Commentary
For reprint orders, please contact [email protected]
Future
Medicinal Chemistry
Photodynamic inactivation of bacteria: finding the effective targets “
In spite of the multi-target nature of photodynamic inactivation it is generally accepted that the main targets in bacteria are the external structures, cytoplasmic membrane and cell walls.
”
Keywords: bacterial resistance • photodynamic inactivation • photosensitizers • reactive oxygen species
The photodynamic inactivation (PDI) of microorganisms in the presence of a photosensitizer (PS), visible light and oxygen is considered a valuable alternative to the use of conventional antibiotic approach [1,2] . While antibiotics act on a specific cellular constituent, such as a key fitting into a lock, PDI, due to the reactive oxygen species formed during the lighting process, acts upon various critical molecular targets such as proteins, lipids and/or nucleic acids [3,4] . In spite of the multi-target nature of PDI it is generally accepted that the main targets in bacteria are the external structures, cytoplasmic membrane and cell walls [2,5–7] . Oxidative stress induced by photodynamic treatment of bacterial cells causes irreversible damages on proteins and lipids of the cytoplasmic membrane and of the cell wall, resulting in leakage of cellular contents or inactivation of membrane transport systems and enzymes [8,9] . Being the main targets of PDI the external structures, the PS does not need to reach the intracellular compartment since specific and proper adhesion to those structures is adequate for its light-activated destruction. As a consequence, the probability of resistance development in target microorganisms by blocking uptake, increasing metabolic detoxification or increasing the export of the drug is minimal [10] . This is without doubts one of the main advantages of PDI over the common antibiotics; the fact that to be a multi-target therapeutic approach, to which photoresistance has not yet been evidenced [11,12] . Moreover, the inactivation of drug-resistant bacteria by
10.4155/FMC.15.59 © 2015 Future Science Ltd
PDI is equally as effective as that of their native counterparts [10,13–14] . In this editorial, we propose that for a better understanding of the photoinactivation process, the knowledge of how the molecular targets are affected by PDI assumes a great importance. For this reason, detailed photophysical and photochemical studies of the interactions between the toxic species generated by the PS and surrounding key biomolecules, such as lipids, proteins and nucleic acids are essential for the knowledge and prediction of the photosensitization process efficiency. Bacterial cell structure In Gram-positive and Gram-negative bacterial cells, the cytoplasmic membrane are structurally similar. It consists of a phospholipid bilayer, some minor lipids and proteins. Since bacteria are devoid of intracellular organelles, inner membrane proteins play vital functions, such as energy production, lipid biosynthesis, protein secretion and transport [15] . Nevertheless, the cell wall of Gram-negative and Gram-positive bacteria is distinct. In Gram-negative bacteria, beside the thin peptidoglycan layer, the presence of an intricate outer membrane creates an impermeable barrier to antimicrobial agents [16] . The outer membrane consists of glycolipids in the outer leaflet, mainly lipopolysaccharides, lipoproteins and β-barrel proteins, lipoteichoic acids, a phospholipid bilayer in the inner leaflet, which anchors these constituents and the peptidoglycan (2–7 nm) [3] . In Gram-positive bacteria the
Future Med. Chem. (2015) 7(10), 1221–1224
Adelaide Almeida Author for correspondence: Department of Biology & Center for Environmental & Marine Studies (CESAM), University of Aveiro, 3810-193 Aveiro, Portugal Tel.: +351 234 370 200 [email protected]
Maria AF Faustino Department of Chemistry & Organic Chemistry, Natural Products & Food Stuffs Research Unit (QOPNA), University of Aveiro, 3810-193 Aveiro, Portugal
João PC Tomé Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal and Department of Organic & Macromolecular Chemistry, Ghent University, B-9000 Gent, Belgium
part of
ISSN 1756-8919
1221
Commentary Almeida, Faustino & Tomé cell wall is formed by only one thick peptidoglycan layer, surface proteins (fibronectin, fibrinogen, elastin) are attached to peptidoglycan, to teichoic acids (adhesins) or to stem peptides within the peptidoglycan layers. Furthermore, there are other proteins involved in immune system evasion, internalization and phage binding sites [3] .
