Abstract

---

We investigated the action of indole-3-acetic acid (IAA) combined with horseradish peroxidase (HRP) on Staphylococcus aureus death. S. aureus is a pathogen microorganism capable of causing several diseases. S. aureus was incubated for 1.5, 3.0, and 6.0 h in the absence (control) and presence of IAA, HRP or both IAA/HRP and was then examined: Colony formations, membrane integrity and membrane depolarization. Hydrogen peroxide (H2O2) and superoxide anion (O2.^-) production by IAA/HRP and their roles in S. aureus  death were also evaluated. The gathered data were tested by ANOVA and Tukey test, using P-.

Introduction

---

Studies report that the combination of indole-3-acetic acid (IAA) and a plant enzyme, horseradish peroxidase (HRP), is cytotoxic to human cancer cells [1, 2] and leukocytes [3, 4] available through lipid peroxidation and DNA oxidation [5], and by chromatin condensation, mitochondrial depolarization, and membrane lesion [4]. However, IAA alone has no cytotoxic effect and can promote an increase in the phagocytic capacity of neutrophils [6, 7] and can provide antioxidant activity of liver [8, 9].The reaction between IAA and HRP proceeds via a complex mechanism that remains to be elucidated[4] producing cytotoxic species [10], it has been reported that IAA/HRP produces free radicals including indolyl, skatolyl, peroxyl radicals, and reactive oxygen species (ROS), such as, superoxide anion (O2.-)and hydrogen peroxide (H2O2) [11-13].
Recently, we reported that system IAA/HRP appears to function as a microbicidal mechanism on Prototheca zopfii [14] and Staphylococcus aureus [15]. Specifically, IAA/HRP causes a drastic reduction in colony formation and cell viability. Membrane integrity plays a critical role in membrane depolarization and is intimately related to microorganism survival [16]. For these reasons, was undertaken to investigate the mechanism action of IAA/HRP on membrane integrity and membrane polarization of S. aureus. Staphylococcus aureus is a pathogen microorganism capable of causing a range of life-threatening and mild diseases, including septicemia, meningitis, toxic shock syndrome, food poisoning, and skin abscess [17, 18]. Infection with S. aureus is a major cause of cow mastitis, and an inflammation of their teats that reduces milk production [19, 20].
To investigate the toxic mechanism of system IAA/HRP on S. aureus, microorganism was incubated for 1.5, 3.0, and 6.0 h in the presence of IAA/HRP and examined for the following parameters: i) colony formations in agar manitol and ii) membrane integrity and membrane depolarization assessed by flow cytometry. To identify the possible involvement of hydrogen peroxide (H2O2) and superoxide anion (O2.-) in cytotoxic actions from IAA/HRP on S. aureus death, the study was carried out in the presence of antioxidant enzymes (catalase and superoxide dismutase).

