Abstract not available.

Haemophilus ducreyi expresses outer-membrane receptors for both heme (TdhA) and hemoglobin (HgbA), but no mechanism yet has been identified for the transport of heme from the periplasm to the cytoplasm. SodC protein in H. ducreyi binds to heme in addition to exhibiting superoxide dismutase activity. We hypothesized that SodC is involved in heme uptake in H. ducreyi. An isogenic H. ducreyi sodC mutant was constructed. The generation time of the sodC mutant was increased compared to the wild type strain (35000). The mutant was complemented in trans with either wild type sodC (sod+heme+), sodCH86E with wild type dismutase activity but less heme binding activity (sod+heme-) or with sodCL81FH82QD83G with diminished dismutase activity but wild type heme binding activity (sod-heme+). The generation times of the complemented strains showed no significant difference when grown under limited heme concentration. We conclude that SodC is unlikely to be involved in heme uptake in H. ducreyi.

MinC is the principle inhibitor of cell division in the Gram negative coccus Neisseria gonorrhoeae. Multiple alignment of 28 minC homologues from prokaryotic organisms revealed the conservation of four glycines in the C-terminus. Radical mutations of these residues, G138D, G157D, G164S, and G174E, were introduced to minCNg using site-directed mutagenesis and were expressed heterologously in Escherichia coli. Microscopic analysis of cellular morphology revealed abrogation of MinCNg function as indicated by the inability of the overexpressed protein to induce filamentation, and flow cytometry substantiated these data. The radical mutation of a non-conserved residue was also performed, and no impact on MinCNg function was observed. Circular dichroism was employed to confirm the preservation of protein structure in all mutants. Using the crystal structure of MinC from Thermotoga maritima, the position of the mutations of conserved and non-conserved residues was ascertained. The glycine residues were thus demonstrated to be of paramount importance to protein functionality and their conservation indicates a role for these residues in MinC function among bacterial species. The N-terminus of MinCNg is not conserved across species, but contains common structural motifs that were similarly mutagenized with moderate effect on the resultant protein as ascertained using similar methodology. Truncations of MinCNg revealed the necessity of the thirteenth amino acid for division inhibition function. Although the first ten amino acids were not necessary for protein function, the resultant protein was expressed and present in abundance, implicating a role for the extreme N-terminal region in protein stability. Plasmids for MinCNg localization and purification were also constructed to facilitate future experimentation.

Our objective was to study gene expression related to cytokines and cytokine receptors in FLS. We were looking for any previously unidentified cytokines and receptors that may play a role in the pathogenesis of RA. Using a microarray-based approach to identify any potential expressed cytokine genes we found that two pro-inflammatory cytokines belonging to the IL-10 family of cytokines, IL-19 and IL-22, are constitutively expressed in cultured FLS, and this expression was independent of length of culture. The other work in this thesis focused on identifying potential cytokine receptors expressed in FLS and other cells. From these studies, it was identified that FLS do not express membrane bound receptors for IL-19 or other IL-10 family members, but they are capable of expressing IL-22BP, a non membrane bound soluble form of the IL-22 receptor. This is the first indication that fibroblasts are capable of transcribing this gene. The work presented in this thesis has addressed gene expression in FLS and the findings may prove to be important in understanding maintenance of inflammation in RA. The finding that FLS can transcribe mRNA for IL-19 and IL-22, may indicate that these cells are important sources of these pro-inflammatory cytokines in vivo. The receptor studies may also prove to be useful in expanding the knowledge of receptor expression in FLS. (Abstract shortened by UMI.)

Cell division mechanisms involving native proteins MinCDE or MinCD and DivIVA have been explored using Esherichia coli (Ec) and Bacillus subtilis (Bs), respectively. My research examines the Gram-positive coccal-shaped Enterococcus faecalis (Ef) protein DivIVA, which functions without any Min proteins. To determine whether E. coli could be used as a suitable indicator system, DivIVAEf overexpression and morphological studies were undertaken. Constructs with unique mutations created along the 699-nucleotide length of the divIVAEf gene to abrogate specific coiled-coil domains at the N-terminal, C-terminal and two central regions were investigated in this model. The original mutations were previously used in different systems and therefore required PCR amplification, double digest with new restriction enzymes prior to subcloning in pUC18 for transformation in E. coli (Ramirez-Arcos et al., 2005; Rigden et al., 2005). (Abstract shortened by UMI.)

Phytochemicals and garlic extracts were evaluated for their potential as complementary treatments against Neisseria gonorrhoeae. The minimum inhibitory concentrations (MICs) of the phytochemicals silibinin and dillapiol, and a panel of garlic natural health products were determined against a panel of resistant and susceptible N. gonorrhoeae strains using agar and broth dilution methods and bacterial killing curves. Combinations of subinhibitory concentrations of silibinin or dillapiol with penicillin elicited a significant reduction in MICs of ∼50% and ∼25% of the tested strains respectively. Ternary combinations of silibinin, dillapiol and penicillin yielded a significant MIC reduction in 88% of the tested strains, including some strains whose penicillin MICs were reduced below the penicillin resistance breakpoint. The garlic extracts exhibited discrepancies in product composition and antimicrobial activity, with few products having comparable activity to fresh garlic. The activity of these phytochemicals against multiple N. gonorrhoeae resistance phenotypes makes them worthy of further investigation.

