Phosphate is an essential but often growth-limiting nutrient for bacteria. At low concentrations of phosphate in the growth medium, bacteria induce high-affinity uptake systems for phosphate, and this is usually the ABC-type phosphate specific transport system Pst. In the fully sequenced genomes of pathogenic species of mycobacteria, several copies of the genes encoding for the Pst system (pstSCAB) have been identified and some of these genes have been shown to be virulence factors. The reasons for the presence of multiple copies of pst genes in pathogenic mycobacteria are not understood, and phosphate transport by these bacteria, as well as the gene regulation involved, is poorly characterised. The fast-growing M. smegmatis contains only a single copy of the pst operon, but we recently identified a gene locus containing three genes, phnDCE, which encode for a putative ABC-type phosphate/phosphonate transport system, and a gene, phnF, which encodes for a putative transcriptional regulator of the HutC subfamily of GntR like regulators. To identify a function for the PhnDCE transport system and to characterise high-affinity phosphate transport in M. smegmatis, we created allelic exchange mutants in phnD and pstS, as well as a phnD pstS double deletion mutant. All three mutants failed to grow in minimal medium containing 10 mM phosphate, while the wildtype was able to grow in the presence of micromolar phosphate concentrations. No differences were observed in complex growth medium. Steady-state levels of [��P]-phosphate uptake were approximately 25% lower in all mutant strains as compared to the wildtype. Kinetics of phosphate uptake in the wildtype strain when grown at low phosphate concentrations (50 [mu]M P[i]) were biphasic, suggesting the presence of two inducible transport systems with apparent K[m] values of 16 [mu]M P[i] and 64 [mu]M P[i], respectively. Analysis of the kinetics of phosphate transport in the mutant strains led us to the proposition that the Pst system has an apparent Km value of ca. 16 [mu]M P[i], and the Phn system has an apparent Km of ca. 60 [mu]M P[i]. A third inducible phosphate transport system, which was active in the double mutant strain, had an apparent K[m] of ca. 90 [mu]M P[i]. Uptake of phosphate in all strains was not inhibited by the presence of excess phosphonates or phosphite, suggesting that all three transport systems were specific for phosphate. The study of phosphate transport in the presence of various metabolic inhibitors revealed that uptake by the Phn and Pst systems is driven by ATP-hydrolysis, consistent with ABC-type transport, while the third, unidentified transport system may be driven by the proton motive force. We showed that phnDCE formed an operon, and that the promoter area of the operon lies within 200 bp of the start of phnD. To investigate the regulation of the phn and pst genes, β-galacosidase activities of strains carrying transcriptional lacZ-fusions of the pstSCAB, phnDCE and phnF promoter areas, and levels of mRNA of the phn and pst genes were studied. All genes were induced when phosphate concentrations fell below a threshold value of 30 [mu]M, which coincided with a shift in the growth characteristics of M. smegmatis. Expression of the pst operon appeared to be controlled directly by the PhoPR two-component regulatory system, while the phn operon may be under direct or indirect control by PhoPR. To identify a role for PhnF in the regulation of phn gene expression, we created a phnF deletion mutant. PhnF appeared to repress transcription of phnDCE and phnF under phosphate-replete conditions. We identified two putative binding sequences for PhnF in the intergenic region between phnD and phnF with the sequence TGGTATAGACCA, which is similar to the proposed recognition consensus for HutC-like transcriptional regulators. Using site-directed mutagenesis of these sequences, we demonstrated that they are required for the repression of phnDCE and phnF. To prove PhnF binding to these potential binding sites, we attempted to express the M. smegmatis PhnF protein in E. coli, but could not obtain soluble recombinant protein. Electrophoretic mobility shift assays of the phnDCE promoter fragment using cell-free crude extracts of M. smegmatis were not successful. We propose that Pst and Phn both constitute high-affinity phosphate specific transport systems of M. smegmatis, and that a third inducible phosphate transport system is present in this bacterium. PhnF is required for repression of phnDCE and phnF transcription under phosphate-replete conditions, while induction of the pst operon, and possibly the phn operon, under phosphate-limited conditions involves the PhoPR system.