chains of cardiolipins were identified [18] . In addition, in Escherichia coli, a Gram-negative bacterium hydroxyl and hydroperoxy derivatives were also identified as oxidized molecular species from unsaturated fatty acyl chains of phosphatidylethanolamines, the major phospholipid component [17] . Proteins
“...the understanding of the photodynamic
inactivation effect in the different biological targets will definitely assist in the development of potent photoactive drugs to photoinactivate not only bacteria but also other microorganisms...
”
The nucleic acid is also structurally similar in Gram-positive and Gram-negative bacteria. The bacterial nucleic acid consists of a double strand DNA molecule. A bacterium usually has a single circular chromosome approximately 1 mm long, which is about 1000-times longer than the typical bacterial cell. Therefore, it is looped, folded and packed tightly inside the cell [16] . Some bacteria also present small circular double-stranded DNA molecules, the plasmids, which are physically separated from the chromosomal DNA and are replicated independently of the chromosome [16] . Plasmids often carry genes that may benefit the survival of the organism, for example, antibiotic resistance. While the chromosomes are big and contain all the essential information for living, plasmids usually are very small and contain additional information [16] . Assessment of bacterial targets Lipids
Although the cytoplasmic and outer membranes of bacteria are rich in lipids, information concerning lipidic oxidation by PDI is scarce, because the identification and characterization of damages on lipids is quite complex. Moreover, given the structural difference of the bacterial cell wall, these damages have a different degree of importance in the photoinactivation process, depending if the bacterium is Gramnegative or Gram-positive. The first lipidomic studies performed on the evaluation of phospholipid oxidation of Gram-positive and Gram-negative bacteria after PDI with a tricationic porphyrin derivative revealed the formation of new oxidized molecular species and changes in the relative amounts of the different phospholipid classes [17] . In the Gram-positive Staphylococcus warneri, there was an increase in the amount of phosphatidylglycerols, and a decrease of cardiolipins and other phospholipids. Furthermore, hydroxyl and hydroperoxy derivatives from unsaturated fatty acyl
1222
Future Med. Chem. (2015) 7(10)
Some studies addressing the oxidative alterations of PDI in bacterial proteins reveal namely attenuation or disappearance of some proteins and increased concentration of high molecular weight products corresponding probably to cross-linked material [3,5,19–20] . Inactivation or loss of enzyme function has been reported for lactate dehydrogenase, NADH dehydrogenase, ATPase and also for succinate dehydrogenase after PDI [5,21–22] . These modifications in outer membrane proteins and enzymes are time dependent and concomitant with a decrease in cell survival. It is also observed that the amount of protein carbonyls also increases with irradiation time [23] . There is, however, only a single report describing the molecular targets of bacterial PDI by proteomics. The results of this study has shown that most of the altered proteins of Staphylococcus aureus by PDI treatment with a porphyrin PS are involved in metabolic activities such as the response to oxidative stress, cell division and sugar uptake [20] . It has also been suggested that the damages induced by PDI are specific and are likely to be dependent on the location of the PS in the bacteria [20] . Alves et al. in another study compared the photo-oxidative effects of two different PS on the protein profiles of different types of bacteria [24] . In this study, the electrophoretic profile of proteins of Gram-positive and Gram-negative bacteria, sensitized by two structurally different porphyrin derivatives with different kinetics of photoinactivation, was evaluated. The results show that changes occurring in the protein pattern during photodynamic treatment are different for the two PS, which helps to explain the different inactivation kinetics of the two bacteria by these PS. This work demonstrated that the bacterial proteins are important targets of PDI. It was clear that, even though the attempt to respond to cell injury by upregulation or synthesis of new proteins of response to stress, the damage was lethal and inhibiting cellular response both during and after photodynamic treatment. Nucleic acids
