<p><em>Chlamydia pneumoniae</em> is a Gram-negative intracellular pathogen that uses type III secretion to invade and survive within eukaryotic cells. The T3SS secretes specific effector proteins during the infection process to facilitate immune evasion and nutrient acquisition. Unfortunately, the genetic intractability and difficult culturing conditions of Chlamydiae has inhibited progress in the chlamydial T3S field. This thesis characterizes fundamental aspects of the <em>C. pneumoniae </em>injectisome such as the ATPase, the inner-membrane export apparatus, and a specific effector protein Cpn0803. Initially, we explored whether <em>C. pneumoniae</em> encodes a functional T3S ATPase and if it associates with other T3S components. We found that CdsN has enzymatic activity consistent with other Gram-negative T3S ATPases, and that CdsN associates with inner-membrane and soluble components such as CdsD, CdsQ, CopN and CdsL. We also found that CdsN has binding surfaces for either structural or putative effector / chaperone T3S proteins. Next, we explored the putative flagellar genes, which were of interest since <em>Chlamydia</em> is a non-motile bacteria that lacks flagellum. We found that the flagellar proteins associate with the T3S apparatus, suggesting that they play a role in T3S during the life-cycle. We extended this observation to show that CdsL, a T3S component, down-regulates both CdsN and FliI enzymatic activity, suggesting that the flagellar proteins are involved in T3S. Furthermore, we characterized Cpn0803 as an exemplary effector, which associates with both CdsN and FliI. We found that Cpn0803 is secreted into host cells upon<em> Chlamydia</em> infection. Cpn0803 was thought to be the T3S needle-tip protein; however, the crystal structure does not support this hypothesis. Presently, the actual role of Cpn0803 in the T3S apparatus remains unknown. Overall, our data suggests that CdsN and FliI both function during the chlamydial life-cycle in the T3S process, possibly coordinating effector proteins (such as Cpn0803) for secretion into host cells.</p> / Doctor of Philosophy (PhD)

<p>Bacteria demonstrate an extraordinary capacity to survive and adapt to changing environments. In part, this ability to adapt can be attributed to horizontal gene transfer, a phenomenon which introduces novel genetic information that can be appropriated for use in particular niches. Nowhere is this more relevant than in pathogenic bacteria, whose acquisition of virulence genes have provided an arsenal that permits them to thrive within their selected host. Regulatory evolution is necessary for timely regulation of these acquired virulence genes in the host environment. <em>Salmonella enterica</em> serovar Typhimurium is an intracellular pathogen which possesses numerous horizontally-acquired genomic islands encoding pathogenic determinants that facilitate its host lifestyle. One island, <em>Salmonella</em> Pathogenicity Island (SPI)-2, encodes a type-III secretion system (T3SS) which is regulated by the two-component regulatory system SsrA-SsrB. This system coordinates expression of the SPI-2 T3SS as well as an array of virulence effectors encoded in horizontally-acquired regions throughout the <em>Salmonella</em> genome. The studies presented here investigated the mechanisms in which the transcription factor SsrB functions to integrate virulence processes through regulatory adaptation. This work identified the regulatory logic controlling SsrB and defined the associated SsrB regulon. Furthermore, SsrB was found to induce a regulatory cascade responsible for the expression of bacteriophage genes encoded within SPI-12, an island that also contributes to <em>Salmonella</em> virulence. These findings demonstrate the important contribution of regulatory evolution in pathogen adaptation to the host, and show that horizontally-acquired genes, once integrated into appropriate regulatory networks, can contribute to pathogen fitness in specific niche environments.</p> / Doctor of Philosophy (PhD)

<p><strong>ABSTRACT</strong></p> <p>As plants are sessile organisms, detecting the presence of neighboring plants and exhibiting competitive behavior to acquire limiting resources is crucial. One of the ways plants respond to belowground competition is by allocation to fine roots in order to acquire the limited resources. However, this phenotypic plasticity can be costly as it assigns resources away from reproduction. Being able to recognize the relatedness of one′s neighbours and preferentially compete with strangers is a beneficial trait that can minimize the costs of competition with relatives and increases inclusive fitness. Many studies have looked at the association between resource availability and competition in plants while others have observed kin recognition in several plants species. However, no one has yet studied the effect of resource availability on kin recognition in plants. Here, I looked at root architecture to test if there is an association between kin recognition and nutrient availability in <em>Cakile edentula</em>.</p> <p>I found that the root system architecture is highly plastic and complex, showing variable responses to neighbour identity signals and resource availability. The results from the four experiments demonstrate that the responses of <em>C. edentula </em>to neighbour relatedness are dependent on nutrient availability. Additionally, this study also indicates that kin recognition in <em>C. edentula</em> does not require root contact; instead it occurs through a signal found in soluble compounds excreted from plants, possibly root exudates, as observed in <em>Arabidopsis thaliana</em> <em>(</em>Biedrzycki et al. 2010).</p> <p>In conclusion, this study provides novel findings regarding the dynamics of root behavior in response to nutrient availability and the relatedness of neighbours.</p> / Master of Science (MSc)

Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCδ-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCδ in platelets. SH2 domain-containing Inositol Phosphatase (SHIP)-1 is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists, SFLLRN and AYPGKF, or GPVI agonist, convulxin. GPVImediated SHIP-1 phosphorylation occurred rapidly at 15 sec whereas PAR-mediated phosphorylation was delayed, occurring at 1 min. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKCδ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCδ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCδ regulates the phosphorylation of SHIP-1. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1, Y311 and Y155 of PKCδ were inhibited. In murine platelets lacking Lyn, or SHIP-1, GPVI-mediated dense granule secretions were potentiated, whereas PAR-mediated dense granule secretions were inhibited. Phosphorylated SHIP-1 associated with phosphorylated-Y155 PKCδ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCδ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets. / Physiology

Antagonistic interactions between competing species of bacteria are an important driver of bacterial community composition in the human gut microbiota. Of particular significance is the role of the type six secretion system (T6SS), which many species of Gram-negative bacteria use to kill competitor bacteria in a contact-dependent manner. T6SSs are syringe-like nanomachines that function to deliver antibacterial toxins into susceptible competitors. Many bacteria present in the human gut microbiota possess an extremely potent T6SS that is capable of rapidly eradicating nearby bacteria. Remarkably, however, species of beneficial bacteria that coexist in the gut are often resistant to T6SS attack by their neighbours. This resistance is mediated by bacterial immunity proteins that block the activity of the antibacterial toxins delivered by the T6SS. Intriguingly, past studies have shown that the widespread T6SS-mediated competition in the gut has led to the acquisition of repertoires of immunity genes across different bacterial strains. By examining available human gut metagenomes, I identified a putative immunity locus, named I2, in a species of gut bacteria. This locus is located downstream of its cognate T6SS toxin-encoding locus, E2, and I show when co-expressed with E2 in E. coli, it protects against E2 mediated-toxicity. Additionally, I show that four gut-derived I2 homologues bearing sequence identity levels to I2 ranging from 38% to 75% are equally capable of abrogating E2 toxicity. Using quantitative biophysical measurements, I also show that these I2 homologues physically bind E2 equally tightly pointing to the potential molecular mechanism of toxin neutralization. Lastly, through mutagenesis experiments, I found that the E2-I2 interaction is likely mediated by electrostatic forces between a small number of residues found in the interaction interface of the two proteins. Overall, these findings demonstrate that a human gut microbiome encoded type VI secretion system effector can be neutralized by divergent immunity proteins. / Thesis / Master of Science (MSc)

No description available.

No description available.

Clostridium perfringens is a spore-forming anaerobic Gram-positive rod which has gliding motility through type IV Pili (TFP). Since the discovery of TFP in Gram-positive bacteria is relatively new, more studies are required to understand the mechanism and interaction of the proteins of this machinery. Moreover, the similarities between TFP and type 2 secretion system (T2SS) suggest that C. perfringens has also a T2SS. We studied the localization of TFP ATPases, PilB1, PilB2 and PilT in Bacillus subtilis to compare the localization in an organism other than C. perfringens and which lacks any known genes similar to TFP. Unlike the case in C. perfringens, PilB1 in B. subtilis localized to the poles in the absence of PilT, with some central foci at the future division sites. Colocalization of PilB1 was also studied with PilT and the results suggested that PilB1 needs PilT to migrate from the poles to the center. Localization of PilB2 in B. subtilis, was similar to the results in C. perfringens and to the localization of PilB1 in B. subtilis. We have not been able to co-express PilB2 with PilT yet. Succeeding in this study will help us better understand the interactions between PilB proteins and PilT. In another project, we studied a von Willebrand factor Type A-Domain Containing protein (vWA) which is secreted from C. perfringens strain 13. We overexpressed and purified this protein and tested the effects on mammalian cells. We found that the vWA is probably not a toxin but since it seems to bind to macrophage membranes, we propose that the vWA could be part of a toxin complex, probably the subunit of the complex that binds to the host cells. / Master of Science

