Abstract
Antimicrobial resistance in Neisseria gonorrhoeae is a major issue of public health, and there is a critical need for the development of new antigonococcal strategies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL; wavelength, 405 nm), an innovative nonpharmacological approach, for the inactivation of N. gonorrhoeae. Our findings indicated that aBL preferentially inactivated N. gonorrhoeae, including antibiotic-resistant strains, over human vaginal epithelial cells in vitro. Furthermore, no aBL-induced genotoxicity to the vaginal epithelial cells was observed at the radiant exposure used to inactivate N. gonorrhoeae. aBL also effectively inactivated N. gonorrhoeae that had attached to and invaded into the vaginal epithelial cells in their cocultures. No gonococcal resistance to aBL developed after 15 successive cycles of inactivation induced by subtherapeutic exposure to aBL. Endogenous aBL-activatable photosensitizing porphyrins in N. gonorrhoeae were identified and quantified using ultraperformance liquid chromatography, with coproporphyrin being the most abundant species in all N. gonorrhoeae strains studied. Singlet oxygen was involved in aBL inactivation of N. gonorrhoeae. Together, these findings show that aBL represents a potential potent treatment for antibiotic-resistant gonococcal infection.
Neisseria gonorrhoeae is a human-specific bacterial pathogen and the cause of the sexually transmitted disease gonorrhea [1, 2]. If not appropriately treated, gonorrhea can result in severe sequelae, such as pelvic inflammatory disease, infertility, and ectopic pregnancy [3]. Furthermore, gonorrhea facilitates the transmission and acquisition of human immunodeficiency virus [4, 5]. In the absence of a gonococcal vaccine, public health control of gonorrhea relies primarily on the availability of effective antibiotic treatment [3]. However, N. gonorrhoeae has become resistant to each of the first-line antibiotics that have been used to treat it [6, 7]. The antibiotic combination of azithromycin and ceftriaxone is the current Centers for Disease Control and Prevention (CDC) recommendation for treating gonorrhea [8]. It is worrying that N. gonorrhoeae strains with decreased susceptibility or resistance to ceftriaxone and concomitant azithromycin resistance are already circulating globally [9, 10]. The CDC has flagged multidrug-resistant N. gonorrhoeae as an urgent threat to human health [11], the highest level of concern.
As the use of antibiotics drives the emergence and spread of multidrug-resistant N. gonorrhoeae [3, 12, 13], there is a critical need for the development of new treatment strategies [14, 15]. Antimicrobial blue light (aBL; wavelength, 405 nm), an innovative nonpharmacological therapy, has attracted increasing attention because of its intrinsic antimicrobial properties without the involvement of exogenous photosensitizers [16, 17]. Although the mechanism of action of aBL is still not fully understood, a common hypothesis is that aBL excites naturally occurring endogenous photosensitizers (eg, iron-free porphyrins) within bacterial cells and subsequently leads to the production of cytotoxic oxidative species [16–18].
In contrast to antibiotics, aBL is an innovative approach that is based on the physical properties of light and bacteria. The advantages of aBL over antibiotics include rapid action and equal inactivation effectiveness independent of antibiotic resistance status [16, 17]. It is also currently thought that bacteria are less able to develop resistance to aBL than to traditional antibiotics because of the multiple-target characteristic of aBL [16, 17, 19]. In comparison to traditional antimicrobial photodynamic therapy (aPDT), aBL is appealing in that it inactivates bacteria (ie, it renders bacteria unable to multiply) without the involvement of exogenous photosensitizers [20–24]. In addition, it is well accepted that aBL is much less detrimental to the host cells than germicidal UV irradiation [25, 26].
Although aBL has been reported to be effective in inactivating a range of nosocomial pathogens [20, 27–29], aBL has never been studied as a treatment for gonorrhea [30]. The superficial nature of gonococcal infection (<0.5 mm in depth [31–33]) renders aBL an amendable candidate treatment for this infection (the aBL penetration depth in human tissue is 0.5–1.0 mm [34, 35]). To be potentially transferable to the clinic, aBL should preferentially inactivate N. gonorrhoeae while sparing normal host cells. In this study, we investigated the effectiveness of aBL inactivation of N. gonorrhoeae, the toxicity of aBL to human epithelial cells, and the potential development of gonococcal resistance to aBL. In addition, we identified and quantified aBL-activatable endogenous photosensitizing chromophores in N. gonorrhoeae.