The main results of the studies done until now in order to evaluate the nucleic acid changes show that PDI do not affect directly Gram-negative and Grampositive bacterial nucleic acids. Bacterial DNA are only affected by PDI when the bacteria are already
future science group
Photodynamic inactivation of bacteria: finding the effective targets
inactivated or nonviable, and thus, the possibility that bacteria can develop resistance mechanisms to this kind of treatment is very low or even absent [7] . Guanine bases [5,8–9,19] have been shown to be particularly reactive and their oxidation by singlet oxygen generates 8-oxo-7,8-dihydro-2′-deoxyguanosine as a major product [19] . In addition to DNA, damage in RNA extracted from bacteria after PDI treatment has also been detected [25] . Usually, agarose gel electrophoresis analysis show time-dependent marked reductions in the nucleic acids from photosensitized Gram-positive and Gram-negative bacteria, and these reductions occur in parallel with the decrease of surviving cells. Nucleic acids photocleavage, specifically single strand breaks, depicted by smearing in genomic DNA [19,26–27] , in total RNA [27] and in plasmid DNA [5,19,27–28] has also been observed. In this last case, conversion of supercoiled DNA (form I) to nicked circular DNA (form II) can occur [26,28] . However, there are exceptions to be highlighted, namely in the extent of damage observed on the same bacterium with different porphyrins [26,28] ; between bacterial strains of the same species [19] and on isolated bacterial plasmid DNA with different PS, including cationic porphyrins with different charge number [26] . Furthermore, contrasting effects were observed in the photosensitized Gram-negative E. coli, intact chromosomal DNA [8] versus drastic reduction in DNA and RNA [27] , along with maximal bacterial killing. These apparently contradictory results could be related with the experimental design followed in each case. In a recent study, Alves et al. [17,18] investigated the effect of tri- and tetra-cationic porphyrinic PS on the concentration of intracellular nucleic acids of E. coli and S. warneri. In this study, nucleic acid changes occurred simultaneously to cell survival decrease. The
binding capacity of the photosensitizers was different between both bacteria (the tri-cationic PS had higher affinity for E. coli and tetra-cationic PS for S. warneri) and was not directly related neither to the extent of inactivation of the cell suspensions nor to the reduction in nucleic acid content. The authors concluded that different irradiation condition (irradiance and exposition time) could be responsible for different DNA reduction profile after photosensitization. The recent data about the understanding of the PDI effect in the different biological targets will definitely assist in the development of potent photoactive drugs to photoinactivate not only bacteria but also other microorganisms, in addition to enabling a full understanding their photoinactivation mechanisms. We believe that the new studies in this area will guide not only the establishment of improved photoinactivation protocols but also the design of enhanced molecules/materials. We are confident that PS will form the next generation of antibiotics, and PDI will, in the future, be recognized as a powerful antibacterial protocol. Financial & competing interests disclosure The authors are thankful to the University of Aveiro, Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, COMPETE and FEDER for funding the Centre for Environmental and Marine Studies (CESAM) research unit (project Pest-C/MAR/LA0017/2013) and the QOPNA research unit (project PEst-C/QUI/UI0062/2013, FCOMP-01-0124FEDER-037296). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. coli by meso-tetra(N-methyl-4-pyridyl)porphine. Biochem. Biophys. Res. Commun. 256, 84–88 (1999).
References 1
Wainwright M. Photodynamic antimicrobial chemotherapy (PACT). J. Antimicrob. Chemother. 42, 13–28 (1998).
2
Hamblin MR, Hasan T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem. Photobiol. Sci. 3, 436–450 (2004).
3
Alves E, Faustino MAF, Neves MGPMS, Cunha A, Tomé JPC, Almeida A. An insight on bacterial cellular targets of photodynamic inactivation. Future Med. Chem. 6, 141–164 (2014).
4
Tavares A, Carvalho CMB, Faustino MA et al. Mechanisms of photoinactivation of Gram-negative recombinant bioluminescent bacteria by cationic porphyrins. Photochem. Photobiol. Sci. 10(10), 1659–1669 (2011).
