Includes bibliographic references. / 2 v. : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A detailed description of possible aetiological mechanisms in sudden infant death syndrome and reports original work investigating a variety of causal mechanisms and pathological markers. / Thesis (M.D.)--University of Adelaide, Dept. of Paediatrics, 1994?

Bibliography: leaves 144-174. / ix, 176, [66] leaves, [22] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Isolates Escherichia coli from Adelaide children and screens for the presence of Shiga-like toxins genes using the polymerase chain reaction and by hybridization with specific DNA and oligodeoxynucleotide probes. Four SLT-producing strains were isolated. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1994

"8th July 2002." / Includes bibliographical references (leaves 276-308) / vi, 308, [57] leaves : charts ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the systematics (taxonomy, phylogeny, and epiemiology) of the genus Candida using a combination of traditional and contemporary methodologies. Assesses these methods to determine their diagnostic potential to unequivocally identify and characterise species and strains of this medically and dentally important yeast genus. / Thesis (Ph.D.)--University of Adelaide, Dept. of Dentistry, 2002

Mycobacterium avium subsp. hominissuis (MAH) is a pervasive environmental bacterium that can cause opportunistic infections in humans. Among the most robust and hardy members of the Mycobacterium genus, M. avium can persist and thrive in a range of challenging environments, including many which place it in direct contact with humans. Surface-exposed proteins are central to the bacterial processes involved in both environmental persistence and pathogenesis. These proteins also play a critical role in how the immune system of the host recognizes and responds to pathogens. Mycobacteria have evolved a specialized mechanism for protein export, a Type VII Secretion System (T7SS), in order to transport their proteins through their thick and impermeable cell envelope. This system is responsible for the export of several classes of proteins, many of which play an integral role in virulence. A central focus of this dissertation is the characterization of a conserved element of the T7SSs in pathogenic mycobacteria, a PPE family protein, whose deletion attenuates virulence in M. avium. Specifically, we examined the localization of this PPE protein (MAV_2928) within the bacterium, screened potential protein-protein interactions with other conserved elements in the adjacent T7SS loci and analyzed the transcriptional regulation of the gene in response to environmental changes. Seeking to more thoroughly characterize the surface-exposed proteome of M. avium, particularly in the context of early infection, we then developed a method, based on selective biotinylation and affinity purification, to profile the of surface-exposed proteome of the bacterium. We employed this method to analyze the surface-exposed proteomes of M. avium 109 that had been exposed to macrophages to those of M. avium 109 that had been cultured in media. This comparison detected several proteins whose presence at the bacterial surface appeared to be dependent on particular growth conditions. Lastly, in order to establish a more efficient method to isolate biotinylated surface proteins from complex mixtures, we developed a testing paradigm to identify modifications to the original method that might improve our coverage of identified proteins. Through this process, we developed a more robust methodology that yielded improved coverage and depth. We then utilized this technology to profile the surface-exposed proteome of another clinical isolate of M. avium subsp. hominissuis, M. avium 104. Beyond improving our understanding of the basic biology of M. avium, this new data provides independent evidence that PPE family proteins are indeed exported to the surface of M. avium, where they remain associated with the bacterial cell envelope. In total, this analysis represents the most comprehensive profile of the surface-exposed proteins of M. avium generated to date. / Graduation date: 2012

