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The characterization of rodent cell mutants hypersensitive to UV light has led to the identification of at least 10 complementation groups all defective in some aspect of the first step in the excision repair of UV damaged DNA. The phenotypic properties of these mutants are thus of considerable importance to our understanding of DNA repair. In recent years five different excision repair cross complementing (ERCC) human genes have been isolated which correct the DNA repair deficiency in a number of Chinese Hamster Ovary (CHO) cell mutants and at least three of these genes also complement the repair deficiency in cells from patients suffering from xeroderma pigmentosum (XP), Cockayne syndrome (CS) and/or Trichothiodystrophy (TTD). Adenovirus (Ad) infection of rodent cells is generally semi-permissive and does not give rise to viral progeny, such that Ad reactivation in CHO cells has not previously been reported. This study utilizes the ability of CHO cells and human cells to replicate viral DNA in order to examine the reactivation of Ad in several CHO as well as human cell DNA repair mutants. Unirradiated and UV-irradiated suspensions of Ad were assayed for their ability to synthesize viral DNA following the infection of several CHO and human cell DNA repair mutants. The cell types examined included CHO cell mutants from complementation groups 1, 2, 3, 4, 5, 6, 9, 10 as well as human XP and tumour cells. The survival of viral DNA synthesis for UV-irradiated Ad was significantly reduced in several of the CHO and human cell mutants compared to that in normal cells. Cell mutants showing a reduced UV survival for this viral function included CHO cell mutants from complementation groups 1 to 6, XP cells and the 2 human tumour cell lines examined. This reduced host cell reactivation (HCR) for Ad indicates a reduced capacity for the repair of viral DNA in these cell types. DNA replication for unirradiated virus was also reduced for some of the mutants, especially the UV20 CHO cell mutant from complementation group 1, suggesting a deficiency for both DNA replication and repair in these cells. This study also used the recombinant viruses Ad5(denV) and Ad5(ERCC1) as vectors to examine the effect of the bacteriophage T 4 denV gene and the human ERCC1 gene on viral reactivation in the various cell mutants. UV survival of Ad5(denV) was increased compared to that of the control Ad5(LacZ) following infection of all the CHO and human cell types examined, indicating the denV gene product increases repair of Ad in both repair-proficient and repair-deficient cells. UV survival of Ad5(ERCC1) was increased compared to that of control Ad5(LacZ) following infection of the CHO mutant UV20 from complementation group 1, as well as all CHO cell types having normal HCR for AdS. However, UV survival of Ad5(ERCC1) was not increased compared to Ad5(LacZ) following infection of CHO mutants from complementation groups 2 to 6 and 10. These results support a specific complementation of the UV20 repair defect by ERCC1 and suggest that the human ERCC1 gene is more efficient than its hamster counterpart in repair-proficient CHO cells or that the ERCC 1 product is rate-limiting for the excision repair process in CHO cells. UV survival of Ad5(ERCC1) was also increased compared to Ad5(LacZ) in the normal human fibroblast cells and human tumour cells, but not in the XP (group D) cells. The kinetics of viral DNA synthesis and viral protein synthesis for unirradiated Ad5(denV) and Ad5(ERCC1) was also investigated following the infection of human and rodent cells. The deficiency in viral DNA synthesis and viral protein synthesis found for AdS(LacZ) following infection of rodent compared to human cells is partially complemented by either denV or ERCC1. The more marked deficiency in viral DNA synthesis of the UV20 CHO mutant was also complemented by either ERCC1 or denV, suggesting an ability of these genes to function in both repair and replication of viral DNA. / Thesis / Master of Science (MS)

There is an increasing incidence of antimicrobial-resistant organisms isolated from food and food products. Coupled with that rising incidence is increased media scrutiny and coverage of outbreaks of foodborne illnesses. Consequently, consumers increasingly demand safer food, and that the antimicrobial measures used be other than antimicrobial drugs. A possible solution is to use bacteriophages, or the purified holin and endolysin proteins that make them lethal and lytic, as antimicrobial food treatments or additives. The bacteriophage Felix O1 is a promising candidate for development as an anti-Salmonella food treatment. This dissertation describes the work done to determine if these proteins could be of value as bioremedial agents. Endolysin treatments of Gram negative bacteria require two agents: the lytic