MATERIALS AND METHODS
Blue Light Sources
A light-emitting diode with peak emissions at 405 nm and a full width at half maximum of 20 nm (M405L2; Thorlabs, Newton, NJ) was used for irradiation. The wavelength of 405 nm is the optimal wavelength targeting porphyrins [46]. The irradiance of 60 mW/cm2, which was optimized on the basis of the log10 bacterial colony-forming units inactivated per unit radiant exposure of aBL (in J/cm2) in a preliminary study, was used throughout the study. Light irradiance was measured using a PM100D power/energy meter (Thorlabs). The radiant exposure of light (in J/cm2) was calculated as the irradiance (in W/cm2) multiplied by the irradiation time of light (in seconds).
N. gonorrhoeae Strains and Culture Conditions
ATCC 700825 (FA 1090) and 4 clinical N. gonorrhoeae isolates were studied. The clinical isolates were obtained through the CDC and Food and Drug Administration Antibiotic Resistance Isolate Bank. Supplementary Materials 2 shows the antibiotic susceptibilities of the 4 N. gonorrhoeae clinical isolates. Bacteria were routinely grown on gonococcal medium base GC (Remel, Lenexa, KS) agar plates containing GCHI enrichment (Remel) and hemoglobin at 37°C and 5% CO2.
Human Vaginal Epithelial Cells and Growth Conditions
Human vaginal epithelial cells VK2/E6E7 (ATCC CRL-2616) were used. Cells were cultured in keratinocyte serum-free medium (Gibco, Grand Island, NY) supplemented with 5 ng/mL recombinant epidermal growth factor, 50 μg/mL bovine pituitary extract (Invitrogen, Grand Island, NY), and 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island) at 37°C and 5% CO2. All experiments were performed in the exponential growth phase of VK2/E6E7 cells (48 hours after plating).
aBL Inactivation of N. gonorrhoeae in Suspensions
Overnight N. gonorrhoeae cultures were collected from GC agar plates, washed using phosphate-buffered saline (PBS), and then resuspended in PBS to an OD600nm of 0.3 (approximately 108 colony-forming units [CFU]/mL). A 3-mL bacterial suspension was added to a 35-mm-diameter Petri dish prior to aBL exposure. After varying aBL exposures (9, 18, 27, 36, 45, 54, 72, 90, and 108 J/cm2) had been delivered, 30-µL aliquots of the bacterial suspension were collected, and the number of N. gonorrhoeae CFU was measured using a colony-forming assay. The experiment was performed in 3 independent replicates for each condition. In addition, bacterial suspensions without exposure to aBL served as negative controls.
Transmission Electron Microscopy (TEM) of aBL-Induced Morphological Changes to N. gonorrhoeae Cells
To examine aBL-induced morphological changes of N. gonorrhoeae cells, untreated and aBL-treated ATCC 700825 N. gonorrhoeae cells were fixed in 1% paraformaldehyde and 1.25% glutaraldehyde immediately after aBL exposures (9 J/cm2 [2.5-minute illumination], 18 J/cm2 [5-minute illumination], and 27 J/cm2 [7.5-minute illumination]) and stored at 4°C for 2 hours. The N. gonorrhoeae cells were then washed 3 times with 0.1 M sodium cacodylate buffer after centrifugation (at 13 500 × g for 10 minutes) and decanting the fixative. The cell pellets were subsequently processed for TEM.
Evaluation of aBL-Induced Toxicity to Human Vaginal Epithelial Cells
To test the toxicity of aBL to normal vaginal epithelial cells, VK2/E6E7 cells were subjected to aBL exposures of 54, 108, and 162 J/cm2. The viability of VK2/E6E7 cells was measured using the MTT assay 24 hours after aBL exposure. The experiment was performed in 6 independent replicates for each condition.
In addition, aBL-induced DNA damage in VK2/E6E7 cells was evaluated using a CometAssay kit (Trevigen, Gaithersburg, MD). The experiments were performed on the basis of the Trevigen standard protocol for single-cell gel electrophoresis. Briefly, aBL-treated and untreated VK2/E6E7 cells were detached using trypsin. The cell pellets were suspended at 37°C with molten LMAgarose after centrifugation and then loaded onto a precoated CometSlide slide (50 µL/well), which was immediately cooled on ice. The embedded cells were lysed in cold Trevigen lysis solution overnight at 4°C and then electrophoresed in cold alkaline buffer (pH 13.3). DNA strands/fragments stained with SYBR Gold were visualized under an Olympus FV-1000 confocal microscope at 496/522 nm.
