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Candida albicans is an opportunistic fungal pathogen. It is the fourth leading cause of nosocomial infections and can endanger immunocompromised patients. Candida has the ability to form biofilms on plastic medical devices, such as catheters and central nervous system shunts. Two clinical isolate series were profiled using a number of phenotyping assays comprising in vivo, ex vivo, and in vitro tests. These tests shed light on host-pathogen relations as well as offer potential information useful in the treatment of these infections. Fluconazole, an antifungal, is the first line of treatment for fungal infections. The incidence of fluconazole-resistant infections is increasing annually, and there are not many other drugs available to treat infections. In 2013, Fazly et al. discovered the drug Filastatin, which prevents adhesion and filamentation of Candida albicans. In our study, two screens were performed to identify the target of Filastatin. Because there is no complete knockout library for Candida, an available, partial knockout library was screened. This library is enriched for transcription factors. We screened for regulators of biological pathways that may be important for adhesion and filamentation in Candida albicans, to identify potential Filastatin targets. The iron-uptake pathway was chosen as the focus for the remainder of this study.

Iron is a trace element required for a range of metabolic reactions in virtually all living organisms. Studies of prokaryotes, plants, yeast, and vertebrates have established detailed information on iron uptake and the role iron plays in metabolic processes. Iron is an essential growth requirement of schistosomes in vitro and schistosomes also express the highly conserved iron storage protein ferritin. However, studies into how this iron is taken up by the parasite have been neglected. This study aims to identify molecules involved in iron uptake and homeostasis in the human parasite Schistosoma japonicum. I have characterised two isoforms of a divalent metal transporter (DMT), a membrane bound protein of schistosomes. These DMTs have significant homology to the mammalian DMT1, the primary ferrous iron uptake protein of the intestinal brush border. Both schistosome isoforms displayed functional iron uptake by rescuing growth in a yeast strain deficient in iron uptake (fet3fet4). Interestingly schistosome DMT1 was localised to the tegument and not the gastrodermis of adult parasites, suggesting surface mediated iron uptake across the tegument. In physiological conditions, iron is abundant as largely insoluble ferric iron and hence ferric reductases are an essential component of iron uptake, reducing iron to the soluble ferrous form. Cytochromes b561 (Cyts-b561) are a family of ascorbate reducing transmembrane proteins found in most eukaryotic cells. Recent observations that Cyts-b561 may be involved in iron metabolism have opened new perspectives for their physiological function. Here, I have identified a new member of the cytochrome b561 family in Schistosoma japonicum that localises to the tegument of this trematode. Expression of the SjCytb561 in a Saccharomyces cerevisiae mutant that lacks plasma membrane ferrireductase activity (fre1fre2) was able to rescue the growth defect in iron deficient conditions, suggesting involvement in iron metabolism. Plasma membrane ferrireductase activities were also quantified using intact transformed yeast cells. These data further support the hypothesised tegumental uptake of host iron. Further, I have identified a putative schistosome transferrin. In mammals, transferrin is a glycoprotein responsible for binding and transporting iron in the bloodstream and delivering iron into cells via a specific transferrin receptor. Preliminary characterisation of the schistosome transferrin sequence has revealed it does not contain all the conserved amino acid residues associated with iron binding, with conservation seen only in the C-terminal lobe, not in both the N and C-lobes observed in mammalian transferrins. This difference makes it unclear whether the schistosome transferrin shares functional homology with its mammalian counterpart. In addition, no transferrin receptor has been identified to support an iron trafficking and uptake function, nor would this function be expected in an acoelomate organism. Further characterisation and localisation of this protein is required to elucidate its biological significance and function. The tegumental location of both the SjDMT1 and the SjCytb561 for the uptake of host iron make it possible to consider these proteins as potential vaccine candidates. A preliminary vaccination study with these proteins elicited only low to moderate protection from infection, and further studies are required to fully assess their potential. The data presented in this thesis provide evidence for surface-mediated uptake of iron by adult schistosomes, and represent the first characterisation of iron uptake proteins in any helminths. These studies show a novel method of iron uptake in schistosomes, and contribute to our understanding of how these parasites are able to survive and thrive by scavenging nutrients, in this case iron, from the host organism.

