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. 2024 Aug 9;10(8):2795-2813.
doi: 10.1021/acsinfecdis.4c00160. Epub 2024 Jul 29.

Deciphering the Intracellular Action of the Antimicrobial Peptide A11 via an In-Depth Analysis of Its Effect on the Global Proteome of Acinetobacter baumannii

Thanit Thitirungreangchai  1 , Sittiruk Roytrakul  2 , Ratchaneewan Aunpad  1
Affiliations

Deciphering the Intracellular Action of the Antimicrobial Peptide A11 via an In-Depth Analysis of Its Effect on the Global Proteome of Acinetobacter baumannii

Thanit Thitirungreangchai et al. ACS Infect Dis. .

Abstract

The potential antimicrobial activity and low propensity to induce the development of bacterial resistance have rendered antimicrobial peptides (AMPs) as novel and ideal candidate therapeutic agents for the treatment of infections caused by drug-resistant pathogenic bacteria. The targeting of bacterial membranes by AMPs has been typically considered their sole mode of action; however, increasing evidence supports the existence of multiple and complementary functions of AMPs that result in bacterial death. An in-depth characterization of their mechanism of action could facilitate further research and development of AMPs with higher potency. The current study employs biophysics and proteomics approaches to unveil the mechanisms underlying the antibacterial activity of A11, a potential candidate AMP, against Acinetobacter baumannii, a leading cause of hospital-acquired infections (HAIs) and consequently, a serious global threat. A11 peptide was found to induce membrane depolarization to a high extent, as revealed by flow cytometry and electron microscopy analyses. The prompt intracellular penetration of A11 peptide, observed using confocal microscopy, was found to occur concomitantly with a very low degree of membrane lysis, suggesting that its mode of action predominantly involves a nonlytic killing mechanism. Quantitative proteomics analysis employed for obtaining insights into the mechanisms underlying the antimicrobial activity of A11 peptide revealed that it disrupted energy metabolism, interfered with protein homeostasis, and inhibited fatty acid synthesis that is essential for cell membrane integrity; all these impacted the cellular functions of A. baumannii. A11 treatment also impacted signal transduction associated with the regulation of biofilm formation, hindered the stress response, and influenced DNA repair processes; these are all crucial survival mechanisms of A. baumannii. Additionally, robust antibacterial activity was exhibited by A11 peptide against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of A. baumannii; moreover, A11 peptide exhibited synergy with levofloxacin and minocycline as well as low propensity for inducing resistance. Taken together, the findings emphasize the therapeutic potential of A11 peptide as an antibacterial agent against drug-resistant A. baumannii and underscore the need for further investigation.

Keywords: A11 peptide; Acinetobacter baumannii; antimicrobial peptides; intracellular actions; proteomic analysis; therapeutic potential.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Time-kill kinetics of A11 peptide against A. baumannii ATCC 19606 at concentrations of 0.5× MIC, 1× MIC, and MBC over a 24 h duration. Data are presented as mean ± SD. An asterisk (*) indicates a statistically significant difference compared to the control with p < 0.05.
Figure 2
Figure 2
Flow cytometry analysis of A. baumannii ATCC 19606 cells treated with A11 peptide. The percentages of fluorescence-positive cells following treatment with A11 peptide at 0.5× MIC (A) and 1× MIC (B) for 0.5, 1, 2, 4, and 6 h were measured. Statistically significant differences compared to the negative control were denoted with an asterisk (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Figure 3
Figure 3
SEM micrographs of A. baumannii ATCC 19606 treated with A11 peptide. (A, B) Untreated bacteria. (C, D) Bacteria treated with A11 peptide at 0.5× MIC for 2 h. SEM imaging was performed with three biological replicates of samples for each condition.
Figure 4
Figure 4
TEM micrographs of A. baumannii ATCC 19606 treated with A11 peptide. (A, B) Untreated bacteria. (C–F) Bacteria treated with A11 peptide at 0.5× MIC for 2 h. Blurred cells with damage membranes, leakage of intracellular content, and cytoplasmic clearance are marked with black, yellow, and red arrows, respectively. TEM imaging was performed with three biological replicates of samples for each condition.
Figure 5
Figure 5
Confocal microscopy imaging of A. baumannii ATCC 19606 treated with TAMRA-labeled A11 peptide at 0.5× MIC for 5 (A), 30 (B), and 120 (C) min and stained with CellBrite Fix 488 and Hoechst 33342. Green, blue, and red fluorescence correspond to the cell membrane stained with CellBrite Fix 488, the DNA stained with Hoechst 33342, and the localization of A11 peptide within the cells, respectively. CLSM imaging was conducted with two biological replicates of samples for each condition.
Figure 6
Figure 6
Gel retardation assay to evaluate the binding of A11 peptide with the genomic DNA of A. baumannii ATCC 19606. Lane 1: DNA Marker, lane 2:400 ng of genomic DNA alone, lanes 3–11:400 ng of genomic DNA incubated with A11 peptide at concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, and 250 μg/mL, respectively. Two replicates of runs were performed.
Figure 7
Figure 7
Proteomics profiling of A. baumannii ATCC 19606 treated with A11 peptide. (A) PLS-DA of untreated (control) and A11 peptide-treated A. baumannii cells. Volcano plots of statistically significant DEPs identified upon treatment of cells with A11 peptide for 0.5 h (B) and 2 h (C) as compared to the control. Red and blue dots represent upregulated and downregulated proteins, respectively (fold change ≥2 and p < 0.05). (D) Numerical representation of DEPs in A. baumannii treated with A11 peptide for 0.5 and 2 h. (E) Venn diagram of DEPs identified upon treatment of A. baumannii cells with A11 peptide for 0.5 and 2 h.
Figure 8
Figure 8
Twenty highly enriched functions of DEPs identified in A. baumannii ATCC 19606 following treatment with A11 peptide for 0.5 h (A) and 2 h (B). The data were sorted based on fold enrichment in functions associated with DEPs, with a false discovery rate (FDR) < 0.05.
Figure 9
Figure 9
Interaction network analysis of biological pathways enriched among DEPs identified upon the treatment of A. baumannii ATCC 19606 with A11 peptide at 0.5× MIC for 0.5 and 2 h using ShinyGO and STRING databases and employing Cytoscape software. Unique pathways observed among DEPs identified upon 0.5 and 2 h of treatment are represented by red rectangles (Edge cutoff = 0.1 and FDR p-value cutoff = 0.05).
Figure 10
Figure 10
Development of resistance in A. baumannii ATCC 19606 to A11 peptide and antibiotics over the course of 30 serial passages. The MICs of A11 peptide, meropenem, levofloxacin, and minocycline were recorded to determine fold changes after each passage. Three biological replicates of samples were performed for each agent.
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