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Transition metals are essential micronutrients for organisms. Excess zinc can be lethal to bacterial survival. Zinc homeostasis is crucial and is regulated by zinc uptake and efflux system. Streptococcus pneumoniae AdcR (Adhesin competence regulator) transcription factor is the member of MarR (multiple antibiotic resistance regulator) family proteins that suppress the zinc upregulation transporter. In the absence of zinc, AdcR has less affinity to bind DNA and uptake genes are expressed in order to transport zinc inside the cell. Streptococcus pneumoniae is a gram-positive human pathogen that is capable of colonizing in the human nasopharynx, especially in young children and leads to severe diseases including pneumonia, septicaemia, and meningitis. In this work, we have transformed AdcR gene in E.Coli (BL21D3) cell, and we have overexpressed and purified AdcR using classical molecular biology and protein purification techniques. We also performed preliminary biophysical characterization of purified zinc(II) dependent transcription factor AdcR.

The clonality of a random sample of 102 invasive pneumococcal serotype 3 strains isolated from Gauteng Province during January 2000 to December 2003 was investigated. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed a heterogeneous population with several PFGE clusters and sequence types (STs). The largest PFGE cluster comprised 36/102 (35%) isolates including seven that belonged to ST458, a clone that is not common in other parts of the world. The global clone (ST180) which is common in the United States and other countries was found in a cluster that represented only 14/102 (14%) of isolates examined. The first multidrug-resistant pneumococcal serotype 19A strain that was isolated in South Africa in 1977 was compared with invasive serotype 19A multidrug-resistant strains isolated in South Africa during June 1999 to December 2004. PFGE analysis of these isolates demonstrated clonal diversity among the isolates. MLST of 16 randomly selected isolates revealed several STs, none of which was the same ST as the 1977 clone. Both serotype 3 and 19A were not associated with increased mortality or HIV seropositivity compared to other serotypes.

Streptococcus pneumoniae is a nasopharyngeal commensal in most healthy individuals. However, it can translocate from this niche to deeper tissues, causing diseases such as otitis media, meningitis, sepsis and pneumonia, which are responsible for significant morbidity and mortality worldwide. At the commencement of this work, inherent difficulties in harvesting sufficient bacterial numbers from experimental animals restricted the examination of pneumococcal gene expression during pathogenesis, and thus virulence gene transcription patterns were largely unknown outside of an in vitro environment. This thesis aimed to investigate such transcriptional patterns in vivo, and to hence gain a better understanding of pneumococcal behaviour during colonisation and disease. This work describes refinement of an intranasal S. pneumoniae infection model in CD-1 mice that enables pneumococci to be harvested from multiple niches with low contamination by nasopharyngeal microflora or host tissue, and minimal crosscontamination with circulating pneumococci in the vascular system. The challenge route simulates the acquisition of S. pneumoniae in the human population, and progression to IPD occurs naturally. RNA extraction, enrichment and linear amplification procedures were optimised so that RNA could be obtained from in vivo site in sufficient quantities and with sufficient integrity to be used in semi-quantitative assays. Linear amplification allowed the examination of gene expression in niches where low bacterial numbers had previously prevented such analyses. Real-time RT-PCR and microarray analyses were used to examine bacterial RNA samples recovered from the nasopharynx, lungs, blood and brains of CD-1 mice, providing the first comparative transcriptional data for pneumococci during carriage and disease, within the same animal model. Two pneumococcal serotypes were examined; a type 2 (D39) and a type 6A (WCH16) strain. CbpA, Ply, and SpxB were shown to be important for carriage in both strains, with pneumococci up-regulating the expression of the genes encoding these virulence proteins in the nasopharynx. This provides in vivo evidence supporting the ascribed roles of these proteins in reducing the level of competing microflora and promoting nasopharyngeal adherence. Similarly, D39 nanA and pspA transcription levels were up-regulated in the nasopharynx. The level of pspA mRNA was also higher in the blood than the lungs, suggesting an increased requirement in the bloodstream, where PspA is involved in reducing complement-mediated opsonisation. Despite the antiphagocytic role of the pneumococcal polysaccharide capsule in the bloodstream, D39 cpsA mRNA was present in similar quantities in the nasopharynx, lungs and blood, which may support previous studies indicating post-transcriptional regulation of capsule expression. However, cpsA expression was up-regulated in the blood for WCH16. These results may indicate the existence of strain-specific differences in virulence gene regulation. Microarray analysis of in vivo-harvested S. pneumoniae D39 found that mRNAs encoding components of phosphotransferase systems, CbpA, a putative neuraminidase, and v-type sodium ATP synthase subunits were significantly higher in bacteria involved in carriage than bacteraemia. Conversely, the expression of genes involved in competence, and dinF (present on a competence-induced operon), were up-regulated in the blood compared to the nasopharynx, providing evidence that competence is induced during bacteraemia. Pneumococci also showed increased expression of genes involved in fatty acid metabolism, pgdA, lytB and cbpG in the blood compared to the nasopharynx. This study used a single pneumococcal strain and infection model and, therefore, overcomes inherent issues of serotype/strain- and animal model- specific gene expression that may have complicated interpretation of data in previous studies. This thesis reports some of the first in vivo pneumococcal gene expression data gained using a single animal model and pneumococcal strain. The data reinforce the putative roles of several virulence factors, and provides novel transcription data for pneumococci during carriage. Results suggest the existence of core genes that are essential for infection in multiple pneumococcal serotypes, whereas other genes appear to have strain-specific roles. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1287056 / Thesis (Ph.D.)-- University of Adelaide, School of Molecular and Biomedical Science, 2007