endolysin, and a second agent to permeabilize the outer membrane of the bacterium. The holin protein was proposed as an outer membrane permeabilization agent. Methods used to locate the holin gene included BLAST analysis, analysis of putative Felix O1 proteins for transmembrane domains, and examination of the lysin sequence for an N-terminal signal sequence. Analyses did not reveal a promising candidate. Cloning of rIIA as a potential holin was attempted without success. Results of various analyses are discussed, as are chemical alternatives to the use of purified holin as a permeabilization agent. The endolysin, LysO1, was successfully cloned and characterized. PHYRE analysis predicted that the enzyme structure is composed of α helices arranged into two lobes, with the active site in a cleft between them. The enzyme lysed all tested strains of Salmonella and a tested strain of the foodborne pathogen Escherichia coli. Campylobacter jejuni susceptibility remains ambiguous, and the enzyme had no effect on Listeria monocytogenes or Micrococcus luteus. LysO1 was most active at alkaline pH and low ionic strength; optimal activity was observed in 25 mM buffer at pH 10. If removed from frozen storage, the enzyme was most thermostable at 30 °C. Lytic activity was adversely affected by the presence of the divalent cations calcium, magnesium, and zinc, and by high ionic strength. Considerable time was devoted to development of the activity assay used to further characterize the enzyme, and details of those experiments are provided. Logical extensions of the research project, such as further characterization and testing needed to obtain government approval for widespread use of the treatment, and possible pursuit of treatment based on an enzyme derivative such as an antimicrobial peptide, are discussed. / Ph. D.

Staphylococcus aureus est l’un des principaux agents étiologiques des infections suppuratives superficielles et profondes ainsi que des syndromes liés à l’action de toxines. Paradoxalement, cette bactérie est un agent commensal qui est présent sur la peau ainsi que dans les cavités nasales notamment. Cela permet de considérer cette bactérie comme un organisme colonisant commensale. Les bases génétiques expliquant la différence entre une bactérie pathogène et une bactérie commensale reste inconnues. En utilisant la technique Optical Maps sur des souches de S. aureus isolées de plaies de pieds diabétiques avec différents niveau de virulence, nous avons pu montrer l’existence d’un prophage insérés dans le génome des souches colonisantes et absent des souches infectantes. Le phage, nommé ROSA, est localisé dans un hotspot d’insertion de phage NM2. Il est aussi localisé en amont du locus isd qui est requis pour l’assimilation du fer essentiel à la bactérie dans sa phase pathogène. Le phage ROSA inactive la voie isd en dérégulant l’activité du régulateur transcriptionnel majeur Fur en absence de fer. Il réduit aussi la virulence de ces souches sur les 2 modèles de virulence (Le ver C. elegans et le Zebrafish). L’expulsion du phage ROSA restaure la régulation du locus isd par Fur et la production de sidérophores en absence de Fer, la formation du biofilm et la virulence des souches. La mutation du gène Fur nous a permis de déduire que le phage ROSA affectait les bactéries de manière indépendante de Fur. Enfin, nous avons étudié la prévalence des souches colonisantes sur les plaies de pieds diabétiques. Nous avons observé que 20% des souches présentait l’insertion ROSA et 89% appartenait au complexe clonal CC8. Les souches colonisantes, avec leur niveau bas de virulence, devraient faire l’objet de détection dans le but de rationnaliser l’utilisation des antibiotiques et ainsi lutter contre l’apparition de bactéries multirésistantes aux antibiotiques. / Staphylococcus aureus is an opportunistic bacterium capable of causing a wide range of severe diseases when it gains access to underlying tissues. Paradoxically, this causative pathogen is a common inhabitant of the skin microflora and colonizes the nares and other human mucosa, and as such, may be considered as a commensal colonizing organism. The genetic basis for the differences in pathogenic/colonizing potential is unknown. By performing optical maps comparisons of a collection of S. aureus strains of defined virulence potential isolated from diabetic foot ulcers at different stages, we brought to light a prophage present in colonizing-causing bacteria. The phage, namely ROSA, was localized in a hotspot region NM2 near the locus isd, the main iron surface determinant that transport iron across the bacterial wall. It induces a deregulation of the activity of the transcriptional regulator Fur involving the biofilm formation of the bacteria in response to low iron environment. It reduced also significantly the virulence of the strain in two in vivo models (the nematode C. elegans