Uptake of anionic and cationic porphyrins into Escherichia coli

NATALIA S. GSPONER; MARIANA B. SPESIA; EDGARDO N. DURANTINI

Córdoba

Congreso; 16th International Congress on Photobiology; 2014

International Union of Photobiology

The growing resistance against antibiotics and others chemotherapeutics has led to find novel antimicrobial treatments. In the last years, photodynamic inactivation (PDI) of microorganisms represents a potential alternative to inactive microbial. However, the high net negative charge on lipopolysaccharides molecules in Gram-negative bacteria provides a polyanionic external surface that is partly neutralized by divalent cations Mg2+ and Ca2+. Consequently, the outer membrane acts as a effective permeability barrier, which is relatively impermeable to neutral or anionic drugs. However, photosensitization of Gram-negative bacteria can be increased by the addition of biological or chemical molecules, which modify the native consistence of the outer membrane. These changes enhance its permeability facilitating the penetration of photosensitizers to the cytoplasmic membrane. In this study we examined the effect of divalent cations (Ca2+ and Mg2+) and a permeabilizing agent (EDTA) on the uptake of an anionic (TPPS4-) and two cationic (MPAP2+ and TMAP4+) porphyrins by Escherichia coli. Thus, cell suspensions (~108 cell/mL) were incubated with 5 uM porphyrin for 10 min in dark at 37°C and irradiated with visible light. Under these conditions, TMAP4+ was the more effective photosensitizer, producing a 7 log decrease of cell survival. In presence of 50 mM Ca2+, the photoinactivation mediated by cationic porphyrins diminished, while the cytotoxic effect increased with TPPS4-. The tendency was similar using 50 mM Mg2+, except for MPAP2+ that caused a higher inactivation. On the other hand, EDTA was used to cause cell wall damage in E. coli by chelating the divalent cations. The addition of 50 mM EDTA produced an enhancement in the photoinactivation mediated by the three porphyrins. This behavior was accompanied by an increase in the amount of photosensitizer bound to E. coli cells. Studies are in progress to evaluate the dependence of the uptake with the temperature and changes in the outer and inner membranes permeabilization. These results show that divalent cations play an important role in the uptake of porphyrins by E. coli cells. The removal of Mg2+ and Ca2+ that bind together the negatively-charged cell wall molecules produced bacterial cell membrane permeable to photosensitizer. The presence of EDTA enhanced PDI of E. coli by facilitating the penetration of cationic and anionic porphyrins through the outer membrane.