Homeostasis of trace metal ions is essential for a variety of cellular processes and virulence mechanisms, including resistance to oxidative stress, DNA replication, and regulation of cell adhesion/invasion. Understanding how pathogenic microorganisms overcome metal starvation and intoxication provides insight into how these mechanisms could be targeted by novel antimicrobial therapies. Streptococcus pneumoniae, or pneumococcus, is a Gram-positive, commensal of the human nasopharynx and upper respiratory tract. Though this organism is primarily an asymptomatic colonizer, it is also the causative agent of infections ranging in severity from reoccurring acute otitis media to life-threatening community acquired pneumonia or bacterial sepsis. This study aims to characterize aspects of pneumococcal physiology and infection that are responsive to changes in micronutrient zinc availability. Two zinc-binding lipoproteins of S. pneumoniae, AdcA and AdcAII, were characterized as playing redundant roles in zinc acquisition; however, this study shows that these proteins are not equally sensitive to zinc starvation and have additional functionality in adhesion and invasion. Mutant strains lacking AdcAII but not AdcA suffer decreased fitness when exposed to a zinc-chelator; and following chelation adcAII was upregulated 42old whereas adcA was only upregulated 4old. Zinc-deficient mutants lacking AdcA and AdcAII show increased invasion at levels reaching 200-300% compared to parental strains. Additionally, AdcAII-deficient strains show decreased ability to adhere to epithelial cells and colonize nasal tissues during murine challenge, suggesting a role for AdcAII or zinc homeostasis in biofilm formation. Analysis of biofilms grown in varying concentrations of metals revealed that increased zinc, specifically, resulted in the formation of larger, more architecturally complex biofilms. The increase in biofilm size was determined to be due to the formation of cell-to-cell aggregates. In addition to encountering high concentrations of metals, pathogens are competing for micronutrients from the host, and are thus adept to surviving metal starvation. A previously uncharacterized operon, SP1434-1438, was found to be sensitive to zinc-starvation and proteomics strongly suggest an importance for these genes in cellular metabolism. These results have identified roles for zinc homeostasis in cell adhesion, colonization, cell and bloodstream invasion, biofilm formation, and the maintenance of cellular metabolism.

Proper regulation of apoptosis during pneumococcal pneumonia is essential for resolution of infection. We hypothesized that reactive oxygen species (ROS) produced during infection causes sufficient DNA damage to alter expression of pro-apoptotic proteins. Despite inducing DNA damage, challenge with pneumococci did not cause alterations the expression of the pro-apoptotic protein Puma (p53 up-modulated regulator of apoptosis) at early (4 and 6 hour) and late (16 and 24 hour) time points tested in this study. Puma-dependent global expression patterns were assessed, and the data demonstrated significant changes in expression of various genes (Prdx2, Ripk1, Api5 and IL-10) involved in cell death and the inflammatory response. In conclusion, although the presence of Puma is necessary for normal apoptotic cellular death and host resolution of infection, Puma expression in bone marrow neutrophils and lung epithelial cells is not dependent on ROS produced during pneumococcal infection.

