Zugl.: Berlin, Humboldt-Univ., Diss., 2009

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows selective and efficient killing of neuroblastoma cell lines in vitro, and delays human neuroblastoma xenograft tumor growth on nude mice. To further enhance the viral oncolytic efficacy, we also switched the fiber 5 to fiber 35 to generate Ad5PTDf35. This vector shows dramatically increased transduction capacity of primary human cell cultures including hematopoietic cells and their derivatives, pancreatic islets and exocrine cells, mesenchymal stem cells and primary tumor cells including primary cancer initiating cells. Ad5PTDf35-based adenovirus could be a useful platform for gene delivery and oncolytic virus development. Viral oncolysis alone cannot completely eradicate tumors. Therefore, we further armed the Ad5PTDf35-D24 virus with a secreted form of Helicobacter pylori Neutrophil Activating Protein (HP-NAP). Expression of HP-NAP recruits neutrophils to the site of infection, activates an innate immune response against tumor cells and provokes a Th1-type adaptive immune response. Established tumor on nude mice could be completely eradicated in some cases after treatment with this virus and the survival of mice was significantly prolonged.

<p>Species of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals / and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42&ordm / C as well as at ambient room temperature (&plusmn / 22&ordm / C). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4oC for up to 297 days.</p>

Nickel is an essential transition metal for the virulence and survival of Helicobacter pylori in the acidic human stomach. The nickel– and proton– dependent transcriptional regulator HpNikR is important for maintaining nickel homeostasis inside the cytosol by regulating multiple H. pylori genes. A previous ChIP-sequencing experiment with H. pylori G27 and HpNikR identified two novel genes currently annotated as putative iron-transporters, HpG27_866 and HpG27_1499. In vitro DNA-binding assays with the promoter sequences of the two genes revealed nickel-dependent HpNikR binding with an affinity of ~10-7 M. The recognition site of HpNikR was identified on HpG27_1499 by footprinting assays, which loosely correlates with the HpNikR pseudo-consensus sequence. Furthermore, HpG27_1499 transcription showed nickel-dependent repression in WT H. pylori, and no changes in an isogenic ΔnikR strain. These data suggest that HpG27_1499 could be a nickel importer that is regulated by HpNikR in a nickel-responsive manner.

Nickel is an essential transition metal for the virulence and survival of Helicobacter pylori in the acidic human stomach. The nickel– and proton– dependent transcriptional regulator HpNikR is important for maintaining nickel homeostasis inside the cytosol by regulating multiple H. pylori genes. A previous ChIP-sequencing experiment with H. pylori G27 and HpNikR identified two novel genes currently annotated as putative iron-transporters, HpG27_866 and HpG27_1499. In vitro DNA-binding assays with the promoter sequences of the two genes revealed nickel-dependent HpNikR binding with an affinity of ~10-7 M. The recognition site of HpNikR was identified on HpG27_1499 by footprinting assays, which loosely correlates with the HpNikR pseudo-consensus sequence. Furthermore, HpG27_1499 transcription showed nickel-dependent repression in WT H. pylori, and no changes in an isogenic ΔnikR strain. These data suggest that HpG27_1499 could be a nickel importer that is regulated by HpNikR in a nickel-responsive manner.

Introduction: Gastric cancer (GC) is ranked as the second most common fatal malignancy worldwide. Although Helicobacter pylori is recognized as a major predisposing factor for non-cardia GC, infection alone is not sufficient to cause cancer. This thesis aimed to determine the variation in host genetic polymorphisms in subjects from Malaysia and Singapore and to examine the role of H. pylori infection, host genetic factors and dietary factors in the etiology of non-cardia GC in Chinese subjects resident in Malaysia. Methods: Functional dyspepsia (FD) controls from three ethnic groups in Malaysia, Chinese (123), Indian (110) and Malay (84) and Singaporean Chinese (127) plus Malaysian Chinese gastric cancer cases (55)were examined. Polymorphisms in IL-1B-511, IL-1RN, IL-10 cluster, TNFA-308 and TLR5+1174 were determined by PCR-RFLP or PCR; H. pylori status by serology, dietary intake by questionnaire and gastric IL-1b levels by real time PCR. Results: 1) Significant differences existed in the frequency of all polymorphisms, except IL-1B-1473 and TNFA-308, in the three Malaysian ethnic groups and in the IL-1B-511 polymorphism in Malaysian and Singaporean Chinese FD 2) Globally, two distinct patterns of IL-1B-511, IL-1RN, IL-10-1082, IL-10-592 and TNFA-308 exist, Western and East-Asian 3) In Malaysian Malays, the IL-10 ATA haplotype was associated with H. pylori susceptibility 4) In Malaysian Chinese an increased risk of GC was associated with carriage of the IL-1B-1473 G allele {OR=4.4(1.3-15.3)} and the IL-1B-511 C allele {OR=1.8(0.8-4.1)} 5) Increased levels of IL-1b were observed in Singaporean and Malaysian Chinese FD subjects carrying the IL-1-511C and IL-1-1473G alleles 6) Malaysian Chinese not consuming fresh fruit and vegetables had the highest risk of GC {OR=10.2 (3.4-30.6)} 7) The highest risk of GC {OR=37.3(3.3-424.8)} was observed in H. pylori positive Malaysian Chinese who carried both the IL-1B-511C and IL-1B-1473G alleles and did not consume fresh fruit and vegetables. Conclusions: In Malaysian Chinese, H. pylori infection, host genetic and dietary factors all contribute to the risk of GC. However the significant difference observed in the frequency of host genetic polymorphisms within and between ethnic groups suggests that a single group of risk factors cannot be used to determine GC risk across all populations.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, İç Hastalıkları ve Doğum Anabilim Dalı, 2006. / Bibliyografya var.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 60-66). Also available for download via the World Wide Web; free to University of Oregon users.