Ultraperformance Liquid Chromatography (UPLC) Analysis of Endogenous aBL-Activatable Photosensitizers in N. gonorrhoeae and Human Vaginal Epithelial Cells
UPLC was used to indentify and quantify the endogenous aBL-activatable photosensitizers (porphyrins) in N. gonorrhoeae and VK2/E6E7 cells. To extract the endogenous porphyrins from the cells, overnight cultures were washed using PBS. The cell pellets were collected after centrifugation (at 13 500 ×g for 6 minutes), resuspended in 1.0 mL of extraction solvent (ratio of ethanol to dimethyl sulfoxide to acetic acid, 80:20:1 [vol/vol/vol]), and then stored at −80°C for 24 hours. The cell walls were then disrupted by sonication for 20 minutes. After centrifugation (at 13 500 ×g for 6 minutes), the supernatant was collected as the whole-cell lysates. The protein content in each sample was measured using the bicinchoninic acid protein assay.
Standard porphyrins (chromatographic marker kit [product no. CMK-1A; Frontier Scientific, Logan, UT] and protoporphyrin IX [PpIX; Sigma Aldrich]) were used as the reference compounds. The chromatographic marker kit was composed of uroporphyrin I, 7-carboxylporphyrin I, 6-carboxylporphyrin I, 5-carboxylporphyrin I, coproporphyrin, mesoporphyrin IX, and mesoporphyrin IX dihydrochloride.
Identification and quantitation of porphyrins in N. gonorrhoeae and VK2/E6E7 cells were performed using a Waters Acquity UPLC system. The system is composed of a binary solvent manager, sample manager, fluorescence detector, and column heater and an Acquity UPLC BEH C18 column (particle size, 1.7 µM; inner diameter, 2.1 mm; length, 100 mm). For detecting porphyrins, the excitation was set at a 404-nm wavelength and an emission at 618 nm.
aBL Inactivation of N. gonorrhoeae in the Presence of the Singlet Oxygen (1O2) Quencher NaN3
To determine whether 1O2 was involved in aBL inactivation of N. gonorrhoeae, sodium azide (NaN3, 10 mM, Sigma Aldrich), a specific quencher of 1O2 [47] was added to a 3-mL suspension of ATCC 700825 before exposure to aBL. Another dish with a 3-mL suspension of ATCC 700825 without NaN3 served as the negative control. Both dishes were irradiated with aBL, and samples were collected using the same procedure as described above. The experiment was performed in 3 independent replicates for each condition.
Assessment of Potential Development of Gonococcal Resistance to aBL
To assess whether N. gonorrhoeae has the potential to develop resistance to aBL, suspensions of ATCC 700825 in PBS were subjected to 15 repeated cycles of subtherapeutic aBL exposures. In the first cycle, the aBL exposure was adjusted to inactivate approximately 4.0-log10N. gonorrhoeae CFU, and the same aBL exposure was then used throughout the successive cycles. In each cycle, the surviving bacterial cells after aBL exposure were collected, subcultured, and grown overnight for the next cycle of inactivation by aBL.
Infection of Human Vaginal Epithelial Cells by N. gonorrhoeae
N. gonorrhoeae has evolved the mechanism to invade into human vaginal epithelial cells, to overcome the host defense barrier [48]. To evaluate the effectiveness of aBL inactivation of intracellular N. gonorrhoeae, VK2/E6E7 cells were seeded into a 35-mm-diameter Petri dish (Transwell, Costar, NY) at a cell density of 2 × 105 cells/dish. The VK2/E6E7 cells were incubated in 2 mL of keratinocyte serum-free medium for 48 hours at 37°C. The supernatant was then discarded, and a 1-mL suspension of ATCC 700825 in PBS was added. A multiplicity of infection (MOI) of 50 bacteria per VK2/E6E7 cell was used according to the optimized MOI from a previous study [49]. The cocultures of VK2/E6E7 cells and N. gonorrhoeae were incubated at 37°C with 5% CO2 for 4 hours. The invasion of N. gonorrhoeae cells into VK2/E6E7 cells was examined by confocal microscopy (Olympus, FV 1000-MPE Confocal) [50].
aBL Inactivation of Intracellular N. gonorrhoeae
Four identical cultures (A, B, C, and D) of VK2/E6E7 cells infected with N. gonorrhoeae were prepared for each experiment. Culture A was treated with aBL followed by incubation with 0.05% saponin for 20 minutes to lyse the VK2/E6E7 cells and liberate the bacteria. The viability of N. gonorrhoeae was then determined using a colony-formation assay. Culture B was treated with the same aBL exposures, and the viability of VK2/E6E7 cells was assessed using the MTT assay 24 hours after aBL exposure. Cultures C and D were not exposed to aBL. The viability of N. gonorrhoeae in culture C was measured using a colony-formation assay, and the viability of VK2/E6E7 cells in culture D was measured using the MTT assay. aBL exposures of 54 and 108 J/cm2 were tested. The experiment was performed in 6 independent replicates for each condition.