Methods

---

Chemicals and enzymes
All chemicals and enzymes were of analytical grade and were obtained from Sigma Chemical Company (St. Louis, MO, USA). Culture medium was obtained from Oxoid (Basingstoke, Hampshire, England).
Reactive species determination
An activated solution of dichlorofluorescein diacetate (DCFH-DA) [2, 21] was used to determine the formation of reactive species. To activate the DCFH-DA, 350 ml of a 10 mmol/l ethanolic solution of DCFH-DA were mixed with 1.75 ml of 0.01 mol/l NaOH and were allowed to stand for 40 minutes. After this period, the assay mixture was prepared in a 25 nmol/l sodium phosphate buffer, pH 7.2, containing 1.75 mmol/l activated DCFH-DA.
The final volume of all assays was 1.0 ml contained assay mixture, 20 ml of IAA (assay concentration = 1 mmol/l) and 10 ml of HRP (assay concentration = 1 mmol/l) in absence or presence of 20 ml of superoxide dismutase (SOD; assay concentration = 1000 IU/ml) or 20 ml of catalase (CAT; assay concentration = 1000 IU/ml) or both SOD and CAT. Absorbance was measured at 490 nm using a spectrophotometer (DU-800, Beckman), against a blank containing an assay mixture. Changes in absorbance were measured every 1 minute for 50 minutes. Similar assays were carried containing only IAA, HRP, SOD, or CAT in an assay mixture. A positive control was performed by H2O2 (3%, w/v) in an assay mixture.
The results showed absorbance measured at 490 nm during 50 minutes from system IAA/HRP (system), IAA/HRP in the presence of superoxide dismutase (system + SOD), IAA/HRP in the presence of catalase (CAT) and controls assays carried in the presence of HRP, HRP + CAT, CAT, IAA, SOD or H2O2. In addition, the relative percentage of the reactive species was calculated from the values of the areas bellow the curves from each assay carried for 50 min, missing the reactive species from HRP alone
Microorganism isolation and identification
The Staphylococcus aureus strain used in this study was obtained from cows with clinical mastitis. Serial dilutions of milk homogenates were plated on Baird Parker agar with 5% egg yolk tellurite emulsion and then incubated at 35 °C for 48 h [22, 23]. Characteristic colonieswere verified by: a) a positive ability to grow and produce acidon mannitol salt agar; b) a positive coagulase test and a positive catalase test, and c) a positive Voges-Proskauer (VP) test.
Experimental protocol
The experimental protocol was performed using the followings steps:
First, S. aureus were inoculated into brain heart infusion (BHI) broth and cultivated aerobically under continual rotation overnight at 37°C. They were then harvested and washed once in PBS (phosphate buffered saline, pH 7.4) sterile. S. aureus were resuspended in PBS at a concentration of 3.0 x 108 colony, forming units per milliliters (CFU/ml) (optical density at 625 nm of 0.2).
Second, microorganism S. aureus (3.0 x 104 CFU/ml) had been incubated in presence of system IAA (1.0 mmol/l)/ HRP (1.0 μmol/l) for different times (0, 1.5, 3.0 and 6.0 hours) aerobically in PBS with constant agitation at 37ºC. Others three assays in PBS were performed following: only microorganism; microorganism plus IAA and microorganism plus HRP. After all assays, the microorganism was then examined: Colony formation, membrane integrity and membrane depolarization. To verify whether H2O2, O2-. or both species were involved in IAA/HRP response upon S. aureus death, all described assays was performed in the presence of only catalase (1000 IU/ml), only superoxidase (1000 IU/ml) or both enzymes.
Microorganism colonies determination
After the time designated for incubation, the S. aureus was diluted to 3.0 x 103 CFU/ml instead of plaque in manitol salt agar and kept aerobically at 37ºC for 48 hours. The number of colony formation units was then determined, and the results were expressed in CFU/ml.
Results showed the effect of system IAA/HRP on the formation of colonies of Staphylococcus aureus (CFU/ml x 103) after incubation of the microorganism (0, 1.5 and 3.0 hours) in the absence (Control) and presence of system IAA/HRP, HRP, IAA catalase (CAT), or IAA/HRP plus catalase cultured in agar mannitol for 48 hours.
Membrane integrity and membrane potential
Cell membrane integrity and membrane potential (or membrane polarization) were analyzed by FACSAria flow cytometry equipment (BD Biosciences, USA). This instrument is equipped with an argon ion laser for excitation of the fluorescent dyes, providing 15 mW at 488 nm. Filter set up was standard.
For cell membrane integrity assay, the suspension of S. aureus (3.0 x 104 CFU/ml) was incubated with thiazole orange (TO) (420 nmol/l) and propidium iodide (PI) (43 mmol/l) for 5 minutes at room temperature according to the BD Cell Viability Kit. TO green fluorescence emitted was collected, using a 530 nm band-pass filter, and PI red fluorescence emitted was collected using a 610 nm band-pass filter. The data were analyzed using the BD FACSDiva Software (BD Biosciences, USA).
For the membrane potential assay, S. aureus (3.0 x 104 CFU/ml) were stained with 30 µmol/l diethyloxacarbocyanine iodide DiOC2(3) for 4 minutes, and membrane potential was estimated from the ratio of red to green DiOC2(3) fluorescence [24]. The green fluorescence of the DiOC2(3) dyes was collected, using a 530 nm band-pass filter, and the red fluorescence emitted was collected using a 610 nm band-pass filter. The data were then analyzed, using the BD FACSDiva Software (BD Biosciences, USA).
To determine differential staining of live and dead microorganism in the presence of PI, TO and DiOC2(3), similar assays were carried out also through a mixture of live microorganism and microorganism that was heat killed by exposure to 70 oC for 10 min [25].
Results showed the number of events of microorganisms with the membrane integrity and the membrane potential from the effect of system IAA/HRP on the membrane of S. aureus after incubation of the microorganism (0, 1.5, 3.0 and 6.0 hours) in the absence (Control) and presence of IAA/HRP, IAA/HRP plus catalase, HRP or IAA.
Expression of results and statistical analysis
Data are expressed as means ± standard deviation. Comparisons between groups were initially performed by analysis of variance (ANOVA). The alpha level (significance level related to the probability of rejecting a true hypothesis) was set at 0.05. Significant differences were then compared using Tukey’s test with a significance coefficient of 0.05.

