Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised. / Doctor of Philosophy / Antibiotic-resistant bacteria are increasingly recognized as a threat to global health, and new antibacterial drugs are urgently needed. Before a chemical compound can advance far in the journey to becoming a new drug it must be tested for toxicity against mammalian cells. A portion of this dissertation research involved testing the toxicity of several organometallic compounds (OMCs) previously shown to have antibacterial potential. Mouse-derived mammalian cells were treated with several of the OMCs, and initial results indicated that one of the OMCs is non-toxic and is likely a safe option for additional analysis. This OMC was further tested to see if it could inhibit mycobacterial growth inside of the mammalian cells. It did not effectively clear bacteria from inside of the mammalian cells, likely because of poor penetration of the cell membrane. Further research with this compound should focus on ways to effectively transport the OMC inside infected mammalian cells so that it can reach the bacteria it is meant to target. A second portion of this research involved using a peptide nucleic acid (PNA) to try and reverse tetracycline antibiotic resistance in the bacterial strain Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Peptide nucleic acids are short linear molecules that can bind strongly to complementary DNA and RNA sequences and thus be used to interfere with expression of specific genes. A PNA was designed to inhibit expression of the bacterial tetA gene that codes for a protein called the TetA tetracycline efflux pump, which imparts resistances to tetracycline. Treating the bacteria with the PNA resulted in a lower dose of tetracycline needed to inhibit bacterial growth, indicating a successful increase in tetracycline susceptibility. By using a molecular analysis technique called reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), it was possible to measure the amount of tetA messenger RNA (mRNA) in cultures of DT104 treated only with tetracycline or with a combination of tetracycline and the PNA. As expected, bacteria treated with both the antibiotic and the PNA had less tetA mRNA than the cultures treated only with tetracycline, supporting the hypothesis that the PNA prevents the bacteria from effectively expressing the tetA gene. The PNA was next used in conjunction with tetracycline as an experimental treatment for mice infected with DT104. The PNA did not provide the expected protective effect under these circumstances. The overall conclusion for this part of the research is that PNAs offer an exciting potential avenue for counteracting antibiotic resistance, but additional experimentation is needed. Future research should focus on investigating more effective ways to get the PNAs inside the bacteria and on understanding more about how the PNAs behave in live animals. Several other PNAs targeting different genes involved in antibiotic resistance or essential bacterial functions were also tested against DT104 with variable success.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/100634 |
| Date | 29 April 2019 |
| Creators | Ernst, Marigold Ellen Bethany |
| Contributors | Biomedical and Veterinary Sciences, Merola, Joseph S., Sriranganathan, Nammalwar, Ehrich, Marion F., Boes, Katie M. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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