Statistical Analysis
Data are presented as means ± standard errors. The differences between different conditions were analyzed using 1-way analysis of variance. P values of <.05 were considered statistically significant.
RESULTS
Susceptibilities of N. gonorrhoeae in Planktonic Suspensions to Inactivation by aBL
To achieve a reduction of 3-log10N. gonorrhoeae CFU in suspensions, aBL exposures from 25 J/cm2 (for ATCC 700825) to 59 J/cm2 (for strain 199) were used (Figure 1A). To eradicate all bacterial CFU in suspensions (>6-log10 CFU reduction), aBL exposures from 45 J/cm2 (for ATCC 700825) to 108 J/cm2 (for strain 199) were used (Figure 1A).
Antimicrobial blue light (aBL) inactivation of Neisseria gonorrhoeae. A, Susceptibilities of N. gonorrhoeae to aBL inactivation. ATCC 700825 and clinical isolates 174, 179, 181, and 199 were studied. Bars, standard errors. B, Transmission electron microscopy images of N. gonorrhoeae (ATCC700825) untreated or exposed to 9 J/cm2, 18 J/cm2, or 27 J/cm2 aBL (wavelength, 405 nm). The scale bar represents 100 nm. Black arrows, bulges formation; blue arrows, breakage of cell wall accompanied by cytoplasmic release; white arrows, loss of outer membrane; blue stars, cellular disintegration. CFU, colony-forming units.
TEM after varying aBL exposures revealed aBL morphological changes in N. gonorrhoeae cells, such as formation of bulges, breakage of cell walls with cytoplasmic release, loss of outer membranes, and cellular disintegration (Figure 1B). Decreased density of cytoplasm was observed in some cells, accompanied by increased cellular size, suggesting that aBL induced damage to the cell wall of N. gonorrhoeae.
Toxicity of aBL to Normal Human Vaginal Epithelial Cells
As shown in Figure 1A, an aBL exposure of <108 J/cm2 eradicated N. gonorrhoeae CFUs in suspensions (>6-log10 CFU reduction). In contrast, study of the cytotoxicity of aBL to normal human vaginal epithelial cells (VK2/E6E7) showed no statistically significant loss of VK2/E6E7 cell viability when aBL exposures of up to 108 J/cm2 were used (P = .67; Figure 2A). Comet assay results exhibited that no aBL-induced DNA damage occurred in the VK2/E6E7 cells at aBL exposures up to 216 J/cm2 (Figure 2B).
Toxicity of antimicrobial blue light (aBL) to normal vaginal epithelial cells. A, Change in viability of normal VK2/E6E7 cells in response to aBL (wavelength, 405 nm) at different exposures. Bars, standard errors. P = .3474 for the difference in viability of VK2/E6E7 cells at 0 J/cm2 versus 54 J/cm2, P = .6676 for the difference between 0 J/cm2 and 108 J/cm2, and P < .0001 for the difference between 0 J/cm2 and 162 J/cm2. B, Comet assay images of normal VK2/E6E7 cell cultures exposed to 0 J/cm2, 54 J/cm2, 108 J/cm2, or 216 J/cm2 aBL (wavelength, 405 nm). The positive control was treated with H2O2, with damaged cellular DNA exhibiting a classic “comet tail.”
Presence and Quantity of Endogenous Porphyrins in N. gonorrhoeae
The UPLC chromatograms identified the presence of several species of endogenous porphyrins in N. gonorrhoeae, including uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, 5-carboxylporphyrin, and coproporphyrin (Supplementary Materials 1). No PpIX was detected in any of the 5 N. gonorrhoeae strains studied. Quantitative analysis revealed that the most-abundant porphyrin species in N. gonorrhoeae was coproporphyrin, with concentrations ranging from 7.93 nmol/g (protein weight; in strain 181) to 19.16 nmol/g (protein weight; in ATCC 700825) in the 5 N. gonorrhoeae strains (Table 1).
