In vitro photodynamic inactivation of conidia of the phytopathogenic fungus Colletotrichum graminicola with cationic porphyrins

Article abstract of DOI:10.1039/c5pp00372e

Photodynamic inactivation (PDI) is an efficient approach for the elimination of a series of microorganisms; however, PDI involving phytopathogenic filamentous fungi is scarce in the literature. In the present study, we have demonstrated the photoinactivating properties of five cationic meso-(1-methyl-4-pyridinio)porphyrins on conidia of the phytopathogen Colletotrichum graminicola. For this purpose, photophysical properties (photostability and ¹O₂ singlet production) of the porphyrins under study were first evaluated. PDI assays were then performed with a fluence of 30, 60, 90 and 120 J cm^-2 and varying the porphyrin concentration from 1 to 25 μmol L^-1. Considering the lowest concentration that enabled the best photoinactivation, with the respective lowest effective irradiation time, the meso-(1-methyl-4-pyridinio)porphyrins herein studied could be ranked as follows: triple-charged 4 (1 μmol L^-1 with a fluence of 30 J cm^-2) > double-charged-trans2 (1 μmol L^-1 with 60 J cm^-2) > tetra-charged 5 (15 μmol L^-1 with 90 J cm^-2) > mono-charged 1 (25 μmol L^-1 with 120 J cm^-2). Double-charged-cis-porphyrin 3 inactivated C. graminicola conidia in the absence of light. Evaluation of the porphyrin binding to the conidia and fluorescence microscopic analysis were also performed, which were in agreement with the PDI results. In conclusion, the cationic porphyrins herein studied were considered efficient photosensitizers to inactivate C. graminicola conidia. The amount and position of positive charges are related to the compounds' amphiphilicity and therefore to their photodynamic activity.

Full text of DOI:10.1039/c5pp00372e

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PAPER
In vitro photodynamic inactivation of conidia of
the phytopathogenic fungus Colletotrichum
graminicola with cationic porphyrins†
Cite this: Photochem. Photobiol. Sci.,
2016, 15, 673
Camila Chevonica Vandresen,a Alan Guilherme Gonçalves,a
Diogo Ricardo Bazan Ducatti,b Fabio Seigi Murakami,a Miguel Daniel Noseda,b
Maria Eugenia Rabello Duarteb and Sandra Mara Woranovicz Barreira*a
Photodynamic inactivation (PDI) is an efficient approach for the elimination of a series of microorganisms;
however, PDI involving phytopathogenic filamentous fungi is scarce in the literature. In the present study,
we have demonstrated the photoinactivating properties of five cationic meso-(1-methyl-4-pyridinio)por-
phyrins on conidia of the phytopathogen Colletotrichum graminicola. For this purpose, photophysical
1
properties (photostability and O2 singlet production) of the porphyrins under study were first evaluated.
PDI assays were then performed with a fluence of 30, 60, 90 and 120 J cm−2 and varying the porphyrin
concentration from 1 to 25 μmol L−1. Considering the lowest concentration that enabled the best photo-
inactivation, with the respective lowest effective irradiation time, the meso-(1-methyl-4-pyridinio)por-
phyrins herein studied could be ranked as follows: triple-charged 4 (1 μmol L−1 with a fluence of 30 J
cm−2) > double-charged-trans 2 (1 μmol L−1 with 60 J cm−2) > tetra-charged 5 (15 μmol L−1 with 90 J
cm−2) > mono-charged 1 (25 μmol L−1 with 120 J cm−2). Double-charged-cis-porphyrin 3 inactivated
C. graminicola conidia in the absence of light. Evaluation of the porphyrin binding to the conidia and flu-
orescence microscopic analysis were also performed, which were in agreement with the PDI results. In
conclusion, the cationic porphyrins herein studied were considered efficient photosensitizers to inactivate
C. graminicola conidia. The amount and position of positive charges are related to the compounds’
amphiphilicity and therefore to their photodynamic activity.