Results

---

Reactive species formation by the system IAA/HRP
Illustration 1 display the generation of the reactive species by the system IAA/HRP. In this study, horseradish peroxidase action upon IAA produced reactive species, and superoxide dismutase failed to block any IAA/HRP-mediated reactive species formation. However, catalase, a specific H2O2 scavenger, partially suppressed (50%) the reactive species generation by IAA/HRP. Since SOD converts O2.- to H2O2, we also measured reactive species formation from the system IAA/HRP in the presence of both SOD and catalase and obtained results similar to the results for catalase alone. These findings indicate that IAA/HRP generates H2O2 (50%) and the other does not identify reactive species (50%). The addition of the enzymes HRP, SOD, CAT or SOD plus CAT to the assay mixture did not show a significant production reactive species. Similarly, IAA alone did not show reactive species production. A positive control was included in this study with H2O2 (Illustration 1), and the results showed that absorbance from this peroxide was completely abolished by catalase addition (data not shown).
Staphylococcus aureus viability
llustration 2 shows the data on S. aureus colony formation that was cultivated in mannitol agar for 48 hours after exposition to IAA/HRP for 1.5, 3.0, and 6.0 h. System IAA/HRP inhibited growth of the microorganism at 96%, 98%, and 99%, respectively, at 1.5, 3.0, and 6.0 hours of incubation in relation to the controls, thus demonstrating the prooxidant effect of the IAA when combined with HRP. The results show that IAA or HRP did not individually, however, modify colony formation of S. aureus.
The results shown in Inllustration 2 indicate that catalase, an efficient destructor of the H2O2 (Illustration 1), did not alter the deleterious effects produced by system IAA/HRP on S. aureus colony formation. Similarly, superoxide dismutase or superoxide dismutase plus catalase did not show alteration on the CFU of S. aureus incubated in the presence of IAA/HRP, to compare with the CFU in absence of these enzymes (data not shown). These results suggest that S. aureus growth was not dependent of H2O2 or O2-. produced by IAA/HRP.
Next, when S. aureus was exposed to IAA/HRP, reduced viability appeared due to changes in membrane integrity and membrane polarization (Illustration 3). Membrane integrity was significantly decreased at 17% and 22%, respectively, after 3.0 and 6.0 h of incubation in the presence of IAA/HRP, to compare with the controls in respective times. However, membrane integrity for S. aureus exposed to IAA/HRP for 1.5 h showed no significant effects. On the other hand, system IAA/HRP negatively affected cell polarization of S. aureus with 38%, 69%, and 99% reduction, respectively at 1.5, 3.0 and 6.0 h when compared with the controls (P ≤ 0.05). The assays performed in the presence of IAA or HRP did not modify the membrane integrity or the membrane polarization of S. aureus. Differential staining of live and dead cells by PI, TO, or DiOC2(3) was confirmed by testing the probes in a microorganism that was not treated and a microorganism that was heated at 70oC for 10 min (data not shown).
These results show that catalase did not alter the deleterious effects produced by system IAA/HRP on S. aureus membrane (Illustration: 3A and 3B). Similarly, superoxide dismutase or both catalase and superoxide dismutase added to a S. aureus incubation medium did not alter the deleterious effects promoted by system IAA/HRP (data not shown). Addition of calatase, superoxide dismutase or both enzymes in an incubation medium did not alter the membrane of S. aureus in the absence of IAA/HRP. These results suggest that the deleterious effect of the IAA/HRP system on S. aureus viability did not require H2O2 or O2-. presence.