Iron is one of the most important micronutrients used by living organisms. Iron is frequently a limiting nutrient for plant growth, and plants are a major source of iron for human nutrition. The most prominent symptom of iron deficiency in plants is interveinal chlorosis, or yellowing between the veins, which appears first in the youngest leaves. Iron deficiency anemia (IDA) is the number one human nutritional deficiency worldwide. In order to solve the problem of iron deficiency, it is desirable to breed plants that have increased iron in those parts that are consumed by humans. To do this, we must first understand the molecular basis of Fe uptake, transport, and storage in plants. In soil, iron is quickly oxidized to Fe(III), and Fe(III) is relatively insoluble, thus difficult for plants to obtain. Our lab has been working on metal ion homeostasis mechanisms in plants and the ultimate goal of our research is to understand the mechanisms by which plants maintain the correct levels of iron, zinc and copper in each cell and tissue. The Yellow Stripe-like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow stripe 1 (YS1). YS1 transports Fe(III) that is complexed by phytosiderophores (PS), strong Fe(III) chelators of the mugineic acid family of compounds. Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species including Arabidopsis thaliana. YSLs in non-grasses have been hypothesized to transport metals that are complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. In this dissertation, Arabidopsis YSL1 and YSL3 are demonstrated to be important in iron transport and also responsible for loading Fe, Cu, and Zn from leaves into seeds. Arabidopsis YSL4 and YSL6 are demonstrated to be involved in iron transport and metal mobilization into seeds. The transport function of Arabidopsis YSL1 and YSL2 are shown be partially overlapping to the function of Arabidopsis YSL3 in vegetative structures, but distinct in reproductive organs. Arabidopsis YSL3 and YSL6 are shown to have distinct functions in planta.

Iron is among the essential micronutrients for all living organisms and is a cofactor for many cellular redox reactions. Although iron is a highly abundant metal element found in the earth’s crust, it is also a limiting factor in plant development when it is present as insoluble ferric oxides. Plants have evolved two strategies to acquire soluble iron referred to as Strategy I and Strategy II. Our lab has focused on the Arabidopsis thaliana double mutant ysl1ysl3. The mutants display a chlorotic phenotype and are unable to correctly respond to iron deficiency. Grafting is a common method for joining different plant tissues and has been used for studies of long distance signaling. Past studies of iron signaling in Arabidopsis have not been able to provide a mechanism for how plants are able to signal the iron status of the shoot, where iron demand is high, to roots, where iron uptake occurs. The iron signaling experiments included in this thesis follow a seedling-graft approach to understand if grafts are capable of properly sensing iron. A longstanding question of iron homeostasis in plants is the identity of the iron sensors in plants. It was hypothesized that YSL1 and YSL3 have both a transporter function and a receptor function, and therefore function as transceptors. In our predicted model it was proposed that YSL1 and/or YSL3 are directly involved in iron status signaling either in perception and/or transmission of the signal. As evidenced through seedling grafting experiments here, YSLs play a critical part of long distance signaling that plant shoots use to signal their iron status to the roots. In this thesis, YSL1 and YSL3 are shown to be required in the shoots in order for signaling to occur correctly in the roots. To facilitate the analysis of gene expression in the grafts, a FRO3promoter:GUS construct was used in the Col-O WT background. The FRO3 promoter was selected because it is expressed in both leaves and roots under iron deficiency. Experiments showed that the genotype of the shoot used in the grafts is critical for Fe-deficiency induced gene expression in the roots. Thus, grafting has revealed that root iron deficiency responses require YSL1 and YSL3 in leaves for signal transmission. This directly links them to long-distance signaling, and supports the idea that these proteins could be acting as transceptors.

