins (AP) 1-3, which function to assist clathrin mediated cargo transport. AP-1 and AP-3 assist viral particle release, whereas AP-2 inhibits viral particle release. How these APs facilitate HIV protein trafficking and particle assembly, and the consequences of HIV infection on the function of these trafficking pathways, has not been elucidated. The interaction of these pathways with the closely related virus, HIV-2, has also not been defined. Confocal immunofluorescence microscopy revealed HIV-1 and HIV-2 Gag have different intracellular localisations; HIV-1 displayed a mainly diffuse cytoplasmic location, whereas HIV-2 predominantly localised to intracellular compartments. Further investigation through microscopy found that HIV-2, unlike HIV-1, heavily colocalised with AP-3, which functions in endosomal cargo trafficking. siRNA mediated knockdown of the APs also deduced that HIV-2 is more dependent on AP-3 than AP-1 to facilitate particle assembly, whereas HIV-1 had similar dependencies on both. HIV-2 also strongly colocalised with, and redistributed clathrin. Initial investigation of adaptor protein dependent trafficking pathways deduced that HIV-2 interrupted the trafficking of Diphtheria toxin, which traffics in an AP-3 dependent manner through endosomal compartments, to a greater extent than HIV-1. Taken together, these results suggested that these viruses adopt different pathways to reach assembly sites, and that particle assembly likely occurs at different localisations between these viruses; the plasma membrane, via an endosomal intermediate, for HIV-1, and predominantly intracellular compartments for HIV-2. Furthermore, it was found that the matrix domain of Gag, which contains AP binding sites, was not sufficient to drive this different Gag localisation in mutant chimeric viruses. Finally, the interruption of AP dependent trafficking pathways was investigated in astrocytes. HIV-1 displayed a striking perturbation of vesicle endocytosis events, indicating a global negative effect on these AP dependent pathways and suggesting a putative mechanism for the development of neurological symptoms, in particular short-term memory loss, during HIV infection.

Synechocystis sp. pee 6803 is a unicellular, freshwater cyanobacterium. Its dependence upon light to support its photoautotrophic lifestyle increases the importance of environmental sensing mechanisms to be able to maximise lightharvesting whilst avoiding the harmful effects of light-mediated cell damage. The sequencing of the Synechocystis sp. pee 6803 genome in 1996 now allows the identification of genes that encode putative proteins with roles in sensing the environment. Two such open reading frames, slr1212 and slrI213, were identified from the genome following computer analysis of the protein sequences. These two proteins encode a putative two-component signal transduction system with a role in sensing the environment. SIr1212 possesses homology to (i) the binding domain of ethylene receptors of higher plants, (ii) P AS/P AC domains, potentially involved in ligand binding and protein dimerisation, (iii) GAF domains, which contain the chromophore binding region of plant phytochromes, and (iv) histidine kinases of two-component signal transduction systems. SIr1213 possesses homology to characterised response regulators, and contains a helix-turn-helix DNA binding motif. This study set out to characterise a physiological role for these enigmatic proteins by analysing interposon mutants. Single and double interposon mutants were generated in these open reading frames. Growth of these mutants was unaffected in different light qualities, but SIr1212 was shown to be involved in the acclimation of the Synechocystis sp. pee 6803 cells to high light irradiance as analysed by 77K fluorescence spectroscopy, which also indicated possible structural alterations in PSI reaction centres ofORF slr1212 mutants. Using laser photo acoustic spectroscopy, it was shown that Synechocystis sp. pee 6803 could release ethylene following incubation with Ace suggesting a possible ethylene biosynthetic route, though genome analysis revealed no obvious homologues of ACC oxidase, an enzyme required for conversion of Ace to ethylene in vascular plants. It is hypothesised that an ethylene signalling mechanism may be present that regulates cell responses to non-specific stress. Site-directed mutagenesis of SIr1213 that caused constitutive activation of the protein had a lethal effect in Synechocystis sp. pee 6803 cells. The mutation consisted of a substitution of the conserved aspartate residue with glutamate, thus mimicking the phosphorylated state of the protein. In summary, SIr 1212 has a role in the acclimation of cells to high light irradiance and binds ethylene, and may act in conjunction with Slr1213 to modulate these responses.

