Identification and Characterization of RK22, a Novel Antimicrobial Peptide from Hirudinaria manillensis against Methicillin Resistant Staphylococcus aureus

Abstract

Staphylococcus aureus (S. aureus) infections are a leading cause of morbidity and mortality, which are compounded by drug resistance. By manipulating the coagulation system, S. aureus gains a significant advantage over host defense mechanisms, with hypercoagulation induced by S. aureus potentially aggravating infectious diseases. Recently, we and other researchers identified that a higher level of LL-37, one endogenous antimicrobial peptide with a significant killing effect on S. aureus infection, resulted in thrombosis formation through the induction of platelet activation and potentiation of the coagulation factor enzymatic activity. In the current study, we identified a novel antimicrobial peptide (RK22) from the salivary gland transcriptome of Hirudinaria manillensis (H. manillensis) through bioinformatic analysis, and then synthesized it, which exhibited good antimicrobial activity against S. aureus, including a clinically resistant strain with a minimal inhibitory concentration (MIC) of 6.25 μg/mL. The RK22 peptide rapidly killed S. aureus by inhibiting biofilm formation and promoting biofilm eradication, with good plasma stability, negligible cytotoxicity, minimal hemolytic activity, and no significant promotion of the coagulation system. Notably, administration of RK22 significantly inhibited S. aureus infection and the clinically resistant strain in vivo. Thus, these findings highlight the potential of RK22 as an ideal treatment candidate against S. aureus infection.

Keywords: Hirudinaria manillensis; RK22; Staphylococcus aureus; antimicrobial peptide.

Figure 1

RK22 kills S. aureus ATCC6538 and MRSA-Z by permeabilizing the cell membrane. After treatment with or without RK22 (100 μg/mL) for 4 h, the membrane morphology of S. aureus ATCC6538 (A) and MRSA-Z (B) was determined by SEM and TEM. Killing kinetics of RK22 against S. aureus ATCC6538 (C) and MRSA-Z (D) were investigated. Data represent means ± SD of 3 individual experiments.

Figure 2

RK22 exhibits biofilm inhibition and eradication activities. Biofilm was measured at 600 nm after staining with 0.1% crystal violet. Inhibitory effects of RK22 on the biofilm formation of S. aureus ATCC6538 (A) and MRSA-Z (B) were analyzed after incubation with RK22 (final concentration 0.5, 1, 2, 4, 8 × MIC) with the bacteria for 24 h. Furthermore, to determine the effects of RK22 on the biofilm eradication, RK22 (final concentration 0.5, 1, 2, 4, 8 × MIC) was added into the wells with an established biofilm of S. aureus ATCC6538 (C) and MRSA-Z (D). Data represent means ± SD of 3 individual experiments. ns: difference was not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Effects of RK22 on the enzymatic activity of coagulation factors and platelet activation. Compared with the enhancement of LL-37 (final concentration 40 nM (0.18 μg/mL), 200 nM (0.9 μg/mL), 1000 nM (4.5 μg/mL)) on the enzymatic activity of thrombin and FXa, RK22 (final concentration 40 nM (0.112 μg/mL), 200 nM (0.56 μg/mL), 1000 nM (2.8 μg/mL)) showed no significant promotion on thrombin (A,B) and FXa (C,D). Furthermore, RK22 or LL-37 (20, 10, 5, 2.5 μg/mL) was incubated with washed platelets for 10 min, and then platelet activation was determined and analyzed by flow cytometry (E,F). Data represent means ± SD of 3 individual experiments. ns: difference was not significant. *** p < 0.001.

Figure 4

RK22 suppresses the dissemination of S. aureus (ATCC6538) and MRSA-Z in vivo. Mice (n = 10–12 mice per group) were injected with S. aureus (ATCC6538 1 × 10⁸ CFU/mouse, ip or MRSA-Z, 2 × 10⁸ CFU/mouse, ip); 1 h later, samples (RK22: 2, 4, 8 mg/kg; vancomycin: 2, 4 mg/kg, saline as the negative control group) were intraperitoneally injected with the concentration indicated. After 4 h of administration, mice were sacrificed, blood (A,D) and lung (B,E) were collected for the measurement of bacterial loads. Additionally, the sections of lung were stained with hematoxylin & eosin (H&E) for histopathological analysis (C,F), scale bar: 100 μm. Data represent mean ± SD values of six independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.