Ferric ABC Transporters. Pathogenic bacteria have evolved specialised iron acquisition systems that allow them to effectively colonise a host. One of these systems is the ferric binding protein (Fbp) complex that is a member of the ATP-Binding Cassette (ABC) superfamily of small molecule transporters. The Fbp complex is made up of three-components (FbpABC) that transports ferric iron from the periplasm to the cytoplasm of many Gram negative bacteria. FbpA binds iron in the periplasm and transports it to the FbpB transporter complex that permeates the cytoplasmic membrane. Here the iron is actively transported by FbpB through the membrane that is powered by ATP hydrolysis catalysed by FbpC, the cytoplasmic ATPase. Burkholderia cenocepacia is an opportunist pathogen that colonises the lungs of cystic fibrosis patients and is particularly resistant to antibiotic treatment. In this study the iron uptake system of B. cenocepacia strain J2315 is investigated. A putative FbpA from B. cenocepacia J2315 was expressed in the periplasm of Escherichia coli cells and the recombinant FbpA B. cenocepacia protein purified. The structural and electrochemical properties of native FbpA B. cenocepacia were investigated using UV Visible spectroscopy, spectro-electrochemistry, mass spectrometry and crystallographic techniques. It appears that FbpA B. cenocepacia is a novel member of the FbpA superfamily that selectively utilises citrate as an exogenous anion in ferric iron co-ordination. This is the first instance that a recombinant ferric binding protein has been documented as preferentially utilising citrate in this manner. The putative ATPase from B. cenocepacia (FbpC B. cenocepacia) was also expressed in E. coli but it was found to be insoluble. A number of expression systems were tested but none were found to be successful in generating sufficient quantities of FbpC B. cenocepacia for structural studies. Human β-defensin 2. Despite daily contact with a range of microorganisms, mammals do not regularly succumb to pathogenic invasion. One reason is the presence of an important defence mechanism uses a reservoir of antimicrobial peptides (AMPs) that are expressed in eukaryotes as a means of innate immunity. The AMP superfamily is composed of over 900 members, displays broad structural and sequence diversity and is active against a wide range of bacteria, fungi and viruses. β-defensins are small (3-5 kDa), cationic peptides that display antimicrobial activity against a range of microbes and have also been shown to act as chemo-attractants (chemokines) within the adaptive immune system. In this study we obtained milligram amounts of pure human β-defensin 2 (HBD2) for functional studies by the development of a method for the rapid expression and purification of the recombinant peptide. A clone encoding a thioredoxin-HBD2 fusion protein was designed for the expression of soluble peptide in E. coli cells that was purified by simple affinity chromatography. The HBD2 peptide was cleaved from the fusion by an efficient protease step and further purified to yield pure HBD2. This recombinant HBD2 defensin was shown to be active against a Mycobacterium tuberculosis mutant strain.

To examine the effect of buttermilk fractions on growth, iron transport, and uptake, Caco-2 cells (human colon adenocarcinoma) were grown in a bicameral chamber. The Caco-2 cell culture system is a useful model to study micronutrient utilization in the human enterocyte, because Caco-2 cells continuously differentiate and form a monolayer, which has high polarity, a well-developed brush border, and a tight junction. Iron bioavailabilty in various milks is very different depending upon milk composition. The fat fraction especially is known to be associated with iron absorption, because the fat fraction has milk fat globule membrane (MFGM), which contains bioactive molecules such as sphingolipids. Composition of buttermilk that was concentrated by 10 K molecular sieving (MS) or by bacterial fermentation (Lactococcus latis PN-l) was reduced in lactose concentration and increased in protein concentration. Percent fat in MS buttermilk was concentrated to two times higher than in the original buttermilk (P < 0.05). Growth of Caco-2 cells with molecular sieved (MS) or fermented (FM) buttermilk in the growth medium was not significantly different. Transport and uptake of 59Fe was performed with/without cold iron (1 mmol/L) by iron-depleted or iron-repleted cells. Molecular sieved or fermented buttermilk and ganglioside or sphingomyelin standards with dimethyl sulfoxide (DMSO) were added to the Hank's balance salt solution (HBSS) in the apical chamber. With cold iron, addition of MS and FM buttermilk (1, 2, or 3 percent) increased 59Fe transport across iron-repleted cells (P < 0.01). Without cold iron, ganglioside depressed 59Fe transport (P < 0.01). Uptake of 59Fe was not significantly affected by buttermilk concentrates; however, more effective uptake was shown across iron-depleted cells. It is not clear from these studies that buttermilk fractions or their components influence iron uptake or transport by Caco-2 cell cultures.

