The antimicrobial peptide Cec4 has therapeutic potential against clinical carbapenem-resistant Klebsiella pneumoniae

Abstract

The rapid increase in carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, along with the cross-resistance of CRKP to other antibiotics, has created an urgent need for novel therapeutic agents. Among the potential options for next-generation antibiotics, antimicrobial peptides (AMPs) show great promise. In this study, we aimed to elucidate the mechanisms underlying the antibacterial activity against CRKP of an antibacterial peptide named Cecropin-4 (Cec4), which we successfully identified previously. Our results demonstrate that Cec4 not only exhibits rapid antibacterial activity but also effectively inhibits and eradicates bacterial biofilm at a low concentration of 8 µg/mL. Additionally, when used in combination with traditional antibiotics, Cec4 enhances their antibacterial effect. Microscopy techniques, including transmission electron microscopy (TEM), confocal laser scanning microscopy, and scanning electron microscopy (SEM), found that Cec4 destroyed bacteria's cell membrane integrity and increased the membrane permeability (flow cytometry instrument technology further characterization of Cec4 against K. pneumoniae bacteria antibacterial effect). Furthermore, in vitro experiments demonstrated that Cec4 binds to bacterial DNA and RNA of CRKP. Moreover, in vivo studies using a mouse skin wound model confirmed the efficacy of Cec4, and transcriptomic analysis shed light on the molecular mechanisms underlying its antibacterial activity. Based on our findings, Cec4 appears to be a promising candidate for combating CRKP infections.IMPORTANCEThe rapid increase in carbapenem-resistant Klebsiella pneumoniae (CRKP) infections and the serious cross-resistance to multiple antibiotics make the development of new therapeutic drugs urgent. Antimicrobial peptides (AMPs) have attracted much attention as a potential option for the next generation of antibiotics. Previous studies have identified the antimicrobial peptide Cecropin-4 (Cec4), and this study further explored its antimicrobial mechanism against CRKP. Studies have found that Cec4 shows high antibacterial activity at low concentrations, can inhibit and eradicate bacterial biofilms, and can also enhance the efficacy of traditional antibiotics. Its mechanism of action, such as destroying cell membranes and binding nucleic acid, has been revealed by various techniques, and its effectiveness has been confirmed in vivo, providing a promising candidate drug for combating CRKP infection.

Keywords: CRKP; Klebsiella pneumoniae; antibacterial activity; antibacterial mechanism; antimicrobial peptide.

Fig 1

Characterization of novel antimicrobial peptide. (A) Helical wheel projection diagrams of Cec4 using HeliQuest analysis (https://heliquest.ipmc.cnrs.fr/). Hydrophilic amino acids are shown in blue and are positively charged. Amino acids are shown in yellow and gray are hydrophobic. (B) The three-dimensional structure of the peptide was predicted via I-TASSER (https://zhanggroup.org/I-TASSER/). (C) The secondary structure of Cec4 was predicted using the secondary structure analysis software NPS@ (https://npsa-prabi.ibcp.fr).

Fig 2

Cec4 shows bactericidal activity against K. pneumoniae and without detectable resistance development. (A) Drug resistance development of K. pneumoniae ATCC 13883 and CRKP 100 after sub-inhibitory dose of Cec4 treatment generations. (B) Time killing curve of Cec4 against CRKP 100. The experiments were conducted in triplicate and presented as means ± SD. (C) Checkerboard assays of Cec4 in combination with two conventional antibiotics against CRKP100. Cec4 and antibiotics were subjected to 1/2 dilution vertically and horizontally from the MIC concentration at the upper left corner. Yellow (FICI = 0.5) and orange (0.5 < FICI < 1) indicate a partial synergistic effect, and gray indicates growth of bacteria. We defined MIC as inhibiting completely over 99% of CRKP 100 bacterial growth. (D) Effects of Cec4 on biofilm formation (D) and established biofilm (E) of CRKP 100. Results were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3

Killing capacity of Cec4 with CRKP100. (A) Bacterial survival after 1.5 h of treatment with different concentrations of Cec4 (8–16 μg/mL) was observed using laser scanning confocal microscopy. (B) Bacterial survival after 1.5 h of treatment with different concentrations of Cec4 (8–16 μg/mL) was observed using flow cytometry. Without the addition of antimicrobial peptides, the samples represent a negative control. Red fluorescence indicates dead bacteria, green and yellow fluorescence indicate viable bacteria.

Fig 4

Membrane active mechanism of action by Cec4. (A) SEM images of CRKP100 treated with Cec4 (16 µg/mL) for 1.5 h. Black arrows exhibited wrinkling, collapsed, and lysed structures on the cell membrane. (B) Confocal laser scanning microscopy (CLSM) shows fluorescein isothiocyanate (FITC)-labeled Cec4-treated K. pneumoniae ATCC 13883. Cec4 colocalized with FM4-64, indicating a membrane-binding propensity at 1 × MIC for 1 h. The scale bar in this figure is 5 µm.

