Naturally Derived Sortase A Inhibitors as an Alternative Treatment for Superbug Infections

Abstract

The development of multi-drug resistant ‘superbugs’ highlights the need to develop new antibiotics, as well as reduce their development and spread (e.g., through antimicrobial stewardship). Sortase A (SrtA) is a recent target for antimicrobial development. It is responsible for anchoring many virulence-associated proteins, which are associated with biofilms, host defence evasion, and host tissue adhesion, to Gram-positive cell membranes. SrtA inhibition is not lethal and thus does not select for resistance but has been shown to improve the efficacy of other antibiotics to potentially overcome antibiotic resistance.

In this work, naturally-derived SrtA inhibitors (SrtAIs; trans-chalcone, curcumin, quercetin, berberine chloride), and their combinations with antimicrobial peptides (AMPs; pexiganan, indolicidin, and [I5, R8] mastoparan), were assessed as alternative antimicrobials. These SrtAIs, however, demonstrate poor water solubility, which complicates their assessment as antimicrobials and commercial viability. In this work, we: i) developed optimized techniques for assessing the antimicrobial and SrtA inhibition activity of these SrtA inhibitors; ii) developed protein nanoparticles, mesoporous silica nanoparticles, and synthetic high-density lipoprotein nanodiscs SrtAI formulations; iii) assessed these formulations against a library of disease-relevant, and antibiotic-resistant bacterial strains, on their own and in combination with AMPs by broth microdilution and checker board tests in the presence of resazurin colorimetric readout, to determine their minimal inhibitory concentrations (MICs) and whether the combinations demonstrated synergy; and iv) assessed if the formulations demonstrated toxicity towards mammalian cells using resazurin assay. With respect to these aims, the thesis was divided into the following chapters:

Chapter 1 provides an up-to-date review of SrtAIs as novel antimicrobials.

In chapter 2, a library of organic cosolvents was assessed at different concentrations for growth inhibition towards bacteria and solubilization of the SrtAIs. The ideal parameters (5% v/v DMSO-media) were used to determine the optimum method (broth microdilution, agar dilution, and disc diffusion) to determine the MIC for these compounds and the effect of these formulations on toxicity towards mammalian cells. This work demonstrated that standard broth microdilution methods and disc diffusion were unsuccessful due to the precipitation of SrtAIs

over time. However, the addition of the colorimetric metabolic indicator resazurin enabled the use of the broth microdilution assay.

In chapter 3, MSNs were synthesized and loaded with the SrtAIs (SrtAIs mesoporous silica nanoparticles) to improve the solubility, and consequently, bioavailability of the SrtAIs. They were then combined with pexiganan, indolicidin, and [I5, R8] mastoparan to determine their MICs and fractional inhibitory concentrations for measuring synergy using the optimized broth microdilution and checkerboard assay, respectively. The in vitro results demonstrated that: i) the MICs of SrtAIs mesoporous silica nanoparticles were improved by more than 2-fold when compared to the SrtAIs alone, ii) AMPs and berberine chloride and trans-chalcone mesoporous silica nanoparticles demonstrated synergy against the tested strains, and iii) AMPs and SrtAIs mesoporous silica nanoparticles combinations with AMPs were safe at their MICs.

In chapter 4, PNPs were produced and loaded with each SrtAI (SrtAI protein nanoparticles) to improve their solubility. In vitro assessment demonstrated that: i) the MICs of SrtAI protein nanoparticles in combination with the AMPs against Gram-positive decreased by 2 and 16-fold when compared to the SrtAIs (without formulation) and SrtAI protein nanoparticles, respectively, ii) PEX with trans-chalcone and berberine chloride protein nanop