Molecular biology has provided new opportunities to understand better the functioning of the immune system and to exploit this information for the construction of specific antibodies against a wide variety of antigens including the pathogens of humans and animals. In spite of the economic importance of cattle, many aspects of the immunology of this animal remain uncharacterised and tools to understand better bovine infections are lacking. This project has addressed aspects of both issues. The bovine immunoglobulin (Ig) system resembles that of other domesticated mammals in some respects, but other properties (eg the length of the third antigen-binding region of the heavy chain) appear unique. The first area for investigation in this project was to characterise the bovine JH locus and to understand why Ig rearrangement apparently favours a single JH segment. PCR was used to recover JH sequence from genomic DNA, either from non-lymphoid tissues or lambda vectors isolated and studied by other investigators. A region of 3200 bp was characterised which included the DQ52 segment, 6 JH segments and the heavy chain enhancer. The bovine DQ52 sequence is longer than those of other species and differs in sequence from a common consensus. For the most part, the JH locus is homologous to that of the sheep. The sixth JH segment identified appears to undergo rearrangement and is expressed in a minority of cattle antibodies. However, none of the segments carried the sequence which is most commonly expressed in bovine Ig. To identify which segment participates in this process, sequence was recovered from the rearranged genomic DNA of isolated bovine B cells using PCR with primers against VH and JH regions. This implicated the rearrangement of the fourth JH segment in the formation of bovine Igs but as the sequence differed between germline and rearranged copies, it appears that a non-conventional process operates in cattle. It is proposed that a gene conversion event or modified rearrangement process introduces sequence to form the fourth framework region of bovine Ig which does not exist at the JH locus in the germline. The mechanism of this modification requires more investigation. The second part of this project aimed to construct a library of recombinant bovine Fab antibodies from the Ig repertoire of a calf vaccinated against Mannheimia haemolytica (previously named Pasteurella haemolyticd).

No description available.

Lysosomes are essential cellular organelles known to be the main site for the breakdown and recycling of endocytosed macromolecules within cells. However, our knowledge of lysosomal function has changed considerably over the last decade with the lysosome now being a recognized Ca2+ signaling organelle with additional roles in plasma membrane repair, clearance of defective organelles and mediating cell death as well as established roles in clearing infection. Our ability to study these unique organelles has however been stymied by a dearth of good techniques for the purification of functional lysosomes not contaminated by other organelles.

Non-typhoidal serotypes of Salmonella enterica remain important foodborne pathogens worldwide and the frequent emergence of epidemic strains in food-producing animals is a risk to public health. In recent years, Salmonella 4,[5],12:i:- isolates expressing only the first phase of the two flagellar antigens (FliC) have emerged and increased in prevalence worldwide. In Europe, the majority of 4,[5],12:i:- isolates belong to phage types DT193 and DT120 of Salmonella Typhimurium and pigs have been identified as the reservoir species. In this study, a number of pig-derived monophasic (4,[5],12:i:-) and biphasic DT193 isolates were characterised for phenotypes relating to virulence, to improve understanding of their ecological success. Additionally, their ability to invade a porcine intestinal epithelial cell line (IPEC-1) and stimulate a pro-inflammatory response from the host cells was investigated, to determine the infection biology of these strains. Monophasic and biphasic isolates were compared throughout, with the aim of identifying an explanation for the selective pressure behind the loss of flagellar phase variation. It was found that the panel of DT193 isolates possessed a heterogeneous repertoire of virulence-related phenotypes and genotypes. A number of isolates demonstrated the ability to form biofilms, however the optimum temperature and time for expression of this phenotype varied among the isolates, which may have implications for bacterial survival in the environment and in the host. Another variation was in the presence of sopE, the gene for an important SPI-1 secreted effector protein associated with virulence, in the genomes of the isolates. The 4,[5],12:i:- isolates exhibited comparable adhesion and invasion to that of the virulent S. Typhimurium isolate 4/74, suggesting that these strains may be capable of colonising the small intestine of pigs in vivo. Infection with 4,[5],12:i:- and biphasic DT193 isolates resulted in approximately the same level of TLR-5 (a flagellin receptor) and IL-8 (a pro-inflammatory chemokine) mRNA upregulation, except in the case of one 4,5,12:i:- isolate that elicited significantly greater upregulation of these genes. The monophasic variants also elicited similar levels of caspase activation and cytotoxicity to the phase variable DT193 isolates. These results suggest that monophasic Salmonella display a similar infection biology to phase variable S. Typhimurium during colonisation of the porcine intestinal tract. Consequently, failure of 4,[5],12:i:- isolates to express a second phase of flagellar antigen (FljB) is unlikely to hamper their pathogenicity during colonisation of the porcine intestinal tract in vivo.

