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Date of Award

Summer 8-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Antimicrobial resistance is a serious public health concern and threatens the effective prevention and treatment of a wide range of infections. The rapid emergence and spread of anti-malarial drug resistance has been a major challenge in malaria control efforts. Likewise, high rates of antibiotic resistance has been observed in common bacterial pathogens. Thus, understanding the complex mechanisms that underlie drug resistance is critical to prevent the further spread of resistance and to develop new drugs in order to diversify the tools available to treat microbial infections. Structural biology enables understanding of the structural and mechanistic details that enable rational drug design and novel therapeutic strategies to overcome resistance.

The methylerythritol phosphate (MEP) pathway for isoprenoid precursor biosynthesis is an attractive target for novel anti-malarial drug development, as compounds that target this pathway lack toxicity concerns for humans. The small molecule compound fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate (DXR) and is in clinical trials for combination therapy with other anti-malarial compounds. Fosmidomycin-resistant Plasmodium falciparum strains were generated in vitro in order to investigate possible mechanisms of resistance to this drug. Genetic analysis of these parasites revealed that they are highly enriched for mutations in PfHAD1. The crystal structure of PfHAD1 was solved in order to determine the effects of the mutations on PfHAD1 structure and function, which revealed that these mutations cause loss of PfHAD1 function via protein misfolding or interference with substrate binding. PfHAD1 was determined to be a sugar phosphatase member of the haloacid dehalogenase (HAD) superfamily with catalytic activity towards a variety of sugar phosphate compounds, including intermediates of glycolysis – which feed into the MEP pathway. Metabolic profiling revealed that fosmidomycin-resistant parasite strains lacking PfHAD1 have substantial increases in MEP pathway metabolites. Together, these results demonstrate that PfHAD1 regulates substrate availability to the MEP pathway and that loss of PfHAD1 function confers fosmidomycin resistance in P. falciparum.

While the metabolic effects and a biological phenotype of PfHAD1 have been elucidated, the structural determinants for diverse substrate recognition by PfHAD1, or HAD superfamily members in general, are unknown. Crystal structures of PfHAD1 in complex with three different upstream MEP pathway precursors reveal how domain movement in PfHAD1 enables diverse substrate recognition and catalysis. These studies further inform the structural and biochemical basis for catalysis within a large superfamily of HAD enzymes with diverse functions.

Tetracyclines are an important class of antibiotics, widely used due to their broad antimicrobial spectrum, oral availability, and low cost. Tetracycline resistance can occur through: (1) efflux, (2) ribosomal protection, or (3) enzymatic inactivation. Surprisingly, enzymatic inactivation has rarely been documented despite its apparent advantage. However, a new family of tetracycline-inactivating flavoenzymes, termed tetracycline destructases, was recently identified from soil functional metagenomic selections and in Legionella longbeachae, a human pathogen. The tetracycline destructases are likely candidates for dissemination to the clinical setting, potentially compromising the efficacy of an entire class of antibiotics. In order to mitigate the spread of tetracycline resistance, there is a dire need for the rational design of better tetracycline antibiotics that can avoid inactivation and novel therapeutic strategies to overcome resistance. The crystal structures of four tetracycline-inactivating enzymes alone and in complex with tetracycline analogs reveal the structural basis for unexpected plasticity in substrate binding. Our results reveal the potential of a novel tetracycline/tetracycline destructase inhibitor combination therapy strategy to overcome resistance. This strategy holds promise for restoring the efficacy of tetracyclines amidst the troublesome spread of tetracycline resistance.

Language

English (en)

Chair and Committee

Niraj Tolia

Committee Members

Daved Fremont, Daniel Goldberg, Joseph Jez, Audrey Odom

Comments

Permanent URL: https://doi.org/doi:10.7936/K7P8499G

Available for download on Saturday, August 15, 2116

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