5
Valduga G, Breda B, Giacometti GM, Jori G, Reddi E. Photosensitization of wild and mutant strains of Escherichia
future science group
Commentary
6
Durantini EN. Photodynamic inactivation of bacteria. Curr. Bioact. Comp. 2, 127–142 (2006).
7
Alves E, Faustino MAF, Tomé JPC et al. Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins. Bioorg. Med. Chem. 21, 4311–4318 (2013).
8
Nitzan Y, Ashkenazi H. Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a cationic hydrophilic porphyrin at various light wavelengths. Curr. Microbiol. 42(6), 408–414 (2001).
9
Caminos DA, Spesia MB, Pons P, Durantini EN. Mechanisms of Escherichia coli photodynamic inactivation by an amphiphilic tricationic porphyrin and 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl) porphyrin. Photochem. Photobiol. Sci. 7(9) 1071–1078 (2008).
www.future-science.com
1223
Commentary Almeida, Faustino & Tomé
1224
10
Winckler KD. Special section: focus on anti-microbial photodynamic therapy (PDT). J. Photochem. Photobiol. B. Biol. 86, 43–44 (2007).
11
Pedigo LA, Gibbs AJ, Scott RJ, Street CN. Absence of bacterial resistance following repeat exposure to photodynamic therapy. Proc. SPIE USA, 7380, 73803H (2009).
20
Dosselli R, Millioni R, Puricelli L et al. Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach. J. Proteomics 77, 329–343 (2012).
21
George S, Kishen A. Influence of photosensitizer solvent on the mechanisms of photoactivated killing of Enterococcus faecalis. Photochem. Photobiol. 84, 734–740 (2008).
22
Packer S, Bhatti M, Burns T, Wilson M. Inactivation of proteolytic enzymes from Porphyromonas gingivalis using light-activated agents. Lasers Med. Sci. 15, 24–30 (2000).
23
Gomes MC, Silva S, Faustino MAF, et al. Cationic galactoporphyrin photosensitisers against UV-B resistant bacteria: oxidation of lipids and proteins by 1(O2). Photochem. Photobiol. Sci. 12, 262–271 (2013).
24
Alves A, Esteves AC, Correia A et al. Protein profiles of Escherichia coli and Staphylococcus warneri are altered by photosensitization with cationic porphyrins. Photochem. Photobiol. Sci. doi:10.1039/C4PP00194 2015) (Epub ahead of print)).
25
Gong X, Tao R, Li Z, Quantification of RNA damage by reverse transcription polymerase chain reactions. Anal. Biochem. 357(1), 58–67 (2006).
26
Caminos DA, Durantini EN. Interaction and photodynamic activity of cationic porphyrin derivatives bearing different patterns of charge distribution with GMP and DNA. J. Photochem. Photobiol. A Chem. 198(2–3), 274–281 (2008).
12
Tavares A, Carvalho CMB, Faustino MAF et al. Antimicrobial photodynamic therapy: study of bacterial recovery viability and potential development of resistance after treatment. Mar. Drugs 8, 91–105 (2010).
13
Jori G, Coppellotti O. Inactivation of pathogenic microorganisms by photodynamic techniques: mechanistic aspects and perspective applications. Anti-Infect. Agents Med. Chem. 6, 119–131 (2007).
14
Almeida J, Tomé JPC, Neves MGPMS et al. Photodynamic inactivation of multidrug-resistant bacteria in hospital wastewaters: influence of residual antibiotics. Photochem. Photobiol. Sci. 13, 626–633 (2014).
15
Silhavy TJ, Kahne D, Walker S. The bacterial cell envelope. Cold Spring Harb. Perspect. Biol. 2, a000414 (2010).
16
Madigan MT, Martinko JM. Brock Biology of Microorganisms. Pearson Prentice Hall, NY, USA (2006).
17
Alves E, Santos N, Melo T et al. Photodynamic oxidation of Escherichia coli membrane phospholipids: new insights based on lipidomics. Rapid Commun. Mass Spectrom. 27, 2717–2728 (2013)
18
Melo T, Alves E, Simões C et al. Photodynamic oxidation of Staphylococcus warneri membrane phospholipids: new insights based on lipidomics Rapid Commun. Mass Spectrom. 27, 1607–1618 (2013).
