Selective killing of Helicobacter pylori with pH-responsive helix-coil conformation transitionable antimicrobial polypeptides

Abstract

Current clinical treatment of Helicobacter pylori infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill H. pylori under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix-coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)_m-r-(PHLG-MHH)_n, bearing randomly distributed negatively charged glutamic acid and positively charged poly(γ-6-N-(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against H. pylori, including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable H. pylori killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill H. pylori in the stomach while not harming the commensal bacteria/normal tissues.

Keywords: H. pylori; antimicrobial peptide; conformational transition; pH sensitiveness; α-helix.

Fig. 1.

HCT-AMPs display pH-sensitive helix–coil transition. (A) Schematic illustration of the pH-responsive conformation transition of HCT-AMP (PGA)₁₈-r-(PHLG-MHH)₂₀. It adopts random coiled conformation at physiological pH to impart low toxicity while transforming to helical conformation under acidic condition in the stomach to induce potent antimicrobial activity against H. pylori. The green balls represent cationic amine groups, the blue balls represent anionic group (-COO), and the black balls represent neutral groups (-COOH). CD spectra of PL2 (4.4 μM) at various pH values adjusted from 7.4 to 2.7 with 1 M HCl (B) and from 2.7 to 7.2 with 1 M NaOH (C).

Fig. 2.

HCT-AMPs selectively kill H. pylori under acidic condition in vitro. (A) The hemolytic activity of polypeptides at pH 7.4. PL1, PL2, and PDL2, dissolved in PBS (pH 7.4) at various concentrations, was incubated with fresh rabbit blood for 1 h. Hemoglobin release was measured by UV absorbance at 576 nm using a microplate reader. (B) The survival rate of H. pylori SS1 after incubation with PL2 for 1 h at various pHs. PL2, dissolved in the Tris⋅HCl buffer at various pHs (pH 7.4, 4.0, 3.0), was incubated with SS1 at corresponding pHs in Brucella broth (BB) medium supplied with fresh urea (10 mM), 10% FBS, and vancomycin (5 µg/mL). The bacterial count was determined by counting colony-forming units (cfu) of alive bacteria with agar plating. Bacteria incubated with Tris⋅HCl buffer only at corresponding pH were served as 100% survival. (C) Extent of ANTS/DPX efflux in negatively charged liposomes after treatment with PL2 at various concentrations at pH 7.4 or pH 4.0. (D) SEM images of SS1 after treatment with Tris⋅HCl buffer or PL2 at pH 7.4 or pH 4.0. SS1 bacterial cells were incubated with or without PL2 (2.2 µM) at pH 7.4 or 4.0 for 30 min. (Scale bar, 0.5 μm.)

Fig. 3.

HCT-AMPs effectively kill clinically isolated drug-resistant H. pylori. (A) The bactericidal activity of PL2 against clinically isolated H. pylori strains. PL2, dissolved in the Tris⋅HCl buffer (pH 3.0), was incubated with clinically isolated strains in BB medium supplied with fresh urea, FBS, and vancomycin (pH 3.0) for 1 h. The final concentration of PL2 was 4.4 µM. (B) The antibacterial activity of triple therapy against J99A-11 at pH 7.4 or 3.0. Concentrations of OAC in OAC1 are 20.0, 6.8, and 1.9 µM, respectively; in OAC2 are 40.0, 13.6, and 3.8 µM, respectively; in OAC3 are 80.0, 27.2, and 7.6 µM, respectively. (C) The antibacterial activity of omeprazole (O) and a combination of amoxicillin and clarithromycin (AC) against J99A-11 at pH 3.0. O1, O2, and O3 represents the concentration of omeprazole at 20.0, 40.0, and 80.0 µM, respectively. AC1, AC2, and AC3 represents the concentration of AC at 6.8 and 1.9; 13.6 and 3.8 µM; and 27.2 and 7.6 µM, respectively.

Fig. 4.

In vivo distribution of Cy5-labeled HCT-AMPs following oral gavage. (A) Representative whole-body fluorescence imaging of C57BL/6J mice treated with Cy5-PL2 and Cy5-PDL2 (2.6 μmol/kg). Cy5-PL2, Cy5-PDL2, and PBS were administrated by oral gavage. Mice were then imaged with the Bruker Xtreme In-Vivo Fluorescence Imaging System at 1, 2, 4, 7, and 24 h postinjection (p.i.). (B) Representative ex vivo fluorescence imaging of major organs (1, 2, 3, 4, 5, 6, and 7 represents lung, heart, liver, kidney, stomach, spleen, and intestines, respectively) from C57BL/6J mice treated with Cy5-PL2 and Cy5-PDL2. Major organs were harvested and imaged ex vivo 4 h or 24 h p.i. of Cy5-PL2 and Cy5-PDL2. (C) Ex vivo fluorescence intensity of stomach harvested from mice receiving the treatment in B. Ex vivo images were quantified by measuring fluorescence intensity at selected region of interest. All values were expressed as means ± SD (n = 3). (D) Retention of Cy5-PL2 and Cy5-PDL2 in mouse stomach 4 and 24 h after oral gavage. Stomach samples were harvested and homogenized, and the lysates were used to determine the amount of Cy5 retained in the tissues with a fluorescence spectrometer. (E) Confocal image of mouse stomach following treatment with Cy5-PL2. (Scale bar, 100 μm.)

Fig. 5.

Anti-H. pylori efficacy of HCT-AMPs in vivo. (A) The study protocol of H. pylori inoculation, infection development, and treatments in C57BL/6J mice. Each mouse was administered with SS1 (OD₆₀₀ = 2, 0.2 mL) intragastrically through oral gavage every other day for four times (on days 1, 3, 5, and 7, respectively), and the infection was allowed to develop for 2 wk. Mice were then treated with control (PBS), triple therapy (OAC, omeprazole 400 µmol/kg, amoxicillin 68 µmol/kg, and clarithromycin 19.1 μmol/kg), PDL2, and PL2 (2.6 μmol/kg) once daily for a consecutive 3 d. (B) Bacterial burden in the stomach of H. pylori-infected mice treated with PBS, triple-control therapy, PDL2, and PL2 (n ≥ 6). (C) Bodyweight change of mice following treatment of various formulations as in B. (D) The killing effect of PL2 against commensal bacteria determined by measuring the bacterial load in the feces and ileal contents of mice after a daily gavage of PL2, control (5% DMSO), and OAC for three consecutive days. The bacterial load was determined by quantitative real-time PCR. The 16S rRNA gene level was normalized to the tissue weight (n ≥ 6). All of the data are represented as average ± SD and analyzed by Student’s t test (*P ≤ 0.05). “ns” represents no significant difference (P > 0.05).