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the United States. Increasing resistance of Campylobacter to clinical antibiotics raises an urgent need for novel strategies to prevent and control infections in humans and animal reservoirs, which necessitates a better understanding of Campylobacter pathogenesis. We hypothesize that multidrug efflux pump CmeABC and ferric enterobactin (FeEnt) iron acquisition systems, which play a critical role in Campylobacter pathogenesis, are novel targets for developing effective measures against Campylobacter. To test this, the molecular, antigenic, functional, and protective characteristics of two outer membrane proteins, CmeC (an essential component of CmeABC drug efflux pump) and CfrA (a FeEnt receptor), were examined. Both CmeC and CfrA are highly conserved and widely produced in C. jejuni strains. Anti-CmeC and Anti-CfrA antibodies inhibited the function of CmeABC efflux pump and CfrA, resulting enhanced susceptibility to bile salts and reduced utilization of FeEnt of C. jejuni, respectively. Immunoblotting analysis also indicated that CfrA is expressed and immunogenic in vivo. Amino acid substitution mutagenesis demonstrated that a highly conserved basic amino acid R327 in CfrA plays a critical role in FeEnt acquisition. The purified recombinant CmeC and a Salmonella live vaccine expressing the protective epitope of CfrA were evaluated as subunit vaccines against Campylobacter infection in the chicken model. CmeC vaccination elicited immune response but failed to reduce C. jejuni colonization in the intestine. However, Salmonella-vectored vaccine conferred significant protection against C. jejuni challenge. To further elucidate the role of iron acquisition in the pathogenesis of Campylobacter, whole genome sequence of a unique C. jejuni strain was determined using a 454 GS FLX sequencer with Titanium series reagents. Comparative genomics analysis led to the identification of a novel Campylobacter Enterobactin Esterase (Cee) that is essential in the CfrB-dependent FeEnt utilization pathway. Extensive genetic manipulation revealed molecular pathways and mechanistic features of the two orchestrated FeEnt acquisition systems in Campylobacter. This project provides critical information about the feasibility of targeting CmeC and CfrA for immune protection against Campylobacter colonization in the intestine, and increases our understanding of the critical role of FeEnt acquisition in the pathophysiology of Campylobacter.

<p>An estimated twenty-five percent of the fifty-seven million annual deaths worldwide can be directly attributed to infectious disease. Mammals contain both adaptive and innate immune systems to deal with invading pathogens. The genetic model organism <em>Caenorhabditis elegans</em> lacks an adaptive immune system, which makes it a powerful model organism to study the innate immune system without the added complexity of an adaptive immune system. Multiple human pathogens can cause lethal infections in <em>C. elegans</em> and several <em>C. elegans</em> innate immune pathways have been identified that are conserved with mammals and protect the nematode from infection. The goal of this work was to identify novel bacterial virulence factors and innate immune defenses against pathogens by using the genetic model organism <em>C. elegans</em>. We established <em>C. elegans</em> as a model for <em>Yersinia pestis</em> infection and used this model to identify novel bacterial virulence factors that were also important for virulence in a mammalian model of infection. Previous studies demonstrated that <em>C. elegans</em> can identify bacterial pathogens using sensory neurons and activate an avoidance response that requires components of G-protein signaling pathways. We screened forty <em>C. elegans</em> strains containing mutations in chemosensory G-protein coupled receptors for altered survival on pathogen and identified <em>npr-1</em> to be required for full <em>C. elegans</em> defense against pathogens. We found that activation of the NPR-1 nervous circuit enhances host susceptibility to microbial infection while inhibition of the circuit boosts innate immunity. This data provides the first evidence that innate immunity in <em>C. elegans</em> is directly linked to the nervous system and establishes the nematode as a novel system to study neuroimmunology. From this work, we have identified <em>Y. pestis</em> virulence-related genes and <em>C. elegans</em> innate immune effector genes required for innate immunity to human bacterial pathogens.</p> / Dissertation

<p>Fungal cells sense the amount and quality of external nutrients through multiple interconnected signaling networks, which allow them to adjust their metabolism, transcriptional profiles and developmental programs to adapt readily and appropriately to changing nutritional states. In organisms ranging from yeasts to humans, the Tor signaling pathway responds to nutrient-derived signals and orchestrates cell growth. While in the baker's yeast <italic>Saccharomyces cerevisiae</italic> Tor responds to nutrient-derived signals and orchestrates cell growth and proliferation, in <italic>Schizosaccharomyces pombe</italic> Tor signaling modulates sexual differentiation in response to nutritional cues. Thus, these differences provide a framework to consider the roles of Tor in other fungal organisms, in particular those that are pathogens of humans. </p><p>In this dissertation, I demonstrate that in the human fungal pathogen <italic>Candida albicans</italic>, Tor signaling also functions to promote growth. This study also uncovered a novel role for the Tor molecular pathway in promoting hyphal growth of <italic>C. albicans</italic> on semi-solid surfaces and in controlling cell-cell adherence. Gene expression analysis and genetic manipulations identified several transcriptional regulators (Bcr1, Efg1, Nrg1, and Tup1) that together with Tor compose a regulatory network governing adhesin gene expression and cellular adhesion. While the Tor kinases are broadly conserved, these studies further demonstrate the contrasting strategies employed by fungal organism in utilizing the Tor signaling cascade.</p><p>While extensive studies have focused on elucidating functions for the Tor signaling cascades among ascomycetes, little is known about the pathway in basal fungal lineages, in particular among zygomycetes and chytrids. Moreover, given that the Tor pathway is the target of several small molecule inhibitors including rapamycin, a versatile pharmacological drug used in medicine, there is considerable interest in Tor signaling pathways and their function. Capitalizing on emerging genome sequences now available for several basal fungal species, we show a remarkable pattern of conservation, duplication, and loss of the Tor signaling cascade among basal fungal lineages. Targeting the pathway with rapamycin results in growth arrest of several zygomycete species, indicating a conserved role for this pathway in regulating fungal growth. In addition, we show a potential therapeutic advantage of using rapamycin in a heterologous model of zygomycosis. Taken together, the Tor signaling cascade and its inhibitors provide robust platforms from which to develop novel antimicrobial therapies, which may include less immunosuppressive rapamycin analogs.</p> / Dissertation

