MECHANISMS OF ACTION:

PHYSIOLOGICAL EFFECTS

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crossm

Heterocycle Thiazole Compounds Exhibit

Antifungal Activity through Increase in
the Production of Reactive Oxygen
Species in the Cryptococcus neoformans-
Cryptococcus gattii Species Complex
Nvea Pereira de S,a Caroline Miranda de Lima,a Cleudiomar Incio Lino,c
Paulo Jorge Sanches Barbeira,b Ludmila de Matos Baltazar,a Daniel Assis Santos,a
Renata Barbosa de Oliveira,c Eleftherios Mylonakis,d Beth Burgwyn Fuchs,d
Susana Johanna
Departamento de Microbiologia, Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais, Belo
Horizonte, Minas Gerais, Brazila; Departamento de Qumica, Instituto de Cincias Exatas, Universidade Federal
de Minas Gerais, Belo Horizonte, Minas Gerais, Brazilb; Departamento de Produtos Farmacuticos,
Faculdade de Farmcia da UFMG, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais,
Brazilc; Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School, Brown University,
Providence, Rhode Island, USAd

ABSTRACT Human cryptococcosis can occur as a primary or opportunistic infection

and develops as an acute, subacute, or chronic systemic infection involving different Received 21 December 2016 Returned for
modication 13 February 2017 Accepted 8
organs of the host. Given the limited therapeutic options and the occasional resis-
May 2017
tance to uconazole, there is a need to develop novel drugs for the treatment of Accepted manuscript posted online 22 May
cryptococcosis. In this report, we describe promising thiazole compounds 1, 2, 3, 2017
and 4 and explore their possible modes of action against Cryptococcus. To this end, Citation S NPD, Lima CMD, Lino CI, Barbeira
we show evidence of interference in the Cryptococcus antioxidant system. The tested PJS, Baltazar LDM, Santos DA, Oliveira RBD,
Mylonakis E, Fuchs BB, Johann S. 2017.
compounds exhibited MICs ranging from 0.25 to 2 g/ml against Cryptococcus neo- Heterocycle thiazole compounds exhibit
formans strains H99 and KN99. Interestingly, the knockout strains for Cu oxidase antifungal activity through increase in the
and sarcosine oxidase were resistant to thiazoles. MIC values of thiazole compounds production of reactive oxygen species in the
Cryptococcus neoformans-Cryptococcus gattii
1, 2, and 4 against these mutants were higher than for the parental strain. After the species complex. Antimicrob Agents
treatment of C. neoformans ATCC 24067 (or C. deneoformans) and C. gattii strain Chemother 61:e02700-16. https://doi.org/10
L27/01 (or C. deuterogattii) with thiazoles, we veried an increase in intracellular re- .1128/AAC.02700-16.
Copyright 2017 American Society for
active oxygen species (ROS). Also, we veried the synergistic interactions among
Microbiology. All Rights Reserved.
thiazoles and menadione, which generates superoxides, with fractional inhibitory Address correspondence to Susana Johann,
concentrations (FICs) equal to 0.1874, 0.3024, 0.25, and 0.25 for the thiazole com- sjohann@icb.ufmg.br.
pounds 1, 2, 3, and 4, respectively. In addition, thiazoles exhibited antagonistic inter-
actions with parasulphonatephenyl porphyrinato ferrate III (FeTPPS). Thus, in this
work, we showed that the action of these thiazoles is related to an interference with
the antioxidant system. These ndings suggest that oxidative stress may be primarily
related to the accumulation of superoxide radicals.

KEYWORDS antifungal, antioxidant, Cryptococcus, thiazoles, ROS, RNS

C ryptococcosis is an opportunistic or primary fungal infection caused by yeasts of

the genus Cryptococcus, mainly C. neoformans and C. gattii. These encapsulated
fungi can cause disease in many animals and humans, leading to severe clinical
conditions, such as meningoencephalitis (1). The treatments for cryptococcosis caused
by C. neoformans and C. gattii are very similar and include the use of antifungal agents
(azoles, polyenes, and nucleoside analogues), cerebrospinal uid drainage, and occa-
sionally, surgical resection (2, 3). C. neoformans has a global distribution, while the

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FIG 1 Chemical structures for thiazole compounds 1, 2, 3, and 4, highlighting the thiazole portion.