27
Salmon-Divon M, Nitzan Y, Malik Z. Mechanistic aspects of Escherichia coli photodynamic inactivation by cationic tetra-meso(N-methylpyridyl)porphine. Photochem. Photobiol. Sci. 3(5), 423–429 (2004).
19
Bertoloni G, Lauro FM, Cortella G, Merchat M. Photosensitizing activity of hematoporphyrin on Staphylococcus aureus cells. Biochim. Biophys. Acta 1475, 169–174 (2000).
28
Nir U, Ladan H, Malik Z, Nitzan Y. In vivo effects of porphyrins on bacterial DNA. J. Photochem. Photobiol. B. Biol. 11(3–4), 295–306 (1991).
Future Med. Chem. (2015) 7(10)
future science group
For reprint orders, please contact [email protected]
Future
Medicinal Chemistry
Photodynamic inactivation of bacteria: finding the effective targets “
In spite of the multi-target nature of photodynamic inactivation it is generally accepted that the main targets in bacteria are the external structures, cytoplasmic membrane and cell walls.
”
Keywords: bacterial resistance • photodynamic inactivation • photosensitizers • reactive oxygen species
The photodynamic inactivation (PDI) of microorganisms in the presence of a photosensitizer (PS), visible light and oxygen is considered a valuable alternative to the use of conventional antibiotic approach [1,2] . While antibiotics act on a specific cellular constituent, such as a key fitting into a lock, PDI, due to the reactive oxygen species formed during the lighting process, acts upon various critical molecular targets such as proteins, lipids and/or nucleic acids [3,4] . In spite of the multi-target nature of PDI it is generally accepted that the main targets in bacteria are the external structures, cytoplasmic membrane and cell walls [2,5–7] . Oxidative stress induced by photodynamic treatment of bacterial cells causes irreversible damages on proteins and lipids of the cytoplasmic membrane and of the cell wall, resulting in leakage of cellular contents or inactivation of membrane transport systems and enzymes [8,9] . Being the main targets of PDI the external structures, the PS does not need to reach the intracellular compartment since specific and proper adhesion to those structures is adequate for its light-activated destruction. As a consequence, the probability of resistance development in target microorganisms by blocking uptake, increasing metabolic detoxification or increasing the export of the drug is minimal [10] . This is without doubts one of the main advantages of PDI over the common antibiotics; the fact that to be a multi-target therapeutic approach, to which photoresistance has not yet been evidenced [11,12] . Moreover, the inactivation of drug-resistant bacteria by
10.4155/FMC.15.59 © 2015 Future Science Ltd
PDI is equally as effective as that of their native counterparts [10,13–14] . In this editorial, we propose that for a better understanding of the photoinactivation process, the knowledge of how the molecular targets are affected by PDI assumes a great importance. For this reason, detailed photophysical and photochemical studies of the interactions between the toxic species generated by the PS and surrounding key biomolecules, such as lipids, proteins and nucleic acids are essential for the knowledge and prediction of the photosensitization process efficiency. Bacterial cell structure In Gram-positive and Gram-negative bacterial cells, the cytoplasmic membrane are structurally similar. It consists of a phospholipid bilayer, some minor lipids and proteins. Since bacteria are devoid of intracellular organelles, inner membrane proteins play vital functions, such as energy production, lipid biosynthesis, protein secretion and transport [15] . Nevertheless, the cell wall of Gram-negative and Gram-positive bacteria is distinct. In Gram-negative bacteria, beside the thin peptidoglycan layer, the presence of an intricate outer membrane creates an impermeable barrier to antimicrobial agents [16] . The outer membrane consists of glycolipids in the outer leaflet, mainly lipopolysaccharides, lipoproteins and β-barrel proteins, lipoteichoic acids, a phospholipid bilayer in the inner leaflet, which anchors these constituents and the peptidoglycan (2–7 nm) [3] . In Gram-positive bacteria the
Future Med. Chem. (2015) 7(10), 1221–1224
Adelaide Almeida Author for correspondence: Department of Biology & Center for Environmental & Marine Studies (CESAM), University of Aveiro, 3810-193 Aveiro, Portugal Tel.: +351 234 370 200 [email protected]
Maria AF Faustino Department of Chemistry & Organic Chemistry, Natural Products & Food Stuffs Research Unit (QOPNA), University of Aveiro, 3810-193 Aveiro, Portugal
João PC Tomé Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal and Department of Organic & Macromolecular Chemistry, Ghent University, B-9000 Gent, Belgium
part of
ISSN 1756-8919
1221
Commentary Almeida, Faustino & Tomé cell wall is formed by only one thick peptidoglycan layer, surface proteins (fibronectin, fibrinogen, elastin) are attached to peptidoglycan, to teichoic acids (adhesins) or to stem peptides within the peptidoglycan layers. Furthermore, there are other proteins involved in immune system evasion, internalization and phage binding sites [3] .