<p>Disease caused by the Gram-negative <italic>Haemophilus</italic> cryptic genospecies begins with colonization of the maternal genital or neonatal respiratory tract. The primary goal of this work was to identify and characterize the molecular determinant(s) of <italic>Haemophilus<i/talic> cryptic genospecies adherence as a means to better understand the specific adaptation of this species to the urogenital tract and neonatal respiratory tract. Using transposon mutagenesis of prototype strain 1595, we identified a locus that is essential for <italic>Haemophilus</italic> cryptic genospecies adherence to a variety of epithelial cell lines of both genital and respiratory origin. This locus encodes a protein called Cha that shares homology with trimeric autotransporters. Trimeric autotransporters are composed of an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a short C-terminal membrane anchor domain and are classically characterized by head-stalk-anchor domain architecture. By generating chimeric proteins, we demonstrated that the C-terminus of Cha trimerizes in the bacterial outer membrane and is capable presenting a heterologous passenger domain (Hia) in a functional form, thus confirming that Cha is a trimeric autotransporter. Southern analysis revealed that <italic>cha</italic> is unique to the <italic>Haemophilus</italic> cryptic genospecies and is ubiquitous among these strains. </p><p>Similar to a number of trimeric autotransporters, the passenger domain of Cha contains scattered clusters of YadA-like head domains associated with head-to-stalk neck adaptor motifs, predicted coiled-coil stalks and a series of identical tandem coding repeats which are not required for adherence. By evaluating the adherence capacity of <italic>H. influenzae</italic> expressing Cha deletion derivatives, we established that the N-terminal 473 residues of Cha harbor the binding domains responsible for Cha-mediated adherence to epithelial cells. In additional studies, we demonstrated that this same N-terminal region mediates bacterial aggregation through inter-bacterial Cha-Cha binding. </p><p>Further analysis revealed that variable Cha-mediated adherence is linked to spontaneous changes in the number of identical tandem repeats predicted to comprise a coiled-coil stalk domain. Variation in repeat copy number has a direct effect on Cha adhesive and aggregative activity, independent of an impact on transcription of the <italic>cha</italic> locus or surface localization of Cha protein. Moreover, length of Cha surface fibers correlates with repeat copy number expansion. We propose two hypotheses to explain how repeat expansion inhibits bacterial aggregation and host cell binding: 1) Variation in the number of 28-amino acid repeats may influence the conformation of Cha, thus changing the surface accessibility of the Cha binding pocket. 2) Repeat expansion results in the formation of long, flexible Cha fibers on the bacterial cell surface that may have a greater propensity to interact with neighboring Cha trimers at the N-terminus, thereby precluding adherence to other bacteria or host epithelial cells. </p><p>In additional studies screening adherent cryptic genospecies isolates for expression of Cha protein, we identified an additional, antigenically-divergent Cha variant that we refer to as Cha2. Amino acid sequence and domain comparison of Cha2 with Cha (now Cha1) revealed that the structurally undefined N-terminal sequences (encompassing the Cha1 adhesive and aggregative domain) are strikingly divergent. Inspite of this, Cha2 mediates efficient adherence to human epithelial cells, similar to Cha1.</p><p>Identification of Cha offers insight into the apparent tissue tropism associated with the <italic>Haemophilus</italic> cryptic genospecies. We speculate that the unique regulation of Cha adhesive activity enhances the adaptive capability of this pathogenic organism in the human host.</p> / Dissertation

