Harnessing light-activated gallium porphyrins to combat intracellular Staphylococcus aureus using an in vitro keratinocyte infection model

Abstract

Staphylococcus aureus (S. aureus) can survive inside nonprofessional phagocytes such as keratinocytes, enabling it to evade antibiotics and cause recurrent infections once treatment stops. New antibacterial strategies to eliminate intracellular, multidrug-resistant bacteria are needed. This study used a keratinocyte model infected with methicillin-resistant S. aureus (MRSA) to test light-activated compounds, specifically heme-mimetic gallium (III) porphyrin (Ga³⁺CHP) and visible light, known as antimicrobial photodynamic inactivation (aPDI), for eliminating intracellular MRSA. Ga³⁺CHP was found to accumulate more in infected cells, particularly within lysosomal structures where MRSA resides. Flow cytometry and fluorescence microscopy revealed significant colocalization of MRSA and Ga³⁺CHP. Under aPDI, MRSA showed reduced adhesion to host cells and a 70% reduction in the GFP signal from intracellular bacteria. Additionally, light-activated Ga³⁺CHP significantly decreased the number of extracellular bacteria, reducing the potential for further infection. This study is the first to analyze aPDI toxicity in real time within an infection model, demonstrating that this method is neither cytotoxic nor phototoxic.

Fig. 1

Strategies to implement light-activated gallium metalloporphyrins to overcome S. aureus infection of human keratinocytes.

Fig. 2

Effect of aPDI (light-activated Ga³⁺CHP) pretreatment on S. aureus USA300 infection – Strategy 1. (A) On day 0, HaCaT cells were seeded in a 24-well plate. On day 1, the cells were infected with S. aureus USA300 that was either untreated or pretreated with aPDI. After 2 h, the growth medium was collected for plating and the extracellular fraction was counted. Host cells were collected for subsequent lysis (intra + adherent fraction) or incubated for 1 h with antibiotics to eliminate adherent bacteria to obtain the intracellular fraction exclusively. All the fractions were serially diluted and plated for CFU/mL enumeration. (B) The number of bacteria counted from each fraction collected after infection with an untreated (‘MOI 10’, ‘MOI 1’) or aPDI-treated S. aureus inoculum (‘Low’, ‘High’). Before infection, the number of viable S. aureus (10⁷ CFU/mL) was reduced by two doses of aPDI: a low dose that resulted in a 1 log₁₀ reduction in CFU/mL and high dose that resulted in a 2 log₁₀ reduction in CFU/mL. Untreated bacteria with an appropriate MOI of 10 or 1 were used as a control. The number of bacteria used for infection was the same whether the bacteria were pretreated with aPDI or were not pretreated (please see ‘Inoculum’ below). After infection, several fractions were collected, such as ‘Extracellular’ – the free-floating S. aureus collected from the medium after infection; ‘Adherent’ - S. aureus attached to the host cell; ‘Intracellular’ - S. aureus accumulated inside the host cell in the presence of antibiotic pressure; ‘Intra + Adherent’ – the combined number of intracellular and adherent S. aureus; and ‘Inoculum’ – the initial number of treated (Low or High) or untreated (MOI 10 or MOI 1) S. aureus used for infection. The data are presented as the means ± SDs of six separate experiments. The significant differences in infected cell viability at the respective p values are indicated with asterisks [**p < 0.01; ***p < 0.001], and were normalized to the respective control for each pretreatment (two-way Anova).

Fig. 3

Different accumulation patterns of two gallium metalloporphyrins in human keratinocytes. (A) Confocal fluorescence microscopy images showing the accumulation of two gallium compounds, Ga³⁺MPIX and Ga³⁺CHP, in HaCaT cells after incubation in the dark (1–24 h) at a concentration of 10 µM. (B) Percentages of cells that accumulated Ga³⁺MPs among the total number of cells measured by flow cytometry. The cells were incubated with 10 µM Ga³⁺MPIX or Ga³⁺CHP and fixed at each time point (1–24 h) in the absence of light, after which the fluorescence signal in the cells was measured by flow cytometry. (C) The number of accumulated Ga³⁺MPIX or Ga³⁺CHP molecules (10 µM) in keratinocytes after incubation for the time indicated on the X axis, as measured by the fluorescence intensity of the cell lysate. After incubation in the dark for a particular time, the cells were harvested, counted, and lysed with 0.1 M NaOH/1% SDS to determine the fluorescence of each accumulated compound. (D, E) Real-time growth analysis of HaCaT cells after incubation in the dark (1–24 h) with 10 µM Ga³⁺MPIX (D) or Ga³⁺CHP (E).

