Harnessing light-activated gallium porphyrins to combat intracellularStaphylococcus aureusin dermatitis: Insights from a simplified model

Staphylococcus aureus can survive inside nonprofessional phagocytes such as keratinocytes, demonstrating a novel strategy for evading antibiotic pressure. When antibiotic treatment ends, reinfection with staphylococci begins from the intracellular inoculum. This phenomenon is responsible for recurrent infections. The development of new antibacterial methods that can eliminate intracellular bacteria, including those with a multidrug-resistant phenotype, is necessary. In this study, we characterized and used a model of keratinocytes (both wild type and mutants with reduced filaggrin expression) infected with methicillin-resistant S. aureus (MRSA) to verify the possibility of using light-activated compounds, exemplified here by heme-mimetic gallium (III) porphyrin (Ga 3+ CHP) and visible light, an approach known as antimicrobial photodynamic inactivation (aPDI), to eliminate intracellular MRSA. We observed that Ga 3+ CHP accumulated more in infected cells than in uninfected cells. Moreover, Ga 3+ CHP accumulated in cells that harbored intracellular S. aureus . Using flow cytometry and fluorescence microscopy, we found that intracellular MRSA and Ga 3+ CHP mainly colocalized in lysosomal structures, and we showed that under the influence of aPDI, MRSA exhibited reduced adhesion to host cells and a significantly reduced (by 70%) GFP signal originating from intracellular bacteria. Moreover, the use of light-activated Ga 3+ CHP resulted in a significant reduction in the number of extracellular bacteria in the infection system, lowering the potential for further infection of host cells. For the first time, we used the infectious model to analyze the toxicity of aPDI in real time, showing that this approach is not significantly cyto-or phototoxic. Author Summary Staphylococcus aureus is a highly virulent pathogen that is responsible for approximately 80% of all skin infections. During antibiotic treatment, one of the defense mechanisms of S. aureus is the invasion of keratinocytes. Intracellular bacteria are not accessible to antibiotics, which poorly penetrate the interior of host cells. Consequently, such bacteria contribute to recurrent infections. In our study, we proposed using a combination of a light-activated porphyrin compound loaded with gallium ions, Ga 3+ CHP, and visible light as a strategy to eliminate intracellular staphylococci. We demonstrated that the tested compound colocalized with the pathogen in the infected cells, which was an essential condition for the effective elimination of intracellular bacteria. We showed that the proposed approach effectively reduced the infection of keratinocytes with methicillin-resistant S. aureus (MRSA), as well as its adhesion to host cells, while maintaining host cells. The results presented here provide a basis for developing an effective therapy against staphylococci.