various species within the C. gattii species complex are geographically limited. The
genotypes AFLP4/VGI (or C. gattii) and AFLP6/VGII (or C. deuterogattii) have a global
occurrence, while AFLP5/VGIII (or C. bacillisporus) is limited to the Americas and
genotype AFLP7/VGIV (or C. tetragattii) is limited to sub-Saharan Africa and India.
Clinical cases are also frequently reported from other continents, not only the Americas
and Africa (4, 5). Cryptococcosis is responsible for approximately 624,000 deaths per
year worldwide (6), with a universal geographic distribution, but clinical cases are more
common in the Americas and Africa (7, 8).
The antifungal options for the treatment of cryptococcosis are largely limited to
three agents: amphotericin B, 5-uorocytosine (5-FC), and uconazole (9). The Infec-
tious Diseases Society of America (IDSA) suggests specic recommendations for the
treatment of cryptococcal meningoencephalitis, which can be managed successfully in
the vast majority of patients (10). However, underdeveloped countries such as sub-
Saharan Africa still rely on uconazole monotherapy (11). In this case, high doses of
uconazole are necessary (10, 12). In addition, each of these drugs presents challenges.
Amphotericin B is noted for causing nephrotoxicity and thus is administered only as an
initial therapeutic (13). Renal failure was noted in 30% of the cases in a study of 707
patients receiving deoxycholate amphotericin B therapy (14), while liposomal formu-
lations cannot be used in most developing countries because of cost. Also, 5-FC
resistance is considered common and prevents its use as a monotherapy against
cryptococcosis (15). Fluconazole is used for secondary prophylaxis, but cases of ac-
quired resistance by C. neoformans have been reported, resulting in treatment failures
(1619). Overall, with the current therapeutic options, the mortality rate for the disease
remains unacceptably high, as much as 30% (15, 20).
The thiazole compounds, which possess sulfur and nitrogen, are widely distributed
in natural products and have promising biological activity, representing a class of
substances that attracts great synthetic interest (21). Some studies have reported the
synthesis of new thiazoles with anti-inammatory, antibacterial, anticancer, and anti-
HIV activities (2225). As one of the groups involved in this work, we investigated novel
thiazole heterocyclic compounds, which are easy to synthesize, have antifungal activity
against C. gattii and C. neoformans with MICs ranging from 0.25 to 1.25 g/ml, and
display low cytotoxicity in Vero cells and murine macrophages, presenting 50% inhib-
itory concentrations (IC50s) up to 32 g/ml (26). In that previous study (26), we provided
evidence that the thiazole fungicides 1, 2, 3, and 4 (Fig. 1) are promising compounds
for the development of new drugs for the treatment of cryptococcosis. Building on our
previous ndings, in the present work, we used a library of mutants of C. neoformans
strain H99 that aided in the investigation of mechanisms of fungal inhibition of these
new compounds. The results from these experiments with mutants directed the studies
with oxidative stress.

RESULTS
In this work, we found that our group of investigational thiazole compounds had no
action on the fungal membrane or cell wall (see Fig. S1 to S4 in the supplemental
material). With evidence that the thiazole compounds can inhibit mechanisms found
within multiple fungi, we endeavored to dene the genetic targets of the compounds.
The screening of a library of C. neoformans var. grubii H99 mutants described by Liu et
al. (27) for reduced sensitivity to our investigational compounds revealed that mutants
for the enzymes L-aminoadipate-semialdehyde dehydrogenase, sarcosine oxidase, and

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TABLE 1 Cryptococcus neoformans strain H99 mutants resistant to thiazoles
MIC (g/ml)
Mutant IDa Gene ID Description 1 2 3 4
D98 CNAG_04615 L-Aminoadipate-semialdehyde 64 1 8 64
dehydrogenase
D694 CNAG_02984 Sarcosine oxidase 32 2 4 8
D1127 CNAG_03464 Cu oxidase 64 8 8 64
b H99 wild type 8 0.5 4 2
aID, identication.
b, not applicable.