The Type III secretion system (T3SS) of Pseudomonas aeruginosa uses a needle-like protein apparatus to detect eukaryotic host cells and translocate effectors directly into the host cell. The effectors are also known as cytotoxins, which cause disruption of a series of signaling events in the host cell, facilitating the infection by P. aeruginosa. As the T3SS is antigenic and the expression of T3SS is energy-consuming, it is highly regulated where several regulatory proteins interact with each other and control the expression of T3SS genes. Among these proteins, ExsA, the master regulator of T3SS in P. aeruginosa, is of great importance as it is a transcriptional activator that activates the expression of all T3SS genes. Also, as ExsA belongs to the AraC protein family which only exists in bacteria and fungi, it makes an excellent potential target for drugs against P. aeruginosa related infections. With a combination of molecular biology tools and structural biology methods, we solved the N-terminal domain structure of the ExsA protein in P. aeruginosa. The model of the ExsA N-terminal domain has enriched our knowledge about ExsA dimerization and can serve as the base for mapping the interaction interfaces on ExsA and ExsD. Further, we have found two homologues of ExsA by structural alignment, which share a lot of similarities and have conserved amino acid residues that are important for ligand binding. The fact that both of these two proteins are regulated by small ligands rather than proteins also raises the possibility that ExsA may have a second regulatory mechanism under which ExsA is regulated by a small ligand, which so far has not been observed or reported by researchers. In order to map the binding site of ExsA on its anti-activator ExsD, we removed the coiled-coil region (amino acid residue 138-202, the potential binding site) of ExsD, based on the  structure of ExsD. We surprisingly found that the ExsD variant without the coiled-coil region readily inhibits ExsA-dependent in vitro transcription. This result rules out other possibilities and makes us focus on the N-terminus and adjacent regions of ExsD for the interface with ExsA. Moreover, in order to gain a comprehensive understanding of the dynamics of the regulation of T3SS in P. aeruginosa, we have begun to build a mathematical model of the T3SS regulatory pathways. We are measuring the cellular concentrations of T3SS regulatory proteins with quantitative molecular biology methods such as quantitative western blot, quantitative PCR and quantitative mass spectrometry. We have determined the cellular level of ExsA and ExsD proteins under different physiological conditions, and found that some factors such as temperature have a significant impact on the levels of ExsA and ExsD. This study has thus unveiled some unknown features of the T3SS of P. aeruginosa and its related infections. / Master of Science

Fusobacterium nucleatum is a Gram-negative, anaerobic bacterium that is a member of the human oral microbiota. Although it is a normal resident of the mouth, it is associated with a number of human diseases including: sepsis, inflammatory bowel disease (IBD), and colorectal cancer (CRC). Despite the important association of F. nucleatum with human health and disease, remarkably little is known about the molecular mechanisms underlying these infections. This knowledge gap can, in part, be attributed to a lack of molecular tools and experimental workflows. Creating the genetic tools to fill this knowledge gap is an imperative undertaking for the future development of treatments for diseases involving F. nucleatum. Previous work in the field has assigned functions to just a handful of Fusobacterium proteins (Fap2, FadA), and only two of those proteins have a well-defined role in the host-pathogen relationship. This dissertation contains work that lays the molecular and genetic foundation for future studies involving F. nucleatum by creating a unique gene deletion system while simultaneously establishing broadly applicable experimental workflows and molecular tools to study initial bacterial attachment and invasion processes crucial to Fusobacterium virulence. Marker-less gene deletions confirm the importance of Fap2 in host-cell attachment and invasion and suggest a lesser role in invasion for FadA, representing a significant revision to the Fusobacterium-host relationship. Also, our system allows for the overexpression and purification of virulence factors directly from Fusobacterium for the first time. This permits us to study aspects of Fusobacterium protein biology that were previously impossible and will provide further insights into the nature of Fusobacterium virulence. A custom suite of molecular tools was also developed to facilitate recombinant expression of these proteins in general laboratory settings using simple E. coli protein expression systems. We have used these new technologies to express and purify a number of potential Fusobacterium virulence factors as detailed in this dissertation. Also contained in this dissertation is the application of these breakthroughs to probe the function of a novel F. nucleatum outer membrane phospholipase, FplA. Phospholipases are important virulence factors in a number of well-studied human pathogens including Pseudomonas aeruginosa and Legionella pneumophila, where they interfere with host cellular signaling processes to increase intracellular bacterial survival. Our data show that FplA is a Class A1 phospholipase (PLA1) with robust catalytic activity capable of binding to and cleaving a number of lipid types. Additionally, we show that it has the ability to bind to important host signaling lipids including phosphatidylinositol 3, 5-bisphosphate and phosphatidylinositol 3, 4, 5-triphosphate. These data suggest FplA may play a role in manipulating the intracellular processes of host cells. Taken together, work in this dissertation provides tools and experimental frameworks for the future study of F. nucleatum pathogenesis while identifying and initially characterizing a new, potentially significant, virulence factor in FplA. / Doctor of Philosophy