Blue light photoinhibition of Streptococcus mutans: potential chromophores and mechanisms

The direct application of blue light (λ = 400–500 nm) provides a promising antimicrobial modality, the effects of which are mediated through generation of reactive oxygen species. Porphyrins are organic compounds essential for bacterial synthesis of heme and are understood to be the main blue light chromophores within bacteria, which are critical to the sensitivity to blue light. However, Streptococcus mutans — the principal etiological species of dental caries — has shown susceptibility towards blue light despite reportedly lacking heme synthesis pathways, raising a question as to how this susceptibility is mediated. S. mutans lacks heme-containing cytochromes for full aerobic respiration, instead relying mainly on flavin adenine dinucleotide enzymes for oxygen-dependent metabolism. This review article investigates the potential target chromophores and mechanisms underpinning the inhibitory effects of blue light in S. mutans. Multiple reports support the proposition that bacteria with blocked heme synthetic pathways still possess the genetic antecedents capable of generating porphyrins and heme proteins under appropriate conditions. Blue light is absorbed by flavins, and hence, the flavoenzymes also represent potential chromophores. In conclusion, depending on in-vitro growth and metabolic conditions, there is more than one blue light chromophore within S. mutans. To optimise clinical application of blue light-induced antimicrobial effects, future investigations should focus on in-vivo models and clinical trials.


Introduction
Direct light phototherapy using blue wavelengths (λ = 400-500 nm) represents a non-invasive modality for inhibiting pathogenic bacteria.Blue light induces the generation of high levels of reactive oxygen species (ROS), which damage the cell wall, proteins, lipids, mitochondria, nucleus, and DNA [1].Conventionally, utilisation of light for bacterial disinfection has focused on the use of photodynamic therapy (PDT), a method of inhibiting bacteria that uses a light-activated sensitiser producing ROS, such as hydroxyl radicals, superoxide anions, and singlet oxygen [2].There are many PDT studies examining the effect of light in the red part of the visible spectrum (λ = 630-660 nm) on the lactic acid-producing bacterium, Streptococcus mutans (S. mutans) -the main causative bacterial species of dental caries [3] -using photo-activated stains/dyes such as toluidine blue (absorption maximum at λ = 630 nm), methylene blue (absorption maximum at λ = 660 nm), and disulfonate phthalocyanine (absorption maximum between λ = 600 and 700 nm) [4][5][6][7][8][9][10][11].However, to overcome diffusion difficulties of photosensitisers in ex vivo caries models [4], another recent approach has been the use of the direct antimicrobial effect of blue wavelengths without the application of a photosensitiser.The mechanism involved does not rely on photo-thermal eradication, as reported using infra-red diode and carbon dioxide lasers, which would be hazardous to host tissues [12,13].This approach has the advantage of direct light delivery to bacteria in order to excite endogenous chromophores/photo-absorbers, resulting in bacterial killing [14].The most accepted theory by which direct light irradiation induces antimicrobial activity is via activation of iron-free porphyrins, which have been proposed to be the main endogenous photosensitisers within bacteria.Porphyrins have an absorption peak within the blue spectral range (absorption maximum at λ = 400-450 nm).Porphyrins are macromolecules essential for bacterial synthesis of heme (ferrous iron (Fe 2+ )-containing porhyrin).They include a range of intermediate compounds in a cascade starting with aminolevulinic acid (ALA) precursor [15][16][17][18].
Interestingly, blue light has also shown antimicrobial effects towards species which do not synthesise or require heme for growth.S. mutans is one such examples, and recent studies have shown that blue light can exert bactericidal and bacteriostatic effects on this cariogenic species, despite the absence of heme or heme-containing proteins (cytochromes/ catalases).These effects can be achieved either directly, by inhibiting the bacteria, or indirectly by interfering with bacterial cell metabolism, preventing bacterial colonisation and biofilm maturation [19][20][21][22][23][24][25][26][27][28].S. mutans relies mainly on metal and flavin adenine dinucleotide (FAD)-bound enzymes/proteins for oxygen metabolism in place of porphyrin-containing heme [29].Accordingly, the mechanism by which blue light effects antimicrobial action against S. mutans is still not fully understood.This review article explores the potential chromophores and mechanisms governing the antimicrobial effects of blue light against S. mutans.