Cu oxidase were resistant to thiazoles (Table 1). Among these strains, the Cu oxidase
mutant was the most resistant to thiazoles, mainly to compounds 1, 2, and 4, gener-
ating MICs greater than 8 the MIC value for the wild type (WT).
To evaluate if these thiazoles could affect any cellular antioxidant system, we tested
the effect of the thiazole compounds against the thioredoxin system, an antioxidant
pathway that was not evaluated in our screen. We investigated the existence of
interactions between these compounds and glutathione, which in C. neoformans (strain
KN99), can function as an antioxidant and complements the thioredoxin system, an
antioxidant mechanism in fungi. If the thioredoxin system were involved, we would
expect glutathione to act as an antagonist to the thiazole compounds. However, the
MIC was not changed by the presence of glutathione, and fractional inhibitory con-
centration (FIC) values were considered to be not different.
Based on the grouping of oxidase mutants that exhibited resistance to the com-
pounds, we quantied reactive oxygen species (ROS) and reactive nitrogen species
(RNS) from cells exposed to thiazoles to determine if they act through changes to the
antioxidant systems of C. gattii strain L27/01 (or C. deuterogattii) and C. neoformans
ATCC 24067 (or C. deneoformans). Interestingly, we observed a signicant increase in
the amount of intracellular ROS after 1 h of treatment with each the thiazole com-
pounds, but there was only a trend towards an increase in RNS production (Fig. 2).
To better dene the antioxidant systems potentially targeted by the thiazole com-
pounds, we performed a checkerboard assay with compounds 1, 2, 3, and 4 combined
with three oxide stressors and one peroxynitrite sequestrant. We did not observe a
synergy between our investigational compounds and diamide, a thiol oxidizing agent,
or hydrogen peroxide. However, we found that menadione demonstrated synergistic
interactions with the thiazoles, since the FIC values were lower than 0.5 (Table 2). These
interactions suggest that the reductase system targeted by the thiazoles is related to
superoxide production. In addition, there were antagonistic interactions among the
thiazoles and parasulphonatephenyl porphyrinato ferrate III (FeTPPS), a highly reactive
nitrogen species, with FICs greater than 4.0 (Table 3).
Systemic infections of cryptococcosis are treated via intravenous delivery of anti-
fungal compounds. Thus, we examined if our group of investigational compounds
could potentially be delivered into the bloodstream without initiating a hemolytic
effect. We tested serial dilutions of compounds 1, 2, 3, and 4 with 2% human erythro-
cytes and found less than 1% hemolysis (Fig. 3). The 50% lethal concentrations (LC50s)
of the compounds against erythrocytes were higher than 64 g/ml.

DISCUSSION
In an earlier study, we presented the thiazole compounds 1, 2, 3, and 4 as promising
antifungal agents with low toxicity to mammalian Vero and peritoneal macrophage
cells and interesting antifungal activity in vitro against C. neoformans-C. gattii species
complex (26). In this work, we were able to correlate in vitro activity with disturbances
of the fungal antioxidant system.
Among the mutants evaluated, the Cu oxidase mutant was the most resistant to
thiazoles, mainly to compounds 1, 2, and 4, generating MICs greater than 8 the MIC
value for the isogenic wild type (WT). This enzyme is part of a group of multicopper

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FIG 2 Production of reactive oxygen species (ROS) (A) and reactive nitrogen species (RNS) (B) in C. gattii
L27/01 and C. neoformans ATCC 24067 after 1 h of treatment with thiazole compounds (at MICs),
amphotericin B (AmpB), and itraconazole (Itra). All treatments with thiazoles increased ROS production
signicantly compared with that in the control group with DMSO at the same concentration used to
dilute the compounds (P 0.01, Bonferroni posttest). *, P 0.05 by Bonferronis posttest.

proteins that is a very important component of the cellular defense that minimizes the
damage caused by ROS (28). There is a wide variety of multicopper proteins that can
be found in plants, fungi, and bacteria, for example, the nitrite reductase, which
catalyzes the reduction of oxygen to water and reduces nitrite to nitric oxide (29, 30).
To observe whether thiazole compounds were actually interfering with the antiox-
idant system, as suggested by the resistance of copper oxidase mutants to thiazoles, we
evaluated the amount of cellular ROS in the cells treated with the thiazole compounds.
We found that thiazoles 1, 2, 3, and 4 promoted a signicant increase in intracellular
ROS, which indicates that these compounds are capable of interfering with the anti-
oxidant system of Cryptococcus spp. The checkerboard assay with oxide stressors
revealed a synergistic interaction between menadione and thiazoles, with increased C.
neoformans (KN99) susceptibility to thiazoles in the presence of menadione. However,
the combination of thiazoles with diamide, a thiol oxidizing agent, and hydrogen
peroxide had no effect, suggesting that oxidative stress is not caused by an accumu-
lation of oxides and peroxides. Menadione is able to generate superoxide (31, 32), and