Discussion

---

Recent studies show that IAA induces microorganism defense systems [26, 27] and can play an important role in bacterial behavior and plant bacterial interaction [28-30]. Others studies showed that some tryptophan metabolites contributed to the antibacterial action [31, 32]. In our study, IAA alone did not affect S. aureus viability. On the other hand, the system IAA/HRP was shown to provide efficient bactericidal action for S. aureus when evaluated by a drastic reduction of colony formation. Similarly other studies were observed as having a microbicidal effect from plant extract [33].
In the present study, the toxic effects produced by system IAA/HRP on S. aureus derived from membrane lesions and membrane depolarization. The loss of membrane integrity leads to a drastic reduction in membrane depolarization; these alterations may play an important role in microorganism viability. Membrane potential is responsible for generation of the proton motive force across the cell membrane during energy production [24]. Recent studies show that telavancin, a bactericidal agent, increase membrane permeability and reduce the membrane potential of the Gram-positive bacteria [16]. Similarly, others antimicrobials, such as Daptomycin, also show a perturbation of bacterial membrane function [34].
In the present study, 50% of reactive species produced by IAA/HRP is H2O2, but this specie does not alter the S. aureus viability. The cytotoxic effects from IAA/HRP on leukocytes are partially attributed to H2O2 and O2-. and these effects are accompanied by mitochondria depolarization, loss of membrane integrity, DNA fragmentation, and chromatin condensation [4]. In addition, H2O2 and O2.- production from system IAA/HRP intermediates and reactive products has been proposed from this system in aerobic conditions, including the radical skatolyl and peroxyl, which degrade to several products [35]. Others studies shown that indole-3-carbinol, a product from IAA/HRP [10] has in vitro antimicrobial activity [14]. Kim et al. showed that system IAA/HRP induces apoptosis in G361 human melanoma cells by H2O2 action despite the catalase activity [1]. In present study, the toxic effect of system IAA/HRP on S. aureus was independently of H2O2 and O2.- from this system; suggesting that S. aureus, a microorganism catalase positive, can be catalase activity sufficiently to cell self-protection.

Conclusion(s)

---

This research demonstrated that IAA/HRP has cytotoxic effects for S. aureus as seen in the reduction of colony formation, the loss of membrane integrity, and membrane depolarization; thus, system IAA/HRP does exert a toxic action on S. aureus by mechanism independently of H₂O₂ and O₂^.- generation.

Acknowledgement(s)

---

The authors want to acknowledge the FAPESP for financial support and CNPq/PIBIC for grant receive.

