Influence of Cationic meso-Substituted Porphyrins on the Antimicrobial Photodynamic Efficacy and Cell Membrane Interaction in Escherichia coli

Abstract

Photodynamic inactivation (PDI) is a non-antibiotic option for the treatment of infectious diseases. Although Gram-positive bacteria have been shown to be highly susceptible to PDI, the inactivation of Gram-negative bacteria has been more challenging due to the impermeability properties of the outer membrane. In the present study, a series of photosensitizers which contain one to four positive charges (1⁻4) were used to evaluate the charge influence on the PDI of a Gram-negative bacteria, Escherichia coli (E. coli), and their interaction with the cell membrane. The dose-response PDI results confirm the relevance of the number of positive charges on the porphyrin molecule in the PDI of E. coli. The difference between the Hill coefficients of cationic porphyrins with 1⁻3 positive charges and the tetra-cationic porphyrin (4) revealed potential variations in their mechanism of inactivation. Fluorescent live-cell microscopy studies showed that cationic porphyrins with 1⁻3 positive charges bind to the cell membrane of E. coli, but are not internalized. On the contrary, the tetra-cationic porphyrin (4) permeates through the membrane of the cells. The contrast in the interaction of cationic porphyrins with E. coli confirmed that they followed different mechanisms of inactivation. This work helps to have a better understanding of the structure-activity relationship in the efficiency of the PDI process of cationic porphyrins against Gram-negative bacteria.

Keywords: E. coli; cationic porphyrin; gram-negative bacteria; photodynamic inactivation.

Figure 1

Chemical structure of the porphyrins used in this work. Four cationic porphyrin derivatives were synthesized: 1–4 with one to four positive charges, respectively. Compound 2 is a mixture of the cis and trans isomer. 5 and 6 are commercially available and used as control compounds.

Figure 2

(A) Normalized absorption and (B) emission spectra for 10 µM solutions of 1 (blue), 2 (red), 3 (black) and 4 (orange) in DMSO. The four Q absorption bands are shown in the inset.

Figure 3

Time-dependent plots for the decay of DMA photosensitized by 1 (blue), 2 (red), 3 (black), 4 (orange), 5 (purple) and TPP (brown) in DMF at irradiation wavelength of 515 nm. Values include the mean and standard deviation of three independent experiments.

Figure 4

Bacterial toxicity with 1 (blue), 2 (red), 3 (black), 4 (orange), 5 (purple) and 6 (green) against E. coli in the absence of light (top) and after light exposure for 20 min (bottom). E. coli was incubated with cationic porphyrin derivatives for 30 min. Minimal dark toxicity was observed at concentrations below 1 µM. Values represent the average of three independent experiments. Error bars represent one standard of deviation. Statistical analysis was performed by one-way ANOVA (**** p < 0.0001).

Figure 5

Micrographs depicting the interaction of cationic porphyrins with E. coli. 3 (top row) and 4 (bottom row). Left: Differential interference contrast image, middle: fluorescence image (medial z slice), and right: merge image. [PS] = 1 µM; Incubation time: 30 min.

Figure 6

Micrographs of cationic porphyrin 4 with E. coli at various incubation times. (A) 5, (B) 10, (C) 15, and (D) 20 min. Left: Differential interference contrast image, middle: fluorescence image (medial z slice), and right: merge image. [PS] = 1 µM.

Figure 7

Trend of cellular interaction between cationic porphyrins 3 (black) and 4 (orange) and E. coli cells as a function of increasing concentration of Mg⁺² ions, source MgCl₂ (0, 10, 25, 50 mM). The percentage of PS attached was calculated as a percentage of the original PS concentration (1 µM). Values represent the average of three independent experiments. Error bars represent one standard of deviation. Statistical analysis was performed by one-way ANOVA (* p < 0.05).