This thesis is about making inference on the host status of feral ferrets in New Zealand for Mycobacterium bovis, the aetiological agent of bovine tuberculosis. The central question addressed is whether the rate of intra-specific transmission of M. bovis among ferrets is sufficient for the disease to persist in ferret populations in the absence of external, non-ferret sources of infection (inter-specific transmission). The question is tackled in three parts�firstly using model selection to identify suitable models for estimating the force of M. bovis infection in ferret populations; secondly applying statistical hypothesis testing to the results of planned manipulative field experiments to test the relationship between M. bovis infection in brushtail possums and that in ferrets; and thirdly using modelling to estimate intra-specific disease transmission rates and the basic reproductive rate (Ro) of M. bovis infection in ferrets. The model selection approach clearly identified the hypothesis of oral infection related to diet was, as modelled by a constant force of infection from the age of weaning, the best approximation of how M. bovis infection was transmitted to ferrets. No other form of transmission (e.g., during fighting, mating, or routine social interaction) was supported in comparison. The force of infection (λ) ranged from 0.14 yr-1 to 5.77 yr-1, and was significantly higher (2.2 times) in male than female ferrets. Statistical hypothesis testing revealed transmission of M. bovis to ferrets occurred from both brushtail possums and ferrets. The force of M. bovis infection in ferrets was reduced by 88% (λ=0.3 yr-1 vs. λ=2.5 yr-1) at sites with reductions in the population density of sympatric brushtail possum populations. A smaller decline in the force of infection resulting from the lethal cross-sectional sampling of the ferret populations was also demonstrated. The modelling approach estimated the basic reproductive rate (Ro) of M. bovis infection in ferrets in New Zealand to vary from 0.17 at the lowest population density (0.5 km-2) recorded to 1.6 at the highest population density (3.4 km-2) recorded. The estimates of Ro were moderately imprecise, with a coefficient of variation of 76%. Despite this imprecision, the Ro for M. bovis infection in ferrets was significantly less than unity for all North Island sites surveyed. Hence it is inferred ferrets are spillover hosts (0<Ro<1) for M. bovis infection in these environments. That is, M. bovis infection will progressively disappear from these ferret populations if the source of inter-specific transmission is eliminated. The estimates of Ro for M. bovis infection in South Island ferret populations were above one (the level required for disease establishment) for a number (5/10) of populations, though the imprecision made it impossible to ascertain whether Ro was significantly greater than one. The estimated threshold population density (Kt) for disease establishment was 2.9 ferrets km-2. It is inferred that, given sufficient population density (>Kt), the rate of intra-specific transmission of M. bovis among ferrets is sufficient for the disease to establish in ferrets in the absence of interspecific transmission. In these areas, ferrets would be considered maintenance hosts for the disease. Active management (e.g., density reduction or vaccination) of ferrets would be required to eradicate M. bovis from ferret populations in these areas, in addition to the elimination of sources of inter-specific transmission, particularly brushtail possums.

<p>This thesis describes the synthesis of structures related to the capsular polysaccharide of <i>Neisseria meningitidis</i> serogroup A and the synthesis of analogues of mycothiol, a compound produced by <i>Mycobacterium</i> <i>tuberculosis</i>. The first part of the thesis describes the synthesis of structural elements present in the native capsular polysaccharide of <i>Neisseria</i> <i>meningitidis</i> serogroup A. In this part, an improved synthesis of 2-azido-2-deoxy-D-mannopyranose is included. The second part of the thesis describes the formation of stable C-phosphonate analogues related to the capsular polysaccharide. The last part outlines the formation of analogues of mycothiol, where the syntheses of a bicyclic analogue and a thioglycosidic analogue are described.</p>

This thesis describes the synthesis of structures related to the capsular polysaccharide of Neisseria meningitidis serogroup A and the synthesis of analogues of mycothiol, a compound produced by Mycobacterium tuberculosis. The first part of the thesis describes the synthesis of structural elements present in the native capsular polysaccharide of Neisseria meningitidis serogroup A. In this part, an improved synthesis of 2-azido-2-deoxy-D-mannopyranose is included. The second part of the thesis describes the formation of stable C-phosphonate analogues related to the capsular polysaccharide. The last part outlines the formation of analogues of mycothiol, where the syntheses of a bicyclic analogue and a thioglycosidic analogue are described.