Fig. 4

The accumulation of Ga³⁺CHP in infected cells. (A) Accumulation of Ga³⁺CHP in infected and uninfected cells. The cells were infected with S. aureus USA300 (MOI 10) for 2 h in medium without antibiotics, and then the cells were washed and covered with medium supplemented with antibiotics. Uninfected cells were cultured in parallel. The next day, 10 µM Ga³⁺CHP was added and the cells were incubated in the dark. After incubation, the cells were washed and collected, and the percentage of cells with a fluorescence signal derived from Ga³⁺CHP was determined by flow cytometry. The accumulation results were compared with those of uninfected cells. (B) The number of infected cells harboring both GFP + and Ga³⁺CHP + signals after incubation in the dark with Ga³⁺CHP. The significant differences between the two compounds were calculated, and the respective p values are marked with asterisks [***p < 0.001] (one-way Anova).

Fig. 5

Colocalization of intracellular S. aureus and Ga³⁺CHP in lysosomes.

Fig. 6

Light-activated Ga³⁺CHP impacts the number of GFP-expressing cells without causing severe host damage. (A) Scheme of the experiment. On day 0, HaCaT cells were seeded in a 24-well plate. On day 1, S. aureus USA300 (MOI 10) was added, and the cells were incubated for 2 hours. Next, the medium was replaced with antibiotic medium (Antibiotic OFF-> ON) to eliminate extracellular bacteria and maintain intracellular invasion. On day 2, Ga³⁺CHP was added to the medium and incubated for 2–6 h in the dark. Then, the cells were washed, and green light was applied at the proper dose. (B) Percentage of infected cells after aPDI. The number of GFP-expressing cells after 2- or 6-hour incubation in the dark followed by green light illumination. The cells were collected and fixed, and the GFP signal was measured by flow cytometry. The results were calculated in reference to the untreated control (cells with no compound and no light exposure) (two-way Anova). (C) Table showing the growth characteristics of infected or uninfected cells subjected to different treatments. The following parameters were accounted for in the analysis: the cell index (CI) immediately after aPDI treatment (at the 55th hour of the experiment); the ΔCI which is characterized by the growth rate immediately after aPDI treatment, was calculated as the difference in the growth of cells after aPDI treatment (from 55 h of the experiment) and cells in the middle of logarithmic growth (at ~ 100 h of the experiment); the CI_max- maximum CI achieved at the beginning of the plateau phase; the time to plateau phase - time at which cells enter stationary growth phase; and the growth rate, which was calculated as the total rate of increase in the logarithmic phase of growth after treatment to the time at which the plateau phase of the analysis curve was reached. (D, E) Real-time growth analysis of HaCaT cells that were not infected (D) or infected (E) after incubation in the dark for 2 hours with 10 µM Ga³⁺CHP and with or without light illumination of 6.36 J/cm². The CI was measured by a real-time cell analysis (RTCA) instrument every 10 min. The experiments were conducted until the cells reached the plateau growth phase.

Fig. 7

Light-activated Ga³⁺MPs effectively reduce the number of S. aureus USA300 released from keratinocytes – Strategy 1. (A) On day 1, HaCaT cells were infected with S. aureus USA300 (MOI = 10, 2 h) in medium without antibiotics (Antibiotic OFF). An antibiotic was added to remove extracellular S. aureus, and the cells were cultured under antibiotic pressure until the next day (Antibiotic OFF-> ON). The antibiotic was then removed (Antibiotic ON-> OFF), and HaCaT cells containing only intracellular S. aureus were left in an incubator for 16 h. During this time, intracellular S. aureus gradually lysed the cells and was released into the medium. S. aureus cells were harvested, washed with DMEM and then resuspended in tryptic soy broth (TSB). The bacterial suspensions were transferred to a 24-well plate, proper photosensitizers were added, and after incubation, they were illuminated with green light. The bacterial cells were then diluted, plated, and counted (B and C). The reduction in the number of S. aureus USA300 bacteria caused by light activation of Ga³⁺CHP (B) or Ga³⁺MPIX (C) was calculated in relation to that in the untreated cells.