Porphyrins, aminolevulinic acid, and heme/hemin
Endogeneous porphyrins have been found in a range of anaerobic and facultative anaerobic bacterial species [30][31][32][33][34]. Porphyrins share common basic structures that include four pyrrole rings linked together by a methine bridge at their alpha carbon bonds.Due to their common structure, they exhibit blue light absorbance and fluorescence in the red spectrum (λ = 600-700 nm).Similar to PDT, different classes of ROS are generated after light absorption.In the type I reaction, hydroxyl and superoxide anion radicals are produced.In type II reactions, the energy released from the photosensitiser is taken up by a triplet oxygen, transforming it into a much more excited singlet oxygen which is highly reactive and understood to have the major effect in the photodisinfection process [35][36][37][38][39][40][41].
ALA is not a known photosensitiser [42] but is a heme/ cytochrome intrinsic precursor [43].ALA stimulates oxidative phosphorylation by upregulating cytochrome c oxidase activity and inhibiting glycolysis in eukaryotic cells [44,45].ALA is also known to similarly increase cytochrome levels in Escherichia coli (E.coli) [46].The supplementation of bacteria capable of synthesising heme with ALA enhances their porphyrin production; examples include Pseudomonas aeruginosa (P.aeruginosa), Staphlycoccus aureus (S. aureus), and E. coli.Increased levels of porphyrins have subsequently been shown to increase the bacteria's susceptibility to light exposure in both the blue (λ = 407-420 nm) and the red (λ = 635 nm) portions of the visible spectrum [47][48][49][50].Interestingly, ALA supplementation has also been shown to be capable of enhancing the inhibitory effect of both blue (λ = 440 nm) and red (λ = 635 nm) light in S. mutans [51,52] even though this bacterium is incapable of synthesising heme.Similarly, Enterococcus faecalis (E.faecalis) -another lactic acid-producing bacterium -did not show any porphyrin synthesis following ALA treatment [47], yet also exhibited photo-inhibitory effects following ALA supplementation and red light (λ = 633 nm) irradiation [53].It has also been shown that depending on the bacterial species, certain concentrations of ALA can induce inhibition without light irradiation.For example, 40 mM ALA decreased the viability of E. coli while 5-10 mM induced similar effects in P. aeruginosa [48,50].Unexpectedly, 30 mM ALA can also induce antimicrobial effects in S. mutans unaccompanied by light exposure [51,52].
It should be noted that heme/hemin (ferric iron (Fe 3+ )-containing porphyrin) acquisition and utilisation by bacteria remains a subject that has not entirely been elucidated and there are still unanswered questions concerning how bacteria utilise exogenous heme.It is unclear as to whether exogenous or endogenously synthesised forms are preferentially taken up by the bacterial cell, and once absorbed do they exist in a free state or as a chaperone inside the bacterial cell.However, heme synthesis is not limiting to heme-protein/cytochrome production, meaning that heme synthesis can take place at much higher rates than hemeprotein production.It has also been suggested that all Grampositive bacteria use non-canonical pathways for protoheme synthesis compared with Gram-negative bacteria and eukaryotic cells.Gram negative bacteria and eukaryotes rely on protoporphyrin as a final intermediate, while Gram-positive bacteria use coproporphyrin [54][55][56].Intriguingly, heme inhibits the growth of species with confirmed endogenous porphyrins, while enhancing the growth and aero-tolerance of lactic acid-producing species which reportedly do not rely on heme for growth and where the presence of porphyrins has not yet been identified [57][58][59][60][61][62].Nitzan et al. reported that hemin affects the growth and viability of S. aureus rapidly.This was evidenced by cessation of both glucose uptake and production of carbon dioxide.The antibacterial effects were reversed in the presence of albumin -which has a binding capacity to hemin -confirming the binding of hemin to bacterial cells.The toxic effect of hemin was not attributed to singlet oxygen or hydroxyl radical formation.This was confirmed by adding scavengers/quenchers such as mannitol and histidine, which failed to prevent the inhibitory effect of hemin on bacterial growth.Furthermore, reduced -but not oxidised -cysteine and glutathione inhibited the antibacterial effect of hemin.These results supported the hypothesis that the toxic effects of hemin are mediated through oxidation of intracellular components [57,58,63].
