Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 154-180). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 181-207). Also available in print.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xvi, 134 p. : ill., (some col.). Includes abstract and vita. Advisor: Kathryn A. Eaton, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 107-134).

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

As a ubiquitous protein-folding helper in bacterial cytosol, SlyD is a peptidylprolyl isomerase (PPIase) of the FK506-binding protein (FKBP) family. It has two important functional domains, the IF (insert-in-flap) domain with chaperone activity and the FKBP domain with PPIase activity. It also possesses a histidine- and cysteine-rich C-terminal metal-binding domain, which binds to selected divalent metal ions (e.g. Ni2+, Zn2+) and is critical for participation in metal trafficking for metalloenzymes. SlyD from Helicobacter pylori was investigated both structurally and functionally by a variety of biophysical, biochemical and molecular biology techniques. HpSlyD was cloned, expressed and purified. It binds to Ni2+ and Zn2+ with dissociation constants (Kd) of 2.74 and 3.79 μM, respectively. Both Ni2+ and Zn2+ can competitively bind to HpSlyD. The C-terminus was demonstrated to convey nickel resistance in vivo. It also binds to Bi3+ with Kd of 4.4 × 10-24 M. Furthermore, Zn2+, Cu2+ and Bi3+ can induce the dimerization or oligomerization of HpSlyD. The solution structure of the C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC) was determined by NMR, which demonstrates that HpSlyDΔC folds into two well-separated, orientation-independent domains. Both the FKBP and IF domains fold into a structure consisting of a four-stranded antiparallel β-sheet and an α-helix. Binding of Ni2+ instead of Zn2+ induced the conformational changes in FKBP domain, where the active sites are positioned, suggesting a regulatory role of nickel on the function of HpSlyD. It was also confirmed that HpSlyD can associate with the Tat (twin-arginine translocation) signal peptide from small subunit of [NiFe] hydrogenase (HydA), an accessory protein HpHypB for [NiFe] hydrogenase mainly by the IF domain. Surprisingly HpSlyD was found to form a complex with HpUreE, a urease chaperone, indicative of the “cross-talk” between [NiFe] hydrogenase and urease. The possible mechanism of HpSlyD for the cooperation with HpHypB was also explored. In the presence of different metal ions, HpSlyD was shown to regulate the GTPase activity of HpHypB, implicating the possible metal transfer induced by HpSlyD. It was suggested that HpSlyD modulates the nickel insertion of [NiFe] hydrogenase by controlling the GTPase activity of HpHypB. In this thesis, the SlyD protein from H. pylori was shown as an important regulator for the activation of both [NiFe] hydrogenase and urease. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Helicobacter pylori is a gram-negative bacterium that infects half the world population and is the etiological cause of numerous gastric pathologies. H. pylori possess numerous mechanisms to promote its survival and modulate host immunity. We propose that H. pylori can modulate intercellular communication by manipulating the host exosome pathway. Exosomes are secreted nanovesicles that contain different proteins and microRNAs that can be transferred between cells to alter cell signaling and gene expression. We demonstrate that H. pylori infection increases host exosome secretion. Furthermore, infection can alter exosome composition as VacA, a bacterial virulence factor, can be exported in exosomes and Argonaute 5, a miRNA effector protein, is upregulated in exosomes during infection. Lastly, we show preliminary evidence that infection-modulated exosomes can modulate immune-regulatory signaling in dendritic cells by activating STAT3. Together, these studies elucidate a novel mechanism by which H. pylori can modulate the host environment and promote its continued survival.

No description available.

Lactoferrin (Lf) is an iron binding protein produced in mammals. It has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonize mammalian hosts have adapted to living in high Lf environments. Helicobacter pylori, which inhabits the human gut, was chosen as a model organism to investigate how bacteria may adapt to Lf. H. pylori was able to use iron from fully saturated human Lf (hLf) in various low iron media, achieving growth levels similar to the ironreplete control. Partially saturated hLf decreased growth, yet both partially saturated bovine Lf (bLf) and hLf were able to increase internalization of bacteria into mammalian tissue culture cells. A substantially larger increase in internalization was seen when bacteria were supplemented with hLf in low iron conditions, possibly mediated by iron-regulated cellular receptors or bacterial lactoferrin binding proteins. In eukaryotes, Lf is known to bind and facilitate internalization of DNA into cells and sometimes the nucleus, and upregulate gene expression. Here, one hundred bacterial genomes were surveyed for known Lf binding sites as an indication that Lf had similar functions using bacterial DNA. While the frequency and location of Lf binding sites suggest they occur at random, their presence in all genomes suggests that Lf may be able to act as a vector for bacterial DNA, and facilitate the movement of genes between species. Lf is being widely considered for commercial and therapeutic uses, with significant interest in producing it in genetically modified organisms (GMO). Widespread production and use of Lf could increase the number of bacteria that are adapted to it. How Lf interacts with bacteria adapted to it, and the ability of it to act as a DNA vector, may have relevance for GMO risk assessment.

No description available.

Lactoferrin (Lf) is an iron binding protein produced in mammals. It has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonize mammalian hosts have adapted to living in high Lf environments. Helicobacter pylori, which inhabits the human gut, was chosen as a model organism to investigate how bacteria may adapt to Lf. H. pylori was able to use iron from fully saturated human Lf (hLf) in various low iron media, achieving growth levels similar to the ironreplete control. Partially saturated hLf decreased growth, yet both partially saturated bovine Lf (bLf) and hLf were able to increase internalization of bacteria into mammalian tissue culture cells. A substantially larger increase in internalization was seen when bacteria were supplemented with hLf in low iron conditions, possibly mediated by iron-regulated cellular receptors or bacterial lactoferrin binding proteins. In eukaryotes, Lf is known to bind and facilitate internalization of DNA into cells and sometimes the nucleus, and upregulate gene expression. Here, one hundred bacterial genomes were surveyed for known Lf binding sites as an indication that Lf had similar functions using bacterial DNA. While the frequency and location of Lf binding sites suggest they occur at random, their presence in all genomes suggests that Lf may be able to act as a vector for bacterial DNA, and facilitate the movement of genes between species. Lf is being widely considered for commercial and therapeutic uses, with significant interest in producing it in genetically modified organisms (GMO). Widespread production and use of Lf could increase the number of bacteria that are adapted to it. How Lf interacts with bacteria adapted to it, and the ability of it to act as a DNA vector, may have relevance for GMO risk assessment.