chains of cardiolipins were identified [18] . In addition, in Escherichia coli, a Gram-negative bacterium hydroxyl and hydroperoxy derivatives were also identified as oxidized molecular species from unsaturated fatty acyl chains of phosphatidylethanolamines, the major phospholipid component [17] . Proteins
“...the understanding of the photodynamic
inactivation effect in the different biological targets will definitely assist in the development of potent photoactive drugs to photoinactivate not only bacteria but also other microorganisms...
”
The nucleic acid is also structurally similar in Gram-positive and Gram-negative bacteria. The bacterial nucleic acid consists of a double strand DNA molecule. A bacterium usually has a single circular chromosome approximately 1 mm long, which is about 1000-times longer than the typical bacterial cell. Therefore, it is looped, folded and packed tightly inside the cell [16] . Some bacteria also present small circular double-stranded DNA molecules, the plasmids, which are physically separated from the chromosomal DNA and are replicated independently of the chromosome [16] . Plasmids often carry genes that may benefit the survival of the organism, for example, antibiotic resistance. While the chromosomes are big and contain all the essential information for living, plasmids usually are very small and contain additional information [16] . Assessment of bacterial targets Lipids
Although the cytoplasmic and outer membranes of bacteria are rich in lipids, information concerning lipidic oxidation by PDI is scarce, because the identification and characterization of damages on lipids is quite complex. Moreover, given the structural difference of the bacterial cell wall, these damages have a different degree of importance in the photoinactivation process, depending if the bacterium is Gramnegative or Gram-positive. The first lipidomic studies performed on the evaluation of phospholipid oxidation of Gram-positive and Gram-negative bacteria after PDI with a tricationic porphyrin derivative revealed the formation of new oxidized molecular species and changes in the relative amounts of the different phospholipid classes [17] . In the Gram-positive Staphylococcus warneri, there was an increase in the amount of phosphatidylglycerols, and a decrease of cardiolipins and other phospholipids. Furthermore, hydroxyl and hydroperoxy derivatives from unsaturated fatty acyl
1222
Future Med. Chem. (2015) 7(10)
Some studies addressing the oxidative alterations of PDI in bacterial proteins reveal namely attenuation or disappearance of some proteins and increased concentration of high molecular weight products corresponding probably to cross-linked material [3,5,19–20] . Inactivation or loss of enzyme function has been reported for lactate dehydrogenase, NADH dehydrogenase, ATPase and also for succinate dehydrogenase after PDI [5,21–22] . These modifications in outer membrane proteins and enzymes are time dependent and concomitant with a decrease in cell survival. It is also observed that the amount of protein carbonyls also increases with irradiation time [23] . There is, however, only a single report describing the molecular targets of bacterial PDI by proteomics. The results of this study has shown that most of the altered proteins of Staphylococcus aureus by PDI treatment with a porphyrin PS are involved in metabolic activities such as the response to oxidative stress, cell division and sugar uptake [20] . It has also been suggested that the damages induced by PDI are specific and are likely to be dependent on the location of the PS in the bacteria [20] . Alves et al. in another study compared the photo-oxidative effects of two different PS on the protein profiles of different types of bacteria [24] . In this study, the electrophoretic profile of proteins of Gram-positive and Gram-negative bacteria, sensitized by two structurally different porphyrin derivatives with different kinetics of photoinactivation, was evaluated. The results show that changes occurring in the protein pattern during photodynamic treatment are different for the two PS, which helps to explain the different inactivation kinetics of the two bacteria by these PS. This work demonstrated that the bacterial proteins are important targets of PDI. It was clear that, even though the attempt to respond to cell injury by upregulation or synthesis of new proteins of response to stress, the damage was lethal and inhibiting cellular response both during and after photodynamic treatment. Nucleic acids