Streptococcus pneumoniae (pneumococcus) is among the commensal bacteria species that asymptomatically colonise the human nasopharynx. However, this species may occasionally invade the host’s sterile sites (lungs, blood, middle of the ear and cerebral spinal fluid) and cause severe life-threatening disease, such as pneumonia, meningitis, acute otitis media and bacteraemia (bloodstream infection). Invasive pneumococcal disease (IPD) is especially common among children under the age of five, the elderly (>65 years) and among individuals with compromised immunity, such as asplenia and among individuals with immunosuppressive illness, particularly those infected with HIV. Each year, IPD accounts for one fifth of these deaths and more than 150 million episodes of pneumococcal pneumonia in children under five years, most of which occur in sub-Saharan Africa (SSA). Although the population genetics and evolutionary biology of pneumococci is well understood, it still remains unclear why some pneumococci strains are associated with meningitis more than others, and whether there are virulence factors that are associated with the tendency to cause meningitis. This thesis explores the differences in genetic diversity among meningitis and bacteraemia associated (blood sepsis) pneumococci to establish whether such differences exist among invasive pneumococcal isolates collected from Malawian patients. It also examines the differences in likelihood and direction of genetic flux (gene acquisition or loss), which mainly occurs by recombination, among meningitisassociated and bacteraemia-associated pneumococcal isolates. Furthermore, the prevalence of the pneumococcal pilus, which greatly enhances initial pneumococcal adhesion at the V nasopharynx, thereby providing a colonisation advantage; and has been identified as a highly immunogenic potential vaccine candidate was examined for the period prior to the 13-valent pneumococcal conjugate vaccine (PCV13) introduction into the childhood immunisation programme of Malawi, in order assess the impact of PCV13 introduction on pilus prevalence. Various innovative, robust and high-throughput bioinformatics techniques, some of which were newly developed, were used to interrogate the genome sequences of these invasive pneumococcal disease isolates. Here, by comparing the differences in the composition of genomic antigen diversity among isolates associated with bacteraemia and meningitis, I provide some evidence from human disease that meningitis causing pneumococci have a distinct “core genome”, which encodes for both known and previously undescribed proteins that are likely to be essential for meningeal invasion and survival. Furthermore, I present evidence suggesting that meningitisassociated pneumococci undergo less genetic flux and have a more conserved genome than those associated with bacteraemia. I also show for the first time in SSA that there is a low prevalence of invasive piliated pneumococci (16.43%), and that these piliated pneumococci are all covered by the PCV13. Interestingly, it was noted that most of the piliated pneumococci were resistant to cotrimoxazole. However, this antibiotic resistance can also be attributed to the widespread use of sulphadoxine pyrimethamine (Fansidar TM), an antimalarial drug that also has the same targets as cotrimoxazole (that is the dihydrofolate reductase and dihydropteroate synthase genes) that has resulted in drug resistance conferring mutations in Plasmodium falciparum, and more conclusive studies that include carriage isolate datasets are required.

The major fraction of microbial communities cannot be cultivated by artificial means in the laboratory. In order to access the full diversity of microbial life in the open environment it is necessary to employ culture independent methods. Molecular biology and now metagenomics have enabled the phylogenetic and functional investigation of microbial communities without isolation and cultivation of organisms and has led to a new appreciation of the breadth of diversity of microbes on Earth and to the discovery and characterisation of new enzymes. Here, molecular biological techniques have been applied to the study of microbial communities specifically in anaerobic environments and with an emphasis on those involved in the primary degradation of plant cellulosic biomass. Quantitative PCR was used to assess the presence of cellulolytic bacteria both in landfill leachate and specifically in association with cotton cellulose “baits” maintained in leachate microcosms. Lineages of clostridia previously associated with cellulose degrading strains were detected in all five of the landfill leachate samples, and Fibrobacter spp. were detected at low abundance (2.3% of total bacteria) in one sample. Clostridia Group III and Fibrobacter spp. were enriched on the surface of a bait (17% and 29% of total bacteria, respectively) that was rapidly degraded by the colonising community and were present in low abundance (< 1%) and absent, respectively, on another colonised by a community which did not exhibit any degradation of the cellulose. The observed correlation between high levels of cellulose degradation and presence Fibrobacter spp. demonstrates a cellulolytic role outside of the gut environment for these organisms the first time. A metatranscriptome was prepared from a set of cotton cellulose baits maintained in a lake sediment for 2-8 weeks, and Illumina sequencing was used to generated ca. 7 million paired-end reads. Just under one million putative protein coding sequences were identified and of these, MEGAN analysis determined that 40% had no blast hit to the NCBI NR database suggesting that a large number of unknown sequences were present. Analysis of this metatranscriptome and a metagenome produced from the same site revealed that bacteria accounted for 75% of the protein coding sequences and 97% of the metagenome. Genes with matches to cellulolytic lineages of clostridia were found to be present and Fibrobacter sequences were also detected in both of these datasets further demonstrating their presence in the wider environment as probable cellulose degraders ORF prediction and HMM searching were used to search for expressed cellulases in the metatranscriptome and identified 503 sequences with high similarity to glycoside hydrolase protein families, representing carbohydrate active enzymes with possible cellulolytic activity. Of these 112 were also found to have representatives in the metagenome with 100% sequence similarity. All of these sequences had a low level of identity to entries in the NCBI NR database indicating the discovery of previously unknown genes. A fosmid library was produced from the same DNA used to generate the metatranscriptome and it is possible that full-length copies of the expressed genes identified in silico will have been captured. This fosmid library can be interrogated accordingly using probe and PCR primer sequences designed using the curated metatranscriptome dataset. In this way, potentially novel cellulases can be discovered for biochemical characterisation, genetic manipulation and biotechnological exploitation.