and the zebrafish). The expulsion of the phage restored the regulation of the locus isd, the siderophore production, the biofilm formation and the virulence of the strain. The mutation of the fur gene within the colonizing strain enabled us to determine that the phage ROSA affect the the bacteria in a Fur-independent manner. Finally we determined the prevalence of these colonizing strains in skin and soft tissue infections (diabetic foot ulcers). We observed that 20% (39/195) of the strains harboured this insertion and 89% belonged to the clonal complex CC8. This colonizing strain by its low virulence potential must be detected in the aim to contribute to a sounder use of antibiotic treatment, an important point in front of the increase of multidrug resistant bacteria.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-hydroxymethyl-dCMP (Hm5CMP) from dCMP and methylene tetrahydrofolate (CH2THF), analogous to the reaction between dUMP and CH2THF catalyzed by thymidylate synthase (TS), an enzyme of known structure. The amino acid sequence identity between invariant TS residues and CH is at least 50%. Most of the residues which contact the dUMP and CH2THF in TS are conserved in CH. It is hypothesized that CH is homologous to TS in both structure and mechanism. The project described in this thesis tests this hypothesis. In-vitro studies on catalysis by CH variants. The roles of three residues in catalysis by CH have been tested using site-directed mutagenesis. Conversion of Cys148 to Asp, Gly or Ser decreases CH activity at least 105 fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of bound dUMP to the side chain of an Asn; the corresponding CH residue is Asp179. Conversion of Asp179 in CH to Asn reduces kcat/KM for dCMP by 104 fold and increases kcat/KM for dUMP 60 fold, changing the nucleotide specificity of the enzyme. Other studies have shown that the specificity of TS was changed from dUMP to dCMP by conversion of the appropriate Asn to Asp. Based on the crystal structure of TS, a Glu residue (also conserved in CH) is proposed to catalyze formation of the N5 iminium ion methylene donor by protonation of N10 of CH2THF. In CH and TS, overall turnover and tritium exchange are tightly coupled. Replacement of Glu60 in CH or Glu58 in TS uncouples these catalytic steps. Conversion the Glu60/58 to Gln or Asp results in a 5-50 fold decrease in the ability to catalyze tritium exchange, consistent with an inability to catalyze formation of the N5 iminium ion, but also results in a 104-105 decrease in product formation. This suggests that Glu60/58is also involved in a step in catalysis after nucleotide and folate binding and proton removal from carbon 5 of the nucleotide. Isotope effect studies. The observed value of the α-secondary tritium inverse equilibrium isotope effect (EIE = 0.8) on formation of the complex between FdUMP, CH2THF and both wild-type CH and CH(D179N) indicates that carbon 6 of FdUMP is sp3 hybridized (tetrahedral) in the ternary complex. This is consistent with the hypothesis that that carbon 6 is bonded to Cys148 in the complex. Removal of Cys148in CH prevents complex formation with FdUMP. Lack of an observed α-secondary tritium kinetic isotope effect (KIE) for position 6 of dCMP for both enzymes suggests that the intrinsic KIE is masked by other rate-limiting steps or that rehybridization follows the first irreversible step. An observed KIE on carbon 6 of dUMP by CH(D179N) suggests the rate-limiting steps for the two nucleotide substrates is different. In-vivo studies catalysis by CH variants. In order to prevent recombination between CH deficient T4 phage and plasmid borne copies of CH variants, the gene coding for CH, gene 42, was deleted from the T4 chromosome. The T4Δ42 phage requires wild-type CH expressed from a plasmid to kill their host cell. CH variants C148G, D179N, E60Q, and E60D, all which exhibit at least 2000 fold lower activity in vitro, do not complement the T4Δ42 phage in vivo. Interchanging the functional domains of CH and TS. It is proposed that shortening the C-terminal loop seen in the structure of TS changes the solvent structure of the CH active-site such that it becomes more hydrated. Differences in the solvent structure of the active-site may account for differences in the catalytic specificity between CH and TS, respectively, hydration versus reduction. In order to test the hypothesis that these catalytic differences between TS and CH lie within the C-terminal portion of the enzyme, the N-terminus of the CH(D179N) variant was fused to the C-terminus of the wild-type TS to create a chimeric CH/TS enzyme. The chimeric enzyme was predicted to have specificity for dUMP and a active-site solvent structure similar to that for wild-type TS. However, the resulting protein cannot be overproduced to significant levels and does not have any detectable TS activity in vivo.

