Anthelmintics, antiparasitic agents, have been developed as a main weapon to control parasitic nematodes of domestic ruminants. Unfortunately, the intensive use of anthelmintics leads to the development of drug resistance in parasite populations. Anthelmintic resistance has compromised the control of nematode parasites and has become a major problem in many countries of the world. Resistance to the newest anthelmintics such as ivermectin (IVM) and related anthelmintics in Haemonchus contortus in sheep has been developing rapidly in recent years. The development of drug resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. However, the mechanism of ivermectin resistance in nematode parasites is unknown. P-glycoprotein (Pgp) has been well documented in mammalian cells as a membrane transporter by actively extruding a variety of structurally and functionally unrelated hydrophobic cytotoxic drugs out of the cell. This study was to determine whether there is an association between specific alleles at the Pgp locus and IVM or moxidectin (MOX) selection in H. contortus, by investigating the genetic variation of the Pgp homologue in unselected and IVM- and MOX-selected strains of H. contortus. (Abstract shortened by UMI.)

Onchocerca volvulus, a filarial nematode, is the causative agent of onchocerciasis. / O. volvulus is a human parasite with no animal model host and is endemic in the tropics. O. volvulus material is scarce and must be conserved as part of the Onchocerciasis Control Program. A genomic library was constructed to provide a substantial source of renewable genetic material, in place of original parasite DNA. / Currently there is only one glutamate-gated chloride channel that has been sequenced from O. volvulus, but this has not yet been characterized. This GluClx partial cDNA sequence isolated by Cully et al., 1997, may be found in GenBank, accession number U59745. Specific primers were designed to amplify this gene from the genomic library. A fragment of this gene was isolated but the primers were non-specific, amplifying genes in addition to GluClx. / A motif is a short recognition sequence within a protein that may allow the modification of the protein. The cysteine loop in the N-terminal of all the ligand-gated ion channels is interesting because it contains the neurotransmitter-gated ion channel signature sequence. (Abstract shortened by UMI.)

Association between albendazole anthelmintic resistance and a panel of life history traits in Heligmosomoides polygyrus was investigated both prior to and during drug-selection. Associations prior to anthelmintic application were studied in ten independent lines isolated without drug treatment from a susceptible stock population by random genetic drift. Variation among lines was detected in several life history traits (i.e., establishment, development and survival), and, despite lack of previous exposure to albendazole, lines also varied in their tolerance to the drug. No significant correlations were detected between drug-tolerance and any of the life history traits after 11 generations of isolation. The apparent lack of fitness differential between lesser and more drug-tolerant individuals of the susceptible population is not in accordance with the assumption that the low frequency of drug tolerant individuals in the susceptible population is explained by their lower fitness, but is consistent with the neutral theory. Associations between life history traits and drug resistance were also studied using two lines selected for albendazole resistance from the stock population, and two control lines exposed to the same monthly passage procedure but not to the drug. After 10 generations of selection, drug resistance increased from an LC50 of 0.48 $ mu$M to 2.03 $ mu$M. In a primary infection, the higher establishment and higher worm numbers one-month post-infection in the resistant parasites compared to the stock parasites occurred only in the drug-selected lines. Changes in these traits were attributed to the drug selection regime. In contrast, both drug-selected lines and passaged lines showed a faster rate of development and higher early egg production compared with the stock parasites; these changes were attributed to the passage procedure that presumably acted as a selective force on early life history traits. In immunized hosts, changes in several traits that o

Active site mutations that disrupt drug binding are an important mechanism of drug resistance. Such resistance causing mutations impair drug binding, thus reducing drug efficacy. Knowledge of potential resistance mutations, before they are clinically observed, would be useful in a number of ways. During the lead prioritization phase of drug development, this knowledge may direct the research team away from candidate drugs that are most likely to experience resistance. In the clinical setting, knowledge of potential resistance mutations could allow the development of treatment regimens, with drug cocktails likely to maximize efficacy. In this thesis I present a structure-based approach to predict resistance and its evolution. This method utilizes a two-pass search, which is based on a novel protein design algorithm, to identify mutations that impair drug binding while maintaining affinity for the native substrate. The approach is general and can be applied to any drug-target system where a structure of the target protein, its native substrate and the drug is available. Furthermore, it requires no training data for predictions and instead predicts resistance using structural principles. Finally, I use approximate force-field calculations from MMPBSA and simple assumptions about the relationship between binding energy and fitness to build fitness landscapes for a target protein under selective pressure from either a single drug or a drug cocktail. I use a Markov-chain based model to simulate evolution on this fitness landscape and to predict the likely evolutionary trajectories for resistance starting from a wild-type. The structure-based method was used to probe resistance in four drug-target systems: isoniazid-enoyl-ACP reductase (tuberculosis), ritonavir-HIV protease (HIV), methotrexate-dihydrofolate reductase (breast cancer and leukemia), and gleevec-ABL kinase (leukemia). This method was validated using clinically known resistance mutations for all four test systems. In all cases, it correctly predicts the majority of known resistance mutations. Furthermore, exploiting the relationship between binding energy, drug resistance and fitness of a mutant, evolution was simulated on the HIV-protease fitness landscape. This hybrid evolutionary model further improves the resistance prediction. Finally, good agreement between these evolutionary simulations and observed evolution of drug resistance in patients was found.