Received 9th October 2015,
Accepted 6th April 2016
DOI: 10.1039/c5pp00372e
www.rsc.org/pps
evaluated as photosensitizers14,16–18against Gram positive
and Gram negative bacteria,5,19–22 yeast14,17,20,23 and filamen-
Introduction
Photodynamic inactivation (PDI) is a technique based on the tous fungi.9,18 It is important to mention that due to the
association of a photosensitizer, light and molecular oxygen resistance offered by the fungal cell structures (e.g. cell wall),
with the objective of eliminating microorganisms by destroy- the inactivation of such organisms usually demands high
ing their cell structures.1–3 PDI can be employed for the inacti- photosensitizer concentrations, as well as high light
vation of various microorganisms, including bacteria,4,5 intensities.18
viruses,6 fungi3,7–9 and parasites.10,11
Fungus Colletotrichum graminicola is the etiologic agent of
Among the photosensitizers employed for PDI, porphyrins corn (Zea mays) anthracnose,24–26 which is the most important
have been extensively evaluated, especially their cationic disease that affects corn plants.25,27–29 Anthracnose causes a
derivatives. The attachment of positively charged groups to substantial decrease in the corn crop yield, which can be as
the macrocycle enhances porphyrin water solubility12,13 and large as 40%.26,29,30 C. graminicola is capable of surviving on
improves its interaction with microorganism cell corn residues previously infected,31 specially in the soil
structures.3,13–15 In this way, cationic porphyrins have been surface,32 which turns a cultivating area into a potential inocu-
lum site.26 In addition, the great genetic variability shown by
C. graminicola makes the employment of hybrid plants ineffec-
tive as a tool for anthracnose control.33–35 The use of fungi-
cides has only been partially effective for the treatment of
anthracnose. In addition, microorganism resistance against
the most common fungicides has also been reported.36,37 Not
least important is the fact that pesticide-based control of plant
aDepartamento de Farmácia, Universidade Federal do Paraná, Av. Lothário Meissner,
3400, Jardim Botânico, Curitiba, Paraná, Brazil. E-mail: sandra.barreira1@gmail.com
bDepartamento de Bioquímica e Biologia Molecular, Universidade Federal do
Paraná, PO Box 19046, Curitiba, Paraná, Brazil
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5pp00372e
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diseases has several negative implications for the environment Dimethyl sulfoxide (DMSO) and methyl iodide were from
and human health.38
Merck®. Thin-layer chromatography (TLC) was performed on
Studies involving PDI of filamentous fungi have been silica gel coated aluminum sheets 60 F254 (Merck®) using
described in the literature.18,39 In the case of phytopathogenic solvent mixtures measured on a v/v basis. Column chromato-
fungi, PDI using hematoporphyrin has been utilized to elimin- graphy was carried out using silicagel 60 35–70 mesh (Fluka®).
ate a number of filamentous fungi (Alternaria alternata, Asper-
Porphyrin characterization was performed by means of 1H
gillus flavus, Fusarium avenaceum, Fusarium poae, Fusarium NMR spectroscopy, MALDI-TOF mass spectrometry and ultra-
culmorum, Acremonium strictum, Trichothecium roseum and Rhi- violet-visible spectroscopy with a Bruker AVANCE III 400 NMR
zopus oryzae) which are generally relevant to the food indus- spectrometer, a MALDI-TOF/MS model Autoflex II Bruker Dal-
try.9 Considering phytopathogens of the genus Colletotrichum, tonics and UV-1800 Shimadzu spectrophotometer, respectively.
there are two reported PDI studies. Menezes et al. (2014)40
Materials used for the culture media preparation were
employed phenothiazine dyes as photosensitizers for the in- oatmeal (Nestlé®), bacteriological agar (VETEC®) and Sabour-
activation of C. acutatum and C. gloeosporioides. The same aud dextrose agar (Kasvi®). Sterile 6-well plates (well
authors41 also utilized the furocoumarins 8-methoxypsoralen volume: 15.53 mL) for photodynamic inactivation were pur-
and isopimpinellin, and a mixture of two coumarins to photo- chased from TPP®.
inactivate C. acutatum.41 As far as we are aware, porphyrins
The white light source used for the photodynamic and
have never been utilized as photosensitizers for the photo- photophysical assays was a Lumacare LC 122A with a compati-
inactivation of Colletotrichum spp. ble fiber optic probe (400–800 nm) attached to a 250 W quartz/
In view of the potential application of photosensitizers in halogen lamp (LumaCare®, USA). Fluorescence measurements
agriculture, as well as the lack of studies devoted to this for the binding assay were performed with a RF-5301PC Shi-
approach, here we evaluated the photoinactivation of the madzu spectrofluorometer. Centrifugation employed for the
conidia of C. graminicola using cationic porphyrins. The photosensitizer binding assay and microscopic analysis was
choice of the photosensitizers herein utilized (Fig. 1) was performed with the centrifuge Sigma, model 1-14. Microscopy
based on the fact that tetra-methylpyridinio porphyrin (5) has images were recorded using
a fluorescence microscope
been largely utilized for the photoinactivation of (Olympus BX51TF) with a FITC filter and with microscope
bacteria,4,19,21,42–46 filamentous fungi18 and yeast.23,47–49 Con- magnification of eighty. Images were evaluated with Cell^F
sidering that different charge numbers and distributions software, 5.0 version (Olympus Europe Software Information).
could greatly affect the photoinactivating properties of this
Porphyrin synthesis
type of porphyrin, we included in the present study all five
possible cationic porphyrin bearing phenyl or 1-methyl-4-pyri- The five cationic porphyrins evaluated (Fig. 1) were synthesized
dinio groups at the macrocycle meso positions.
