The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant Mycolicibacterium smegmatis Containing Endogenous Porphyrins
Abstract
:1. Introduction
2. Results
2.1. Influence of Metal Ions on the Accumulation of Porphyrins in Dormant Forms of M. smegmatis and Their Photosensitivity
2.2. Influence of Metals on the Representation of Proteins of Porphyrin Metabolism in Dormant M. smegmatis Cells
2.3. Primary Protein Targets for Photodynamic Inactivation of M. smegmatis
2.3.1. Enzymes of the Central Metabolism
2.3.2. Oxidoreductases
2.3.3. Various Metabolic Enzymes (Table 2)
2.3.4. Biopolymer Synthesis Enzymes
2.3.5. Regulatory Proteins (Table 3)
2.3.6. Enzymes of Defense Systems
- -
- Trigger factor/MSMEG_4674 (tig), which is involved in protein export. It acts as a chaperone by maintaining the newly synthesized protein in an open conformation.
- -
- MSMEG_3932/alpha-crystallin stress protein induced by anoxia. It has a proposed role in the maintenance of long-term viability during dormancy and latent infections.
- -
- Thioredoxin/MSMEG_4917 participates in various redox reactions through the reversible oxidation of its active center dithiol to a disulfide, and dithiol–disulfide exchange reactions. Copper/zinc superoxide dismutase/MSMEG_0835 (sodC), which destroys radicals which are normally produced within the cells and are toxic to biological systems. AhpC/TSA family protein/MSMEG_4753, which is a putative antioxidant protein.
- -
- Glyoxalase family protein/MSMEG_5680 which is important in the detoxification of methylglyoxal.
2.4. Enzymes Stable under Light Exposure
2.5. Influence of Photodynamic Inactivation on DCPIP Reduction by M. smegmatis Cells
3. Discussion
4. Materials and Methods
4.1. Formation of the Dormant Forms of M. smegmatis upon the Medium Self-Acidification
4.2. Viability Estimation by MPN
4.3. Infection of Macrophages with Dormant M. smegmatis Forms
4.4. Pigment Extraction from the Cells
4.5. Spectrophotometric and Fluorescence Analysis of Porphyrin in Extracts
4.6. Porphyrin Estimation by HPLC
4.7. Photodynamic Inactivation
4.8. Sample Preparation for SDS Electrophoresis
4.9. Protein Amount Determination
4.10. SDS Electrophoresis
4.11. Protein Identification by MALDI-TOF
4.12. Respiration, DCPIP Reduction
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Name | Product | Time of Illuminations | ||
---|---|---|---|---|
0 min | 5 min | 15 min | ||
MSMEG_0777 | F420-dependent glucose-6-phosphate dehydrogenase | + | − | − |
MSMEG_1030 | (NAD(P)/FAD-dependent oxidoreductase | + | − | − |
MSMEG_3106 | quinone oxidoreductase | + | + | − |
MSMEG_3232 | cytochrome D ubiquinol oxidase subunit II | + | + | + |
MSMEG_3233 | cytochrome D ubiquinol oxidase subunit I | + | − | − |
MSMEG_4645 | alpha oxoglutarate ferredoxin oxidoreductase, beta subunit | + | − | − |
MSMEG_4935 | epsilon subunit ATP synthase F1-F0 | + | + | + |
MSMEG_4936 | beta subunit ATP synthase F1-F0 | + | − | − |
MSMEG_4937 | gamma subunit ATP synthase F1-F0 | + | + | + |
Gene Name | Product | Time of Illuminations | ||
---|---|---|---|---|
0 min | 5 min | 15 min | ||
MSMEG_0029 | anthranilate synthase component 2 | + | − | − |
MSMEG_1342 | (3R)-hydroxyacyl-ACP dehydratase subunit HadC | + | − | − |
MSMEG_3564 | bacterioferritin | + | + | − |
MSMEG_3767 | acyl-CoA synthetase | + | − | − |
MSMEG_4340 | NAD/mycothiol-dependent formaldehyde dehydrogenase | + | − | − |
MSMEG_5136 | pyridoxine 5’-phosphate (PNP) oxidase | + | − | − |
MSMEG_5191 | siderophore binding protein | + | + | − |
MSMEG_5243 | pyridoxine 5’-phosphate (PNP) oxidase | + | − | − |
MSMEG_5471 | UTP--glucose-1-phosphate uridylyltransferase | + | − | − |
MSMEG_5485 | molybdopterin biosynthesis protein | + | + | − |
MSMEG_5702 | molybdenum cofactor synthesis domain protein | + | − | − |
MSMEG_5824 | phosphoribosylformylglycinamidine synthase II | + | − | − |
MSMEG_5892 | trehalose-6-phosphate synthase | + | − | − |
MSMEG_6392 | polyketide synthase | + | − | − |
MSMEG_6520 | orotate phosphoribosyltransferase | + | − | − |
Gene Name | Product | Time of Illuminations | ||
---|---|---|---|---|
0 min | 5 min | 15 min | ||
MSMEG_0937 | DNA-binding response regulator RegX3 | + | − | − |
MSMEG_0994 | regulators DNA-binding response regulator ResD | + | − | − |
MSMEG_3246 | sensor part of a two-component regulatory system | + | − | − |
MSMEG_3647 | glycogen accumulation regulator GarA | + | − | − |
MSMEG_5488 | the DNA-binding response regulator | + | + | − |
MSMEG_6091 | negative regulator of genetic competence ClpC/mecB | + | − | − |
MSMEG_6896 | the single-stranded DNA-binding protein | + | + | − |
Gene Name | Product | Time of Illuminations | ||
---|---|---|---|---|
0 min | 5 min | 15 min | ||
MSMEG_0835 | copper/zinc superoxide dismutase | + | − | − |
MSMEG_0880 | chaperonin GroEL | + | − | − |
MSMEG_3932 | alpha-crystallin stress protein | + | − | − |
MSMEG_4674 | trigger factor | + | − | − |
MSMEG_4753 | antioxidant protein | + | − | − |
MSMEG_4917 | thioredoxin | + | − | − |
MSMEG_5680 | glyoxalase family protein | + | − | − |
LED | Power Density (mW/cm2) | Time of Illuminations (min) and Light Doses (J/cm2) | |||
---|---|---|---|---|---|
565 nm | 180 | 5 min | 15 min | 30 min | 60 min |
54 J/cm2 | 162 J/cm2 | 324 J/cm2 | 648 J/cm2 |
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Shashin, D.M.; Demina, G.R.; Linge, I.A.; Vostroknutova, G.N.; Kaprelyants, A.S.; Savitsky, A.P.; Shleeva, M.O. The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant Mycolicibacterium smegmatis Containing Endogenous Porphyrins. Int. J. Mol. Sci. 2023, 24, 13968. https://doi.org/10.3390/ijms241813968
Shashin DM, Demina GR, Linge IA, Vostroknutova GN, Kaprelyants AS, Savitsky AP, Shleeva MO. The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant Mycolicibacterium smegmatis Containing Endogenous Porphyrins. International Journal of Molecular Sciences. 2023; 24(18):13968. https://doi.org/10.3390/ijms241813968
Chicago/Turabian StyleShashin, Denis M., Galina R. Demina, Irina A. Linge, Galina N. Vostroknutova, Arseny S. Kaprelyants, Alexander P. Savitsky, and Margarita O. Shleeva. 2023. "The Effect of Antimicrobial Photodynamic Inactivation on the Protein Profile of Dormant Mycolicibacterium smegmatis Containing Endogenous Porphyrins" International Journal of Molecular Sciences 24, no. 18: 13968. https://doi.org/10.3390/ijms241813968