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

Abstract

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.

Keywords: Mycobacterium smegmatis; dormant mycobacteria; photodynamic inactivation; porphyrin.

Figure 1

Survival of dormant M. smegmatis cells in macrophages under illumination. Dormant Msm cells obtained under standard (D_st) and elevated (D_Me) concentrations of Mg²⁺ and Zn²⁺ were added to peritoneal macrophages and incubated for 16 h. After washing macrophages with captured Msm, they were illuminated with LED 565 nm for 30 min at a power density of 180 mW. After illumination, macrophages were lysed and the concentration of viable bacteria was estimated with an MPN assay. This experiment was repeated twice. Bars represent SD.

Figure 2

Amount of porphyrins extracted from dormant M. smegmatis cells. Dormant Msm cells obtained under standard (D_st) and elevated (D_Me) concentrations of Mg²⁺ and Zn²⁺ were extracted with chloroform–methanol and 2% triton X-100X100. Porphyrin concentration in supernatant (A), chloroform–methanol (B) and 2% triton X-100 (C). Asterisks indicate that the results are significantly different from the control (D_st) according to Student’s t-test. Student’s test assuming unequal variance was performed for estimation of significance for comparative data. P-values are indicated as follows: * = p < 0.05, *** = p < 0.001.

Figure 3

Absorption and fluorescence spectra of chloroform–methanol extract of dormant M. smegmatis cells. Dormant Msm cells were obtained under standard (D_st) and elevated (D_Me) concentrations of Mg²⁺ and Zn²⁺ as described in the Methods. Absorption spectra of extracts (A) and acidificated extract (B). Fluorescent spectra (excitation 400 nm) of extract from D_st cells (C) and D_Me cells (D). The arrow indicates a shoulder appeared in the Soret band.

Figure 4

HPLC analysis chloroform–methanol extract of dormant M. smegmatis cells. Dormant Msm cells were obtained under (A) standard (D_st) and (B) elevated (D_Me) concentrations of Mg²⁺ and Zn²⁺ as described in the Methods. A C18 Tosoh Bioscience column was used; elution rate 1 mL min⁻¹ and injection sample size 20 μL, gradient mode and gradient program as described in the Methods. The spectrofluorometer was set at 400 nm for absorption, fluorescence excitation wavelength was 400 nm and fluorescence emissions were detected at wavelengths of 580 and 620 nm. The numbers point to peaks observed at 400 nm.

Figure 5

SDS electrophoresis of proteins obtained from dormant M. smegmatis cells before and after illumination with LED 565 nm. Dormant Msm cells obtained under standard (D_st) and an elevated (D_Me) concentration of Mg²⁺ and Zn²⁺ were subjected to LED 565 nm illumination for 5- and 15-min. Total cell proteins were extracted by 2% SDS and used for SDS gel electrophoresis followed by Coomassie staining. Concentration of proteins for each sample was identical (70 mg). 0′—corresponds to unilluminated samples. M—protein standards. Diagrams below represent mean density of protein bands between 130 kDa and 10 kDa for each protein track. Mean density—mean grey value of pixels extracted from monochrome image of electrophoresis gel with background correction.

Figure 6

The number of proteins of dormant forms of M. smegmatis grown under standard conditions (D_st) and at an elevated concentration of metal ions (D_Me) which are stable to PDI for 5 min and 15 min of illumination. The number of stable proteins was calculated as a percentage of total number of protein visible in SDS gel (Figure 4). The 100% represents protein numbers in unilluminated samples.

Figure 7

The stability of proteins in different functional categories of dormant forms of M. smegmatis under illumination for 5 min and 15 min. The number of stable proteins was calculated as a percentage of total number of visible proteins in SDS gel for every functional category (Figure 4). Protein number reached 100% in the unilluminated samples. The bacterial cells were grown under standard conditions.

Figure 8

Influence of illumination with 565 nm on enzymes of the glycolytic pathway, citrate cycle and the glyoxylate shunt of dormant M. smegmatis.

Figure 9

Influence of illumination with 565 nm on ribosomal proteins of dormant M. smegmatis. The scheme of ribosome was taken from KEGG.

Figure 10

Influence of illumination with 565 nm on the respiratory chain activity of M. smegmatis. The 72-h-grown Msm culture in the presence 3 mM ALA was subject to illumination under 565 nm for 15 min. (A) The rate of total oxygen consumption and (B) the rate of reduction of the redox indicator 2,6-dichlorophenolindophenol (DCPIP). 1—control, 2—after illumination.