The main results of the studies done until now in order to evaluate the nucleic acid changes show that PDI do not affect directly Gram-negative and Grampositive bacterial nucleic acids. Bacterial DNA are only affected by PDI when the bacteria are already
future science group
Photodynamic inactivation of bacteria: finding the effective targets
inactivated or nonviable, and thus, the possibility that bacteria can develop resistance mechanisms to this kind of treatment is very low or even absent [7] . Guanine bases [5,8–9,19] have been shown to be particularly reactive and their oxidation by singlet oxygen generates 8-oxo-7,8-dihydro-2′-deoxyguanosine as a major product [19] . In addition to DNA, damage in RNA extracted from bacteria after PDI treatment has also been detected [25] . Usually, agarose gel electrophoresis analysis show time-dependent marked reductions in the nucleic acids from photosensitized Gram-positive and Gram-negative bacteria, and these reductions occur in parallel with the decrease of surviving cells. Nucleic acids photocleavage, specifically single strand breaks, depicted by smearing in genomic DNA [19,26–27] , in total RNA [27] and in plasmid DNA [5,19,27–28] has also been observed. In this last case, conversion of supercoiled DNA (form I) to nicked circular DNA (form II) can occur [26,28] . However, there are exceptions to be highlighted, namely in the extent of damage observed on the same bacterium with different porphyrins [26,28] ; between bacterial strains of the same species [19] and on isolated bacterial plasmid DNA with different PS, including cationic porphyrins with different charge number [26] . Furthermore, contrasting effects were observed in the photosensitized Gram-negative E. coli, intact chromosomal DNA [8] versus drastic reduction in DNA and RNA [27] , along with maximal bacterial killing. These apparently contradictory results could be related with the experimental design followed in each case. In a recent study, Alves et al. [17,18] investigated the effect of tri- and tetra-cationic porphyrinic PS on the concentration of intracellular nucleic acids of E. coli and S. warneri. In this study, nucleic acid changes occurred simultaneously to cell survival decrease. The
binding capacity of the photosensitizers was different between both bacteria (the tri-cationic PS had higher affinity for E. coli and tetra-cationic PS for S. warneri) and was not directly related neither to the extent of inactivation of the cell suspensions nor to the reduction in nucleic acid content. The authors concluded that different irradiation condition (irradiance and exposition time) could be responsible for different DNA reduction profile after photosensitization. The recent data about the understanding of the PDI effect in the different biological targets will definitely assist in the development of potent photoactive drugs to photoinactivate not only bacteria but also other microorganisms, in addition to enabling a full understanding their photoinactivation mechanisms. We believe that the new studies in this area will guide not only the establishment of improved photoinactivation protocols but also the design of enhanced molecules/materials. We are confident that PS will form the next generation of antibiotics, and PDI will, in the future, be recognized as a powerful antibacterial protocol. Financial & competing interests disclosure The authors are thankful to the University of Aveiro, Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, COMPETE and FEDER for funding the Centre for Environmental and Marine Studies (CESAM) research unit (project Pest-C/MAR/LA0017/2013) and the QOPNA research unit (project PEst-C/QUI/UI0062/2013, FCOMP-01-0124FEDER-037296). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. coli by meso-tetra(N-methyl-4-pyridyl)porphine. Biochem. Biophys. Res. Commun. 256, 84–88 (1999).