according to a methodology that has been previously
described,50 with modifications. Briefly, a mixture of pyrrole,
benzaldehyde, 4-pyridinecarboxaldehyde (4 : 1 : 3) in propionic
acid was refluxed for 1 h. After the reaction time, the reaction
mixture was concentrated by heating and porphyrins were preci-
pitated by adding acetone to the concentrated mixture. The
purple solids obtained corresponded to a mixture of six com-
pounds, including tetraphenylporphyrin and the desired meso-
pyridyl porphyrins. The porphyrin mixture was fractionated by
flash chromatography on a silica gel column with chloroform :
methanol (99 : 1) as the mobile phase. Porphyrins containing
one (0.9% yield), two-trans (1.1% yield), two-cis (1.0% yield),
three (3.1% yield) and four (2.5% yield) meso-pyridyl groups were
then obtained separately. Each of the meso-pyridyl porphyrins
was subsequently subjected to a methylation reaction with
excess methyl iodine, in DMF, to give cationic meso-(1-methyl-4-
pyridinio)porphyrins 1 (iodide salt, 90% yield), 2 (diiodide salt,
38% yield), 3 (diiodide salt, 27% yield), 4 (triiodide salt, 60%
yield) and 5 (tetraiodide salt, 64% yield).51 Synthesis procedures
and compound characterization are detailed in the ESI.† Spectral
properties of porphyrins 1–5 coincided with literature data.52,53
Experimental
Reagents, materials and general methods
All reagents and solvents utilized were of reagent grade and
they were used as obtained. Pyrrole, 4-pyridinecarboxaldehyde,
N,N-dimethylformamide (DMF) and 3-diphenylisobenzofuran
(DPBF) were purchased from Sigma-Aldrich®. Benzaldehyde,
propionic acid and ethyl ether were purchased from VETEC®.
Photosensitizer stock solutions
Photosensitizer stock solutions used in the photophysical and
biological studies were prepared in DMSO at a concentration
of 1 mmol L−1 and stored at 4 °C.
Fig. 1 Structure of the porphyrins used for the photoinactivation
studies of Colletotrichum graminicola conidia.
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Photostability assays and singlet oxygen generation
included in each irradiation experiment: a light control (LC)
subjected to the same fluence as the samples, but without a
photosensitizer, and a dark control (DC) containing the photo-
sensitizer at the highest concentration evaluated, in the
absence of light. After initial experiments, higher concen-
trations, 50 and 75 μmol L−1, demonstrated complete inacti-
Photostability experiments were performed as previously
described,18 differing in the concentration of the stock solu-
tions for the porphyrin derivatives (1 mmol L−1 in DMSO).
Photostability was estimated by irradiating the porphyrins dis-
solved in distilled water (2 mL) in a quartz cuvette under stir-
ring, with the following parameters coinciding with those
utilized for the PDI assays: irradiance of 100 mW cm−2, at
room temperature. After periods of 0, 1, 2, 3, 4, 6, 8, 10, 15 and
20 min, the absorbance of the Soret band was measured spec-
trophotometrically and referenced in percentage from the orig-
inal absorbance (100%).
Singlet oxygen generation was determined using DPBF as the
singlet oxygen quencher, with an irradiance of 9 mW cm−2 in a
glass cuvette, using a cut-off filter for wavelengths ≤540 nm. A
solution of each porphyrin (0.5 mmol L−1) and DPBF (50 mmol
L−1) in DMF/H2O (9 : 1) was utilized for the experiments. The
absorption decay of DPBF at 415 nm was measured at
irradiation intervals up to 20 min. The percentage of the DPBF
absorption decay is proportional to the production of 1O2.18
vation after
a fluence of 30 J . Therefore, the
cm−2
concentrations evaluated were 5, 10, 15 and 25 μmol L−1 for all
derivatives under study. Samples of 100 µL were collected in
the beginning of the test and after application of a fluence of
30, 60 and 90 J cm−2 and spread-plated on Sabouraud dextrose
agar for the determination of the concentration of viable
conidia. Colonies formed after 48 h of incubation at 27 °C
were counted. The average value of the duplicates was used as
an estimate of the concentration of viable conidia in the sus-
pension and expressed as CFU per mL. Three independent
assays were conducted for each porphyrin under study, the
profile of inactivation of different photosensitizers being con-
structed with the average and the standard deviation of the
results obtained. The survival of conidia of C. graminicola was
plotted as the logarithm of the concentration of viable conidia
(log CFU per mL) versus fluence (J cm−2).18
Preparation of conidia stock suspension
The strain of Colletotrichum graminicola was isolated at Campo Fluorescence microscopy studies
Mourão, Paraná State, Brazil and it was obtained from the col-
A suspension of 107 conidia per mL was incubated with
lection of phytopathogenic fungi at EMBRAPA Milho e Sorgo,
Minas Gerais, Brazil. Fungus access was authorized by Con-
selho de Gestão do Patrimônio Genético, with the number:
010850/2013-9. Microorganisms were cultivated in oatmeal
agar (FAA, 60 g oatmeal flour, 15 g bacteriological agar,
1000 mL distilled water). Cultures were incubated at 27 °C for
approximately one week until the development of a mycelium.
