Members of the Legionellaceae cause respiratory infections in man; the most severe, pneumonic form is known as Legionnaires' disease. Of the 39 species described to date 16 have been associated with human disease, however the majority of reported cases of legionellosis are caused by Legionella pneumophila serogroup 1. A number of pathogenic bacteria regulate their virulence gene expression in response to environmental stimuli. Temperature and the availability of iron are considered to be stimuli which signal entry to a host environment. The first part of this study utilised chemostat culture to investigate the influence of growth temperature and the availability of iron on the physiology, morphology and virulence of L. pneumophila serogroup 1. This study demonstrated, for the first time, that the virulence of L. pneumophila was significantly reduced (P < 0.05) when the culture temperature was lowered from 37 to 24°C and this modulation was reversed by returning the temperature to 37°C which resulted in a statistically significant (P < 0.05) increase in virulence. Further experiments demonstrated that the concentration of iron in the growth medium also had an effect on virulence. Contrary to expectations iron-limited cultures were less virulent than those grown iron-replete. This modulation was also reversible with a return to virulence when iron-replete conditions were restored. The physiology and morphology of L. pneumophila were also influenced by both growth temperature and iron-limitation. At 24°C cultures consisted of flagellated short rods, whereas cultures grown at 37°C were pleomorphic and flagella were not evident. It was demonstrated that L. pneumophila accumulates the intracellular carbon storage compound, polyhydroxybutyrate, and that the proportion of the cell dry weight which it comprised varied with growth temperature, being maximal at 24°C. The ratio of saturated to unsaturated fatty acids in L. pneumophila decreased as the temperature was reduced to 24QC; this is a common strategy designed to maintain membrane fluidity. Siderophore production was detected in iron-limited cultures but not in iron replete cultures. Protease production was also affected by both growth temperature and iron-limitation. The BIOLOG bacterial identification system was modified for use with legionellae and this was used to investigate the metabolic versatility of these bacteria. A database containing substrate utilisation profiles of Legionella species was constructed using the modified system; this was then used to identify legionella isolates to species level. Evaporative cooling towers are a significant source of Legionnaires' disease accounting for the majority of outbreak cases in the United Kingdom. In the second part of this study a microbiologically-contained, fully-functional evaporative cooling tower was constructed and used to investigate factors that could influence the growth of L. pneumophila in such systems. The mode of operation of the cooling tower was found to influence the multiplication of legionellae in the system. Low-usage situations resulted in enhanced growth of L. pneumophila. Growth of L. pneumophila demonstrated a significant positive correlation with water temperature but its concentration decreased with increased conductivity. The concentrations of calcium, magnesium, potassium and zinc and the total hardness of the water all exhibited inverse relationships with legionella population size. The protocol for the emergency disinfection of cooling systems recommended in the Report of the Department of Health Expert Advisory Committee on Biocides did not eradicate L. pneumophila from the experimental cooling tower.

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2006. / Title from second page of PDF document. Includes bibliographical references (p. 123-144).

No description available.

No description available.

No description available.

Group A Streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces and is capable of producing a variety of diseases. This dissertation investigates the function of a metalloregulator named MtsR in GAS physiology and disease process. An mtsR mutant was constructed and analyzed. Consistent with MtsR role in iron uptake regulation, the mtsR mutant accumulates more iron (80 ± 22.5%) than the wild type strain. Inactivation of mtsR results in constitutive transcription of the sia (Streptococcal Iron Acquisition) operon, which is negatively regulated by iron in the parent strain. We identified the promoter that controls the expression of the sia operon (Pshr) and used it as a model to study MtsR interaction with DNA. Electrophoretic mobility gel shift assays (EMSAs) demonstrated that MtsR binds to the shr upstream region specifically and in an iron and manganese dependent manner. DNase I footprint analysis revealed that MtsR protects a 69 bp segment in Pshr that includes 2 inverted repeats, overlapping the core promoter elements. A global transcriptional analysis determined that MtsR modulates the expression of 64 genes, of which 44 were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including immune evasion, colonization, dissemination, metal homeostasis, nucleic acid and amino acid metabolism, and protein stability. MtsR functions as a dual regulator as it binds to the promoters of the repressed genes ska, aroE, and nrdF.2, as well as the upstream region of the positively regulated genes mga, emm49, and pyrF. A 16 bp MtsR-binding consensus region was identified in all of the promoters that are directly regulated by MtsR. In conclusion, we have demonstrated that MtsR is a global regulator in GAS that controls the expression of vital virulence factors and genes involved in metal transport, virulence and metabolic pathways.

According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. Current detection and treatment strategies for invasive aspergillosis, as well as other invasive fungal infections, are poor. Iron has been shown to be essential for Aspergillus fumigatus virulence. Furthermore, mechanisms in the iron regulatory network are believed to be potential drug targets since iron management in fungi is vastly different from that in mammals and other eukaryotes. Therefore a better understanding of iron homeostasis in Aspergillus fumigatus could help improve drug therapies for invasive aspergillosis. In this research a discrete model of iron uptake, storage and utilization in Aspergillus fumigatus with particular focus on siderophore-mediated iron acquisition is constructed. The model predicts oscillations in gene expression as the fungus adapts to a switch from an iron depleted to an iron replete environment. The model is validated via in vitro experiments. / Master of Science

No description available.