Enterococci are Gram-positive bacteria that inhabit the gastrointestinal tract of humans and animals as commensal flora. In recent years two species, Enterococcus faecalis and Enterococcus faecium, have become an increasing medical concern by virtue of their ability to gain and spread antibiotic resistance. In this study, genomes of vancomycin-resistant isolates of E. faecium from pig, chicken and calf were sequenced using 454 and PacBio platforms. The assembled genomes were annotated and compared with human E. faecium isolates to identify their repertoire of genes potentially associated with colonising each host. Phylogenomics of E. faecium was used to investigate the relationship between animal and human strains. The genomes of the chicken, pig and calf isolates differed in size (2.5 Mb to 3.3 Mb) with the size difference due to horizontally-acquired elements (mostly phage, transposons and insertion sequences); the chicken isolate genome contained five prophages. A mega-plasmid present in each of the sequenced E. faecium was revealed to be integrated into the genome of the chicken isolate. Comparison of the three genomes identified putative niche adaptation genes with a variety of proposed functions, particularly carbohydrate utilisation. Possible factors that explain E. faecium sub-populations, including clinical, commensal and animal isolate clades were examined. Use of the PhenoLink relationship tool to examine the E. faecium sub-populations identified that putative niche specific genes include carbohydrate utilisation genes and mobile genetic elements. Temperate bacteriophages are known to be important drivers of genome plasticity in E. faecium species. The diversity of prophages and their relationship between was investigated after locating 56 prophage elements containing integrase and lysin genes encoded in the 139 publicly available E. faecium genomes. Comparative analysis of these prophages identified eight sequence types, which differed in size and gene content. The prophage genomes comprised between 17 to 72 ORFs and their size ranged from 13.9 to 55.1 kb with 35% to 37.9% average G+C content. Based on alignment analyses of the major functional proteins encoded in the prophage genomes (integrase, terminase large subunit, tail protein and holin) each was assigned a sequence type. All of the prophage integrases were identified to be tyrosine (XerC) recombinases and many of their respective attP/attR sequences were identified. The mosaic nature of E. faecium prophage genome sequence types supports previous hypotheses that extensive genetic recombination drives chimeric phage types.

Antimicrobial lipids on skin are proposed to form a barrier against microbial colonisation. Skin lipids, such as unsaturated fatty acids and sphingosines, cause membrane permeabilisation and/or proton motive force disruption . These lipids may be crucial in determining the diversity and degree of staphylococcal skin colonisation. Specifically, antimicrobial lipids may inhibit skin colonisation by Staphylococcus aureus while permitting the growth of Staphylococcus epidermidis. Here it was shown that skin fatty acids sapienic acid and linoleic acid are more active against S. aureus than S. epidermidis. This supports a role for fatty acids in the prevention of S. aureus skin colonisation. The most anti-staphylococcal skin lipid tested was D-sphingosine; no differences in resistance levels between S. aureus and S. epidermidis to D-sphingosine were observed. The genetic response and basis for resistance to skin antimicrobial lipids of S. epidermidis and S. aureus was investigated using next generation sequencing. The transcriptomic response of both species to sapienic acid was determined using RNA-Seq. Additionally, S. epidermidis and S. aureus were passaged in sapienic acid or D-sphingosine. Isolates with increased lipid resistance after passaging were genome sequenced, and mutations associated with increased resistance were characterised. From these approaches, several genes and pathways potentially involved in the responses of both species to skin lipids became apparent. These components included cell wall biosynthesis, transport and production of small molecules, ammonia production, albumin binding proteins and putative lipid efflux pumps. Cellular components identified as specifically involved in S. aureus resistance to sapienic acid included capsule and staphyloxanthin biosynthesis. Cellular components involved specifically in S. epidermidis resistance to sapienic acid were also speculatively identified, though the functions of these components were not resolved. This study has increased our understanding of staphylococcal molecular interactions with host antimicrobial lipids, which could lead to applications in the design of novel antimicrobial compounds.