References 1
Wainwright M. Photodynamic antimicrobial chemotherapy (PACT). J. Antimicrob. Chemother. 42, 13–28 (1998).
2
Hamblin MR, Hasan T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem. Photobiol. Sci. 3, 436–450 (2004).
3
Alves E, Faustino MAF, Neves MGPMS, Cunha A, Tomé JPC, Almeida A. An insight on bacterial cellular targets of photodynamic inactivation. Future Med. Chem. 6, 141–164 (2014).
4
Tavares A, Carvalho CMB, Faustino MA et al. Mechanisms of photoinactivation of Gram-negative recombinant bioluminescent bacteria by cationic porphyrins. Photochem. Photobiol. Sci. 10(10), 1659–1669 (2011).
5
Valduga G, Breda B, Giacometti GM, Jori G, Reddi E. Photosensitization of wild and mutant strains of Escherichia
future science group
Commentary
6
Durantini EN. Photodynamic inactivation of bacteria. Curr. Bioact. Comp. 2, 127–142 (2006).
7
Alves E, Faustino MAF, Tomé JPC et al. Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins. Bioorg. Med. Chem. 21, 4311–4318 (2013).
8
Nitzan Y, Ashkenazi H. Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a cationic hydrophilic porphyrin at various light wavelengths. Curr. Microbiol. 42(6), 408–414 (2001).
9
Caminos DA, Spesia MB, Pons P, Durantini EN. Mechanisms of Escherichia coli photodynamic inactivation by an amphiphilic tricationic porphyrin and 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl) porphyrin. Photochem. Photobiol. Sci. 7(9) 1071–1078 (2008).
www.future-science.com
1223
Commentary Almeida, Faustino & Tomé
1224
10
Winckler KD. Special section: focus on anti-microbial photodynamic therapy (PDT). J. Photochem. Photobiol. B. Biol. 86, 43–44 (2007).
11
Pedigo LA, Gibbs AJ, Scott RJ, Street CN. Absence of bacterial resistance following repeat exposure to photodynamic therapy. Proc. SPIE USA, 7380, 73803H (2009).
20
Dosselli R, Millioni R, Puricelli L et al. Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach. J. Proteomics 77, 329–343 (2012).
21
George S, Kishen A. Influence of photosensitizer solvent on the mechanisms of photoactivated killing of Enterococcus faecalis. Photochem. Photobiol. 84, 734–740 (2008).
22
Packer S, Bhatti M, Burns T, Wilson M. Inactivation of proteolytic enzymes from Porphyromonas gingivalis using light-activated agents. Lasers Med. Sci. 15, 24–30 (2000).
23
Gomes MC, Silva S, Faustino MAF, et al. Cationic galactoporphyrin photosensitisers against UV-B resistant bacteria: oxidation of lipids and proteins by 1(O2). Photochem. Photobiol. Sci. 12, 262–271 (2013).
24
Alves A, Esteves AC, Correia A et al. Protein profiles of Escherichia coli and Staphylococcus warneri are altered by photosensitization with cationic porphyrins. Photochem. Photobiol. Sci. doi:10.1039/C4PP00194 2015) (Epub ahead of print)).
25
Gong X, Tao R, Li Z, Quantification of RNA damage by reverse transcription polymerase chain reactions. Anal. Biochem. 357(1), 58–67 (2006).
26
Caminos DA, Durantini EN. Interaction and photodynamic activity of cationic porphyrin derivatives bearing different patterns of charge distribution with GMP and DNA. J. Photochem. Photobiol. A Chem. 198(2–3), 274–281 (2008).
12
Tavares A, Carvalho CMB, Faustino MAF et al. Antimicrobial photodynamic therapy: study of bacterial recovery viability and potential development of resistance after treatment. Mar. Drugs 8, 91–105 (2010).
13
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Future Med. Chem. (2015) 7(10)
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