Conversely, hemin showed opposite effects on lactic acidgenerating bacteria, such as Lactococcus Lactis (L.lactis), resulting in enhanced growth and glucose utilisation.Hydrogen peroxide (H 2 O 2 ) levels were also supressed after hemin addition, despite a lack of catalase activity [59].Exogenous heme and hemin have also promoted cytochrome formation in L. lactis, as well as catalase and cytochrome bd activity in E. faecalis.Notably, heme was consumed only to synthesise cytochromes and not to alter the mode of respiration [64].All these outcomes support the proposition that due to their unique potential to adapt to various environmental changes, the ancestors of all lactic acid-producing bacteria had the cytochrome gene -cyd -but multiple gene loss has occurred over time [61].The cytochrome bd genes (cydABCD) within L. lactis improve cellular survival and oxidative stress resistance by inducing a proton motive force.Genes involved in heme biosynthesis have been identified in species not requiring heme for growth.E. faecalis contains the gene HemH, while L. Lactis has the HemZ gene.These genes encode ferrochelatase, the only function of which is to insert Fe 2+ into protoporphyrin IX -to form heme [60,65].However, these genes have not been identified in other lactic acid-producing bacteria, including S. mutans [60][61][62].

Streptococcus mutans growth, oxygen metabolism, and antioxidative defence
Since the photochemical mechanism of blue light is mediated through generating toxic levels of ROS, it is essential to understand how S. mutans survives different oxidative challenges.S. mutans is a Gram-positive, facultative anaerobic bacterium that grows optimally in a microaerophilic atmosphere; however, it can also survive under aerobic conditions.It grows rapidly at a temperature of 37 °C, ferments a range of sugars (i.e.lactose, sorbitol, mannitol, raffinose, mannose, salicin, and trehalose) to produce lactic acid.Compared to other streptococci, it is the most acidogenic and cariogenic species.It can synthesise extracellular polysaccharides (e.g.glucan-fructan) from sucrose, which enhance bacterial colonisation and growth on the tooth surface [66][67][68].Even though S. mutans does not perform oxidative phosphorylation, it utilises an incomplete tricarboxylic acid (TCA) cycle and depends mainly on glycolysis for adenosine triphosphate (ATP) generation [69][70][71][72].
Superoxide dismutase (SOD), peroxidase, and catalase are the main enzymes that bacteria use to manage oxidative stress [73].SOD converts superoxide anions to less reactive H 2 O 2 and molecular oxygen, while catalase is the enzyme responsible for degrading H 2 O 2 to water and oxygen [60].Since S. mutans lacks heme-bound catalase and cannot synthesise cytochromes (heme-containing redox proteins) for full aerobic respiration and energy metabolism, it depends mainly on a set of proteins/enzymes that are either metal-or FAD-bound.Regarding metal-bound enzymes, S. mutans has SOD that requires either iron or manganese cofactors [74,75].S. mutans also has an ability to resist oxidative stress by means of a DNA-binding protein from starved cells (Dps)-like peroxide resistance protein (Dpr), albeit, not through a direct interaction with ROS.Having ferritinlike properties, Dpr sequestrates free iron, which prevents further hydroxyl radical formation through the Fenton reaction [73,76].
Flavins, which are essential for growth and energy metabolism, have also been reported to mediate several redox functions in the process of oxygen metabolism [77].S. mutans adapts to aerobic environments using FAD-bound enzymes such as reduced nicotinamide adenine dinucleotide (NADH) oxidases (Nox), alkyl hydroperoxide reductases (Ahp), and thioredoxin reductase (TRX).Nox-1 (AhpF homologue) and Nox-2 enable lactate dehydrogenase to oxidise NADH by reducing molecular oxygen.However, Nox-1 generates H 2 O 2 , while Nox-2 generates water and plays a greater role in the regeneration of NAD + for the steady operation of glycolysis.H 2 O 2 generated by both SOD and Nox-1 is eliminated by AhpC (non-FAD).TRX maintains proteins in their reduced state by eliminating reversable disulphide bridges formed following oxidative stress (see Fig. 1).Notably, S. mutans can also reduce oxidised proteins using a non-FAD glutaredoxin system [29,72,78,79].To combat oxidative stress, it is understandable that there is a rise in the levels of the antioxidant enzymes once blue light is absorbed in its designated chromophore(s).Interestingly however, if an entire group of these enzymes is bound to a blue light absorbing molecule (FAD) [80], this means that blue light might be ironically inhibiting S. mutans through its own defence system.

