In-depth biological characterization of two black soldier fly anti- Pseudomonas peptides reveals LPS-binding and immunomodulating effects

Abstract

As effector molecules of the innate immune system, antimicrobial peptides (AMPs) have gathered substantial interest as a potential future generation of antibiotics. Here, we demonstrate the anti-Pseudomonas activity and lipopolysaccharide (LPS)-binding ability of HC1 and HC10, two cecropin peptides from the black soldier fly (Hermetia Illucens). Both peptides are active against a wide range of Pseudomonas aeruginosa strains, including drug-resistant clinical isolates. Moreover, HC1 and HC10 can bind to lipid A, the toxic center of LPS and reduce the LPS-induced nitric oxide and cytokine production in murine macrophage cells. This suggests that the peptide-LPS binding can also lower the strong inflammatory response associated with P. aeruginosa infections. As the activity of AMPs is often influenced by the presence of salts, we studied the LPS-binding activity of HC1 and HC10 in physiological salt concentrations, revealing a strong decrease in activity. Our research confirmed the early potential of HC1 and HC10 as starting points for anti-Pseudomonas drugs, as well as the need for structural or formulation optimization before further preclinical development can be considered. IMPORTANCE The high mortality and morbidity associated with Pseudomonas aeruginosa infections remain an ongoing challenge in clinical practice that requires urgent action. P. aeruginosa mostly infects immunocompromised individuals, and its prevalence is especially high in urgent care hospital settings. Lipopolysaccharides (LPSs) are outer membrane structures that are responsible for inducing the innate immune cascade upon infection. P. aeruginosa LPS can cause local excessive inflammation, or spread systemically throughout the body, leading to multi-organ failure and septic shock. As antimicrobial resistance rates in P. aeruginosa infections are rising, the research and development of new antimicrobial agents remain indispensable. Especially, antimicrobials that can both kill the bacteria themselves and neutralize their toxins are of great interest in P. aeruginosa research to develop as the next generation of drugs.

Keywords: LPS binding; Pseudomonas aeruginosa; antimicrobial peptides; black soldier fly; lipopolysaccharide.

Fig 1

Salt sensitivity of HC1 and HC10. (a) The activity of HC1 and HC10 against P. aeruginosa PAO1 strongly decreases in simulated lung fluid conditions (Gamble’s medium). (b and c) The anti-Pseudomonas activity of HC1 and HC10 slightly decreases in the presence of high (≥75 mM) concentrations of NaCl. (d and e) The addition of CaCl₂ has a clear, negative impact on the antimicrobial activity of HC1 and HC10. At physiological concentrations (2.5 mM), the activity strongly decreased. Data are presented as mean ± standard deviation. Data were analyzed using a one sample t-test to test for significant difference from 1. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. All experiments were carried out in biological triplicate.

Fig 2

LPS neutralization by HC1, as determined by a chromogenic LAL assay. HC1 shows a concentration-dependent neutralization of standard E. coli endotoxin, with high neutralization from 4 µM onwards. Data for HC10 are not included in the graph, as this peptide intrinsically induced the LAL enzyme cascade, leading to high background signals. The experiment was carried out in biological triplicate. Data are represented as mean + standard deviation of all technical and biological replicates.

Fig 3

BODIPY-TR cadaverine displacement assay. (a and b) HC1 and HC10 are able to displace BC in a concentration-dependent manner, signified by the increase in fluorescence over time. As a control, samples with BC and LPS (100 ng/mL) without peptide treatment were included. At 32 µM of AMP, there is close to a 300% increase in fluorescence compared to the control. (c) BC displacement assay for the peptide antibiotic reference polymyxin B. The LPS binding of polymyxin B follows a similar concentration-dependent trend over time, but for 16 and 32 µM, the polymyxin-induced LPS binding is lower than for HC1 and HC10. Fluorescence was read during a 1-hour cycle. Data are represented as mean ± standard deviation. Data sets at the last timepoint were compared with a one-way ANOVA test, ****P ≤ 0.0001. The assay was carried out in biological triplicate.

Fig 4

Effect of HC1 and HC10 on nitrite production by LPS-stimulated RAW264.7 macrophages as determined by a Griess reaction. HC1 and HC10 have a concentration-dependent effect on the macrophage activation after LPS addition, presumably through their LPS-binding ability. Both peptides achieve high, near 100% inhibition of nitrite formation at concentrations of 32 µM. As a positive control, LPS-stimulated macrophages without peptides were used. The experiment was carried out in biological triplicate. Data are represented as mean + standard deviation.

Fig 5

Effect of HC1 and HC10 on the release of the pro-inflammatory cytokines TNF-α and IL-6. (a) The release of TNF-α by RAW264.7 macrophages after peptide monotreatment or LPS-AMP co-treatment for 24 h was investigated using ELISA. (b) The release of IL-6 by RAW264.7 macrophages after peptide monotreatment or LPS-AMP co-treatment for 24 h was investigated using ELISA. (c) qPCR analysis of the release of TNF-α by RAW264.7 macrophages after peptide monotreatment or LPS-AMP co-treatment for 4 h. (d) qPCR analysis of the release of IL-6 by RAW264.7 macrophages after peptide monotreatment or LPS-AMP co-treatment for 4 h. LPS-treated cells (100 ng/mL) were included as positive controls. Data are represented as mean + standard deviation and statistically analyzed using a Kruskal-Wallis test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ***P ≤ 0.0001. All experiments were carried out in biological triplicate.