The cultures were then removed from incubation and
mycelium was scrubbed to stimulate fungus sporulation. A
new incubation using the same period of time was performed.
each photosensitizer, in the dark for 20 min, at selected
concentrations (based on preliminary microscopy results):
25 μmol L−1 for compounds 1 and 5 and 10 μmol L−1 for com-
pounds 2, 3 and 4. Then, the unbound photosensitizer was
removed by centrifugation at 11 000g for 5 min. Two washings
with PBS were performed before fresh centrifugation. After the
addition of 1 mL of PBS in tubes, the slides were prepared
with 50 μL of the resulting suspension, inserted between the
slide and coverslip.
After this period, for the preparation of conidia suspensions, a Photosensitizer binding
volume of 5 mL of phosphate buffered saline (PBS, 4 g of
Photosensitizer binding experiments were performed accord-
NaCl, 0.1 g of KCl, 0.72 g of Na2HPO4 and 0.12 g of KH2PO4 to
a final volume of 500 mL and pH 7.4 0.2)18 was added to the
sterile glass tube and a portion of conidia was inserted and
homogenized. To determine the concentration of conidia in
the initial suspension, a hemocytometer was used and the con-
centration of conidia was expressed as colony-forming unit per
milliliter (CFU per mL). The photoinactivation assays were per-
formed with a concentration of conidia of 105 CFU per mL.
ing to a methodology previously described,18 with modifi-
cations. The conidia (suspension of 106 conidia per mL) were
incubated in the dark at 30 °C in the presence of 10 μmol L−1
(2, 3 and 4) or 25 μmol L−1 (1 and 5) of cationic porphyrins (as
defined for the fluorescence microscopy assay). After the incu-
bation periods (0, 20 and 60 min), the unbound photosensiti-
zer was removed out of the suspension by centrifugation for
5 min at 11 000g. In order to evaluate the strength of the
attachment of the porphyrin to the biological material, two
series of aliquots were prepared: one set of conidia was
PDI experimental setup
Initially, a series of experiments using porphyrin 5 were con- digested immediately after centrifugation and the other was
ducted in order to determine the most suitable concentration further washed with PBS, prior to digestion. For digestion, the
for the following comparative photoinactivation assays. Ali- pellets were resuspended in 1 mL of the digesting solution
quots of conidia suspension (0.1 mL) were transferred to (aqueous NaOH 0.1 mol L−1) and incubated at room tempera-
6-well plates. Different volumes of the stock solution of 5 were ture for 24 h.18 The fluorescence of the resulting solutions was
added in order to obtain final concentrations of 5, 25, 50 and then directly measured. The excitation wavelengths were
75 μmol L−1 in the final volume of 5 mL, fulfilled with PBS. 445 nm (1), 419 nm (2), 421 nm (3), 422 nm (4) and 426 nm
The resulting mixtures were kept under stirring on melting ice (5), with the emission being measured in between
to prevent heating during irradiation. Two controls were 600–750 nm.
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Statistical analysis
Statistical analysis was performed by using Graphpad Prism
5.0 for Windows. Data normality was evaluated and attested by
the Shapiro–Wilk test. The significance of the PDI effect for
each porphyrin derivative and of the irradiation time on the
inactivation of conidial cells was assessed by the one-way uni-
variate analysis of variance (ANOVA) model with the Newman–
Keuls multiple comparison test. A value of p < 0.05 was con-
sidered significant.
Fig. 2 Controls and preliminary PDI tests on Colletotrichum gramini-
cola using cationic porphyrins 1–5 as photosensitizers. LC: light control
using a fluence of 120 J cm−2. DC: dark control, conducted with a por-
phyrin concentration of 25 μmol L−1. PDI experiments were performed
with the same concentration and fluence used for controls. Results are
expressed as the logarithm of viable colony-forming unit per milliliter
(log CFU per mL). The average values are indicated above the bars.
Asterisks indicate the level of significance of conidia inactivation (***: p <
0.001). Error bars represent standard deviation.
