Targeted photodynamic inactivation of Salmonella Typhimurium based on nanobody-cell-penetrating peptide conjugates

Abstract

Photodynamic bacterial inactivation (PDI) is a promising non-thermal antibacterial technology for food preservation. However, its effectiveness against gram-negative bacteria is limited by the presence of outer membrane. As one of the most prevalent serotypes responsible for salmonellosis outbreaks worldwide, S. Typhimurium represents a major gram-negative foodborne pathogen. Thus, this study aimed to develop a PDI strategy by combining a specific nanobody (VHH) and a cell-penetrating peptide (CPP), along with curcumin to control S. Typhimurium. A VHH-CPP conjugate was prepared via site-specific coupling of an anti-Salmonella nanobody with the cationic CPP moiety, divalent Tat (di.Tat). With the introduction of VHH-di.Tat, the photosensitizer, hydroxypropyl-β-cyclodextrin-curcumin complex (HP-β-CD-Cur) accumulated and aggregated within bacterial cells, mediating effective PDI. This treatment resulted a 6.69 ± 0.29 Log10 CFU/mL reduction in bacterial viability and achieved 61.0% biofilm destruction. Mechanistic investigations revealed that upon light irradiation, VHH-di.Tat facilitated penetration of HP-β-CD-Cur, leading to enhanced generation of reactive oxygen species (ROS), disruption of cell morphology, and damage to bacterial membranes, thereby impairing normal physiological functions of S. Typhimurium. Overall, this study demonstrates efficient photodynamic inactivation of S. Typhimurium and presents a novel, targeted strategy for precise control of gram-negative pathogens with potential applications in food monitoring and safety