Porphyrins and flavin adenine dinucleotide are potential blue light chromophores in Streptococcus mutans
Even though there are many studies in the literature confirming the inhibitory effects of blue light against S. mutans, no studies have investigated the mechanism underpinning these antimicrobial effects.Earlier reports in the literature stated that streptococci have an endogenous chromophore with two absorbance peaks (λ = 455 and 504 nm) that have variable intensities at different pH, while having an isosbestic absorbance at λ = 400 nm [81].Recently, flavins have been reported as blue light chromophores -besides porphyrins -in both S. aureus and E. coli [34].In this section, we discuss the evidence supporting the hypothesis that porphyrins and flavins are the potential chromophores in S. mutans.
There is evidence supporting the hypothesis that blue light might be absorbed by porphyrins in S. mutans even though this species does not require heme for growth.First, S. mutans has a hemin binding capacity, which is mediated through membrane proteins and not siderophores.Notably, the iron ion bound to hemin is Fe 3+ (oxidised), unlike that bound to heme which is Fe 2+ (reduced).In all cases, both forms of iron have been identified in S. mutans.It binds Fe 3+ prior to transporting it intracellularly in its Fe 2+ form; however, it is unclear what the fate of the iron-free porphyrin is after hemin binding and Fe 3+ acquisition, and why iron is only transported in the Fe 2+ form.When grown aerobically, the Dpr protein binds both forms of iron, limiting the free iron pool.The iron-active SOD is also upregulated when S. mutans are grown aerobically (see Fig. 1).Interestingly however, Fe 3+ uptake and intracellular Fe 2+ iron concentrations are both higher under anaerobic conditions [72,73,75,76,82,83].In E. coli, porphyrins/heme synthesis enzymes are slightly upregulated when bacteria are grown anaerobically [84].This means that S. mutans utilises Fe 2+ for other functions when growing anaerobically.S. mutans cultures showed red fluorescence (λ = 630-670 nm) -following λ = 405 nm blue light excitation -when grown anaerobically, and the fluorescence peaks were matching those of specific porphyrin classes, such as protoporphyrin IX [85,86].Secondly, it has been reported that knocking out the genes involved in the porphyrin enzymatic cascade in species capable of synthesising heme (E.coli) made the bacteria more sensitive to blue light compared to their wildtype counterparts.The phenomenon was associated with the accumulation of certain classes of porphyrins through an incomplete cascade [87,88].Porphyrin accumulation might explain why bacterial species with incomplete or blocked heme synthesis pathways are still susceptible to blue light irradiation.Thirdly, although there are few studies investigating the effects of ALA and blue light combined on S. mutans [51], it is conceivable that ALA might potentiate the effects of blue light due to porphyrin induction.Further investigations to confirm porphyrin accumulation were not carried out in this study.However, there are other possible explanations as to why ALA synergistically potentiated the effects of blue light or even induced inhibition in the absence of light.This evidence is related to the similarity between prokaryotes and eukaryotes in some steps of ) dismutase (SOD) is eliminated by AhpC.TRX maintains proteins in their reduced state through eliminating disulphide bridges.Iron (Fe) from exogenous heme compounds is utilised by S. mutans as a cofactor for SOD; however, Fe is also bound to the (Dps)-like peroxide resistance protein (Dpr) to control hydroxyl radical ( • OH) formation from H 2 O 2 .While exogenous aminolevulinic acid (ALA) and heme compounds can upregulate intracellular porphyrins and heme proteins / cytochrome synthesis, S. mutans is possibly relying on the glutamyl t RNA endogenous pathway for porphyrin synthesis the heme synthesis pathway [55,56].Cyclic diadenosine monophosphate (C-di-AMP) helps S. mutans grow into biofilms and combat oxidative stress through upregulating the glucan-producing enzyme, glucosyltransferases.Moreover, S. mutans relies on F-type adenosine triphosphatase (F-ATPase) to fight acid stress by pumping out hydrogen ions to maintain