Results
Porphyrin photostability and singlet oxygen generation
Table 1 shows the photostability and 1O2 generation results
presented by porphyrins 1–5. Photostability was estimated
according to the percentages of the Soret band decay, which
were recorded under an irradiance of 100 mW cm−2. The
photostability results in Table 1 refer to the remaining absor-
bance after an irradiance of 100 mW cm−2, where lower per-
centages reflect lower photostabilities. All porphyrins
evaluated were considerably photostable. Porphyrin 1 was the
least stable photosensitizer evaluated, which showed 88% of
remaining absorbance after 20 min of irradiance.
shown in Fig. 2. These first experiments were also performed
in order to evaluate if porphyrins 1–5 would be adequate
photosensitizers against C. graminicola conidia, namely, if the
tested porphyrins were active only in the presence of light and
photosensitizers, simultaneously. For this purpose, conidia
susceptibility was first evaluated with light, in the absence of a
photosensitizer (light control) and without light, in the pres-
ence of a photosensitizer (dark control). At this stage, light
The capacity of 1O2 production was determined by the
indirect photooxidation method, which was based on the
measurement of the DPBF absorbance decay at 415 nm, in the
presence of each of the porphyrins herein studied, over 20 min
at 9 mW cm−2 of irradiance. According to Table 1, porphyrins
1 and 4 were the best 1O2 producers, which decreased DPBF
absorbance by 87 and 88%, respectively.
control and PDI were conducted with a fluence of 120 J cm−2
.
This evaluation indicated that porphyrins 2, 4 and 5 efficiently
eliminated C. graminicola in the PDI assay, showing no signifi-
cant conidia killing in the dark. Porphyrin 1 presented a
decrease in conidia viability in the PDI assay that was con-
sidered to be low, taking into account the high porphyrin con-
centration utilized.
Differently, porphyrin 3 eliminated C. graminicola for both
PDI and dark control assays. Porphyrin 3 was further evaluated
under lower concentration values (1–15 μmol L−1) in the dark;
however, a complete conidia killing was observed for all cases
(data not shown). In this way, this porphyrin was not con-
sidered an appropriate photosensitizer and it was not
employed for the following PDI assays.
Photodynamic inactivation of the conidia of Colletotrichum
graminicola
Results of the preliminary PDI experiments, which were per-
formed using 25 μmol L−1 of the porphyrin concentration, are
Table 1 Photostability and singlet oxygen generation of porphyrins 1–5
DPBF absorbance
Compound
Photostabilitya (%)
decayb (%)
Based on the results shown in Fig. 2, porphyrins 2, 4 and 5
were then more thoroughly studied (Fig. 3) by varying por-
phyrin concentrations with lower values (5, 10 and
15 μmol L−1) and using fluences of 30, 60 and 90 J cm−2. Por-
phyrins 2 and 4 promoted complete conidia inactivation under
all porphyrin concentrations and fluences evaluated. On the
other hand, porphyrin 5 promoted a photoinactivation that
was effective for most of the conditions tested, although it was
considerably lower in comparison with porphyrins 2 and 4,
especially for fluences studied in Fig. 3.
In order to differentiate between the photosensitizing
efficiency of porphyrins 2 and 4, even lower concentrations of
these porphyrins were employed for the PDI experiments
(1 μmol L−1 and 2.5 μmol L−1) (Fig. 4). This evaluation indi-
DPBF
—
88
95
90
96
99
12
87
82
80
88
78
1
2
3
4
5
a Photostability results are expressed as percentages from the initial
absorbance of the porphyrin Soret band, after 20 min of irradiance
(100 mW cm−2). Higher percentage values reflect higher
photostabilities. b Singlet oxygen generation was assessed by the DPBF
photooxidation (50 mmol L−1; in DMF/H2O 9 : 1) upon irradiation for
20 minutes with white light filtered through a cut-off filter for wave-
lengths <540 nm (9 mW cm−2), with or without a photosensitizer
(0.5 mmol L−1). Percentage values indicate DPBF absorbance decay
from the initial absorbance. Higher percentage values reflect higher
1O2 generation. Complete monitoring (1–20 min) of photostability and
1O2 assays, is shown in the ESI.
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Fig. 4 Controls and PDI of Colletotrichum graminicola employing por-
phyrins 2 and 4 at concentrations of 1 μmol L−1 (a) and 2.5 μmol L−1 (b),
with a fluence of 0, 30, 60 and 90 J cm−2. Results are expressed as the
logarithm of viable colony-forming unit per milliliter (log CFU per mL).
