Date

8-1-2018

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Chemical Engineering

First Adviser

Brown, Angela C.

Abstract

Treatment of bacterial illnesses has become increasingly difficult as the development of new antibiotics is being outpaced by the increasing number of antibiotic-resistant organisms. This has led us to search for alternative therapeutic approaches to help combat these illnesses. A viable approach in treating these illnesses is to focus on inhibiting protein toxins, which are one of the many virulence factors that bacteria secrete. Many toxins recognize and bind to cholesterol (Chol) on the host cell membrane as an initial step in their mechanism; however, a viable method of inhibiting this interaction has yet to be uncovered.For our model toxin, which recognizes and binds to Chol, we have chosen the repeats-in-toxin (RTX) toxin leukotoxin A (LtxA) secreted by the Gram-negative bacterium Aggregatibacter actinomycetemcomitans. LtxA functions by resisting the host’s immune response by binding to and killing white blood cells via Chol within their membrane. This association with Chol is regulated by a Chol recognition amino acid consensus (CRAC) motif, with a sequence of 334LEEYSKR340, in the N-terminal (hydrophobic) region of the toxin.Here, we have demonstrated LtxA’s requirement for Chol; removal of Chol from the plasma membrane of leukocytes inhibits the activity of the toxin. We have shown that a peptide designed from LtxA’s CRAC motif (CRACWT) has a similar affinity for Chol and can inhibit LtxA cytotoxicity by binding to Chol and preventing subsequent LtxA binding and internalization. Utilizing biophysical techniques, we characterized the interaction between CRACWT and Chol and found that the hydroxyl group within Chol is key to this interaction and that CRACWT does not disrupt membrane packing, suggesting that CRACWT primarily sits near the water-membrane interface.To further improve upon the interaction between CRACWT and Chol-containing membranes, we investigated the effect of altering the net charge of CRACWT to create a peptide that binds to Chol with a stronger affinity. We synthesized four CRACWT mutants that corresponded to an increase or decrease in the overall net charge of CRACWT. To measure the affinities of these mutants for Chol-containing membranes, as well as their ability to inhibit LtxA cytotoxicity, we employed localized surface plasmon resonance (LSPR) measurements and cell-based assays respectively. We found that neither decreasing nor increasing the net charge of CRACWT led to an increase in the peptide’s affinity for Chol-containing membranes, but mutants with high net charges were incapable of inhibiting LtxA cytotoxicity.Next, to determine the significance that each residue within the CRAC motif has on the peptide’s ability to bind to membrane Chol, we synthesized 10 CRAC peptide mutants. Each peptide mutant had one residue within the CRAC domain substituted with an alanine residue. We found that seven of the ten residues within the CRAC motif have a significant effect on the peptide’s affinity for Chol-containing membranes, with the most prominent residues being the three highlighted in the CRAC domain definition, leucine (Leu), tyrosine (Tyr), and arginine (Arg).Finally, to test the efficacy of CRACWT in vitro against other Chol-binding pathogens we utilized two Streptococcal toxins, streptolysin O (SLO) and pneumolysin O (PLO), which are considered important virulence factors for this genus. We investigated the inhibitory effect of CRACWT on the cytotoxic and hemolytic activity of SLO and PLO and found that CRACWT inhibited the cytotoxicity of SLO and PLO, as well as the hemolytic activity of PLO in a concentration-dependent fashion.These results suggest that CRACWT holds potential clinical applicability to treat not just bacterial illnesses but potentially other viruses that utilize Chol during pathogenesis since features of the CRAC motif have been implicated in the function of proteins relevant to the human immunodeficiency virus (HIV), the influenza virus (flu), and the herpes simplex virus.

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