References

---

1. Kim DS, Jeon SE., Jeong YM, Kim SY, Kwon SB, Park KC. Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis. FEBS Lett. 2006; 580(5): 1439-1446.
2. Kim DS, Jeon SE, Park KC. Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells. Cell. Signal. 2004; 16(1): 81-88.
3.De Melo MP, Pithon-Curi TC, Curi R. Indole-3-acetic acid increases glutamine utilization by high peroxidase activity-presenting leukocytes. Life Sci. 2004; 75(14): 1713-1725.
4.De Melo MP, De Lima TM, Pithon-Curi TC, Curi R. The mechanism of indole acetic acid cytotoxicity. Toxicol. Lett. 2004; 148(1-2): 103-111.
6. Lins PG, Valle CR, Pugine SMP, Oliveira DL, Ferreira MSL, Costa EJX, De Melo MP. Effect of indole acetic acid administration on the neutrophil functions and oxidative stress from neutrophil, mesenteric lymph node and liver. Life Sci. 2006;   78(6): 564-570.
7. Pugine SMP, Brito P, alba-Loureiro TC, Costa EJX, Curi R, De Melo MP. Effect of indole-3-acetic acid administration by gavage and by subcutaneous injection on rat lelukocytes. Cell Biochem. Funct.  2007; 25(6): 723-730.
8. Oliveira DL, Pugine SMP, Ferreira MSL, Lins PG, Costa EJX, De Melo MP. Influence of indole acetic acid on antioxidant levels and enzyme activities of glucose metabolism in rat liver. Cell Biochem. Funct. 2007; 25(2):  195-201.
9. Mourão LRB, Santana RSS, Paulo LM, Pugine SMP, Chaible LM, Fukumasu H, Dagli M LZ, de Melo MP. Protective action of indole-3-acetic acid on induced hepatocarcinoma in mice. Cell Biochem. Funct., 2009; 27(1): 16-22.
10. Folkes LK, Wardman P. Oxidative activation of indole-3-acetic acids to cytotoxic species - a potential new role for plant auxins in cancer therapy. Biochem. Pharmacol. 2001; 61(2): 129-136.
11. Candeias LP, Folkes LK, Porssa M, Parrick J, Wardman P. Enhancement of lipid peroxidation by indole-3-acetic acid and derivatives: substituent effects. Free Radic. Res., 1995; 23(5): 403-418.
12. De Melo M, Pithon-Curi TC., Miyasaka CK., Palanch AC, Curi R. Effect of Indole Acetic Acid on Oxygen Metabolism in Cultured Neutrophils Gen. Pharmacol. 1998; 31(4): 573–578.
13. Kawano, T., Kawano, N., Hosoya, H., Lapeyrie, F. Fungal auxin antagonist hypaphorine competitively inhibits indole-3-acetic acid-dependent superoxide generation by horseradish peroxidase Biochem. Biophys. Res. Commun. 2001; 288(3): 546-551.
14. Cunha LT, Pugine, S. M. P., Silva, M. R. M., Costa, E.J., De Melo, M.P. Microbicidal action of Iindole-3-acetic acid combined with horseradish peroxidase on Prototheca zopfii from bovine mastitis.  Mycopathologia 2009; 169(2): 99-105.
15. Pugine SM P. Dissertation, Pirassununga, S. P., Brazil, Faculdade de Zootecnia e Engenharia de Alimentos (FZEA), Universidade de São Paulo (2008).
16. Higgins DL, Chang R, Debabov DV, Leung J, Wu T, Krause KM, Sandvik E, Hubbard JM, Kaniga K, Schmidt JrDE, Gao Q, Cass RT, Karr DE, Benton BM, Humphrey PP. Telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 2005; 49(3): 1127-1134.
17. Murray RJ. Recognition and management of Staphylococcus aureus toxin-mediated disease. Intern. Med. J. 2005; 35(2): 106–109.
18. Todd JK. Staphylococcal infections Pediatr. Rev. 2005; 26(2): 444–450.
19. Rich M, Staphylococci in animals: prevalence, identification and antimicrobial susceptibility, with an emphasis on methicillin-resistant Staphylococcus aureus. Br. J. Biomed. Sci. 2005; 62(2): 98-105.
20. Barkema HW, Schukken YH, Zadoks RN. The role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. J. Dairy Sci., 2006; 89(6), 1877–1895.
21. Valkonen M, Kuusi T. Spectrophotometric assay for total peroxyl radical-trapping antioxidant potential in human serum. J. Lipid Res. 1997; 38(11-12): 823-833.