Antibiotic resistance has become a major problem in the United States. Approximately 2 million people are affected by hospital-acquired infections. Each year about 90,000 people are killed from them. Of the infections 70% of them are resistant to at least one drug. In order to trigger antibiotic resistance in bacteria, the antibiotics need to be detected by sensors in the bacteria. Riboswitches may act as toxin sensors in bacteria. Riboswitches are RNA aptamers that regulate gene expression via allosteric structural changes triggered by binding of a small molecule. Most identified riboswitches specifically recognize the metabolic product of the gene to be regulated. When the concentration of the metabolite reaches its threshold it binds to the riboswitch causing a structural change that in most cases turns off transcription or translation of the metabolite-producing gene. The ykkCD riboswitch appears to recognize the antibiotic, tetracycline to up-regulate expression of an efflux pump (also called ykkCD) that exports toxic drugs from the bacterial cell. In this work we present initial characterization of the previously uncharacterized ykkCD riboswitch. With the help of tetracycline derivatives and mutagenesis studies on the riboswitch we will (1) determine the substrate specificity of this riboswitch; (2) assess the importance of aromatic character and/or functional groups in antibiotic recognition. To achieve this goal we have developed a fluorescent binding assays. The binding assays will measure the binding affinity (Kd) of the riboswitch-antibiotic complex. Since substrates of the efflux pump are toxic to the bacterial cell, we posit that the ykkCD riboswitch might work as a toxin sensor and could serve as a target in the fight against bacterial pathogens. Afterwards we will evaluate how the ykkCD riboswitch sensor works in vivo. In order to do this we will have to quantify the amount of protein produced in the presence of tetracycline derivatives and mutant sensors. However quantifying the level of a particular protein in the cell is difficult so instead we replace the sequence of the efflux pump with the B-gal gene in B subtilis cell and quantify B-gal enzymatic activity using a colorimetric assay. This is a widely used technique in which the fluorescence corresponds to how much protein is produced. / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Riboswitches are naturally occurring RNA aptamers that form a precise three-­dimensional structure and selectively bind to cellular target molecules. Binding of the target molecule initiates an allosteric structural change in the riboswitch that in turn regulates expression of a relevant target gene. Most riboswitches specifically recognize the metabolic product of the gene that is being regulated. Expression may be regulated at either transcription or translation stage of gene expression. Most riboswitches are off switches meaning they turn off expression of metabolite producing gene when metabolite concentration is high enough. The ykkCD putative riboswitch appears to increases production of an efflux pump that expels toxic drugs from the cell by binding to the antibiotic tetracycline. Based on previous data collected the ykkCD putative riboswitch seems to regulate the efflux pump at the transcriptional level. To confirm this hypothesis we want to map the structural change that takes place upon binding of the antibiotic tetracycline to the mRNA. Nucleic acid footprinting studies will be used to map the binding site of tetracycline and the allosteric change that takes place upon tetracycline binding. / Department of Chemistry

A significant challenge of the 21st century is the growing health threat stemming from drug-resistant infectious diseases. There is an undeniable need to discover new, safe and effective drugs with novel mechanisms of action to combat this threat. A study of drugs currently on the market showed that natural products account for approximately 75% of new anti-infective drugs, either as new agents or analogs based upon their structure. Unfortunately, major pharmaceutical companies have cut back tremendously in natural products research in part due to the frustrating obstacle of frequent rediscovery of compounds. Fungi in particular are difficult to work with in that they do not always produce the same variety and quantities of secondary metabolites under laboratory conditions. One of the groundbreaking discoveries evolving from genomics research is the observation that many fungi possess more gene clusters encoding for the production of secondary metabolites than the reported number of natural products isolated from those organisms. Simple epigenetic modifications such as DNA methlytransferase or histone deacetylase inhibition can activate silenced genes leading to the genesis of novel chemistry from the fungus. The work presented herein is a study of the isolation and characterization of anti-infective compounds from Floridian mangrove endophytes. In addition, epigenetic modifications were explored in order to increase the production of secondary metabolites as well as for the purpose of generating new analogs not found in the controls. Finally, structure activity relationship studies were performed in order to maximize the anti-malarial and antibiotic activity of cytosporone E.

Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’-dipyridyl, rob promoter activity was observed to be slightly increased in the early and mid-log phase by 1.4 and 1.1 fold, respectively. We also showed that sodium decanoate and sodium salicylate can reduce the transcription of rob at 30°C and 37°C. This reduction was observed more potently when rob was exposed to sodium decanoate at 30°C. Minimum inhibitory concentration (MIC) for various antibiotics of the S. marcescens rob knock-out demonstrated a decrease in susceptibility to nalidixic acid, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, and ofloxacin. Over-expression of rob resulted in an increased resistance by 4, 2, and 2-fold to nalidixic acid, tetracycline and chloramphenicol, respectively. In addition, rob over-production displayed 8, 4, and 4-fold increase in resistance to ciprofloxacin, norfloxacin, ofloxacin, respectively. To discover the role of rob in the efflux mechanism, we performed ethidium bromide accumulation assays on over-expressing and knock-out strains. Organic solvent tolerance assays were carried out using n-hexane to determine if rob is involved in expression of efflux pumps. We found the rob null mutant to be sensitive to n-hexane while the over-expression of rob resulted in resistance to n-hexane. RT-PCR of the rob knock-out strain showed a decrease in expression of micF, ompC, sdeXY, sdeAB and tolC, respectively, and an increase in the expression of ompF. To conclude, we identified a rob homolog in S. marcescens which contributes to resistance to multiple antibiotics and tolerance to organic solvent.