Antimicrobial Peptide Induced-Stress Renders Staphylococcus aureus Susceptible to Toxic Nucleoside Analogs

Abstract

Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and are also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty of reaching sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defense peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied whether sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that Staphylococcus aureus is highly susceptible to antimetabolite nucleoside analogs when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides.

Keywords: antibiotic resistance; antibiotics; antimetabolites; antimicrobial peptides; nuceloside analogs; pexiganan.

Figure 1

Heatmap of relative protein expression based on label-free quantification detected by liquid chromatography-mass spectrometry (LC-MS). Only the 50 most significantly up-regulated proteins compared to the control are shown (log2 fold-change). Red rectangle highlights proteins that participate in or depend on nucleotide metabolism. Proteins were extracted after 30 min of pexiganan addition (0.125, 0.25, 0.5 and 1 fractions of the minimal inhibitory concentration). log2 fold-changes are given from highest (green) to lowest (red).

Figure 2

Functional characterization of pathways of up-regulated proteins in S. aureus SH1000 at different concentrations of pexiganan (0.125, 0.25, 0.5, and 1 fractions of the minimal inhibitory concentration, MIC). For this analysis, only the 100 most highly differentially expressed proteins for each concentration of pexiganan were used. On top of each chart the number of identified genes and the number of pathway hits (number of genes used for the enrichment analysis) is visible. The analysis was carried out using the online gene ontology analysis software PANTHER (62).

Figure 3

Isobolographical response of pexiganan combination with different antimetabolite nucleosides. The crosses indicate the presence of bacterial growth in the unique concentration combinations of each well. Blue rectangles indicate the MIC value for single drug situations (pexiganan or antimetabolites) and is marked as a reference to visually compare with the actual level of inhibition for each pexiganan-antimetabolite combination.

Figure 4

Pexiganan-nucleoside antimetabolite combinations drastically increases the killing capacity of pexiganan. (A) Killing dynamic of S. aureus SH1000 at different concentrations of pexiganan using the MIC as the starting point. (B) Example data of time-kill experiment exposing mid-exponential phase bacterial cultures to pexiganan-nucleoside antimetabolite combinations (both at 1/2x MIC concentrations). The combination has a dramatic effect on the killing ability of pexiganan. Data points were determined by counting colony-forming units (CFU) at different time points. Mean ± SDM, n = 5. Asterisks represent significant differences (R package nparLD, one asterisk for p < 0.05 and two asterisks for p < 0.01 and three asterisks for p < 0.001). Only comparisons between pexiganan (1/2x MIC) and pexiganan-analogs combinations are shown.

Figure 5

A general model illustrating the positive interaction between pexiganan and nucleoside antimetabolites against S. aureus. The interactions of pexiganan with the membrane at sub-inhibitory concentrations lead to transient permeability changes in the envelope that promote leakage of small molecules such as nucleotides, nucleobases or nucleosides. Simultaneously, other small molecules such as toxic nucleoside analogs can increase the diffusion rate toward the intracellular compartment. This stress is sensed by the cell that responds by activating nucleoside metabolism creating an intervention opportunity. In this situation, toxic nucleoside antimetabolites are more efficiently incorporated into RNA, DNA, and other nucleotide depending reactions that may include envelope synthesis, enhancing toxicity and leading to faster cell killing.