The average values are indicated above the bars. Asterisks indicated the
level of significance of conidia inactivation (***: p < 0.001). Error bars
represent standard deviation.
intense fluorescence when using concentrations higher than
10 μmol L−1, which impaired a good visualization of the coni-
dial structures. The images collected from the microscopic
evaluation of the C. graminicola conidia and their interactions
with the cationic porphyrins 1–5 are shown in Fig. 5. The
green image areas are related exclusively to the conidial struc-
tures and denoted the absence of porphyrins. The red color is
related to the fluorescence emitted by the porphyrin molecules
that are not interacting with conidial structures. The yellow-
colored areas denote interaction between porphyrin and coni-
dial structures. Fig. 5b indicates that porphyrin 1 was heavily
present on the surface of and inside the conidia; however, the
whole content of 1 was not necessarily interacting with coni-
dial structures. All the other porphyrins seem to interact with
conidial structures to some extent, with porphyrin 3 (Fig. 5d)
causing evident conidial disruption.
Fig. 3 Controls and PDI of Colletotrichum graminicola employing por-
phyrins 2, 4 and 5 at concentrations of 5 μmol L−1 (a), 10 μmol L−1 (b),
15 μmol L−1 (c) and 25 μmol L−1 (d), with a fluence of 0, 30, 60 and 90 J
cm−2. Results are expressed as the logarithm of viable colony-forming
unit per milliliter (log CFU per mL). The average values are indicated
above the bars. Asterisks indicated the level of significance of conidia
inactivation (**p < 0.01, ***: p < 0.001). Error bars represent standard
deviation.
Porphyrin binding to the conidia of Colletotrichum
graminicola
cated that porphyrin 4 photoinactivated C. graminicola with a
higher efficiency in comparison with 2. Porphyrin 4 was able
to provide a complete inactivation of the conidia, even at
1 μmol L−1 under a fluence of 30 J cm−2 while porphyrin 2
required 60 J cm−2 using the same concentration.
The porphyrin concentrations utilized for the binding experi-
ments were exactly the same as those utilized for the fluo-
rescence microscopy experiments, namely, porphyrins 1 and 5
were assayed at 25 μmol L−1 and porphyrins 2, 3 and 4 at
10 μmol L−1. Compounds 2 and 3 presented the higher
Fluorescence microscopic analysis
The fluorescence microscopy experiments shown in Fig. 5 were binding values after 60 min of incubation, which were 4.35 ×
performed with 25 μmol L−1 (porphyrins 1 and 5) or with 109 and 5.16 × 109 molecules per CFU, respectively. Porphyrin
10 μmol L−1 (porphyrins 2, 3 and 4). These concentrations 4, at 10 μmol L−1, showed comparable binding values with por-
were defined based on preliminary experiments that showed phyrins 1 and 5, both at 25 μmol L−1, which were 8.46 × 108,
that the most active porphyrins (2, 3 and 4) promoted an 1.48 × 109, and 1.20 × 109 molecules per CFU (60 min of incu-
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Fig. 5 Fluorescence microscopy assays of Colletotrichum graminicola conidia in the absence (a) and in the presence of cationic porphyrins 1 (b), 2
(c), 3 (d), 4 (e), 5 (f) at a concentration of 10 μmol L−1 for porphyrins 2, 3 and 4 and 25 μmol L−1 for 1 and 5. The green color is related to the conidial
structures, red to porphyrins and yellow to the interaction between the microorganisms and porphyrins.
bation), respectively. The washing procedure served to elimin- specifically for C. graminicola. As 5 presents four identical
ate those photosensitizer molecules that were not firmly cationized meso-pyridyl groups, we synthesized porphyrins
associated with the fungal structures. As shown in Fig. 6, the having a different number or positioning of the phenyl and
washings caused a reduction in the binding values for all por- 1-methylpyridinio groups at the meso positions of the macro-
phyrins and incubation times evaluated.
cycle. It is well known that the above-mentioned structural
differences play an important role in porphyrin physico-
chemical, photophysical and photodynamic properties.54
Preliminary PDI assays (Fig. 2), which were performed with
a high porphyrin concentration (25 μmol L−1) demonstrated
the suitability of the fluence utilized and also proved that por-
phyrins 1, 2, 4 and 5 could be considered suitable photosensi-
tizers, since they were active only in the presence of light. On
the other hand, porphyrin 3 promoted a complete inactivation
of C. graminicola in the absence of light, even using lower por-
phyrin concentrations. This behavior suggests a light-indepen-
dent mode of action (and consequently a 1O2-independent
action) that also operates for this porphyrin. This observation
was supported by the results shown in Fig. 6, which indicated
a large binding of porphyrin 3 to C. graminicola conidia, with
subsequent cell disruption (Fig. 5d). Simões et al. (2016),55
Alves et al. (2009)5 and Kessel et al. (2003)56 also comparatively
studied the PDI of cationic porphyrins presenting different
charge numbers and positioning. For the three studies men-
tioned, the porphyrin presenting two positive charges at adja-
cent meso-positions was the most active photosensitizer. This
behavior was explained by the distortion of the porphyrin ring,
which was directly caused by the electrostatic repulsion of the
two close positive charges.55,56 In the present work, the high
photoactivity expected for the doubly-charged cis-porphyrin (3)
was somehow translated as the cytotoxicity observed in the
dark. Considering the different PDI/dark susceptibilities
shown by distinct microorganisms, a previous study9 reported
that Aspergillus flavus was inactivated by hematoporphyrin, in
the absence of light, at a concentration of 5 μmol L−1, while
the other fungi evaluated were only eliminated under PDI con-
Discussion
Cationic porphyrins have been shown to be suitable photosen-
sitizers for the inactivation of diverse microorganisms by PDI.