In Haemophilus ducreyi, heme uptake likely proceeds via a receptor-mediated process. The initial event involves binding to either of two outer membrane receptors, TdhA and HgbA. Once heme is deposited into the periplasmic space, we hypothesize that a heme-dedicated periplasmic binding protein (hHBP) is responsible for transporting heme across the periplasmic space to the inner membrane. To identify the hHBP, periplasmic extracts were generated from H. ducreyi 35000 grown under high and low heme conditions and subjected to proteome mapping. Peptide sequences of upregulated proteins grown under heme-restrictive conditions were determined by mass spectroscopy. A candidate hHBP was identified as a periplasmic binding protein homologous to YfeA of Yersinia pestis. The gene encoding this protein appears to be in a typical ABC transporter operon. Under iron-limiting conditions, no upregulation of the hHBP expression was observed; however, the purified hHBP was shown to specifically bind heme in a concentration-dependent manner.

Pulp and paper mills generate biosolids as a by-product of their effluent treatment systems. These biosolids show excellent potential as a soil conditioner. However, the detection of high levels of E. coli (10 2 to 105 CFU/gdw), in the absence of any significant fecal loading, has caused concern among effluent treatment system operators, land applicators and regulators. This research examines the sources and ecology of E. coli strains comprising the population of E. coli in the biosolids of an eastern Ontario pulp and paper mill by applying the molecular microbial source-tracking tool repetitive sequence-based polymerase chain reaction. Confirmed E. coli were successfully isolated from 2 forested sites, treated mill process feed water, storm effluent, mill fibres, wood chips, primary and secondary clarifier effluents and sludges, and biosolids. While laboratory isolates could be accurately distinguished from forest and industrial isolates, cluster and jackknife analyses were incapable of differentiating reliably among isolates from the forest, on-site inputs, effluent treatment system and biosolids (average rates of correct classification values ranged from 43.9% to 72.9%). No input could be excluded as a source of E. coli to the effluent treatment system. Many fingerprint types were unique to the primary and secondary clarifiers; however, only a subset of the fingerprint types recovered from the primary and secondary clarifiers were detected in the biosolids. This suggests that the E. coli strains found in mill biosolids were those able to survive the hot, desiccating conditions of dewatering. Highly similar fingerprint types (> 80% similarity) were recovered repeatedly over an 8-month period from the effluent treatment system (primary and secondary clarifiers) and biosolids. These results are consistent with growth within the effluent treatment system and biosolids, rather than fecal loading. Over a 3-year period the pulp and paper mill effluent treatment system underwent large-scale changes, including a 50% reduction in effluent volume upon the elimination of on-site pulping activities. New fingerprint types were detected in the biosolids following the restructuring, and the Shannon index of diversity increased. Disruption of established operating conditions could have opened niches, allowing new strains to colonize the mill effluent treatment system successfully.

Xenotransplantation with pig organs is being considered to alleviate donor organ shortages; however, the risk of introducing porcine viruses to humans is exaggerated in this setting. Accordingly, the goal of this study was to determine the infectious potential of porcine cytomegalovirus (PCMV), a xenozoonotic virus of interest, in human fibroblasts in vitro. Confluent cells were incubated with either live PCMV or controls. Infection was investigated by light microscopy/neutral red staining, by RT-PCR and sequencing, and by Western blotting. Neutralization experiments were also performed. Cells incubated with PCMV demonstrated significant cytopathic effect by 7 days post-infection. Also, RT-PCR sequencing identified PCMV DNA polymerase in infected cells. In Western blots, monoclonal antibodies (mAbs) to human CMV glycoprotein B and pig serum presumed to contain anti-PCMV antibodies detected PCMV proteins by 19 days post-infection. Furthermore, one of these mAbs and the pig serum neutralized PCMV infection. Western blots and neutralization studies using a mAb to a human herpes virus 6 membrane glycoprotein provided no concrete evidence for a biological relationship between PCMV and human herpes virus 6. These results demonstrate that PCMV can infect human fibroblasts in vitro.

Listeria monocytogenes is a human foodborne pathogen capable of causing invasive and non-invasive infections. Ubiquitous in the environment, strains are often classified into lineages I (serovars 1/2a, 1/2c, 3a; food); II (containing serovars 1/2b, 4b, 3b; human illness); and III (4a, 4c; food-production). It is not clearly understood how serovars become tropic to a particular niche. The objective of this project was to investigate strain differences in clinical, environmental and food isolates, with the aim of identifying genomic features responsible for the tropism of the microorganism for clinical, food and environmental niches. Comparative genomic techniques, namely mixed-genome microarrays, dot-blot hybridization and suppressive subtractive hybridization were used to compare the genomes of L. monocytogenes isolates of different serotypes from a variety of different sources. Over 30 genome sequences, potentially involved in the tropism of the microorganism, were identified using Versions 2 and 3 of a Listeria mixed-genome array. These included genes with similarities to cystathionine beta-lyase and homoserine O-acetyltransferase, a putative outer-membrane lipoprotein, conserved hypothetical proteins, a peptidoglycan lytic protein and a glycosyltransferase family 65 enzyme. The glycosyltransferase family 65 gene was found to be present in isolates belonging to lineage II but was absent in lineage I isolates. By creating an isogenic deletion mutant of this gene, it was found that this enzyme is involved in cell wall biosynthesis and could be responsible for increased survival of lineage II strains in the host environment, thus rendering these isolates more prevalent in cases of human listeriosis.