Photostability studies of GarKS peptides for topical formulation development.

There is a growing interest in the use of antimicrobial peptides (AMPs) as potent alternatives for conventional antibiotics, especially in chronic infected wounds. The development of a suitable topical formulation requires a thorough assessment of the photostability profiles of AMPs. In this study, we sought to investigate the photostability of novel Garvicin KS (GarKS; composed of three peptides GakA, GakB, and GakC) peptides either as an individual peptide or in combinations. The photostability of the aqueous peptide solution was determined using Suntest (indoor and outdoor conditions). Furthermore, the antimicrobial efficacy of the peptides was evaluated following UVA irradiations. Photodegradation of the peptides under indoor and outdoor conditions followed first-order kinetics. Individual peptides (GakA, GakB, and GakC) were more prone to photodegradation as compared to combination peptides (GakA+GakB, GakB+GakC, and GakA+GakC) both under indoor and outdoor conditions where the GakA+GakB combination was the most photostable. A combination of GakA+GakB+GakC enhanced photostability under indoor conditions but was reduced under outdoor conditions. A combination of three peptides with an antioxidant (glutathione) or superoxide/hydrogen peroxide scavenger (trehalose) enhanced the photostability of peptides with the highest stability achieved at a peptide:photostabilizer molar ratio of 1:0.8 for glutathione. A nominal increase in the MIC value for the peptide combinations as opposed to a larger increase for individual peptides further supports the photostability effects of combination peptides following UVA irradiations. These results suggest that the GakA+GakB or GakA+GakB+GakC combinations exhibited the highest photostability with excellent antimicrobial efficacy deemed suitable for the development of a potent AMP formulation for topical applications.

Preformulation studies on novel garvicin KS peptides for topical applications.

Antimicrobial peptides (AMPs) are emerging as a viable alternative to antibiotics attributable to their potent antimicrobial effects and low propensity for resistance development, especially in chronic infected wounds. The development of an optimized topical formulation of AMPs is thus warranted. Preformulation studies for determination of the suitability and optimization requirements of AMPs in topical formulation development are important. Therefore, we sought to investigate the preformulation studies for a novel bacteriocin garvicin KS (GarKS), which is composed of three peptides (GakA, GakB, and GakC). The effects of physiological fluids and varying temperatures on GarKS peptide stability were determined. The antimicrobial effects of the peptides and their combinations were evaluated in Staphylococcus aureus (methicillin sensitive and resistant strains). Furthermore, their effects on fibroblast viability and proliferation were determined. The GarKS peptides were stable in water and PBS at room and physiological temperatures, however, the peptides were significantly degraded in simulated wound fluid. The antimicrobial and fibroblast cell viability/proliferation effects of either individual GarKS peptides or their combinations varied. A careful consideration of the peptide stability, antimicrobial efficacy, and fibroblast viability/proliferation effects suggests GakA+GakB as a potent combination for the development of an optimized topical formulation of GarKS peptides.

Dual Transcriptomics of Host-Pathogen Interaction of Cystic Fibrosis Isolate Pseudomonas aeruginosa PASS1 With Zebrafish.

Pseudomonas aeruginosa is a significant cause of mortality in patients with cystic fibrosis (CF). To explore the interaction of the CF isolate P. aeruginosa PASS1 with the innate immune response, we have used Danio rerio (zebrafish) as an infection model. Confocal laser scanning microscopy (CLSM) enabled visualization of direct interactions between zebrafish macrophages and P. aeruginosa PASS1. Dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the host during P. aeruginosa infection. Following establishment of infection in zebrafish embryos with PASS1, 3 days post infection (dpi), there were 6739 genes found to be significantly differentially expressed in zebrafish and 176 genes in PASS1. A range of virulence genes were upregulated in PASS1, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase and fimbrial subunits. Additionally, iron and phosphate acquisition genes were upregulated in PASS1 cells in the zebrafish. Transcriptional changes in the host immune response genes highlighted phagocytosis as a key response mechanism to PASS1 infection. Transcriptional regulators of neutrophil and macrophage phagocytosis were upregulated alongside transcriptional regulators governing response to tissue injury, infection, and inflammation. The zebrafish host showed significant downregulation of the ribosomal RNAs and other genes involved in translation, suggesting that protein translation in the host is affected by PASS1 infection.