Problem: Shigella is a gram-negative enteropathogen that, when passed through fecal particles from one host to the oral cavity of another host, causes an infectious disease known as shigellosis. One of the distinctive features of the infection by Shigella is its ability to bypass its host’s autophagic defenses. It does this through the use of a Type III secretion system, found in gram-negative pathogens like Shigella, which injects virulent proteins into the host cell. One of these proteins is IcsB; however, its exact function is not well understood. This study aims to better understand the role of this protein in the infection. Methods: A yeast two-hybrid screening test is used in this case to examine the interactions between variations of the protein IcsB, and a library of host proteins. Given IcsB’s high yeast toxicity and that resulted in the total absence of yeast colony formation, the first aim was to identify IcsB variants which expression would not prevent yeast growth. The second aim was to use the mutant with reduced cytotoxicity to perform a Y2H screen that will allow for the identification of candidate host proteins interacting with IcsB. Results: Two mutations of the IcsB protein grew in the Y2HG yeast strain, indicating a significant reduction in the protein’s toxicity. Of the cultures that reacted, high stringency and strong interaction was observed between four genes and IcsB proteins. Among the four identified clones that grew, three corresponded to the gene RNF2, while the last one corresponds to a non-coding sequence. Key control experiments revealed that the interaction of IcsB with RNF2 is likely false-positive. Thus, when screened full-length IcsB using new epithelial cells cDNAVI libraries, strong interaction was observed between three genes and our IcsB proteins. All the three genes DDX3X, FANCL, and SGT1 passed the false-positive interaction tests. It is interesting to notice that DDX3X and SGT1 interacted with catalytically active and inactive IcsB, suggesting that the interactions established between IcsB and prey proteins does not require the catalytic - C306A mutation and that IcsB most likely does not function as a protease against these two proteins. By contrast, FANCL bound catalytically inactive, but not catalytically active IcsB, suggesting it could be a substrate of IcsB. The literature provides some support for the putative role of DDX3X, FANCL, and SGT1 in regulating the vacuole escape of Shigella through IcsB action. Conclusion: The aim of this study was to determine the functional of IcsB in the vacuole escape of Shigella. This study successfully identified three candidates interacting partner proteins for IcsB. Key control experiments confirmed the interaction of IcsB with DDX3X, FANCL and SGT1. This study provides a basis for further research, with further study aimed at confirming these results during Shigella infection

There are 5 distinct aspects to this study. (i) Two histological stains for collagen were compared against each other for the first time, namely Herovici's technique and picrosirius-polarization. (ii) Skin samples from embalmed cadaveric tissue from human cadavers were compared against samples taken from surgical patients. (iii) Skin samples were studied from different regions of the body to assess if dermal structure correlates with scarring potential. (iv) Skin samples were sectioned in a plane parallel to the epidermis to gain further insight into dermal structure. (v) A novel basement membrane stain was produced. Type I and type III collagen are important structural constituents of dermis and play a crucial role in wound healing. Only two traditional histological methods are thought to differentiate between them, so avoiding the need for antibodies. These were compared against each other for the first time in order to establish differences in image quality and discrimination between Type I and type III collagen. Neither technique requires antibodies, however picrosirius requires polarisation microscopy. to result in a clearer, consistently reproducible collagen staining pattern than the picrosirius method and more importantly did not require elaborate apparatus to analyze. Additionally other cellular elements were visible. Skin samples for research are often obtained from surgical excision. This clearly limits which tissues are available for comparative study to those areas operated on. Studying samples from embalmed medical school cadavers has the great advantage of studying areas of the body not routinely available from common surgical procedures. It was therefore desirable to assess whether embalmed cadaveric tissues exhibited different properties by virtue of their age and the embalming process compared to fresh surgical specimens, in order to give confidence that studies utilising the former would be equally valid. To test this, 58 skin samples from embalmed medical school cadavers were compared to skin samples from 38 fresh operative specimens. The levels of tissue preservation and processing artefacts were similar in both groups. Embalmed medical school cadavers clearly offer an opportunity to study tissue areas not routinely available during surgery. This is the first time such a comparison has been made. Many things will affect the final appearance of the scar, but the single most important determinant is the body region affected. The most common areas for unfavourable scarring, specifically keloid or hypertrophic scarring have been shown to be the ear, deltoid and sternal areas. To test the hypothesis that there is no difference in histological structure of skin that correlates to body region, comparative histology was undertaken exploring the regional variations of skin characteristics in 58 cadaveric samples. Closely comparable samples were taken from the deltoid (9), abdomen (13), sternum (10), post-auricular (5), earlobe (12) and eyelid (9). Epidermal thickness, epidermal appendage density and collagen fibre orientation were examined and qualitative structural differences were assessed for each region Skin samples were then grouped by both topographical location of the body and scarring potential. Skin samples exhibited qualitative and quantifiable regional variations in the characteristics studied. Epidermal thickness and appendage counts did not correlate with scarring potential. Both however were statistically significantly higher in skin sampled from the head compared to the trunk. Bundles of collagen fibres in the reticular dermis were grouped according to their orientation in relation to the coronal plane; either parallel, oblique or perpendicular. The ratio of oblique to parallel fibres was statistically significantly higher in body areas with poorer scarring prognosis. This corresponds to a more disorganised arrangement of collagen fibres in these areas. Further qualitative understanding of dermal collagen fibres came from perpendicular to conventional histological samples. This new method stained basement membranes purple, cytoplasm was stained greenish-brown and nuclei dark brown. Collagen fibres were either thin and blue or thick and green. This method was compared to PAS staining and although required more preparative steps allows greater identification of other cellular structures.