TABLE 2 FIC indices for thiazole compound combinations against C. neoformans KN99
FIC index with:
Compound Glutathione Diamide Menadione Hydrogen peroxide
1 3 1.125 0.1874 1
2 3 0.74 0.3024 0.5156
3 3 1.125 0.25 1.0156
4 2 1.125 0.25 1

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TABLE 3 FIC index for thiazole compounds in combination with FeTPPS against
C. neoformans ATCC24067 and C. gattii L27/01
FIC index with FeTPPS against:
Compound C. neoformans ATCC24067 C. gattii L27/01
1 4.125 4.25
2 3 4.083
3 4.25 4.25
4 4.25 4.25

the synergism may indicate that the thiazoles lead to superoxide accumulation inside
the cell, culminating in yeast death.
Additionally, we sought to determine if there was synergy with RNS by testing
peroxynitrite, a highly reactive nitrogen species formed from the reaction of nitric oxide
(NO) and superoxide, and FeTPPS, a ferric porphyrin complex that causes the decom-
position of peroxynitrite by catalytic isomerization to produce nitrate both in vitro and
in vivo. We observe that when FeTPPS sequestered peroxynitrite, Cryptococcus yeasts
were more resistant to thiazoles. Thus, a relationship could exist between the accumu-
lation of superoxide and peroxynitrite production, culminating in fungal death. Similar
to our results, Ferreira et al. (33) demonstrated that amphotericin B causes lipid
peroxidation in C. gattii cells through a greatly enhanced production of oxidative and
nitrosative radicals with increased peroxidase activity. Those authors also observed that
antifungals in combination with FeTPPS results in an antagonistic interaction, which
conrmed the importance of peroxynitrite in the activity of amphotericin B.
Thorpe et al. (28) indicated that superoxide anions play a protective role in stress
caused by hydrogen peroxide to a certain extent, because the oxidative stress caused
by both radicals is lethal to the cell. Thus, there is a possibility that the high concen-
tration of superoxide protects against the action of hydrogen peroxide. This would
explain the result found by our group, where there is an antagonistic interaction
between these thiazoles and amphotericin B against various isolates of C. neoformans
and C. gattii (Table 4) (26). Some studies have demonstrated that amphotericin B, in
addition to the action on the membrane ergosterol, also acts by promoting an increase
in intracellular H2O2 and lipid peroxidation, which helps the fungicide action of this
drug and prevents the emergence of resistant organisms (33, 34).
Another example that shows the involvement of hydrogen peroxide in the antifun-
gal activity of amphotericin occurs with Aspergillus terreus, where it plays a role in the
intrinsic resistance to amphotericin B, related to its high activity as a catalase that

FIG 3 Evaluation of the toxicity of thiazole compounds in human erythrocytes. (A) Picture showing wells
with total hemolysis caused by treatment with Triton X-100 and the absence of hemolysis after treatment
with thiazoles. (B) Percentages of hemolysis of human erythrocytes treated with thiazoles and Triton
X-100. Results are from two independent experiments performed in triplicates.

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TABLE 4 MICs of thiazole analogs
MIC (g/ml)a
Fungi Strain AMB FLC ITC 1 2 3 4
C. neoformans H99 0.5 2 0.03 2 0.5 2 2
C. neoformans KN99 0.5 2 0.5 2 0.5 2 2
C. neoformans ATCC24067 0.5 4 0.125 1 0.25 0.25 0.5
C. gattii L27/01 0.5 16 0.0156 1 0.25 0.25 1
aAMB, amphotericin B; FLC, uconazole; ITC, itraconazole.

detoxies hydrogen peroxide and transforms it into water and O2 (35). The catalase is
one of the main tools of the eukaryotic antioxidant system, which also includes
superoxide dismutase, peroxidase, glutathione peroxidases, peroxiredoxins, and the
glutathione and thioredoxin systems (36, 37).
Although the compounds cause the accumulation of ROS, a state that can trigger
apoptosis and be lethal, they did not adversely affect red blood cells at the tested
concentrations. Our evaluation with human erythrocytes showed that thiazole com-
pounds 1, 2, 3, and 4 are not hemolytic, which in principle, enables the intravenous
administration of these compounds. Previously, our group showed that these com-
pounds also do not exhibit toxicity against murine macrophages and Vero cells (26),
providing further evidence that they do not exhibit mammalian toxicity.
In conclusion, in this work, we showed that the action of thiazole compounds 1, 2,
3, and 4 is related to the interference in the antioxidant system; however, the thiazoles
do not target thioredoxin reductase. It is also suggested that oxidative stress may be
primarily related to the accumulation of superoxide radicals and that, perhaps, copper
oxidase may be a superoxide scavenger.

