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- All Subjects: Porphyromonas gingivalis
- All Subjects: 2 hybrid system
- Creators: Shi, Yixin
Description
Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.
ContributorsLeonetti, Cori (Author) / Shi, Yixin (Thesis advisor) / Stout, Valerie (Committee member) / Nickerson, Cheryl (Committee member) / Sandrin, Todd (Committee member) / Arizona State University (Publisher)
Created2013
Description
Colorectal cancer is the third most common type of cancer that affects both men and women and the second leading cause of death in cancer related deaths[1, 2]. The most common form of treatment is chemotherapy followed by radiation, which is insufficient to cure stage four cancers[3]. Salmonella enteric has long been shown to have inherent tumor targeting properties and have been able to penetrate and exist in all aspects of the tumor environment, something that chemotherapy is unable to achieve. This lab has developed a genetically modified Salmonella typhimurium (GMS) which is able to deliver DNA vaccines or synthesized proteins directly to tumor sites. These GMS strains have been used to deliver human TNF-related apoptosis inducing ligand (TRAIL) protein directly to tumor sites, but expression level was limited. It is the hope of the experiment that codon optimization of TRAIL to S. typhimurium preferred codons will lead to increased TRAIL expression in the GMS. For preliminary studies, BALB/c mice were subcutaneously challenged with CT-26 murine colorectal cancer cells and treated with an intra-tumor injection with either PBS, strain GMS + PCMV FasL (P2), or strain GMS + Pmus FasL). APC/CDX2 mutant mice were also induced to develop human colon polyps and treated with either PBS, strain GMS + vector (P1), P2, or P3. The BALB/c mouse showed statistically significant levels of decreased tumor size in groups treated with P2 or P3. The APC/CDX2 mouse study showed statistically significant levels of decreased colon polyp numbers in groups treated with P3, as expected, but was not significantly significant for groups treated with P1 and P2. In addition, TRAIL was codon optimized for robust synthesis in Salmonella. The construct will be characterized and evaluated in vitro and in vivo. Hopefully, the therapeutic effect of codon optimized TRAIL will be maximal while almost completely minimizing any unintended side effects.
ContributorsCrawford, Courtney Rose (Co-author) / Crawford, Courtney (Co-author) / Kong, Wei (Thesis director) / Shi, Yixin (Committee member) / Fu, Lingchen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Porphyromonas gingivalis (P. gingivalis) is an oral pathogen known for causing periodontal diseases like periodontitis and alveolar bone loss. In this study, we investigate the molecular mechanisms of P. gingivalis with focus of the molecular cloning of the two DNA strains of the bacteria PGN_1740 and PGN_0012 in the Ampr pTCow. PGN_1740 is an RNA polymerase ECF-type sigma factor used for transcription. PGN_0012 is a two-component system regulator gene that is important in signal transduction. We demonstrated the cloning mechanism through transformation and confirmed the results through gel electrophoresis and using a positive transformant as a control. The process of cloning the DNA inserts into the bacteria followed a polymerase chain reaction for the amplification of the DNA fragments, digestion of the plasmid and DNA fragments with the restriction endonucleases (BamHI and HindIII), ligation and finally heat shock transformation are presented in this thesis. The effectiveness of these procedures was observed through agarose gel electrophoresis and ethanol precipitation for the purification of the PCR products. In this investigation, we discuss molecular and biological characterization of the P. gingivalis bacteria in regard to cloning and ampicillin resistance.
ContributorsOkeyo, Diana (Author) / Shi, Yixin (Thesis director) / Liu, Wei (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Porphyromonas Gingivalis (P.G.) is a gram-negative anaerobic bacillus that is commonly implicated in periodontal disease in humans via invasion of oral epithelial cells. Characterizing the intracellular mechanisms that allow for these infections to take place is important for future attempts to stop or halt the spread of infection. Given the complexity of bacterial virulence, research on the subject often necessitates precise measurements of very specific biochemical pathways. In this study, we focus on the type IX secretion system utilized by P.G. to initiate colonization of host cells. Specific to this secretion system is the PorX-PorY two-component regulatory system. Here we use the bacterial adenylate cyclase based 2 hybrid system to test if two specific domains of the PorX-PorY system communicate intracellularly with each other; and hence gain further knowledge on how this infection occurs.
ContributorsKrautz, Zackary (Author) / Shi, Yixin (Thesis director) / Lynch, John (Committee member) / Barrett, The Honors College (Contributor) / Dean, W.P. Carey School of Business (Contributor) / School of Life Sciences (Contributor)
Created2024-05