intracellular pH [29,89,90].ALA inhibits that action of both ATPase and cAMP in eukaryotic cells [91,92]; therefore, it is possible that ALA also inhibits both C-di-AMP and F-Atpase in S. mutans through a similar biochemical process.Fourthly, S. mutans possess an incomplete TCA cycle [70,71], and bacteria having incomplete TCA cycle can synthesise and utilise ALA through alternative pathways [93].However, concentrations and distributions of intermediate compounds in the pathway differ according to growth conditions and between species.In all cases, the pathway is directly or indirectly linked to both the TCA cycle and cytochrome synthesis.Lactic acid produced can also reverse the function of the TCA cycle from catabolic to anabolic pathways promoting cytochrome synthesis [94].Glutamyl tRNA (ALA precursor) is an alternative pathway for heme synthesis depending on glutamine [95,96], and S. mutans depends on glutamine for biofilm growth and acid tolerance [97,98] (see Fig. 1).Interestingly, stimulating intrinsic cytochrome formation through supplementing cultures with exogeneous ALA is significantly more effective than exogenous heme supplementation [43].ALA increases the expression of cytochromes irrespective of intracellular heme levels [54].Although porphyrins maximally absorb blue light wavelengths and ALA is the precursor of porphyrin production [15,35], some investigations rely on red light (λ = 633-635 nm) after incubating bacterial cells with ALA.Intriguingly, bacterial growth inhibition was successful with both E. faecalis and S. mutans [51][52][53], which suggests that there might be other chromophores alongside porphyrins; cytochromes are possible candidates.E. faecalis cytochrome bd has absorption peaks at 561 and 626 nm [99].H 2 O 2 forming Nox is mainly found in species possessing cytochromes and heme-proteins [78].Bacterial species that generated cytochromes after heme supplementation were also capable of producing flavins when grown in media devoid of exogeneous heme [64].S. mutans contains an extracellular electron transport system, which likely contains both flavins and cytochromes for full operation -flavo-cytochromes or flavo-porphyrins [100][101][102].Accordingly, it is possible that S. mutans might possesses the potential of generating porphyrins, heme-proteins, and cytochromes.However, the underlying pathways are not elucidated yet.
With regard to FAD, not only does FAD absorb blue light (absorption maximum at λ = 450 nm) but also induces ROS production which is essential for the inhibitory effects of light to occur [80,103].Besides the fact that ROS can be generated from FAD on its own, there are several flavoenzymes in S. mutans that are also strong candidates for mediating blue light-induced toxicity.Regarding the Nox system, it could be hypothesised that Nox-1 acts as a blue light chromophore since it is triggered by increasing FAD concentrations and generates H 2 O 2 [78].However, the FAD in Nox-2 cannot be excluded since in the process of reducing diatomic oxygen to water, other ROS are generated such as hydroxyl radicals and superoxide anions [72].On the other hand, and following a different mechanism, TRX also represents a potential candidate for blue light absorption in S. mutans.This enzyme has been deactivated by blue light (λ = 460 nm) in L. lactis.Deactivation effects declined when an oxygen scavenger (an oxidase/catalase combination) and potassium iodide (a quencher for photoexcited FAD) were used, confirming the role of both oxygen and excited FAD in the process.Intriguingly, the blue light deactivated TRX in L. Lactis shares similar crystal structures with its homologue AhpF in E. coli [104,105].Therefore, deactivating the Nox-1 (AhpF) in S. mutans -and hence blocking its ability to reduce molecular oxygen -is also a possibility.Consequently, there is also strong evidence supporting FADbound enzymes as blue light chromophores, but the process might be more complex compared to the photoinhibition mediated through porphyrins.It is difficult to determine whether the mechanism of blue light inhibition takes place due to ROS production from a photo-excited FAD molecule solely, Nox-1/Nox-2 excitation, TRX/ Nox-1 inhibition, or a combination of all of these mechanisms (see Fig. 2).