Concentrations of Porphyrins in the Extracts of Neisseria gonorrhoeae Strains
| Porphyrin | Strain 174 | Strain 179 | Strain 181 | Strain 199 | Strain ATCC 7007825 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | |
| Uroporphyrin | 0.06 | 0.61 | 0.08 | 0.54 | 0.18 | 1.94 | 0.58 | 3.21 | 0.37 | 1.68 |
| 7-Carboxylporphyrin | 0.58 | 5.52 | 0.70 | 4.45 | 0.51 | 5.44 | 1.11 | 6.09 | 1.22 | 5.48 |
| 6-Carboxylporphyrin | 0.27 | 2.63 | 0.41 | 2.60 | 0.19 | 2.06 | 0.50 | 2.72 | 0.41 | 1.84 |
| 5-Carboxylporphyrin | 0.56 | 5.35 | 0.46 | 2.93 | 0.52 | 5.57 | 1.06 | 5.82 | 1.10 | 4.94 |
| Coproporphyrin | 8.97 | 85.89 | 14.01 | 89.48 | 7.94 | 85.00 | 14.93 | 82.16 | 19.16 | 86.06 |
| Protoporphyrin IX | ND | … | ND | … | ND | … | ND | … | ND | … |
| Sum | 10.45 | 100 | 15.65 | 100 | 9.34 | 100 | 18.18 | 100 | 22.26 | 100 |
| Porphyrin | Strain 174 | Strain 179 | Strain 181 | Strain 199 | Strain ATCC 7007825 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | |
| Uroporphyrin | 0.06 | 0.61 | 0.08 | 0.54 | 0.18 | 1.94 | 0.58 | 3.21 | 0.37 | 1.68 |
| 7-Carboxylporphyrin | 0.58 | 5.52 | 0.70 | 4.45 | 0.51 | 5.44 | 1.11 | 6.09 | 1.22 | 5.48 |
| 6-Carboxylporphyrin | 0.27 | 2.63 | 0.41 | 2.60 | 0.19 | 2.06 | 0.50 | 2.72 | 0.41 | 1.84 |
| 5-Carboxylporphyrin | 0.56 | 5.35 | 0.46 | 2.93 | 0.52 | 5.57 | 1.06 | 5.82 | 1.10 | 4.94 |
| Coproporphyrin | 8.97 | 85.89 | 14.01 | 89.48 | 7.94 | 85.00 | 14.93 | 82.16 | 19.16 | 86.06 |
| Protoporphyrin IX | ND | … | ND | … | ND | … | ND | … | ND | … |
| Sum | 10.45 | 100 | 15.65 | 100 | 9.34 | 100 | 18.18 | 100 | 22.26 | 100 |
Abbreviation: ND, not detected.
aProtein weight.
Concentrations of Porphyrins in the Extracts of Neisseria gonorrhoeae Strains
| Porphyrin | Strain 174 | Strain 179 | Strain 181 | Strain 199 | Strain ATCC 7007825 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | |
| Uroporphyrin | 0.06 | 0.61 | 0.08 | 0.54 | 0.18 | 1.94 | 0.58 | 3.21 | 0.37 | 1.68 |
| 7-Carboxylporphyrin | 0.58 | 5.52 | 0.70 | 4.45 | 0.51 | 5.44 | 1.11 | 6.09 | 1.22 | 5.48 |
| 6-Carboxylporphyrin | 0.27 | 2.63 | 0.41 | 2.60 | 0.19 | 2.06 | 0.50 | 2.72 | 0.41 | 1.84 |
| 5-Carboxylporphyrin | 0.56 | 5.35 | 0.46 | 2.93 | 0.52 | 5.57 | 1.06 | 5.82 | 1.10 | 4.94 |
| Coproporphyrin | 8.97 | 85.89 | 14.01 | 89.48 | 7.94 | 85.00 | 14.93 | 82.16 | 19.16 | 86.06 |
| Protoporphyrin IX | ND | … | ND | … | ND | … | ND | … | ND | … |
| Sum | 10.45 | 100 | 15.65 | 100 | 9.34 | 100 | 18.18 | 100 | 22.26 | 100 |
| Porphyrin | Strain 174 | Strain 179 | Strain 181 | Strain 199 | Strain ATCC 7007825 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | Concentration, nmol/ga | Percentage of Total Porphyrin Content | |
| Uroporphyrin | 0.06 | 0.61 | 0.08 | 0.54 | 0.18 | 1.94 | 0.58 | 3.21 | 0.37 | 1.68 |
| 7-Carboxylporphyrin | 0.58 | 5.52 | 0.70 | 4.45 | 0.51 | 5.44 | 1.11 | 6.09 | 1.22 | 5.48 |
| 6-Carboxylporphyrin | 0.27 | 2.63 | 0.41 | 2.60 | 0.19 | 2.06 | 0.50 | 2.72 | 0.41 | 1.84 |
| 5-Carboxylporphyrin | 0.56 | 5.35 | 0.46 | 2.93 | 0.52 | 5.57 | 1.06 | 5.82 | 1.10 | 4.94 |
| Coproporphyrin | 8.97 | 85.89 | 14.01 | 89.48 | 7.94 | 85.00 | 14.93 | 82.16 | 19.16 | 86.06 |
| Protoporphyrin IX | ND | … | ND | … | ND | … | ND | … | ND | … |
| Sum | 10.45 | 100 | 15.65 | 100 | 9.34 | 100 | 18.18 | 100 | 22.26 | 100 |
Abbreviation: ND, not detected.
