The emergence of multidrug resistant Staphylococcus aureus strains has increased the difficulty of treating these infections. Bacteriophages can eliminate bacterial infections and some bacteriophage tails are lethal to their bacterial hosts. Interestingly, strains of Pseudomonas produce specialized phage tail-like molecules, called pyocins, which are lethal to bacterial strains. The central goal of my project was to explore the use of phages lacking DNA (ghosts) and genetically engineered phage tail variants as anti-staphylococcal disinfectants or therapeutic agents. I tested a variety of methods to generate staphylococcal phage ghosts and tails. For one of these, engineered tails derived from staphylococcal phage 77, I tested its ability to effectively kill S. aureus by measuring their effect on cell growth and survival, their ability to bind staphylococcal cells and induction of potassium efflux. These genetically created tails bound staphylococcal cells, but no effect on growth or survival was observed from tails or ghosts.

The emergence of multidrug resistant Staphylococcus aureus strains has increased the difficulty of treating these infections. Bacteriophages can eliminate bacterial infections and some bacteriophage tails are lethal to their bacterial hosts. Interestingly, strains of Pseudomonas produce specialized phage tail-like molecules, called pyocins, which are lethal to bacterial strains. The central goal of my project was to explore the use of phages lacking DNA (ghosts) and genetically engineered phage tail variants as anti-staphylococcal disinfectants or therapeutic agents. I tested a variety of methods to generate staphylococcal phage ghosts and tails. For one of these, engineered tails derived from staphylococcal phage 77, I tested its ability to effectively kill S. aureus by measuring their effect on cell growth and survival, their ability to bind staphylococcal cells and induction of potassium efflux. These genetically created tails bound staphylococcal cells, but no effect on growth or survival was observed from tails or ghosts.

Macromolecular complexes are important in almost all cellular processes. The bacteriophage head-tail connector complex offers a model with which to study the mechanisms that control their formation and the interactions that govern their assembly. The head-tail connector joins DNA-filled heads with mature tails. This thesis describes the structures, functions, and evolution of two connector proteins, HK97 gp6 and Lambda gpFII. Middle-ring connector proteins act as head-stabilizing proteins after the packaging of DNA in the heads. I found that gp6 is the middle-ring connector protein of bacteriophage HK97. I determined that gp6 is part of a large family of middle-ring connector proteins that share common sequence and structure elements. I also show that the mechanism for the addition of gp6 to the connector is mediated by limiting gp6 expression by strictly controlling translation initiation. I also describe a three-dimensional model for the assembly of the head-tail joining protein of bacteriophage Lambda, gpFII. This model correctly predicts regions of the protein predicted to interact with the Lambda head and tail. It also provides evidence for the description of a new family of proteins that evolved from a tail tube protein. This family includes members from both contractile and non-contractile phage tails and the bacterial type VI secretion system.

Candida species are the fourth most common cause of hospital-acquired blood-stream infections in the United States with mortality rates approaching 50%. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. I established a new role for the essential molecular chaperone Hsp90 and the protein phosphatase calcineurin in regulating resistance to cell wall stress exerted by the echinocandins, the only new class of antifungal drug to reach the clinic in decades, in both Candida albicans and Candida glabrata, the two leading causes of candidiasis. Through reciprocal co-immunoprecipitation studies, calcineurin activation studies, and protein stability assays, I established calcineurin as the first client protein of Hsp90 in C. albicans. I found that the calcineurin downstream effector Crz1 plays only a partial role in mediating tolerance to the echinocandins in C. albicans, implicating additional downstream effectors. Complementary studies in the model organism Saccharomyces cerevisiae revealed a divergence in the requirement of Hsp90 and calcineurin in this species’ ability to tolerate echinocandins despite a conserved functional relationship ii between Hsp90 and calcineurin. I also provided the first global view of mutations that accompany the evolution of fungal drug resistance in a human host. I found an association of mutations in CDC6 and MOH1 with echinocandin resistance in unrelated C. glabrata clinical isolates, genes previously not implicated in echinocandin resistance. I propose a new model by which Hsp90 and calcineurin regulate echinocandin resistance by controlling expression of the resistance determinant FKS2. Taken together, my research reveals new mechanisms mediating antifungal drug resistance and suggests new therapeutic strategies to save human lives.