Clostridium difficile is the most common bacterial cause of infectious diarrhoea in healthcare environments and in 2014 was responsible for 13,785 infections in the UK. C. difficile infection (CDI) is spread via the faecal-oral route and by contact with contaminated surfaces. However, despite the healthcare concerns no tests are available to validate if sufficient cleaning has been conducted. In addition, Polymerase Chain Reaction (PCR) and Enzyme Immunoassays (EIAs)-based tests used to diagnose CDI lack sensitivity and specificity and hence false negative results are commonly obtained. To overcome these concerns the aim of the PhD research has been to develop the first diagnostic test that exploits the specific interactions of C. difficile bacteriophages (phages), viruses that specifically infect and kill C. difficile. In order to develop a C. difficile phage-based test, first suitable phages that can be used for the test were identified and this was conducted by screening 35 different C. difficile phages against 160 clinically relevant C. difficile isolates. Five phages were found to infect the most number of isolates and were investigated further to identify whether a phage-based diagnostic could be developed based on phages binding (adsorption) to different C. difficile subgroups. However, for all five phages, adsorption rates were not consistently high for C. difficile subgroups in comparison to other common bacteria found in similar locations to C. difficile. Therefore, to increase specificity of the phage-based diagnostic test a new approach was taken by tagging two phages with luminescence luxAB genes (reporter phages), which would be expressed once C. difficile cells were infected with the phages. To design the C. difficile reporter phages, non-essential phage genes were replaced with the luxAB genes, but this study revealed mutagenesis of C. difficile was troublesome and extensive optimisation was required. In addition, once the reporter phages had successfully been constructed the luxAB genes were unstable within the phage genome and were lost during phage replication. Despite extensive optimisation and due to time constrains the luxAB genes were not stabilised within the phages but future work will focus on stabilising the genes.

Viruses has a relatively recently been discovered in high abundances in aquatic systems. Their possible importance has therefore been largely overlooked. In aquatic food webs there should be large differences in carbon and nutrient fluxes depending on whether the main cause of bacterial mortality is viral infection or grazing, where lysis following viral infection should result in a release of organic carbon and nutrients from the lysed bacteria and hence not reach higher trophic levels. Recent research on aquatic viruses has mainly focused on marine environments and the number of studies on freshwater viral ecology is limited. Hence, there is a need for more studies on the importance and functioning of viruses in freshwater systems. The aims of this thesis were to explore the functioning and diversity of viruses that infects bacteria (phages) in freshwater systems. To effectively address this I conducted two experiments and three field studies in 23 lakes in different parts of Sweden. The results show that viral infection and subsequent lysis of the host cell can partly explain the formation of non-nucleoid-containing bacteria and further that viruses can respond to increases in phosphorus concentrations without any net increase in bacterial abundance. Generally, a larger part of bacterial production in lakes were grazed by flagellates than lysed by viruses, but a larger fraction of the total bacterial mortality can be attributed to viruses in hypolimnion compared to in epilimnion. Further, the largest impact of phages on bacterial production may be in humic lakes, which have a relatively high frequency of visibly infected bacterial cells, but low flagellate abundance. Community composition of bacteria and viruses were only weakly coupled in the studied systems. The most important factors for predicting viral community composition were temperature and concentrations of dissolved organic carbon, total phosphorus and soluble reactive phosphorus. The viral community composition changed over the season and temperate phages could be detected by incubations with mitomycin C showing that a large fraction of the viruses detected appeared to be temperate phages. The most important environmental factor co-varying with viral community composition was again concentrations of total phosphorus. To summarize, bacteriophages, as a bacterial mortality factor, are important in freshwater microbial food webs and phosphorus supply has a potential central function in the regulation of the importance of bacteriophages and additionally for viral diversity.

published_or_final_version / Physiology / Master / Master of Philosophy