aProtein weight.
Presence and Quantity of Endogenous Porphyrins in Human Vaginal Epithelial Cells
Quantitative analysis by UPLC showed that the total protein concentration of endogenous porphyrins in VK2/E6E7 cells is 0.0176 nmol/g (protein weight; Table 2), which is hundreds of times lower than the total amount of porphyrins in N. gonorrhoeae (range, 9.34–22.26 nmol/g [protein weight]; Table 1).
Concentrations of Endogenous Porphyrins in VK2/E6E7 Cells
| Porphyrin | Concentration, nmol/ga | Percentage of Total Porphyrin Content |
|---|---|---|
| Uroporphyrin | 0.0074 | 41.95 |
| 7-Carboxylporphyrin | ND | … |
| 6-Carboxylporphyrin | ND | … |
| 5-Carboxylporphyrin | ND | … |
| Coproporphyrin | 0.0102 | 58.05 |
| Protoporphyrin IX | ND | … |
| Sum | 0.0176 | 100 |
| Porphyrin | Concentration, nmol/ga | Percentage of Total Porphyrin Content |
|---|---|---|
| Uroporphyrin | 0.0074 | 41.95 |
| 7-Carboxylporphyrin | ND | … |
| 6-Carboxylporphyrin | ND | … |
| 5-Carboxylporphyrin | ND | … |
| Coproporphyrin | 0.0102 | 58.05 |
| Protoporphyrin IX | ND | … |
| Sum | 0.0176 | 100 |
Abbreviation: ND, not detected.
aProtein weight.
Concentrations of Endogenous Porphyrins in VK2/E6E7 Cells
| Porphyrin | Concentration, nmol/ga | Percentage of Total Porphyrin Content |
|---|---|---|
| Uroporphyrin | 0.0074 | 41.95 |
| 7-Carboxylporphyrin | ND | … |
| 6-Carboxylporphyrin | ND | … |
| 5-Carboxylporphyrin | ND | … |
| Coproporphyrin | 0.0102 | 58.05 |
| Protoporphyrin IX | ND | … |
| Sum | 0.0176 | 100 |
| Porphyrin | Concentration, nmol/ga | Percentage of Total Porphyrin Content |
|---|---|---|
| Uroporphyrin | 0.0074 | 41.95 |
| 7-Carboxylporphyrin | ND | … |
| 6-Carboxylporphyrin | ND | … |
| 5-Carboxylporphyrin | ND | … |
| Coproporphyrin | 0.0102 | 58.05 |
| Protoporphyrin IX | ND | … |
| Sum | 0.0176 | 100 |
Abbreviation: ND, not detected.
aProtein weight.
Role of 1O2 in aBL Inactivation of N. gonorrhoeae
In the presence of 10 mM NaN3, a significant decrease in the aBL-induced reduction in the number of N. gonorrhoeae CFU in suspensions was observed (Figure 3). For example, at an aBL exposure of 48 J/cm2, the aBL-induced reduction decreased from approximately 7.0 log10 to 2.5 log10 CFU (P < .01).
Effects of a singlet oxygen quencher (NaN3) on the efficacy of Neisseria gonorrhoeae (ATCC 700825) inactivation by antimicrobial blue light. Bars, standard errors. CFU, colony-forming units.