Streptococcus pneumoniae is a significant human pathogen and the leading cause of community-acquired pneumonia and acute otitis media. One of the primary defense mechanisms of the human immune system against pneumococcal infection involves granule-mediated killing of bacterial cells by neutrophils. While this mechanism has previously been shown to kill about half of pneumococci in vitro, we hypothesized that some pneumococcal strains have evolved to be more resistant to this granule-mediated killing. Clinical isolates demonstrated a varying range of sensitivity to neutrophil granules. Additionally, we established that the absence of the capsule may affect sensitivity as unencapsulated isolates showed a higher average survival than encapsulated isolates. Finally, pneumococcal surface protease HtrA was found to potentially serve as a protective factor as many knockouts were more sensitive than the wildtypes, recombinant HtrA protected wildtype TIGR4, and a resistant isolate showed higher htrA expression levels than sensitive isolates.

Vaccination is one of the most effective public health interventions in the world, saving millions of lives and preventing the onset of debilitating diseases. With widespread emergence of multi-drug resistant pathogens, the importance of preventative medicine has become even more apparent. However, one of the limiting factors in developing novel vaccines that are both safe and highly immunogenic is the availability of adjuvant delivery systems licensed for human use. The purpose of this study was to investigate the role gold nanoparticles could play as an effective vaccine delivery system. A variety of coupling chemistries were explored for their ability to conjugate protein and polysaccharide antigens onto the surface of gold nanoparticles for the development of vaccines against a number of biologically important human pathogens including Y. pestis, B. mallei and S. pneumoniae. Retention of antigenicity and coupling efficiency of conjugated molecules was measured using characterisation techniques such as localised surface plasmon resonance and immunoblotting. Gold nanoparticle coupled antigens were then used to immunise mice and to measure the protective efficacy and the immunological response induced. The findings indicate antigen-specific immune responses are elevated when an antigen is coupled onto gold nanoparticles. Moreover, immunological data from nanoparticle coupled glycoconjugate vaccines against B. mallei and S. pneumoniae indicate the likely presence of a strong T cell immune response which is essential for providing immunological memory. Finally, an intracellular trafficking assay was carried out to identify some of the mechanisms that might be involved in uptake of gold nanoparticles into professional phagocytes. Confocal imaging of receptors associated with endosomal compartments revealed that gold nanoparticles may enter cells through multiple pathways. The findings reported in this study suggest that gold nanoparticles may be an excellent candidate for further investigation as a novel vaccine delivery system.

Control of retrotransposon expression in the mammalian germline is regulated by Argonaute family PIWI proteins and their associated small non-coding RNAs known as PIWI-interacting RNAs (piRNAs). To date, no study has demonstrated clear PIWI protein expression nor identified a cellular function(s) for PIWI proteins in the mammalian soma. In contrast to the germline-restricted expression of piRNA associated proteins, we observed that Miwi2 mRNA was induced specifically in epithelial cells during pneumococcal pneumonia. Further investigation showed that similar to its mRNA, MIWI2 protein was indeed expressed outside of the mammalian germline, and was localized to the cytoplasm of a discrete population of multiciliated lung epithelial cells. Immunoprecipitation of MIWI2 from whole lung lysates indicated that it was bound to a small RNA that was longer than a traditional piRNA. Microarray analysis revealed that depletion of MIWI2 in a murine epithelial cell line or in a whole animal model had no effect on retrotransposon expression, further suggesting that lung MIWI2 is independent of nuclear piRNA silencing pathways. Under basal conditions, MIWI2 was required for the normal maintenance of airway epithelial cell fate. In fact, Miwi2 deficiency resulted in an increase in club cells and decrease in ciliated cells indicating that MIWI2 could play a primary role in mucociliary homeostasis or clearance. Similarly, as MIWI2 is induced during lung infection we sought to determine if it participated in host innate immune responses to bacterial infection. Using a clinically relevant model of community acquired pneumonia, Miwi2 deficient mice exhibited an increased expression of inflammatory mediators and immune cell recruitment thus leading to enhanced bacterial clearance. Taken together, these data support the notion that MIWI2 exerts piRNA-independent functions outside of the germline in the ciliated lung epithelium to regulate innate immunity during pneumonia. More broadly, these studies shed light on new areas in PIWI protein and lung ciliated cell biology, and may have implications for multiple diseases including cancer, inflammatory disorders, and infectious diseases.