Fig 5

Interaction of Cec4 and Gram-negative bacterial membranes in a molecular dynamics simulation system. (A) The root-mean-square deviation (RMSD) value of Cec4. (B) The root-mean-square fluctuation (RMSF) value of Cec4. (C) Distance from each amino acid to the centre of mass of the bacterial membrane. (D) Binding energy contributions of Cec4 residues to Gram-negative bacteria. (E and F) MD simulation images of Cec4 interacting with a 3POPG: 1POPE lipid bilayer at representative time points (10 and 100 ns).

Fig 6

Cec4 binds to cell membrane components and alters membrane permeability. Whole cell membrane permeability of K. pneumoniae ATCC 13883 and CRKP 100, which were assessed by fluorescence probes NPN (A) (excitation 350 nm and emission 420 nm) and PI (B) (excitation 535 nm and emission 615 nm), respectively. CLSM shows Cec4 in K. pneumoniae ATCC 13883 and CRKP 100 after treatment with Cec4 at 8–16 μg/mL for 1.5 h. (C) Exogenous addition of LPS impaired the antibacterial activity of Cec4 against K. pneumoniae ATCC 13883 and CRKP 100 in a dose‐dependent manner. (D) Increased MICs of Cec4 against K. pneumoniae ATCC 13883 and CRKP 100 in the presence of CL, PG, PC, and PE, ranging from 1 to 512 µg/mL.

Fig 7

Effects of Cec4 on bacterial cell membrane and oxidative stress. (A) Cec4 dissipates the ΔΨ component of the PMF. Cec4 dissipated the membrane potential of K. pneumoniae ATCC 13883 and CRKP 100 determined by monitoring the fluorescence intensity of 3,3'-dipropylthiadicarbocyanine iodide (DiSC3(5), excitation at 622 nm and emission at 622 nm). (B) Membrane fluidity changes in K. pneumoniae ATCC 13883 and CRKP 100 under exposure to Cec4. Membrane fluidity was determined using 10 µM Laurdan, and the fluorescence intensities were detected with emission wavelengths of 435 and 490 nm upon excitation at 350 nm. (C) Cec4 triggers the production of ROS in K. pneumoniae ATCC 13883 and CRKP 100. Before the fluorescence assay, probe-labeled cells were incubated with Cec4 at 37°C for 1.5 h. (D) NAC supplementation abolished the antibacterial activity of Cec4 against K. pneumoniae ATCC 13883 and CRKP 100. All data were presented as mean ± SD, and the statistical significance was determined by non‐parametric one‐way analysis of variance (NS, no significant; results were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001).

Fig 8

Effects of Cec4 on bacterial cell membrane and oxidative stress. (A) TEM images of CRKP 100 treated with Cec4 (16 µg/mL) for 1.5 h. (B) Gel retardation analysis of the binding of Cec4 to CRKP 100 DNA. (C) Gel retardation analysis of the binding of Cec4 to CRKP 100 RNA. Lane M: DL2000 DNA Marker; Lanes 1–6: the genomic DNA/RNA treated with Cec4 of 0–512 μg/mL. Lane 7: the genomic RNA treated with Cec4 at 1,024 µg/mL.

Fig 9

In vivo antibacterial assay. (A) Schematic illustration of the in vivo bacterial infection and treatment using Cec4. (B) Bacterial infection was treated 1 day after infection, with photographs taken on days 1, 3, 7, and 11 after treatment. (C) Quantification of the process of wound healing for all groups (n = 6). (D) The subeschar bacterial colonies on the 11th day. (E) HE and Masson-stained (F) histologic sections of wounds on the 11th day. Abbreviations: f, follicle; s, sebaceous gland; k, keratinized layer; e, epidermis layer; d, dermis. Original magnification: ×100. *** denotes P < 0.001.

Fig 10

Transcriptome analysis of K. pneumoniae ATCC 13883 after exposure to Cec4. (A) Volcano plot annotation analysis of the differential expression genes (DEGs) in K. pneumoniae ATCC 13883 after exposing Cec4 (8 µg/mL) for 1.5 h. Significantly differentially expressed genes were treated with red dots (up-regulated) or green dots (down-regulated). The abscissa represents fold change, and the ordinate represents statistical significance. (B) Gene Ontology (GO) annotation analysis of the DEGs in K. pneumoniae ATCC 13883. (C) The classification of genes into Kyoto Encyclopedia of Genes and Genomes (KEGG) terms after enrichment analysis. Up-regulated genes are labeled red, and down-regulated genes are labeled green. (D) Comparison of eight gene expression levels between RNA-Seq and qRT-PCR. The red line represents the FPKM value of the gene in RNA-Seq, and the blue columns represent the relative expression value calculated by qRT-PCR. The experiments were performed in three biological replicates, and data are presented as means ± SD.

Fig 11

Schematic representation of the mode of action of Cec4 against K. pneumoniae. Cec4 may kill the K. pneumoniae by crossing the cell wall and destroying cell membrane integrity, further resulting in membrane dysfunction, or acting on intracellular targets, to affect the transcriptional regulation of DNA and ribosomal biosynthesis, which in turn affects the normal physiological activities of bacteria. The picture was drawn with Figdraw.