Potential Development of Gonococcal Resistance to aBL
Figure 4 shows the reduction in the log10 ATCC 700825 CFU after 15 successive cycles of subtherapeutic exposure to aBL. Correlation analysis found a weak tendency toward increased susceptibility to aBL among N. gonorrhoeae as the number of cycles of aBL exposure increased (correlation coefficient = 0.24). However, the correlation was not statistically significant (P = .40), indicating that development of gonococcal resistance to aBL did not occur after 15 successive cycles of subtherapeutic aBL exposure.
Efficacy of antimicrobial blue light in inactivating Neisseria gonorrhoeae (ATCC 700725) during 15 successive cycles of subtherapeutic exposures. Bars, standard errors. CFU, colony-forming units.
Effect of Bacterium–Host Cell Interaction on the Tolerance of Human Vaginal Epithelial Cells to aBL and the Susceptibility of N. gonorrhoeae to Inactivation by aBL
Confocal microscopy indicated that N. gonorrhoeae cells either adhered to the cell walls or invaded the cytoplasm of the VK2/E6E7 cells in their cocultures (Figure 5A). Both VK2/E6E7 cells and N. gonorrhoeae were stained by SYTO9 (Figure 5A). Figure 5B and 5C display changes in the viability of VK2/E6E7 cells and N. gonorrhoeae, respectively, in their cocultures in response to aBL. Compared with noninfected VK2/E6E7 cells (Figure 2), infected VK2/E6E7 cells were more vulnerable to aBL inactivation. The decrease in viability among infected VK2/E6E7 cells was 11.3% (0.05-log10 CFU) at an exposure of 54 J/cm2 (P = .146) and 24.9% (0.12-log10 CFU) at an exposure of 108 J/cm2 (P = .0011; Figure 5B). Under the same aBL exposures, reductions of 4.18- and 5.07-log10N. gonorrhoeae CFU were observed, respectively (Figure 5C), indicating that the selectivity of aBL inactivation of N. gonorrhoeae over VK2/E6E7 cells still existed in the cocultures.
Effect of bacterium–host cell interaction on the tolerance of human vaginal epithelial cells to antimicrobial blue light (aBL) and the susceptibility of Neisseria gonorrhoeae to aBL inactivation. A, Confocal image of vaginal epithelial cells (VK2/E6E7) infected with N. gonorrhoeae (ATCC 700825). Both VK2/E6E7 cells and N. gonorrhoeae were stained green by SYTO9, and N. gonorrhoeae cells are depicted as green dots (arrows). N. gonorrhoeae cells either adhered to the cell walls or invaded the cytoplasm of the VK2/E6E7 cells. B, Viability changes among human vaginal epithelial cells (VK2/E6E7) infected by N. gonorrhoeae in response to aBL. C, Viability changes among intracellular N. gonorrhoeae in response to aBL.
DISCUSSION
In this study, we explored the potential use of aBL for the treatment of gonorrhea, a disease that is becoming untreatable because of increasing antibiotic resistance. Our findings demonstrated that N. gonorrhoeae, regardless of its antibiotic resistance status, was highly susceptible to 405-nm aBL. TEM revealed aBL-induced cellular damage in bacterial cells. Previous studies reported that, in contrast to most other bacterial species, N. gonorrhoeae does not contain superoxide dismutase, which is the major antioxidant defense system in bacteria [36]. This might explain the high susceptibility of N. gonorrhoeae to inactivation by aBL, which is dominantly mediated by singlet oxygen. Unpublished data in our laboratory showed that N. gonorrhoeae is on average 7.32-, 5.87-, and 28.0-fold more susceptible to aBL than Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli, respectively.
The UPLC analysis in this report ascertained the presence of endogenous porphyrins in N. gonorrhoeae. The most abundant porphyrin species in N. gonorrhoeae was found to be coproporphyrin, which is a highly efficient 1O2 generator [37]. The significant decrease in aBL activity in the presence of the 1O2 quencher NaN3 indicated the participation of 1O2 in inactivating N. gonorrhoeae by aBL. Interestingly, PpIX, which is commonly present in bacteria, was not detected in any of the 5 N. gonorrhoeae isolates studied in this report. In addition, by comparing the data in Figure 1A and Table 1, we found that there was no correlation between the concentrations of endogenous porphyrins and the corresponding aBL susceptibility of the 5 strains of N. gonorrhoeae. This might be due to the variations in the antioxidant properties of different N. gonorrhoeae strains.