22. Lancette GA, Bennett RW. Staphylococcus aureus and staphylococcal enterotoxins. In: Downes, F.P., and Ito, K., Editors, Compendium of Methods for the Microbiological Examination of Foods, Apha, Washington, 2001:387–403.
23. Rall VLM., Vieira FP, Rall R, Vieitis RL, Fernandes JrA., Candeias JMG., Cardoso KFG, Araújo JrJP. PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk. VETMIC 2008; 132(3-4): 408-413.
24. Novo DJ, Perlmutter NG, Hunt RH, Shapiro HM. Accurate flow cytometric membrane potential measurement in bacteria using diethylcarbocyanine and a ratiometric technique. Cytometry 1999; 35(1):55-63.
25. Hasui M, Hirabayashi Y, Kobayashi Y. Simultaneous measurement by flow cytometry of phagocytosis and hydrogen peroxide production of neutrophils in whole blood. J. Immunol. Meth. 1989; 117(1): 53-58. 
26. Remans R., Spaepen S, Vanderleyden J. Auxin signaling in plant defense. Science 2006; 313(5784): 171-171.
27. Spaepen S, Vanderleyden J, Remans R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol. Rev. 2007; 31(4): 425-448. 
28. Bianco C, Imperlini E, Calogero R, Senatore B, Amoresano A, Carpentieri A, Pucci P, Defez R. Indole-3-acetic acid improves Escherichia coli’s defences to stress. Arch. Microbiol. 2006a; 185(5): 373–382.
29. Bianco C, Imperlini E, Calogero R, Senatore B, Pucci P, Defez R.  Indole-3-acetic acid regulates the central metabolic pathways in Escherichia coli. Microbiology, 2006b; 152(8): 2421–2431.
30. Camerini S, Senatore B, Lonardo E, Imperlini E, Bianco C, Moschetti G, Rotino GL, Campion B, Defez R.Introduction of a novel pathway for IAA biosynthesis to rhizobia alters verch root nodule development.  Arch. Microb. 2008; 190(1): 67-77.
31. Sung WS, Lee DG. Mechanism of decreased susceptibility for Gram-negative bacteria and synergistic effect with ampicillin of Indole-3-carbinol. Biol. Pharm. Bull. 2008; 31(9): 1798-1801.
32. Narui K., Noguchi A. N., Saito A. A., Kakimi B. K., Motomura C. N., Kubo B. K., Takamoto D. S., Sasatsua M., Anti-infectious Activity of Tryptophan Metabolites in the L-Tryptophan–L-Kynurenine Pathway. Biol. Pharm. Bull. 2009; 32(1): 41-44.
33. Liu M.,  Otsuka N, Noyori K, Shiota S, Ogawa W, Kuroda T, Hatano T, Tsuchiya T. Synergistic effect of kaempferol glycosides purified from Laurus nobilis and fluoroquinolones on methicillin-resistant Staphylococcus aureus. Biol. Pharm. Bull. 2009; 32(3): 489-492.
34. Cottagnoud P. Daptomycin: a new treatment for insidious infections due to gram-positive pathogens. Swiss Med. Wkly. 2008; 138(7-8): 93-99.
35. Dunford HB. Heme peroxidases. In: Dunford HB ed. New York: Wiley-VCH. 1999: 45-55.

Source(s) of Funding

---

none

Competing Interests

---

none

Disclaimer

---

This article has been downloaded from WebmedCentral. With our unique author driven post publication peer review, contents posted on this web portal do not undergo any prepublication peer or editorial review. It is completely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscript but also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions before submitting any information that requires obtaining a consent or approval from a third party. Authors should also ensure not to submit any information which they do not have the copyright of or of which they have transferred the copyrights to a third party.
Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater to the needs of an individual patient. The web portal or any content(s) therein is neither designed to support, nor replace, the relationship that exists between a patient/site visitor and his/her physician. Your use of the WebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harm that you may suffer or inflict on a third person by following the contents of this website.