Antimicrobial effects of Photodynamic Therapy to gram negative bacteria envelope revealed by Cryo-electron tomography

Abstract

Cryo-electron tomography (cryo-ET) is an emerging technology that enables thin samples, including small intact prokaryotic cells, to be imaged in three dimensions in a near-native 'frozenhydrated' state to a resolution sufficient to recognize very large macromolecular complexes in situ. This methodology has been fundamental to provide information about cellular ultrastructure. This study used cryo-ET to evaluate the photodynamic effect on the viability and envelope architecture of a Gram-negative bacteria. Bacterial suspension of E. coli minicells were submitted to photodynamic treatment with methylene blue solution (100μM) and a 100mW low power diode laser emitting at 660nm with 6 and 18J of energy. As a control group, a suspension of minicells were submitted to 462 IU/mL penicillin G for 60 min at 30 °C, to compare the damage in cell wall structure. After treatment bacteria were immediately plunge-frozen across EM grids and standard cryo-ET tilt series were collected, 3D images reconstructions were calculated and recorded. The imagens showed detachment of the bacterial cell walls and mesosome-like structures. In addition, some sites showed interrupted stretches in both inner and outer membranes and cell wall degradation, indicating bacterial envelope damage. Cryo-electron tomography revealed that the effects of photodynamic therapy on Gram negative bacteria was based on damage to the outer membrane, cell wall and inner membrane and occurs in an energy-dependent manner.