The biosynthesis of photosensitizing porphyrins in bacteria occurs in the pathway of heme synthesis [38]. It is generally believed that the heme synthesis pathway of most bacteria begins with charged glutamyl-tRNAGlu to form the universal precursor ALA and that porphyrins are formed through a series of conserved enzymatic steps. The PpIX pathway was believed to be the classical pathway. However, the lack of endogenous PpIX and the abundant presence of endogenous coproporphyrin in N. gonorrhoeae indicates that, unlike most other gram-negative bacteria, N. gonorrhoeae synthesizes heme via the coproporphyrin III pathway [39, 40]. In addition to synthesizing its own heme, N. gonorrhoeae is able to internalize and use exogenous heme within host epithelial cells for growth [41].
The comparison of the aBL susceptibility between N. gonorrhoeae and human vaginal epithelial cells suggested that there is a therapeutic window where N. gonorrhoeae could be selectively inactivated while human vaginal epithelial cells are preserved. This can be explained by our UPLC finding that the content of aBL-activatable endogenous photosensitizers in the vaginal epithelial cells is >500-fold lower than that in N. gonorrhoeae.
N. gonorrhoeae has evolved mechanisms to infect a variety of host cells and subvert clearance by the host immune response. Infection of genital mucosa by N. gonorrhoeae involves adherence to and invasion of epithelial cells. Therefore, to be an effective therapeutic agent, aBL must also be able to inactivate intracellular N. gonorrhoeae. This study demonstrated that aBL inactivated N. gonorrhoeae both adherent to and inside the vaginal epithelial cells. Although the infection of the vaginal epithelial cells by N. gonorrhoeae slightly reduced the tolerance of the vaginal epithelial cells to aBL, we showed that there still existed a therapeutic window of aBL for preferentially inactivating N. gonorrhoeae over human vaginal epithelial cells.
The hypothesis that bacteria are unlikely to develop resistance to aBL is supported by the findings in the present study. It is well accepted that photo-oxidative stress reacts with several cellular macromolecules, including proteins, lipids, DNA, and RNA, and subsequently results in cell damage [42]. This multitarget feature of aBL minimizes the potential that bacteria will develop resistance to aBL [16, 17]. We experimentally investigated the potential development of aBL resistance by N. gonorrhea by performing 15 successive cycles of subtherapeutic aBL inactivation. No development of aBL resistance by N. gonorrhea was observed. This finding is also consistent with that of a recent study showing that 15 repeated sublethal exposures of Staphylococcus aureus suspension did not affect the susceptibility of this organism to 405-nm light [19].
How can aBL be delivered to treat gonorrhea in the clinic? In our ongoing animal study, we have developed a novel polymeric optical fiber to deliver aBL intravaginally to the infection sites in female mice (Supplementary Materials 3). The optical fiber can deliver axially uniform irradiation over a desired length. The fiber emission profile is retained upon deformation or surrounding changes. In a clinical study, the investigators used cylindrical fiber diffusers to deliver light intraurethrally in PDT for urethral condylomata [43]. In another study of aPDT for urinary tract infections in female mice, the authors used a glass cylindrical diffuser to deliver light intravascularly to the infection sites [44]. With the development of waveguide technology, it is now possible to deliver light to almost any anatomical region, via endoscopes, interstitially inserted microneedles, or fiber optics [45].
In conclusion, this study provides novel fundamental information regarding the use of aBL for treating gonorrhea. aBL preferentially inactivates N. gonorrhoeae while preserving the host cells. Endogenous aBL-activatable porphyrins are present in N. gonorrhoeae cells. The development of gonococcal resistance to aBL is unlikely. Further studies are warranted to investigate whether vaginal flora might impact the usefulness of aBL, to assess the effectiveness and safety of aBL therapy for gonococcal infection in animal models, and to explore potential clinical applications.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Notes
Acknowledgments. Y. W., R. F. E., Y. B., K. D. H., Y. H. G., Y. G., J. A. F., and T. D. designed the experiments. Y. W., R. F. E., Y. B., and X. S. G. performed the experiments. Y. W., Y. B., and T. D. analyzed the data and wrote the manuscript. R. F. E., K. D. H., Y. H. G., and J. A. F. reviewed the manuscript.
Financial support.This work was supported by the National Institutes of Health (R01AI123312 to T. D.), the US Department of Defense (FA9550-17-1-0277 to T. D.), the National Natural Science Foundation of China (61575222 to Y. W.), and the China Scholarship Council (201603170004 to Y. W.).
Potential conflicts of interest.All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
