Increasing Staphylococcus Aureus Antibiotic Susceptibility Through Membrane Charge Manipulation Using Peptides and Small Molecules

Abstract

With the rapid evolution of antibiotic resistance, the need for more effective antibiotics is imminent. Bacterial membranes are an appealing target due to their accessibility and relatively conserved structures. Membrane targeting antibiotics, especially cationic antimicrobial peptides (CAMPs) such as host defense peptides, have been increasingly explored as novel antibiotics and tunable innate antimicrobials. The latter could be achieved by treatment with an antibiotic adjuvant: a compound that would increase the potency of host CAMPs without killing the bacteria on its own. Boosting the host’s own immune system with an adjuvant is beneficial over using antibiotics and would theoretically avoid triggering bacterial resistance. One mechanism of bacterial resistance is increasing the cationic charge of the membrane. As CAMPs are electrostatically attracted to anionic bacterial membranes, making the membrane more cationic decreases that attraction, rendering CAMPs less effective. To target this resistance mechanism chemically, two antibiotic adjuvant strategies were explored as co-treatments with various CAMPs: membrane targeting peptides used to bind and block surface amines, and small molecules used to either acetylate surface amines or convert a cationic membrane phospholipid to an anionic phospholipid. Co-treatment of the Staphylococcus aureus (S. aureus) membrane targeting peptide KAM-CT and various CAMPs increased S. aureus susceptibility to those CAMPs. Bacterial surface acetylation using sulfo-NHS-acetate followed by CAMP treatment caused up to 10 times increased CAMP potency. Hydrazine and hydroxylamine were shown to cleave the lysine moiety from the lysyl-phosphatidylglycerol (Lys-PG) phospholipid to generate phosphatidylglycerol (PG) in liposome models. S. aureus was treated with a hydroxylamine-CAMP conjugate, but it showed decreased antibiotic activity compared to the CAMP alone. To better understand what was happening in the bacteria, a novel Lys-PG quantification protocol was created by fluorophore labeling Lys-PG and quantifying the labeled Lys-PG via normal phase high-performance liquid chromatography (NP-HPLC). Cyclic peptides, such as KAM-CT, represent complex yet synthetically attainable moieties that could be used as novel antibiotics adjuvants. Expanding the repertoire of reversible covalent chemistries, especially those applied to peptide cyclization, is desirable due to the high potency and selectivity of such interactions. Herein, we also describe a novel reversible covalent chemistry between 2-formylphenylboronic acid (FPBA) and 2,3-diaminopropionic acid (Dap): the imidazolidino boronate (IzB) conjugate. It was found to be potent (Kd = 100 μM) and quickly reversible (t1 = ~6 sec) under physiological conditions. IzB formation was successfully employed as a peptide cyclization strategy as there was little interference from biologically relevant small molecules, except cysteine. Cysteine interference was utilized to create “smart” peptides that can linearize upon increasing cysteine concentrations via thiazolidino boronate (TzB) formation with the FPBA moiety in the peptide. Such “smart” peptides could be used as pH-responsive peptides or cysteine sensors able to report on the cysteine concentration in complex media.