The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant Mycolicibacterium smegmatis Containing Endogenous Porphyrins

During transition into a dormant state, Mycolicibacterium (Mycobacterium) smegmatis cells are able to accumulate free porphyrins that makes them sensitive to photodynamic inactivation (PDI). The formation of dormant cells in a liquid medium with an increased concentration of magnesium (up to 25 mM) and zinc (up to 62 µM) resulted in an increase in the total amount of endogenous porphyrins in dormant M. smegmatis cells and their photosensitivity, especially for bacteria phagocytosed by macrophages. To gain insight into possible targets for PDI in bacterial dormant mycobacterial cells, a proteomic profiling with SDS gel electrophoresis and mass spectrometry analysis were conducted. Illumination of dormant forms of M. smegmatis resulted in the disappearance of proteins in the separating SDS gel. Dormant cells obtained under an elevated concentration of metal ions were more sensitive to PDI. Differential analysis of proteins with their identification with MALDI-TOF revealed that 45.2% and 63.9% of individual proteins disappeared from the separating gel after illumination for 5 and 15 min, respectively. Light-sensitive proteins include enzymes belonging to the glycolytic pathway, TCA cycle, pentose phosphate pathway, oxidative phosphorylation and energy production. Several proteins involved in protecting against oxygen stress and protein aggregation were found to be sensitive to light. This makes dormant cells highly vulnerable to harmful factors during a long stay in a non-replicative state. PDI caused inhibition of the respiratory chain activity and destroyed enzymes involved in the synthesis of proteins and nucleic acids, the processes which are necessary for dormant cell reactivation and their transition to multiplying bacteria. Because of such multiple targeting, PDI action via endogenous porphyrins could be considered as an effective approach for killing dormant bacteria and a perspective to inactivate dormant mycobacteria and combat the latent form of mycobacteriosis, first of all, with surface localization.