MATERIALS AND METHODS

Antifungal compounds. Thiazoles compounds 1, 2, 3, and 4 were synthesized according to
methodology previously described by S et al. (26) (Fig. 1).
Strains and culture conditions. C. neoformans ATCC 24067 (AFLP2/VNIV, or C. deneoformans,
according to the name proposed by Hagen et al. [5]) was obtained from the culture collection of the
University of Georgia (Atlanta, GA, USA). C. neoformans strain KN99 and C. neoformans strain H99
(AFLP1/VNI, both serotype A) were obtained from the culture collection of the Division of Infectious
Diseases, Brown University (Providence, RI, USA). C. gattii strain L27/01 (VNII, by PCR-ngerprinting using
[GTG]5 primer [unpublished data], or C. deuterogattii, according to the name proposed by Hagen et al.
[5]) was deposited in the Culture Collection of Microorganisms and Cells of the Universidade Federal of
Minas Gerais under code UFMG-CM-Y6141 (Belo Horizonte, MG, Brazil). All the yeast strains were stored
frozen at 80C. In this work, we refer to the isolates of different molecular species as the C.
neoformans-C. gattii species complex, as a proposed by Kwon-Chung et al. (38).
In vitro susceptibility testing. Broth microdilution testing was performed in accordance with the
guidelines in the CLSI document M27-A3 (39). Susceptibility was determined by the broth microdilution
method, which was performed in sterile at-bottom 96-well microplates. Briey, Cryptococcus neoformans
species complex (H99 and KN99) and C. gattii (L27/01, or C. deuterogattii) were cultured in yeast
extract-peptone-dextrose (YPD) broth at 37C for 48 h, and the inoculum concentration was 2.5 105
CFU/ml. The thiazole compounds 1, 2, 3, and 4 were dissolved in dimethyl sulfoxide (DMSO) (Sigma-
Aldrich, St. Louis, MO, USA) and diluted in RPMI 1640 medium supplemented with L-glutamine and
buffered to pH 7.0 with 0.165 M morpholine propanesulfonic acid ([MOPS] Sigma-Aldrich, St. Louis, MO,
USA). Compounds were tested at concentrations ranging from 0.125 to 64 g/ml. Growth and sterility
controls were also performed. Fluconazole (Sigma-Aldrich, St. Louis, MO, USA) and amphotericin B
(Sigma-Aldrich, St. Louis, MO, USA) were included as the positive antifungal controls and were tested
from 0.125 to 64 g/ml. The plates were incubated at 35C for 72 h. MIC values were determined using
a spectrophotometer (Bio-Rad Benchmark Plus spectrometer) at 595 nm.
Screening of cryptococcal mutants. We screened the Madhani collection of C. neoformans mutants
available from the Fungal Genetic Stock Center (27) in the search for strains with resistance to our group
of compounds. Briey, the library of 1,265 strains of C. neoformans H99 mutants and H99 wild type were
cultured in YPD broth at 30C overnight. The thiazole compounds 1, 2, 3, and 4 were dissolved in DMSO,
diluted in RPMI 1640, and evaluated for MIC (2, 0.25, 2, and 2 g/ml, respectively) and 6 MICs by adding
200 l to sterile at-bottom 96-well microplates. The inoculum was added using a stamp replicator. The
plates were incubated at 37C for 48 h. All experiments were carried out using controls for H99 and
mutant isolates, and the isolates that did not present similar growth to that of H99 at 30C and 37C were
disregarded. Growth within the wells was evaluated using a spectrophotometer (Bio-Rad Benchmark Plus
spectrometer) at 595 nm. The isolates that exhibited growth in the test compounds were further