Siderophores are low molecular weight, iron chelating compounds produced by many bacteria for uptake of iron in case of iron scarcity. Vicibactin is a trihydroxamate type siderophore produced by Rhizobium leguminosarum bv. trifolii ATCC 14479. This work focuses on identifying an outer membrane receptor involved in the transport of vicibactin. We have confirmed the presence of the putative fhuA gene in R. leguminosarum bv. trifolii ATCC 14479. This bacteria shows mutualistic symbiosis with the red clover plant Trifoliium prantense. Leghemoglobin, with its cofactor heme is present in the plant root nodules that surrounds the infecting organism present in the nodules. This work attempts to elucidate the ability of Rhizobium leguminosarum bv. trifolii ATCC 14479 to utilize heme-bound iron and genes involved in the transport. We have also elucidated the role of energy transducing proteins TonB- ExbB-ExbD on the heme-bound iron uptake system.

Escherichia coli is an incredibly diverse group of bacteria that consist of both commensal and pathogenic strains that cause disease in a wide variety of tissues in many different animals. The current dogma, based on years of extensive molecular and genetic studies, is that individual strains have adapted to specific environments through acquisition of specific genes or come from lineages that are particularly suited to a unique tissue or host. However, mastitis-associated E. coli (MAEC) have thus far resisted such descriptions. The fitness and virulence factors of MAEC are poorly understood and molecular tools are rarely applied. This dissertation reports new approaches to assess virulence of MAEC strains, enabling comparative genomic studies across multiple strains as well as genome-wide analysis of specific successful MAEC isolates. I outline the identification of the first virulence factor of MAEC, a ferric dicitrate receptor that is essential for colonization of a lactating mammary gland in a murine model. Genes previously studied in the contexts of other extraintestinal E. coli infections were also implicated in mastitis. These include a type III capsule found in the MAEC strain M12, which is crucial for dissemination from the mammary gland to the spleen. A mutant unable to produce capsule had diminished lethality in Galleria mellonella and decreased kidney colonization in a mouse urinary tract infection. I also report a link between zinc uptake, bile salts, and capsule production. I have utilized a transposon mutant library paired with deep sequencing of transposon junctions to elucidate the fitness factors needed to grow in milk and colonization of both murine and insect models. This analysis implicates a broad set of genes and metabolic pathways pertinent to these conditions. In addition to Tn-seq, I sequenced 94 MAEC genomes and identified genes associated with disease severity, growth in milk, and colonization of mammary glands in cow and mouse models. Employing bioinformatic tools to interrogate the pan-genome, I identified genes that are involved in biofilm formation and adhesion that were specifically associated with either mild or severe disease. In summary, I have employed several powerful genetic, genomic, computational, and molecular approaches to the characterization of mastitis associated E. coli. This work provides the groundwork for future experiments to better understand the host-pathogen interface and a model for mastitis-associated E. coli.

Rhizobium leguminosarum is a Gram negative, motile, nitrogen-fixing soil bacterium. Due to the scarcity of iron in the soil bacteria have developed a wide range of iron scavenging systems. The two types of iron scavenging systems used are indirect and direct. In-silico analysis of the genome identified a unique direct iron scavenging system the Hmu operon. This system has been identified in other closely related rhizobium species and is believed to be involved in utilizing heme compounds as a sole source of iron. We have attempted to characterize the role of the Hmu operon in iron utilization by monitoring the growth of R. leguminosarum ATCC 14479 in hemin supplemented media. Growth curves show that it is capable of using hemin as a sole source of iron. The outer membrane profiles were analyzed for the presence of hemin binding proteins.