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- Creators: Schubert, Franz, 1797-1828
- Creators: School of Life Sciences
- Creators: Ravel, Maurice, 1875-1937
Description
The prevalence of antibiotic resistant bacterial pathogens has increased since the introduction of penicillin in the 1940s. Insufficient development of novel antibacterial agents is leaving us with a failing arsenal of therapies to combat these pathogenic organisms. We have identified a clay mineral mixture (designated CB) that exhibits in vitro antibacterial activity against a broad spectrum of bacterial pathogens, yet the antibacterial mechanism of action remains unknown. Antibacterial susceptibility testing of four different clay samples collected from the same source revealed that these natural clays had markedly different antibacterial activity. X-ray diffraction analyses of these minerals revealed minor mineralogical differences across the samples; however, ICP analyses demonstrated that the concentrations of many elements, Fe, Co, Cu, Ni, and Zn in particular, vary greatly across the four clay mixture leachates. Supplementation of a non-antibacterial leachate containing lower concentrations of Fe, Co, Ni, Cu, and Zn to final ion concentrations and a pH equivalent to that of the antibacterial leachate resulted in antibacterial activity against E. coli and MRSA, confirming the role of these ions in the in vitro antibacterial clay mixture leachates. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to CB-L. Supplementation of CB-L with ROS scavengers eliminated oxidative damage in E. coli, but did not rescue the cells from killing, indicating that in vitro killing is due to direct metal toxicity and not to indirect oxidative damage. Finally, we ion-exchanged non-antibacterial clays with Fe, Co, Cu, and Zn and established antibacterial activity in these samples. Treatment of MRSA skin infections with both natural and ion-exchanged clays significantly decreased the bacterial load after 7 days of treatment. We conclude that 1) in vitro clay-mediated killing is due to toxicity associated directly with released metal ions and not to indirect oxidative damage and 2) that in vivo killing is due to the physical properties of the clays rather than metal ion toxicity.
ContributorsOtto, Caitin Carol (Author) / Haydel, Shelley (Thesis advisor) / Stout, Valerie (Committee member) / Roberson, Robby (Committee member) / Sandrin, Todd (Committee member) / Rege, Kaushal (Committee member) / Arizona State University (Publisher)
Created2014
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
ContributorsSchubert, Franz, 1797-1828 (Composer)
ContributorsSchubert, Franz, 1797-1828 (Composer)
ContributorsSchubert, Franz, 1797-1828 (Composer)
ContributorsSchubert, Franz, 1797-1828 (Composer)
ContributorsSchubert, Franz, 1797-1828 (Composer)
ContributorsSchubert, Franz, 1797-1828 (Composer)
ContributorsSchubert, Franz, 1797-1828 (Composer)
ContributorsSchubert, Franz, 1797-1828 (Composer)