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evaluated by broth microdilution testing to determine the MIC of the compounds against the mutant
strain, performed in accordance with the guidelines in the CLSI document M27-A3 (39).
Cellular ROS and RNS. The C. neoformans strain ATCC 24067 (or C. deneoformans) and C. gattii strain
L27/01 (or C. deuterogattii) were cultured in Sabouraud-dextrose agar for 48 h at 37C. Microplate wells
received 100 l inoculum containing 105 CFU/ml in RPMI medium without phenol red, and then the
probes were added. Briey, the probes for ROS (2=,7=-dichlorouorescein diacetate; Sigma-Aldrich, St.
Louis, MO, USA) and RNS (dihydrorhodamine 123) were prepared in methanol and phosphate-buffered
saline (PBS), respectively, at 100 M, and 20 l was distributed to each well for a nal concentration of
10 M. Hydrogen peroxide (4 M), amphotericin B, and itraconazole were included as controls. The drug
treatment solutions were evaluated at the respective MICs, as published by S et al. (26), in nal volumes
of 200 l. The plates were incubated at 37C in the dark. After 1 h, the uorescence was measured with
a uorometer (Synergy 2 SL luminescence microplate reader; BioTek) using excitation at 485 nm and an
emission wavelength of 530 nm (33).
Checkerboard assay. Eight serial 2-fold dilutions of thiazole compounds (0 to 16 g/ml) and
glutathione (0 to 500 g/ml), diamide (2.65 to 170 g/ml), menadione (15.6 to 1000 g/ml), hydrogen
peroxide (0.53 to 34 g/ml), and FeTPPS (3.18 to 204 g/ml) were arrayed in serial concentrations. The
checkerboard was prepared in a microtiter plate for multiple combinations of two molecules. Each row
(x axis) in the plate contained the 2-fold serially diluted concentrations of the thiazole compounds.
Similarly, each column (y axis) in the plate contained 2-fold serially diluted concentrations of the oxide
stress agents. The concentration of the drug combination at which growth was completely inhibited was
taken as the effective MIC for the combination. Inoculum suspensions were performed as described by
S et al. (26), and 100 l of inoculum was added to each well and cultured for 48 to 72 h. FICs were
calculated as the MIC of the combination of drugs divided by the MIC of the drug alone. The same
calculation was performed for all experimental compounds. The FIC index (FICI) was calculated by adding
both FICs, and this was interpreted in the following manner. An effect was classied as synergistic when
its value was 0.5, as no interaction when the value fell within the 0.5 to 4.0 range, and as antagonistic
when its value was 4.0 (40).
Erythrocyte toxicity assay. The erythrocyte toxicity assay was performed as described previously by
Evans et al. (41) with modications. We evaluated the compounds for hemolytic effects using red blood
cells. Human erythrocytes (Rockland Immunochemicals, Pottstown, PA, USA) cells were diluted to 2% in
PBS and 190-l aliquots were added to a 96-well plate. An aliquot of the thiazole compounds, 10 l in
PBS, was added to the wells with red blood cells. The nal concentrations of the investigational
compounds in the wells ranged from 0.125 to 64 g/ml. As a positive control, Triton X-100 was included,
and PBS was used as a negative control; a control with DMSO was also included. The plate was incubated
at 37C for 1 h and then centrifuged at 500 g for 5 min to pellet the intact erythrocytes. One hundred
microliters of the supernatant from each well was transferred to a new 96-well plate. Percentages of
hemolysis were determined by measuring the absorbance at 540 nm (Bio-Rad Benchmark Plus spec-
trometer, Bio-Rad Laboratories, USA) and then normalizing the mean absorbance values to the mean
absorbance of the 1% Triton X-100-treated samples, which represented 100% hemolysis. Two indepen-
dent experiments were performed in triplicates.
Statistical analysis. Statistical analysis was performed using GraphPad Prism statistical software 5.0
(GraphPad Software, Inc., San Diego, CA, USA). A two-way analysis of variance (ANOVA) with Bonferronis
posttest was used to statistically compare the differences among groups, and a 95% condence interval
(CI) was considered in all experiments.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AAC
.02700-16.
SUPPLEMENTAL FILE 1, PDF le, 0.3 MB.

ACKNOWLEDGMENTS
Funding was provided by a grant to B.B.F. and E.M. from the Brown/Brazil Initiative.
This work was also supported by Conselho Nacional de Desenvolvimento e Tecnolgico
(CNPq), Fundao de Amparo Pesquisa Estado de Minas Gerais (FAPEMIG), Coordena-
o de Aperfeioamento de Pessoal de Nvel Superior (CAPES), and Pr-Reitoria de
Pesquisa da UFMG.
We declare no conict of interest.

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