New Insights into the Mechanism of Antibacterial Action of Synthetic Peptide Mo-CBP3-PepI against Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is a multidrug-resistant opportunistic human pathogen related to various infections. As such, synthetic peptides have emerged as potential alternative molecules. Mo-CBP₃-PepI has presented great activity against K. pneumoniae by presenting an MIC₅₀ at a very low concentration (31.25 µg mL^-1). Here, fluorescence microscopy and proteomic analysis revealed the alteration in cell membrane permeability, ROS overproduction, and protein profile of K. pneumoniae cells treated with Mo-CBP₃-PepI. Mo-CBP₃-PepI led to ROS overaccumulation and membrane pore formation in K. pneumoniae cells. Furthermore, the proteomic analysis highlighted changes in essential metabolic pathways. For example, after treatment of K. pneumoniae cells with Mo-CBP₃-PepI, a reduction in the abundance of protein related to DNA and protein metabolism, cytoskeleton and cell wall organization, redox metabolism, regulation factors, ribosomal proteins, and resistance to antibiotics was seen. The reduction in proteins involved in vital processes for cell life, such as DNA repair, cell wall turnover, and protein turnover, results in the accumulation of ROS, driving the cell to death. Our findings indicated that Mo-CBP₃-PepI might have mechanisms of action against K. pneumoniae cells, mitigating the development of resistance and thus being a potent molecule to be employed in producing new drugs against K. pneumoniae infections.

Keywords: antibacterial peptides; multidrug-resistant bacteria; proteomic analysis; synthetic peptides.

Figure 1

Assessment of toxicity of Mo-CBP3-PepI to human cell lines. (A) L929, (B) HaCaT, and (C) MRC-5 lines were incubated with synthetic peptide at a concentration of 1 mg mL⁻¹ to evaluate the damage to DNA by comet assay. (D–F) Cell lines were incubated with peptides as described and evaluated for viable cells and cells in apoptosis. MMS (4 × 10⁻⁵ M) was employed as a positive control for cell toxicity, and healthy cells as a negative control for toxicity. Data are shown as mean ± standard deviation of three independent experiments. * p < 0.05. Small letters indicate statistical significance.

Figure 2

Evaluation of toxicity of Mo-CBP3-PepI to human cell lines by MTT viability assay. In (A) L929, (B) HaCaT, and (C) MRC-5 lines were incubated with synthetic peptides at a concentration of 1 mg mL⁻¹. Methyl methanesulfonate (MMS; 4 × 10⁻⁵ M) was employed as a positive control for cell toxicity, and healthy cells as a negative control for toxicity. Data are shown as mean ± standard deviation of three independent experiments with significance of p < 0.05. Small letters indicate statistical significance.

Figure 3

Mechanism of action of Mo-CBP3-PepI assessed by fluorescence microscopy. In all experiments, Mo-CBP3-PepI was used at a concentration of 31.26 μg mL⁻¹. (A,B) control K. pneumoniae cells for PI uptake assay. (E,F) Mo-CBP3-PepI-treated K. pneumoniae cells for PI uptake assay. Red fluorescence (F) indicates pore formation induced by Mo-CBP3-PepI. (C,D) control K. pneumoniae cells for FITC-Dextran uptake assay. (G,H) Mo-CBP3-PepI-treated K. pneumoniae cells for FITC-Dextran uptake assay. Green fluorescence (H) indicates 10-kDa -sized pore formation induced by Mo-CBP3-PepI. (I,J) control K. pneumoniae cells for ROS overproduction assay. (K,L) Mo-CBP3-PepI-treated K. pneumoniae cells for ROS overproduction assay. Green fluorescence (L) indicates pore formation induced by Mo-CBP3-PepI. Bars indicate 100 μm.

Figure 4

Scanning electron microscope of K. pneumoniae cells treated with Mo-CBP3-PepI. (A) control K. pneumonia cells treated with DMSO-NaCl solution. (B–H) K. pneumoniae cells treated with Mo-CBP3-PepI 31.26 μg mL⁻¹.

Figure 5

Distribution of K. pneumoniae proteins obtained after treatment with Mo-CBP3-PepI. (A) Venn diagram shows the dispersion of total proteins of control and Mo-CBP3-PepI-treated cells. (B) Represents the fold-change value of overlapping proteins, which were found in both groups. The vertical bar indicates the color scale according to each protein’s fold-change value.

Figure 6

Classification proteins from K. pneumoniae cells identified by LC-ESI-MS/MS analysis. The overlapping proteins (found in both control and treated) were classified based on biological process and molecular function.

Figure 7

Scheme showing the mechanisms of antibacterial action of Mo-CBP3-PepI against K. pneumoniae. This scheme was produced only with proteins that decreased in abundance. The numbers explain important process. All them are explained in the text.