<p>Many studies have reported low cure rates for multidrug-resistant tuberculosis (MDRTB) patients and MDR-TB patients co-infected with human immunodeficiency virus (HIV). However, little is&nbsp / known about the effect of HIV infection and antiretroviral therapy on the treatment outcomes of MDR-TB in South Africa. Therefore, the objectives of the study are: to find out whether HIV infection&nbsp / and interactions between ARVs and second line anti-TB drugs have an impact on the following MDR-TB treatment outcomes: cure rate and treatment failure at Brewelskloof Hospital. MDR-TB&nbsp / patients were treated for 18-24 months. The study was designed as a case-control retrospective study comparing MDR-TB treatment outcomes between HIV positive (cases) and HIV negative&nbsp / patients (controls). Patients were included in the study only if they complied with the following criteria: sensitivity to second line anti-TB drugs, MDR-TB infection, co-infection with HIV (for some&nbsp / of them), male and female patients, completion of treatment between 1 January 2006 and 31 December 2008. Any patients that presented with extreme drug-resistant tuberculosis (XDR-TB)&nbsp / were excluded from the study. Data were retrospectively collected from each patient&rsquo / s medical records. There were a total of 336 patients of which 242 (72%) were MDR-TB patients and 94&nbsp / (27.9%) MDRTB co-infected with HIV patients. Out of the 242 MDR-TB patients, 167 (69.2%) were males and 75 (30.7%) were females. Of the 94 patients with MDR-TB co-infected with HIV, 51&nbsp / (54.2%) males and 43 (45.7%) females. Patients with multidrug-resistant tuberculosis co-infected with HIV who qualify for antiretroviral therapy were treated with stavudine, lamivudine and&nbsp / efavirenz while all MDR-TB patients were given kanamycin, ethionamide, ofloxacin, cycloserine and pyrazinamide. The cure rate of MDR-TB in HIV (+) patients and in HIV (-) patients is 34.5%&nbsp / and 30 % respectively. There is no significant difference between both artes (pvalue = 0.80). The MDR-TB cure rate in HIV (+) patients taking antiretroviral drugs and in HIV (+) patients without&nbsp / antiretroviral therapy is 35% and 33% respectively. The difference between both rates is not statistically significant. The study shows that 65 (28.0%) patients completed MDR-TB treatment but&nbsp / could not be classified as cured or failure, 29 (12.5%) patients failed, 76 (32.7%) defaulted, 18 (7.7%) were transferred out and 44 (18.9%) died. As far as treatment completed and defaulted is concerned,&nbsp / there is no significant statistical difference between HIV (+) and HIV (-) The number of patients who failed the MDR-TB treatment and who were transferred out is significantly higher in the HIV (-)&nbsp / group than in the HIV (+) group. Finally the number of MDR-TB patients who died is significantly higher in the HIV (+) group). The median (range) duration of antiretroviral therapy before starting&nbsp / anti-tuberculosis drugs is 10.5 (1-60) months. According to this study results, the MDR-TB treatment cure rate at Brewelkloof hospital is similar to the cure rate at the national level. The study also&nbsp / hows that HIV infection and antiretroviral drugs do not influence any influence on MDR-TB treatment outcomes.</p>

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "December 2000." Typescript. Vita. Includes bibliographical references (leaves 114-120). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 114-120). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 91-108).