Factors affecting photoinhibition success in vitro
The variability identified in the inhibitory effects of blue light on different bacterial strains is due to the difference in the photo-absorber types/levels, the antioxidant potential, and the light irradiation parameters (light source, wavelength, irradiance, irradiation time, and energy density) [14,106].The chromophore levels in bacteria differ greatly in wild-type strains cultured in their natural environment [107], and the antioxidative potential of each strain fluctuates depending on culture conditions [108].Following blue light excitation (λ = 405 nm), S. mutans exhibited red fluorescence when tryptone soy cultures were supplemented with either a spinach extract (λ = 674 nm) or a mixture of blood, hemin, and vitamin K (λ = 636 nm).Conversely however, there was no fluorescence signal in non-supplemented tryptone soy or when cultures were supplemented with blood only [85].Non-supplemented casein peptone soybean cultures, as well as those supplemented with chlorophyll, showed fluorescence peaks (λ = 630-670 nm) attributed to hematin, hematoporphyrin, and protoporphyrin IX.However, peaks of much less intensity (only at λ = 630 nm) resulted when blood was added to the media, which likely took place due to limited porphyrin production via a negative feedback loop [86].The Nox system also operates at the converging point of several pathways involved in oxidative stress responses and energy metabolism.It controls and is itself affected by these pathways to maintain S. mutans survival [109].Nox-2-deficient S. mutans cannot utilise the lactate dehydrogenase pathway due to the inability of these bacteria to oxidise the NADH generated [78].Additionally, different sugars affect Nox levels differently.Mannitol generates more NADH compared to glucose, and hence, more Nox is produced to balance the NADH/NAD + levels [79].Conversely, Nox is less active throughout biofilms and acidic/low pH environments [110].Importantly, light absorption within a chromophore does not necessarily mean that bacterial killing will take place.The chromophore has to be situated within the cell at the correct biochemical orientation.Previously, blue light (λ = 450 nm) did not show any signs of inhibition of Streptococcus agalactiae growth although it contains granadaene -a blue light chromophore (peak absorption at λ = 438-485 nm).Furthermore, exogenous FAD only minimally enhanced the inhibitory effects of blue light.This also highlights the issue with PDT; it is not clear whether ROS is generated inside the cell or generated in the media post irradiation to cause bacterial killing [111,112].It is worth mentioning that S. mutans flavoenzymes are localised in the cytoplasm [78]; however, if it utilises flavo-cytochromes/ flavo-porphyrins for its electron transport system at the membrane level [100][101][102], then membrane depolarisation remains a possibility.Conversely, recent studies have confirmed that -sequentially -intracellular chromophores (porphyrins and FAD) are responsible for the production of ROS which in turn leads to a reduction in transmembrane potential of S. aureus [113,114].It is also important to consider other aspects such as the half-life and the diffusibility of the ROS generated.For example, singlet oxygen has a very short half-life in aqueous solutions (4 μs) and can only diffuse a distance of 100 nm.Accordingly, it is essential to produce cytotoxic levels of ROS relative to the amount of photons absorbed [115][116][117].

Conclusion
To summarise, both porphyrins and flavins represent potential target chromophores mediating blue light inhibition of S. mutans.However, the exact structural changes taking place in the flavoenzymes are not fully elucidated.The concept that more than one chromophore -including cytochromes -is involved in the process cannot be ruled out.It is also ) and hydrogen peroxide (H 2 O 2 ).H 2 O 2 leads to hydroxyl radical (•OH) formation through the Fenton reaction (ii) inhibition of thioredoxin reductase (TRX) and Nox-1 impeding their abilities to eliminate proteins disulphide bridges and reduce molecular oxygen (O 2 ), respectively (iii) cytotoxic levels of reactive oxygen species (ROS) generated after the excitation of flavin adenine dinucleotide (FAD), porphyrins and cytochromes crucial to acknowledge that different strains, metabolic states, and light irradiation parameters are major modulating factors in-vitro, especially if more than one chromophore/ mechanism of action is involved.Overall, direct blue light phototherapy provides a non-invasive modality for caries prevention and treatment.To optimise translation into clinical practice, future work should focus on investigating light irradiation protocols in in-vivo models and clinical trials.