Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Abstract

Commonly used antimicrobials show poor cellular uptake and often have limited access to intracellular targets, resulting in low antimicrobial activity against intracellular pathogens. An efficient delivery system to transport these drugs to the intracellular site of action is needed. Cell-penetrating peptides (CPPs) mediate the internalization of biologically active molecules into the cytoplasm. Here, we characterized two CPPs, α1H and α2H, derived from the Yersinia enterocolitica YopM effector protein. These CPPs, as well as Tat (trans-activator of transcription) from HIV-1, were used to deliver the antibiotic gentamicin to target intracellular bacteria. The YopM-derived CPPs penetrated different endothelial and epithelial cells to the same extent as Tat. CPPs were covalently conjugated to gentamicin, and CPP-gentamicin conjugates were used to target infected cells to kill multiple intracellular Gram-negative pathogenic bacteria, including Escherichia coli K1, Salmonella enterica serovar Typhimurium, and Shigella flexneri Taken together, CPPs show great potential as delivery vehicles for antimicrobial agents and may contribute to the generation of new therapeutic tools to treat infectious diseases caused by intracellular pathogens.

Keywords: CPP-translocated antimicrobials; antimicrobial drug delivery; cell-penetrating peptides; gentamicin; intracellular pathogenic bacteria.

FIG 1

α1H and α2H peptide sequence analysis and modeling. (A) YopM consists of two N-terminal, antiparallel α-helices (green) and a variable number (13 to 22) of leucine-rich repeats (LRR) (orange). The YopM-derived CPPs are highlighted in the red squares: the first α-helix, α1H (YopM_34–51), the second α-helix, α2H (YopM_53–71), and both α-helices, 2αH (YopM_1–86). The 3D structure was generated using PyMOL. (B) PSIPRED prediction (Pred) of the YopM_34–73 secondary structure (42). Secondary structure predictions are defined by letter code: C, coiled coil; H, helix. The confidence (Conf) of the prediction is indicated by different scale bar levels. (C and D) Computational analysis for the prediction of secondary structure in water and in a lipid bilayer (membrane) environment. The analyzed sequence comprises the first α-helix (aa 34 to 51 of YopM) (C) and the second α-helix (aa 53 to 73 of YopM) (D) of the N-terminal domain of YopM. The average helical content of the peptides in water or in the membrane and the simulation of the residue distribution on the membrane surface are shown. The prediction was performed by using MCPEP software (43). The orientation of each peptide toward the membrane bilayer additionally is shown in the 3D conformation model. The residues that interact with the membrane according to the membrane simulation graph are highlighted in red. The 3D models were generated using PyMOL. (E) Helical wheel projection of α1H and α2H peptides were generated using HeliQuest (44). The arrows indicate the helical moment.

FIG 2

α1H, α2H, and Tat uptake and localization in eukaryotic cells. (A and B) Fluorescence-activated cell sorting (FACS) analysis of CPP-FITC uptake in HeLa cells (A) and HBMEC (B). CPP-FITC (750 nM) was incubated continuously with cells at 37°C, and the fluorescence intensity was measured by using flow cytometry. External fluorescence was quenched by adding 0.2% trypan blue. Bars represent means ± standard errors of the means (SEM). AU, arbitrary units. (C) Fluorescence microscopy analysis of CPP-FITC in HeLa cells and HBMEC. Cells were incubated with 1 μM CPP-FITC (green). Actin was counterstained with phalloidin-TRITC (red) and nuclei with Draq5 (blue). Topical and orthogonal views are shown and marked as yellow lines at that point of the field of cells. Scale bar, 10 μm.

FIG 3

CPP-FITC enters cells via endocytosis and partially via direct translocation. (A and B) FACS analysis of CPP-FITC uptake in HeLa (A) and HBMEC (B) cells. CPP-FITC (750 nM) was incubated continuously with cells at 4°C, and the fluorescence intensity was measured by using flow cytometry. External fluorescence was quenched by adding 0.2% trypan blue. AU, arbitrary units. (C) Effect of endocytosis inhibitors on the uptake of CPP-FITC in HeLa cells. Cells were preincubated with different endocytosis inhibitors for 1 h before adding 750 nM CPP-FITC for 6 h. The amount of intracellular CPP-FITC was determined by using flow cytometry. External fluorescence was quenched by adding 0.2% trypan blue. Significance was calculated for each condition to the respective control by using the Student t test: *, P < 0.05; **, P ≤ 0.01; ***, P < 0.005. Bars represent means ± SEM. (D and E) Membrane integrity assay using HeLa cells (D) and HBMEC (E) incubated with CPP-FITC. All cell lines were incubated with 750 nM CPP-FITC and coincubated with 1 μg/ml propidium iodide (PI) at 37°C. Samples were obtained during the ongoing incubation, and PI fluorescence was measured by using flow cytometry. Triton X-100 (0.2%) was added as a positive control for membrane disruption.