The photoinactivating properties of porphyrin 5 have been
extensively evaluated in the literature.4,17,18,21,23 For this
reason, we decided to study porphyrin 5, together with other
closely related porphyrins, in order to verify if cationic meso-(1-
methyl-4-pyridinio)porphyrins could be potentially useful as
an alternative for the control of fungal phytopathogens,
Fig. 6 Binding of the tested porphyrins to Colletotrichum graminicola
conidia after incubation in the dark for 0, 20 and 60 min at 30 °C (with
and without washings with PBS), using 25 μmol L−1 of porphyrins 1 and
5 and 10 μmol L−1 of porphyrins 2, 3 and 4. Binding values are expressed
as the number of porphyrin molecules per colony-forming unit (PS per
CFU). PS: photosensitizer. Error bars represent standard deviation.
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ditions. These literature data could indicate that the light inde- (2 and 3) were the ones that bound effectively to
pendent inactivation promoted by porphyrins is related to C. graminicola, two methylpyridinio groups (hydrophilic) and
species of the microorganism under study.
two phenyl groups (hydrophobic) being the best ratio of those
The preliminary PDI results (Fig. 2) also showed porphyrin substituents at meso-positions of the macrocycle. The fact that
1 as exhibiting a photoactivity that was considerably lower porphyrin 2 gave the best binding value reinforces the impor-
than the other porphyrins. This compound (1), also differently tance of the amphiphilic character, which is more pronounced
from the other porphyrins evaluated, is poorly soluble in for the cis-porphyrin that possesses both hydrophobic and
aqueous medium. This property is directly related to the pres- hydrophilic groups on the same side of the molecule.
ence of only one positively charged group attached to the
The number of bound molecules herein determined by the
highly hydrophobic tetrapyrrole ring. The lack of water solubi- binding assay was from 846 million up to 5.16 billion of mole-
lity can trigger an aggregative behavior, which has been recur- cules per conidia. These numbers are substantially higher
rently observed for this type of compound.12,18,55,57 In this way, than the ones determined by Gomes et al. (2011)18 for Penicil-
the lower photostability and the high estimate of 1O2 singlet lium chrysogenum, which reached approximately 20 million per
production presented by 1 (Table 1) could be, in part, artefacts conidia. Considering that porphyrin 5 was included in the
raised from aggregation events. Aggregation could also explain cited paper, it is presumable that the type of microorganism
the lower photoactivity observed for 1. Even though this por- plays an important role in terms of photosensitizer binding.
phyrin presented a considerable binding value (Fig. 6), most of
PDI studies utilizing the same structurally related porphyr-
the photosensitizers aggregated on the surface of the conidia ins herein evaluated are described in the literature, mainly for
(Fig. 5b). This probably caused an inaccessibility of 1O2 or bacteria. Porphyrins 3, 4 and 5 were evaluated in terms of their
other oxygen reactive species to important conidial structures, photoinactivating properties against the marine bacteria Entero-
which reflected the low photoactivity observed.
coccus seriolicida and Vibrio anguillarum, and Escherichia
Considering PDI experiments with varying fluence and por- coli.46 In this case, compounds 3 and 4 were efficient in elimi-
phyrin concentration (Fig. 3 and 4), porphyrins 2 and 4 were nating V. anguillarum, while only compound 4 effectively
arguably more photoactive than 5. Porphyrin 5 required a eliminated E. coli. The photoinactivation of E. coli and
fluence of 90 J cm2 at 15 μmol L−1 in order to completely in- Staphylococcus warneri,19 which used porphyrin 5 as a photo-
activate C. graminicola, while porphyrins 2 and 4 provided the sensitizer, served as the reference for the evaluation of an
same effect under all concentrations and fluences outlined in analog containing three meso methylpyridino cationic groups
Fig. 3. Fig. 4 experiments ultimately allowed the differentiation and one pentafluorophenyl ring. For E. coli, both photosensiti-
between porphyrins 2 and 4, which indicated the latter as the zers completely inactivated bacteria at 5 µmol L−1. In the case
most efficient photosensitizer herein evaluated. Taking into of S. warneri, only the tricationic derivative was effective at a
account the lowest concentration that enabled the best photo- concentration of 0.5 µmol L−1. In general, these studies are
inactivation with the respective lowest effective fluence, the consistent with a higher photoinactivating efficiency of the
photosensitizers could be ranked as follows: 4 (1 μmol L−1 unsymmetrical cationic porphyrins, which is in agreement
with a fluence of 30 J cm−2) > 2 (1 μmol L−1 with 60 J cm−2) > 5 with the study herein presented.