Candida species are the fourth most common cause of hospital-acquired blood-stream infections in the United States with mortality rates approaching 50%. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. I established a new role for the essential molecular chaperone Hsp90 and the protein phosphatase calcineurin in regulating resistance to cell wall stress exerted by the echinocandins, the only new class of antifungal drug to reach the clinic in decades, in both Candida albicans and Candida glabrata, the two leading causes of candidiasis. Through reciprocal co-immunoprecipitation studies, calcineurin activation studies, and protein stability assays, I established calcineurin as the first client protein of Hsp90 in C. albicans. I found that the calcineurin downstream effector Crz1 plays only a partial role in mediating tolerance to the echinocandins in C. albicans, implicating additional downstream effectors. Complementary studies in the model organism Saccharomyces cerevisiae revealed a divergence in the requirement of Hsp90 and calcineurin in this species’ ability to tolerate echinocandins despite a conserved functional relationship ii between Hsp90 and calcineurin. I also provided the first global view of mutations that accompany the evolution of fungal drug resistance in a human host. I found an association of mutations in CDC6 and MOH1 with echinocandin resistance in unrelated C. glabrata clinical isolates, genes previously not implicated in echinocandin resistance. I propose a new model by which Hsp90 and calcineurin regulate echinocandin resistance by controlling expression of the resistance determinant FKS2. Taken together, my research reveals new mechanisms mediating antifungal drug resistance and suggests new therapeutic strategies to save human lives.

Macromolecular complexes are important in almost all cellular processes. The bacteriophage head-tail connector complex offers a model with which to study the mechanisms that control their formation and the interactions that govern their assembly. The head-tail connector joins DNA-filled heads with mature tails. This thesis describes the structures, functions, and evolution of two connector proteins, HK97 gp6 and Lambda gpFII. Middle-ring connector proteins act as head-stabilizing proteins after the packaging of DNA in the heads. I found that gp6 is the middle-ring connector protein of bacteriophage HK97. I determined that gp6 is part of a large family of middle-ring connector proteins that share common sequence and structure elements. I also show that the mechanism for the addition of gp6 to the connector is mediated by limiting gp6 expression by strictly controlling translation initiation. I also describe a three-dimensional model for the assembly of the head-tail joining protein of bacteriophage Lambda, gpFII. This model correctly predicts regions of the protein predicted to interact with the Lambda head and tail. It also provides evidence for the description of a new family of proteins that evolved from a tail tube protein. This family includes members from both contractile and non-contractile phage tails and the bacterial type VI secretion system.

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.

The enteropathogenic bacteria Salmonella are the main cause of food borne gastroenteritis worldwide. The activation of Toll-like receptor 4 (TLR4) by LPS triggers an immune response to counter infection. Signaling by TLR4 requires the adaptor proteins, TIRAP and TRAM. Recruitment and activation of these molecules is dependent on the membrane lipid, PIP2. The Salmonella effector, SigD, is a 4-phosphatase that depletes PIP2 from the host plasma membrane during invasion. Thus, we investigated if SigD could lead to the interruption of the TLR4 pathway. We observed that SigD expression caused the disappearance of TIRAP from the Salmonella containing vacuoles (SCVs) in HeLa cells. Furthermore, we demonstrated that SigD attenuates NF-κB activation, implicating SigD in the disruption of the MyD88 dependent pathway. In addition, the observed inhibition of PKCε phosphorylation suggests SigD may also block the other branch of the TLR4 signaling cascade, the MyD88 independent pathway.

Bisphosphonate-induced osteonecrosis of the jaw (BPONJ) has been identified as a severe complication of dental treatment in 1-10% of patients previously treated with intravenous bisphosphonates. The mechanism by which bisphosphonates induce BPONJ is uncertain. It has been noted that necrotic bone from BPONJ sites display signs of bacterial infection that suggests that an immune defect may play a role in the pathophysiology of BPONJ. The purpose of this thesis examined the effect of a potent bisphosphonate, zoledronate, on the innate immune system, specifically, neutrophil function, differentiation and survival with in vitro and in vivo murine models. Zoledronate exposure leads to decreased neutrophil migration, neutrophil NADPH oxidase activity, circulating neutrophil counts, as well as neutrophil survival, however does not appear to affect neutrophil differentiation. We present evidence that bisphosphonates have the potential to depress the immune system in mice and a subset of patients, possibly contributing to the pathogenesis of BPONJ.

Legionella pneumophila (Lp) is the pathogen responsible for Legionnaires disease. Lp invades and survives in human macrophages to form an intracellular compartment, called the Legionella containing vacuole (LCV). Within the LCV, Lp avoids degradation and replicates, before killing its host and releasing its virulent progeny. Lp is pleomorphic, exhibiting a combination of short rod phenotypes and filaments. Filamentous Lp has been reported in patient samples, but current studies of Lp virulence are exclusively focused on rods. Our results show that filamentous Lp can invade and replicate in human and murine macrophages. Filaments are phagocytosed gradually into tubular phagocytic cups. Interestingly, the formation of the LCV starts at this stage of phagocytosis, and LCV markers were detected before the sealing of the phagosome occurred. We present evidence that the filamentous morphology acts cooperatively with Lp effectors to subvert the microbicidal activities of the macrophage, contributing to the survival of Lp.