FIG 4

Immunofluorescence analysis of infected cells after treatment with CPP-gentamicin in LAMP1-positive compartments. (A) CLSM of HBMEC and infected HBMEC with E. coli K1 either left untreated or treated with the indicated CPP-FITC conjugates for 1 h. (B and C) CLSM of HeLa cells and infected HeLa cells with Salmonella enterica serovar Typhimurium (B) or with Shigella flexneri (C) either left untreated or treated with the indicated CPP-FITC conjugates for 1 h. Pictures were taken using a laser-scanning microscope (LSM 510 META microscope equipped with a Plan-Apochromat 63×/1.4-numeric-aperture oil immersion objective; Carl Zeiss). All three fluorescence images were merged and consisted of one optical section of a z-series with a pinhole of 1 airy unit. The indicated single fluorescence channels are shown on the right and the merged images are on the left (red, LAMP-1; green, CPP-FITC; blue, nuclei; yellow, colocalized red and green signals). Scale bars, 10 μm.

FIG 5

CPP-gentamicin conjugates maintain gentamicin antibacterial activity and are not cytotoxic. (A) Schematic representation of CPP-gentamicin conjugation. Gentamicin and the cross-linker SMCC were mixed in PBS and incubated for 2 h at RT to form a stable amide bond. Subsequently, the CPPs were added and incubated for an additional 2 h at RT to form the thioether bond. (B) LDH release cytotoxicity assays in HeLa cells and HBMEC. CPP-gentamicin (200 μg/ml of gentamicin; 600 μg/ml of CPP and SMCC), 200 μg/ml of gentamicin, 600 μg/ml of CPPs or SMCC, and 6% DMSO were incubated in HeLa cells or HBMEC for 2 h. Bars represent means ± SEM. Significance was calculated by using the Student t test: *, P < 0.05; **, P ≤ 0.01. (C) BrdU proliferation assays in HeLa cells and HBMEC. CPP-gentamicin (200 μg/ml of gentamicin; 600 μg/ml of CPP and SMCC), 200 μg/ml of gentamicin, 600 μg/ml of CPPs or SMCC, and unconjugated CPPs and gentamicin (CPP + gentamicin) were incubated in HeLa cells or HBMEC for 90 min. After incubation, fresh medium was added and cells were allowed to recover for 72 h. Bars represent means ± SEM. Significance was calculated by using 2-way analysis of variance (ANOVA): *, P < 0.05; **, P ≤ 0.01. (D to F) CPP-gentamicin (50 μg/ml gentamicin, 150 μg/ml CPP and SMCC), 50 μg/ml gentamicin, 150 μg/ml of CPPs or SMCC, and 6% DMSO were added to liquid cultures of E. coli K1 RS218 (D), Shigella flexneri (E), and Salmonella enterica serovar Typhimurium (F). The OD₆₀₀ was measured after 4 h of incubation. Measurements for gentamicin, α2H, and α1H in panels E and F were performed in duplicate; in all other cases, the experiments were performed at least in triplicate. Bars represent means ± SEM. Significance was calculated using Student's t test and referred to untreated cultures (Ctr): *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0005.

FIG 6

CPP-gentamicin efficiently reduced the amount of intracellular E. coli K1, Shigella, and Salmonella in infected cells. (A) HBMEC were infected with E. coli K1 RS218 before adding CPP-gentamicin (200 μg/ml gentamicin, 600 μg/ml CPP and SMCC), 200 μg/ml gentamicin and doxycycline, or 600 μg/ml SMCC. Intracellular bacteria titers were determined by plating cell lysates. (B) HBMEC were infected with E. coli K1 RS218 and treated with 200 μg/ml gentamicin and 600 μg/ml CPPs, briefly premixed before adding to the cells (CPP + gentamicin), or with 600 μg/ml CPPs alone. Intracellular bacterial titers were determined by plating cell lysates. (C) HeLa cells were infected with Shigella flexneri before adding CPP-gentamicin (200 μg/ml gentamicin; 600 μg/ml CPP and SMCC), 200 μg/ml gentamicin and doxycycline, and 600 μg/ml CPP or SMCC. Intracellular bacterial titers were determined by plating cell lysates. The experiment was performed in triplicate, except for Tat, α2H, and α1H (performed in duplicate). (D) HeLa cells were infected with Salmonella enterica serovar Typhimurium and subsequently treated with CPP-gentamicin, gentamicin, CPP, or SMCC as described for panel C. Intracellular bacterial titers were determined as before. Bars represent means ± SEM. (A to D) Significance was calculated using one-way ANOVA and compared to PBS treatment unless specified otherwise: *, P ≤ 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

FIG 7

Electron microscopic analysis of infected cells after treatment with CPP-gentamicin. (A) HeLa cells and HeLa cells infected with Salmonella enterica serovar Typhimurium and Shigella flexneri before adding CPP-gentamicin. (B) HeLa cells infected with Salmonella or Shigella after addition of different CPP-gentamicin conjugates for 1.5 h. (C) HBMEC and HBMEC infected with E. coli K1 RS218 before adding CPP-gentamicin. (D) HBMEC infected with E. coli K1 RS218 after addition of different CPP-gentamicin conjugates for 1.5 h. All cells were counterstained with uranyl acetate, and ultrathin sections of the samples were analyzed with an FEI-Tecnai 12 electron microscope. Scale bars, 200 nm.