Rationally Engineered D-Amino Acid Peptide DT7-3 Combats Multidrug-Resistant Helicobacter pylori via a Novel “Triple-Hit” Mechanism

Helicobacter pylori (H. pylori) is the primary etiological agent for chronic gastritis, peptic ulcers, and gastric adenocarcinoma. The alarming rise in multidrug-resistant (MDR) strains, particularly against clarithromycin (CLR), metronidazole (MNZ), and levofloxacin (LVX), has severely compromised standard therapies. Thus, there is an urgent clinical need for novel antimicrobial agents that operate through distinct mechanisms to bypass resistance pathways and mitigate gastric cancer risk. We designed and synthesized a series of antimicrobial peptides, focusing on the proteolytically stable all-D-amino acid enantiomer, DT7-3, derived from a probiotic-sourced template. Minimum inhibitory concentrations (MICs) were determined against standard strains and 11 clinical MDR isolates via the broth microdilution method. Antimicrobial mechanisms were elucidated using scanning electron microscopy (SEM) for morphology, fluorescence-based assays for anti-adhesion activity, and real-time qPCR to quantify virulence gene expression (babA, ureA, and vacA). Biocompatibility was assessed using defibrinated sheep erythrocytes, gastric epithelial cells (GES-1), and representative beneficial gut microbiota. Analysis of the clinical isolates revealed resistance rates of 63.6% for CLR/LVX and 81.8% for MNZ, with 54.5% identified as MDR. DT7-3 exhibited superior potency (MIC 1–32 µg/mL) against all strains, significantly outperforming its L-enantiomer counterparts. Mechanistic studies unveiled a “triple-hit” mechanism: (1) rapid membrane disruption; (2) potent inhibition of bacterial adhesion to host cells (~60% reduction at 0.5 × MIC); (3) significant downregulation of critical virulence factors (babA, ureA, and vacA). Furthermore, DT7-3 showed an excellent safety profile, with negligible hemolysis (<5% at 32 µg/mL) and minimal cytotoxicity toward GES-1 cells, yielding a high selectivity index (SI, MHC/MIC) > 32 relative to mammalian cells. Crucially, DT7-3 showed high selectivity for the pathogen over beneficial gut microbiota (MIC > 128 µg/mL, SI > 16). Crucially, DT7-3 maintained potent bactericidal activity (MIC ≤ 16 µg/mL) even under cholesterol-enriched conditions. The engineered D-peptide DT7-3 is a potent candidate for combating MDR H. pylori. Its multifaceted mechanism, targeting bacterial viability while suppressing core virulence factors, positions it as a robust lead compound for next-generation eradication therapies aimed at reducing the burden of H. pylori-associated diseases.