No description available.

No description available.

No description available.

No description available.

Bacteria have developed various means of secreting proteins that can enter the host cell membrane. In this work I focus on two systems: cholesterol-dependent cytolysins and Type III Secretion. Cholesterol is a molecule that is critical for physiological processes and cell membrane function. Not only can improper regulation lead to disease, but also the role cholesterol plays in cell function indicates it is an important molecule to understand. In response to this need, probes have been developed that detect cholesterol molecules in membranes. However, it has been recently shown that there is a need for probes that only respond to cholesterol that is accessible at the membrane surface. Perfringolysin O (PFO) is a toxin secreted by Clostridium perfringens that has been developed into a probe capable of detecting accessible cholesterol. Recently, researchers have been expanding the capabilities of this probe by substituting residues, modifying residues, truncating the probe, or a combination of the three. However, lack of characterization of these new probes has led to controversial results. To understand the role of a conserved Cys residue, here we perform cholesterol binding assays and measure the pore formation activity of a Cys modified PFO derivative. The Type III Secretion (T3S) system is a syringe-like apparatus used by various pathogens to inject effector proteins into target cells. The apparatus spans both the inner and outer bacterial membrane, extending to make contact with the host cell where it forms a pore known as the translocon. In Pseudomonas aeruginosa, the translocon is made up of two proteins, PopB and PopD. While recent advances have been made on the structure of the needle and injectisome, information on the translocon remains sparse. In this work, the P. aeruginosa T3S translocon is analyzed using both in vivo and in vitro methods.

<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> Four stucturally distinct types of macromolecular antifreezes have been previously isolated from the sera of polar marine fish. When the water temperature surrounding these organisms drops below -0.7°C, the freezing point of their bodily fluids, any contact with surrounding ice will nucleate internal ice crystal growth. The antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) synthesized by the fish act to inhibit the growth of existing ice crystals in their sera through direct adsorption to the ice lattice. The α-helical structure of type I AFP from winter flounder has been solved to atomic resolution and its mechanism of ice binding has been proposed. The NMR solution structure of a type III AFP from ocean pout has identified proteins in this class to exist in a β-sandwich conformation, however their mechanism of action remains uncertain.</p> <p> To facilitate the pursuit of an x-ray crystal structure solution, we subcloned the gene for a type III AFP (HPLC6) into pET15b and expressed recombinant His-rHPLC6 AFP in E. coli. Purified rmHPLC6 product has been successfully crystallized, and heavy atom soaks were performed in order to attempt a structure solution by multiple isomorphous replacement. The lone tyrosine in this recombinant AFP has been successfully derivatized in solution with iodine, and the modified protein was crystallized. In order to optimize the measurement of anomalous scattering information, modifications to our data collection system were required. Cryocrystallography techniques were employed to improve the quality of collected data.</p> <p> The expression, purification, crystallization and optimized data collection on an iodine-derivatized type III AFP from ocean pout will be presented here. This work has been instrumental in providing the high quality x-ray data required to solve the crystal structure to atomic resolution. Future examination of the solved structure will promote an increased understanding of the ice-binding mechanism exhibited by this class of proteins.</p> / Thesis / Master of Science (MSc)

No description available.