(15 μmol L−1 with 90 J cm−2) > 1 (25 μmol L−1 with
The PDI studies involving the genus Colletotrichum avail-
able in the literature indicated that phenothiazine dyes and
120 J cm−2).
Taking into account the 1O2 production and binding shown coumarines require a concentration ranging from 5 to 50 µmol
by porphyrins 2, 4 and 5, compound 4 owes most of its activity L−1 to photoinactivate these microorganisms.40,41 In this way,
to the production of high levels of 1O2 (Table 1). This obser- cationic porphyrins 2 and 4 can be considered better photo-
vation is sustained by the low binding results presented by sensitizers than the compounds previously evaluated for the
this porphyrin (Fig. 6). In this way, it is presumable that the genus Colletotrichum.
high level of killing provided by a small amount of porphyrin 4
Regarding the potential application of porphyrin derivatives
inside the conidia was made possible by its outstanding 1O2 in agriculture, it is important considering the alleged photo-
production per molecule. On the other hand, in comparison damage to the host plant (Zea mays). There are a number of
1
with porphyrin 4, compound 2 presented a lower level of O2 publications indicating that superior plants are quite resistant
production, which was counterbalanced by an excellent por- in the face of light-activated photosensitizers. In fact, photo-
phyrin binding to the conidia. The lower photoactivity of por- sensitizing processes naturally occur in plants – via endogen-
phyrin 5, in comparison with 2 and 4, could be explained by ous phytoalexins,59 thiophens,60 root ketoalkenes and
its 1O2 production (comparable to 2) and its low binding ketoalkynes61 as defense mechanisms against phytopathogens
values (comparable to 4).
and as natural physiological processes.62 To avoid photo-
According to Engelmann and coworkers (2007)58 and damage, vegetal cells minimize oxygen reactive species and singlet
Simões et al. (2016),55 a suitable photosensitizer should oxygen action via superoxide dismutase, ascorbate peroxidase
present both a hydrophilic and hydrophobic character in order and glutathione reductase, which are present in the plant
to trespass the biological membranes. In the present work, the chloroplasts.63 In addition, xanthophylls (plant secondary
sole evaluation of the binding results (Fig. 6) clearly indicated metabolites) dissipate the excess of energy from light through
that the porphyrins presenting two positively charged groups non-photochemical mechanisms.64 These natural protecting
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Photochemical & Photobiological Sciences
mechanisms presented by plants could spare the host plant
against the photodamage promoted by porphyrins, which
could favour the exclusive photoinactivation of the infecting
C. graminicola. Nevertheless, these are just indications of the
potentiality of porphyrins for such a purpose. The in vivo
evaluation of porphyrins using Zea mays plants affected by
Colletotrichum is an ongoing project in our laboratory and it
will be reported soon.
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Concluding remarks
The present study demonstrated that cationic porphyrins
structurally related to tetra-charged porphyrins (5), especially
the trans-doubly-charged (2) and triply-charged (4) porphyrins, 11 K. Kassab, D. Dei, G. Roncucci, G. Jori and O. Coppellotti,
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17 S. A. G. Lambrechts, M. C. G. Aalders and J. Van Marle,
Antimicrob. Agents Chemother., 2005, 49, 2026–2034.
These results confirm the applicability of cationic porphyr-
ins for the PDI of filamentous fungi and open the possibility 18 M. C. Gomes, S. M. W. Barreira, M. A. F. Faustino,
of using these types of compounds as photosensitizers to be
applied in agriculture.
R. Fernandes, M. G. P. M. S. Neves, A. C. Tom,
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Acknowledgements
Thanks are due to Empresa Brasileira de Pesquisa Agro-
pecuária (EMBRAPA) – Milho e Sorgo for providing the micro- 20 S. Beirão, S. Fernandes, J. Coelho, M. A. F. Faustino,
organisms. This work was supported by grants from Fundação
Araucária (37925), CAPES and PRONEX-Carboidratos
J. P. C. Tomé, M. G. P. M. S. Neves, A. C. Tomé, A. Almeida
and A. Cunha, Photochem. Photobiol., 2014, 90, 1387–1396.
(14669). C. C. V. is grateful to the scholarships from CAPES, 21 K. Komagoe, H. Kato, T. Inoue and T. Katsu, Photochem.
M.E.D and M.D.N.
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