Tomato bushy stunt virus (TBSV) provides a good model system to investigate molecular virus-host interactions in plants. P22 and P19 proteins encoded by TBSV contribute to multiple invasion-associated functions. Green fluorescence-mediated visualization of TBSV invasion in this study suggests that virus exit from inoculated epidermal cells is a crucial event. Close examination of one P22 mutant showed that it had lost the capacity to move between epidermis and mesophyll which was possibly due to an altered subcellular localization. P19 is a potent suppressor of RNA interference (RNAi) in various systems by forming dimers that bind 21-nucleotide (nt) duplex siRNAs (short interfering RNAs), to affect the programming of the RNA-induced silencing complex (RISC). P19 is attractive for biotechnological and research purposes to prevent RNAi of certain value-added genes in plants. To obtain a good plant-based expression platform, a suppression-active mutant P19 was expressed in transgenic N. benthamiana lines. This is the first example of P19 accumulating to detectable levels in a transgenic plant and initial results suggest it is actively suppressing RNAi. Furthermore, to investigate the correlation between siRNA binding of P19 and its various biological roles, predicted siRNA-interacting sites of TBSV P19 were modified, and the corresponding TBSV mutants were used to inoculate plants. Substitutions on siRNA-contact sites on the central domain of P19 resulted in more severe symptoms in N. benthamiana compared to those affecting peripheral regions. All tested combinations of siRNA-binding mutations were associated with reduced accumulation of total TBSV-derived siRNAs, and loss of siRNA sequestration by P19. Additionally, some modifications were found to cause RNAi-mediated disappearance of viral and host materials in N. benthamiana but not in spinach. In conclusion, exit out of epidermal cells is a key host range determinant for TBSV and particular amino acids on P22 may influence this by regulating the proper subcellular localization. Mutant P19 transgenic plants were successfully established with minor physiological effects to be applied as a platform to study RNAi and to over-express proteins. Finally, a compromised P19-siRNA binding impacts symptom development, systemic invasion, integrity of virus plus host RNA and proteins, and that all in a hostdependent manner.

Abstract S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals that, as with other IM-HK TCS, it responds to cell-envelope damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, CCCP and penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. Phenotypically, nsaS mutants display a 200-fold decreased ability to develop resistance to another cell-wall targeting antibiotic, bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS, a decrease in intracellular divalent metal ions was observed in an nsaS mutant, when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants also have alterations in cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell wall damage in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. Interestingly, in our microarray analysis, we observed a more than 30-fold decrease in transcription of an ABC transporter, SACOL2525/2526, in the nsaS mutant. This transporter bears strong homology to nsaAB, and is currently uncharacterized. Exploration of the role of SACOL2525/2526 revealed that, along with NsaRS, it too responds to cell-envelope damaging antibiotics. Specifically, its expression was induced by phosphomycin, daptomycin, penicillin G, ampicillin, oxacillin, D-cycloserine and CCCP. Mutation of this transporter results in increased sensitivity to the antibacterial agent daptomycin, and decreased sensitivity to free fatty acids. These findings are perhaps explained by altered membrane fluidity in the mutant strain, as the transporter null-strain is more readily killed in the presence of organic solvents, such as toluene. In addition, SACOL2525/2526 mutants have a decreased ability to form spontaneous mutants in response to several other peptidoglycan synthesis targeting antibiotics, suggesting a role for SACOL2525/2526 in antibiotic resistance. Inactivation of this transporter alters the cell envelope, and produces similar effects to those observed with the nsaS mutant, with increased capsule production, that may provide resistance to lysostaphin. Interestingly, the nsaS microarray revealed that this TCS negatively regulates only 34 genes, including 6 out of the 10 major secreted proteases. Despite a number of reports in the literature describing these enzymes as virulence factors, the data is often conflicting. Therefore, the contribution of proteases to CA-MRSA pathogenesis was investigated, by constructing a strain lacking all 10 extracellular protease genes. Analysis of this strain using murine models of infection reveals secreted proteases significantly impact virulence in both localized and systemic infections. Additionally, inactivation of these enzymes strongly influences survival in whole human blood, and increases sensitivity to antimicrobial peptides. Using a proteomics approach, we demonstrate that the contribution of secreted proteases to pathogenicity is related to differential processing of a large number of surface-associated virulence factors and secreted toxins. Collectively these findings provide a unique insight into the role of secreted proteases in CA-MRSA infections.