The persistent increase of bovine tuberculosis (bTB) over the past twenty years has put a substantial strain on both the British economy and the welfare of livestock. However, the development of an effective bTB vaccine has been continually hindered by the lack of knowledge on the immune response following Mycobacterium bovis (M. bovis) infection. In collaboration with the TB Research Group at the Veterinary Laboratories Agency (VLA, Surrey), this thesis is part of a much wider strategy managed by the Department of Environment, Food and Rural Agency (DEFRA) aimed at elucidating the immunopathogenesis of M. bovis and to develop more effective infection control measures. The specific focus of this thesis was to enable a stronger understanding of the bovine immune response over different periods of M. bovis infection and to apply this new knowledge in evaluating the efficacy of a novel BCG vaccination. Time Course Study: Knowledge of time dependent cytokine expression following M. bovis infection would aid vaccine development by revealing potential correlates of protection. Interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α), interleukin (IL) 4 and 10 expression were analysed using quantitative (q) PCR in formalin fixed bovine lymph nodes following five, twelve and nineteen weeks of M. bovis infection. A strong pro-inflammatory/ T helper 1 (TH1) lymphocyte response was evident at five weeks post M. bovis infection, represented by IFN-γ and TNF-α expression (log2 copies of 6.5 and 2.15, respectively) in the absence of IL4. Between five and twelve weeks of infection, a significant increase was observed in IL10 (log2 copies from 5.97 to 8.27, p<0.01, Mann Whitney test), accompanied by an increase in both IFN-γ (log2 7.53) and TNF-α (log2 3.94). This data conformed to a recently described aspect of TH1 lymphocytes, a ‘self-limiting’ nature in which cells produced both IFN-γ and IL10 with the aim of controlling the heightened pro-inflammatory response. The role of IL10 as an immunosuppressive became evident when comparing cytokine expression between four different types of thoracic lymph node; the left bronchial (LB), cranial mediastinal (CRM), caudal mediastinal (CM) and cranial tracheobronchial (CRT) nodes. The LB and CRM lymph nodes produced significantly higher levels of IFN-γ expression (log2 copies between 8.2 and 10) as compared to the CM and CRT (log2 copies between 2.6 and 5.5, p<0.001, Mann Whitney test). Further analysis of the data as a profile of cytokine expression for each lymph node type revealed that IFN-γ was dominantly expressed within the LB and CRM nodes, whereas within the CM and CRT nodes, IL10 was the dominant cytokine. The former nodes also displayed a higher level of pathological damage (represented by mean percentage area coverage of granuloma, 33.6 and 20%, respectively) as compared to the CM (13%) and the CRT lymph node types (10.8 %). This suggests conflicting roles for IFN-γ and IL10 in the development of immune-associated pathology. Following nineteen weeks of infection, the expression levels of IFN-γ, TNF-α and IL10 reduced (log2 6.22, 3.02 and 7.03, respectively) implying a loss of the cellular response. The later stages of bovine tuberculosis have been shown within the literature to display characteristics of a humoral rather than cell mediated response. However, within this study at nineteen weeks post infection IL4 (an important cytokine in the development of the humoral response) remained undetectable. The results from this study therefore confirm the importance of the cell mediated immune profile in response to M. bovis infection as well as the integral role of IFN-γ in both protection and pathology. It also further demonstrates the involvement of IL10 in controlling the IFN-γ response and highlights this cytokine as being potentially important in future immunologybased vaccination studies. BCG Vaccination Study: The current vaccine used against human tuberculosis, BCG, has provided variable results on protection against infection in experimental bovine studies. The BCG bacterium has lost a comparatively large quantity of genomic DNA through attenuation since its primary production in 1921, of which the majority represented genes encoding antigenic proteins. MPB70 and MPB83 are differentially expressed between BCG sub-strains due to a single nucleotide polymorphism in the alternative sigma factor K (SigK). BCG Pasteur has been shown to produce low levels of these antigenic proteins; however complementation of BCG Pasteur with a copy of sigK from BCG Russia resulted in up-regulating expression. It was therefore hypothesised that the recombinant BCG (sigK) Pasteur would prove more efficient in controlling M. bovis infection by inducing a stronger protective immune response post vaccination. IFN-γ, TNF-α, IL 4 and 10 expression were analysed using qPCR within the freshly dissected lymph nodes of five experimental cattle groups; BCG Pasteur vaccinated M. bovis challenged, BCG (sigK) Pasteur vaccinated challenged, non-vaccinated infected, non-vaccinated noninfected and BCG Pasteur vaccinated non-infected. Five weeks following infection, a strong IFN-γ mRNA response was detected in both the non-vaccinated and vaccinated cattle (mean log2 copies between 9.6 and 10.5 as compared to between 7.84 and 8.58 in the non-infected cattle). M. bovis infection also induced a significant reduction in IL10 mRNA levels in both vaccinated and non-vaccinated cattle (mean log2 14.4 in the infected groups compared to 15.5 in the non-infected cattle, p<0.005, Mann Whitney test) although there was little difference in TNF-α expression (mean log2 copies between 11.06 and 11.8 in all five groups). Interestingly, IL4 mRNA was detectable only within the two non-infected control groups (mean log2 12.4), further supporting the concept of a strong cell mediated response after five weeks of infection. Vaccination prior to challenge had an effect on IFN-γ mRNA levels only, as both the BCG Pasteur and BCG (sigK) Pasteur vaccinated groups displayed a smaller increase in IFN-γ mRNA following challenge (mean log2 10.3 and 9.6, respectively) as compared to the nonvaccinated group (mean log2 10.5). This reflected the role of vaccination in priming the immune response to enable more rapid elimination of the bacteria and subsequently inducing a lesser pro-inflammatory response. Interestingly, the BCG Pasteur vaccinated group appeared to control the immune response to a greater extent, as IFN-γ mRNA was significantly similar to that observed in the non-vaccinated non-infected group (mean log2 8.58, p>0.05, Mann Whitney test). In addition to the qPCR data, levels of IFN-γ and TNF-α protein (represented by the number of cells producing these proteins) were also analysed by immunohistochemistry. IFN-γ protein in the five experimental groups displayed the same pattern as that observed for IFN-γ mRNA expression (p<0.001, Spearmans correlation coefficient). However, analysis of TNF-α protein revealed significant differences between the five groups (p<0.005, Kruskal Wallis test) in contrast to that observed for the mRNA levels (p>0.05, Spearmans correlation coefficient) suggesting that posttranscriptional controls may play an important role in TNF-α translation. The difference in IFN-γ mRNA and protein expression between the two vaccination groups was also reflected within the pathological data. Although both BCGs reduced levels to below that of the non-vaccinated group (represented by mean percentage area coverage of granuloma, 59%), the BCG Pasteur group displayed less pathology (mean 6%) compared to the BCG (sigK) Pasteur cattle (mean 35%). It was suggested that the increased antigenic repertoire of the recombinant BCG (sigK) Pasteur did result in a stronger stimulation of the immune response post vaccination but that, as a consequence the bacterial threat was eliminated more rapidly.

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy