Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents
Identifieur interne : 000821 ( Ncbi/Merge ); précédent : 000820; suivant : 000822Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents
Auteurs : Tatiana Takahasi Komoto [Brésil] ; Tamires Aparecida Bitencourt [Brésil] ; Gabriel Silva [Brésil] ; Rene Oliveira Beleboni [Brésil] ; Mozart Marins [Brésil] ; Ana Lúcia Fachin [Brésil]Source :
- Evidence-based Complementary and Alternative Medicine : eCAM [ 1741-427X ] ; 2015.
Abstract
Url:
DOI: 10.1155/2015/180535
PubMed: 26257814
PubMed Central: 4516844
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PMC:4516844Le document en format XML
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during Coculture on Keratinocytes Exposed to Antifungal Agents</title>
<author><name sortKey="Komoto, Tatiana Takahasi" sort="Komoto, Tatiana Takahasi" uniqKey="Komoto T" first="Tatiana Takahasi" last="Komoto">Tatiana Takahasi Komoto</name>
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<author><name sortKey="Silva, Gabriel" sort="Silva, Gabriel" uniqKey="Silva G" first="Gabriel" last="Silva">Gabriel Silva</name>
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<author><name sortKey="Beleboni, Rene Oliveira" sort="Beleboni, Rene Oliveira" uniqKey="Beleboni R" first="Rene Oliveira" last="Beleboni">Rene Oliveira Beleboni</name>
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<author><name sortKey="Fachin, Ana Lucia" sort="Fachin, Ana Lucia" uniqKey="Fachin A" first="Ana Lúcia" last="Fachin">Ana Lúcia Fachin</name>
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<country xml:lang="fr">Brésil</country>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Gene Expression Response of <italic>Trichophyton rubrum</italic>
during Coculture on Keratinocytes Exposed to Antifungal Agents</title>
<author><name sortKey="Komoto, Tatiana Takahasi" sort="Komoto, Tatiana Takahasi" uniqKey="Komoto T" first="Tatiana Takahasi" last="Komoto">Tatiana Takahasi Komoto</name>
<affiliation wicri:level="2"><nlm:aff id="I1">Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP, Brazil</nlm:aff>
<country xml:lang="fr">Brésil</country>
<wicri:regionArea>Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP</wicri:regionArea>
<placeName><region type="state">État de São Paulo</region>
</placeName>
</affiliation>
</author>
<author><name sortKey="Bitencourt, Tamires Aparecida" sort="Bitencourt, Tamires Aparecida" uniqKey="Bitencourt T" first="Tamires Aparecida" last="Bitencourt">Tamires Aparecida Bitencourt</name>
<affiliation wicri:level="2"><nlm:aff id="I1">Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP, Brazil</nlm:aff>
<country xml:lang="fr">Brésil</country>
<wicri:regionArea>Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP</wicri:regionArea>
<placeName><region type="state">État de São Paulo</region>
</placeName>
</affiliation>
</author>
<author><name sortKey="Silva, Gabriel" sort="Silva, Gabriel" uniqKey="Silva G" first="Gabriel" last="Silva">Gabriel Silva</name>
<affiliation wicri:level="2"><nlm:aff id="I1">Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP, Brazil</nlm:aff>
<country xml:lang="fr">Brésil</country>
<wicri:regionArea>Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP</wicri:regionArea>
<placeName><region type="state">État de São Paulo</region>
</placeName>
</affiliation>
</author>
<author><name sortKey="Beleboni, Rene Oliveira" sort="Beleboni, Rene Oliveira" uniqKey="Beleboni R" first="Rene Oliveira" last="Beleboni">Rene Oliveira Beleboni</name>
<affiliation wicri:level="2"><nlm:aff id="I1">Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP, Brazil</nlm:aff>
<country xml:lang="fr">Brésil</country>
<wicri:regionArea>Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP</wicri:regionArea>
<placeName><region type="state">État de São Paulo</region>
</placeName>
</affiliation>
</author>
<author><name sortKey="Marins, Mozart" sort="Marins, Mozart" uniqKey="Marins M" first="Mozart" last="Marins">Mozart Marins</name>
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<country xml:lang="fr">Brésil</country>
<wicri:regionArea>Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP</wicri:regionArea>
<placeName><region type="state">État de São Paulo</region>
</placeName>
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</author>
<author><name sortKey="Fachin, Ana Lucia" sort="Fachin, Ana Lucia" uniqKey="Fachin A" first="Ana Lúcia" last="Fachin">Ana Lúcia Fachin</name>
<affiliation wicri:level="2"><nlm:aff id="I1">Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP, Brazil</nlm:aff>
<country xml:lang="fr">Brésil</country>
<wicri:regionArea>Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP</wicri:regionArea>
<placeName><region type="state">État de São Paulo</region>
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<series><title level="j">Evidence-based Complementary and Alternative Medicine : eCAM</title>
<idno type="ISSN">1741-427X</idno>
<idno type="e-ISSN">1741-4288</idno>
<imprint><date when="2015">2015</date>
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<front><div type="abstract" xml:lang="en"><p><italic>Trichophyton rubrum</italic>
is the most common causative agent of dermatomycoses worldwide, causing infection in the stratum corneum, nails, and hair. Despite the high prevalence of these infections, little is known about the molecular mechanisms involved in the fungal-host interaction, particularly during antifungal treatment. The aim of this work was to evaluate the gene expression of <italic>T. rubrum</italic>
cocultured with keratinocytes and treated with the flavonoid <italic>trans</italic>
-chalcone and the glycoalkaloid <italic>α</italic>
-solanine. Both substances showed a marked antifungal activity against <italic>T. rubrum</italic>
strain CBS (MIC = 1.15 and 17.8 <italic>µ</italic>
g/mL, resp.). Cytotoxicity assay against HaCaT cells produced IC<sub>50</sub>
values of 44.18 to <italic>trans</italic>
-chalcone and 61.60 <italic>µ</italic>
M to <italic>α</italic>
-solanine. The interaction of keratinocytes with <italic>T. rubrum</italic>
conidia upregulated the expression of genes involved in the glyoxylate cycle, ergosterol synthesis, and genes encoding proteases but downregulated the ABC transporter <italic>Tru</italic>
MDR2 gene. However, both antifungals downregulated the ERG1 and ERG11, metalloprotease 4, serine proteinase, and <italic>Tru</italic>
MDR2 genes. Furthermore, the <italic>trans</italic>
-chalcone downregulated the genes involved in the glyoxylate pathway, isocitrate lyase, and citrate synthase. Considering the urgent need for more efficient and safer antifungals, these results contribute to a better understanding of fungal-host interactions and to the discovery of new antifungal targets.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Evid Based Complement Alternat Med</journal-id>
<journal-id journal-id-type="iso-abbrev">Evid Based Complement Alternat Med</journal-id>
<journal-id journal-id-type="publisher-id">ECAM</journal-id>
<journal-title-group><journal-title>Evidence-based Complementary and Alternative Medicine : eCAM</journal-title>
</journal-title-group>
<issn pub-type="ppub">1741-427X</issn>
<issn pub-type="epub">1741-4288</issn>
<publisher><publisher-name>Hindawi Publishing Corporation</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">26257814</article-id>
<article-id pub-id-type="pmc">4516844</article-id>
<article-id pub-id-type="doi">10.1155/2015/180535</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Gene Expression Response of <italic>Trichophyton rubrum</italic>
during Coculture on Keratinocytes Exposed to Antifungal Agents</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Komoto</surname>
<given-names>Tatiana Takahasi</given-names>
</name>
<xref ref-type="aff" rid="I1"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Bitencourt</surname>
<given-names>Tamires Aparecida</given-names>
</name>
<xref ref-type="aff" rid="I1"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Silva</surname>
<given-names>Gabriel</given-names>
</name>
<xref ref-type="aff" rid="I1"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Beleboni</surname>
<given-names>Rene Oliveira</given-names>
</name>
<xref ref-type="aff" rid="I1"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Marins</surname>
<given-names>Mozart</given-names>
</name>
<xref ref-type="aff" rid="I1"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Fachin</surname>
<given-names>Ana Lúcia</given-names>
</name>
<xref ref-type="aff" rid="I1"></xref>
<xref ref-type="corresp" rid="cor1"><sup>*</sup>
</xref>
</contrib>
</contrib-group>
<aff id="I1">Unidade de Biotecnologia, Universidade de Ribeirão Preto, Avenida Costábile Romano 2201, 14096-900 Ribeirão Preto, SP, Brazil</aff>
<author-notes><corresp id="cor1">*Ana Lúcia Fachin: <email>afachin@unaerp.br</email>
</corresp>
<fn fn-type="other"><p>Academic Editor: Giuseppe Esposito</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><year>2015</year>
</pub-date>
<pub-date pub-type="epub"><day>14</day>
<month>7</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>14</day>
<month>7</month>
<year>2015</year>
</pub-date>
<pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
. </pmc-comment>
<volume>2015</volume>
<elocation-id>180535</elocation-id>
<history><date date-type="received"><day>27</day>
<month>4</month>
<year>2015</year>
</date>
<date date-type="accepted"><day>25</day>
<month>6</month>
<year>2015</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2015 Tatiana Takahasi Komoto et al.</copyright-statement>
<copyright-year>2015</copyright-year>
<license license-type="open-access"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
</license>
</permissions>
<abstract><p><italic>Trichophyton rubrum</italic>
is the most common causative agent of dermatomycoses worldwide, causing infection in the stratum corneum, nails, and hair. Despite the high prevalence of these infections, little is known about the molecular mechanisms involved in the fungal-host interaction, particularly during antifungal treatment. The aim of this work was to evaluate the gene expression of <italic>T. rubrum</italic>
cocultured with keratinocytes and treated with the flavonoid <italic>trans</italic>
-chalcone and the glycoalkaloid <italic>α</italic>
-solanine. Both substances showed a marked antifungal activity against <italic>T. rubrum</italic>
strain CBS (MIC = 1.15 and 17.8 <italic>µ</italic>
g/mL, resp.). Cytotoxicity assay against HaCaT cells produced IC<sub>50</sub>
values of 44.18 to <italic>trans</italic>
-chalcone and 61.60 <italic>µ</italic>
M to <italic>α</italic>
-solanine. The interaction of keratinocytes with <italic>T. rubrum</italic>
conidia upregulated the expression of genes involved in the glyoxylate cycle, ergosterol synthesis, and genes encoding proteases but downregulated the ABC transporter <italic>Tru</italic>
MDR2 gene. However, both antifungals downregulated the ERG1 and ERG11, metalloprotease 4, serine proteinase, and <italic>Tru</italic>
MDR2 genes. Furthermore, the <italic>trans</italic>
-chalcone downregulated the genes involved in the glyoxylate pathway, isocitrate lyase, and citrate synthase. Considering the urgent need for more efficient and safer antifungals, these results contribute to a better understanding of fungal-host interactions and to the discovery of new antifungal targets.</p>
</abstract>
</article-meta>
</front>
<body><sec id="sec1"><title>1. Introduction</title>
<p><italic>Trichophyton rubrum</italic>
is the most prevalent dermatophyte, accounting for about 80% of dermatomycoses that affect keratinized tissues such as skin, hair, and nails [<xref rid="B1" ref-type="bibr">1</xref>
]. Moreover, these infections play an important role in the scenario of opportunistic infections in immunocompromised patients [<xref rid="B2" ref-type="bibr">2</xref>
]. Despite the high prevalence, little is known about the molecular mechanisms involved in the fungal pathogen-host interaction, much less when the infection is treated with antifungal agents.</p>
<p>For a better understanding of this process, culture medium supplemented with keratin and coculture of<italic> T. rubrum</italic>
on human keratinocytes have been used to mimic skin infection with dermatophytes. The results of gene expression analysis have shown the involvement of important genes in the process of infection, which may contribute to the discovery of new antifungal targets and subsequent new antifungal agents [<xref rid="B3" ref-type="bibr">3</xref>
–<xref rid="B5" ref-type="bibr">5</xref>
].</p>
<p>The understanding of the mode of action of antifungal agents, such as the flavonoid<italic> trans</italic>
-chalcone and the glycoalkaloid <italic>α</italic>
-solanine, is important since these compounds can act on different specific targets of the fungus, a fact that could render these compounds more effective against resistant strains. In a screening study of natural compounds inhibiting the enzyme fatty acid synthase,<italic> trans</italic>
-chalcone was the flavonoid exhibiting the best antifungal activity against<italic> T. rubrum</italic>
strain MYA 3108, reducing ergosterol levels and affecting fatty acid synthesis [<xref rid="B6" ref-type="bibr">6</xref>
]. Pinto et al., 2011, [<xref rid="B7" ref-type="bibr">7</xref>
] demonstrated expressive antifungal activity of glycoalkaloids. In fact, solamargine and solasonine exhibited strong activity against dermatophytic fungi, including<italic> T. rubrum</italic>
. A major mode of antifungal action of glycoalkaloids is the alteration of cell membrane integrity through binding to 3-beta-hydroxy sterol groups, causing an increase in membrane permeability, pore formation, and the consequent breakdown and loss of cell integrity [<xref rid="B8" ref-type="bibr">8</xref>
].</p>
<p>The aim of this work was to evaluate the gene expression modulation of target genes in<italic> T. rubrum</italic>
in response to coculture on human keratinocytes and coculture exposed to the antifungal agents<italic> trans</italic>
-chalcone and <italic>α</italic>
-solanine. To our knowledge, this is the first study describing the modulation of gene expression in<italic> T. rubrum</italic>
during the interaction with keratinocytes exposed to antifungal compounds.</p>
</sec>
<sec id="sec2"><title>2. Methods</title>
<sec id="sec2.1"><title>2.1. Strains, Media, and Growth Conditions</title>
<p><italic>Trichophyton rubrum strain CBS 118892 </italic>
was cultured on Sabouraud dextrose agar (Oxoid, Hampshire, England) for 15 days at 28°C until suitable conidiation.</p>
</sec>
<sec id="sec2.2"><title>2.2. Chemicals</title>
<p><italic>Trans</italic>
-chalcone and <italic>α</italic>
-solanine were purchased from Sigma-Aldrich (St. Louis, MO, USA) and diluted in 10% aqueous DMSO. Terbinafine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and diluted in 5% aqueous DMSO. The final concentration of DMSO used in the antifungal and coculture assays was fixed at a maximum of 0.5%. Terbinafine was used as positive control (commercial antifungal involved in ergosterol synthesis inhibition) [<xref rid="B9" ref-type="bibr">9</xref>
]. Negative controls consisted of DMSO without the tested compounds at a final concentration of 0.5%.</p>
</sec>
<sec id="sec2.3"><title>2.3. Determination of Minimum Inhibitory Concentration and Minimum Fungicidal Concentration</title>
<p>Susceptibility of<italic> T. rubrum</italic>
strain CBS 118892 (1.0 to 3 × 10<sup>5</sup>
CFU/mL) was assessed by determining the minimum inhibitory concentration (MIC) of different concentrations of<italic> trans</italic>
-chalcone, <italic>α</italic>
-solanine, and terbinafine, using the M38-A microdilution technique proposed by the Clinical and Laboratory Standards Institute [<xref rid="B10" ref-type="bibr">10</xref>
] and described by Bitencourt et al. [<xref rid="B6" ref-type="bibr">6</xref>
] The MIC<sub>100</sub>
was defined as the lowest concentration of the drug that completely inhibited the growth of<italic> T. rubrum</italic>
. Microtiter trays were incubated at 28°C and MICs were recorded after 7 days of incubation.</p>
<p>The minimum fungicidal concentration (MFC) was determined from the results of the MIC tests. An aliquot of 100 <italic>μ</italic>
L of the respective wells in which the tested concentrations of the antifungals showed inhibition was seeded in duplicate onto the surface of plates containing Sabouraud dextrose agar and incubated with<italic> T. rubrum</italic>
for 24 h at 28°C. The MFC was defined as the lowest concentration of the tested drug that inhibited fungal growth. The MIC and MFC assays were carried out in three independent experiments performed in triplicate.</p>
</sec>
<sec id="sec2.4"><title>2.4. Keratinocytes, Media, and Growth Conditions</title>
<p>The cell line of immortalized human keratinocytes (HaCaT) was provided by Professor Dr. Ana Paula de Souza Pardo (University of Dentistry of Piracicaba, UNICAMP) and Professor Dr. João Ernesto de Carvalho (Division of Pharmacology and Toxicology, Multidisciplinary Center for Chemical, Biological and Agricultural Research, CPQBA). Keratinocytes were cultured in RPMI medium (Sigma) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO<sub>2</sub>
. Penicillin (100 U/mL) and streptomycin (100 <italic>μ</italic>
g/mL) were added to the medium.</p>
</sec>
<sec id="sec2.5"><title>2.5. Cytotoxicity Testing of the Antifungal Agents</title>
<p>The keratinocytes were cultured in RPMI medium at 37°C in a humidified atmosphere containing 5% CO<sub>2</sub>
until 90% confluence. The cells were trypsinized (0.15% trypsin and 0.02% EDTA), counted in a hemocytometer (2.5 × 10<sup>5</sup>
cells/well), and incubated in a 96-well plate for 24 h. A stock solution (10 mg/mL) of the antifungal agents was prepared in 10% DMSO and each agent was directly diluted in RPMI medium. After addition of the antifungals in fresh medium, the cells were cultured at 37°C in a 5% CO<sub>2</sub>
atmosphere for 24 h and cytotoxicity was analyzed by the MTT assay [<xref rid="B11" ref-type="bibr">11</xref>
], with modifications, as described by Rizo et al., 2013 [<xref rid="B12" ref-type="bibr">12</xref>
]. Briefly, 20 <italic>μ</italic>
L MTT/well (5 mg/mL in Hanks solution) was added to the 96-well plate and incubated for 4 h under the same conditions. All treatments were carried out in triplicate. The formazan dye was extracted with 200 <italic>μ</italic>
L isopropanol and quantified spectrophotometrically at 550 nm (BioTek ELx800, Singapore). The absorbance of untreated cells was used as a reference.</p>
</sec>
<sec id="sec2.6"><title>2.6. Coculture Conditions and Exposure to Natural Compounds</title>
<p>Keratinocytes were collected, washed, and counted in a hemocytometer. A total of 2.5 × 10<sup>5</sup>
cells/mL were plated on RPMI plus 2% FBS in 250 mL tissue culture flasks and cultured for 24 h at 37°C in 5% CO<sub>2</sub>
. The<italic> T. rubrum</italic>
solution (1 × 10<sup>7</sup>
conidia/mL) was grown in 5 mL liquid Sabouraud medium for 7 h under gentle shaking. The keratinocyte culture was recovered by centrifugation, washed in saline, and resuspended in RPMI plus 2% FBS. The conidia solution and 0.288 <italic>μ</italic>
M<italic> trans</italic>
-chalcone or 4.32 <italic>μ</italic>
M <italic>α</italic>
-solanine or 0.0162 <italic>μ</italic>
M terbinafine were added to the keratinocyte cultures and incubated for 24 h at 37°C in 5% CO<sub>2</sub>
. One control and one coculture flask per treatment were reserved for staining with May-Grünwald and Giemsa for verification of infection. Fungi and human cells were recovered by scraping and centrifuged at 1,730 g for 10 min. RNA was prepared directly from the recovered cells as described in the next item.</p>
</sec>
<sec id="sec2.7"><title>2.7. RNA Isolation</title>
<p>The coculture was treated with lysis solution (20 mg/mL of lysing enzymes from<italic> Trichoderma harzianum</italic>
purchased from Sigma-Aldrich; 0.7 M KCl and 1 M MgSO<sub>4</sub>
, pH 6.8) for 1 h at 28°C under gentle shaking, followed by centrifugation at 1,000 g for 10 min. Total RNA was extracted from the coculture using the Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). The RNA preparations were analyzed for the absence of proteins and phenol by UV spectrophotometry, while RNA integrity was confirmed by denaturing electrophoresis. The RNA samples were used for RT-PCR. Uninfected cultured keratinocytes and<italic> T. rubrum</italic>
conidia were included as controls.</p>
</sec>
<sec id="sec2.8"><title>2.8. Quantitative RT-PCR</title>
<p>A set of nine genes were selected to evaluate the level of gene expression in<italic> T. rubrum</italic>
cocultivated with keratinocytes in presence or absence of natural compounds by quantitative RT-PCR (<xref ref-type="table" rid="tab1">Table 1</xref>
). The genes were chosen based on their potential as antifungal targets such as glyoxylate pathway [<xref rid="B13" ref-type="bibr">13</xref>
], ergosterol biosynthesis [<xref rid="B14" ref-type="bibr">14</xref>
, <xref rid="B15" ref-type="bibr">15</xref>
], response to drugs [<xref rid="B16" ref-type="bibr">16</xref>
], and proteases related to the infection process [<xref rid="B5" ref-type="bibr">5</xref>
]. Complementary DNA was synthesized from 500 ng total RNA in a 20 <italic>μ</italic>
L reaction volume using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas). Quantitative RT-PCR experiments were performed in duplicate using the SYBR Taq ReadyMix Kit (Sigma) in an Mx3300 QPCR system (Stratagene). The cycling conditions were initial denaturation at 94°C for 10 min, followed by 40 cycles at 94°C for 2 min, at 60°C for 60 s, and at 72°C for 1 min. A dissociation curve was constructed at the end of each PCR cycle to verify single product amplification. Gene expression levels were calculated by the comparative Ct method using beta-tubulin as normalizer gene [<xref rid="B17" ref-type="bibr">17</xref>
] and<italic> T. rubrum</italic>
conidia and<italic> T. rubrum</italic>
coculture without the tested drugs as reference. The results are reported as the mean ± standard deviation of two experiments.</p>
</sec>
<sec id="sec2.9"><title>2.9. Data Analysis</title>
<p>The cytotoxicity results were analyzed by two-way ANOVA (<italic>p</italic>
< 0.05). IC<sub>50</sub>
values were calculated by nonlinear regression analysis.</p>
</sec>
</sec>
<sec id="sec3"><title>3. Results </title>
<sec id="sec3.1"><title>3.1. Antifungal and Cytotoxic Activity of the Inhibitory Compounds</title>
<p><italic>Trans</italic>
-chalcone (MIC = 1.15 <italic>μ</italic>
M) and <italic>α</italic>
-solanine (MIC = 17.28 <italic>μ</italic>
M) exhibited marked inhibitory activity against<italic> T. rubrum</italic>
. The compounds <italic>α</italic>
-solanine and terbinafine exerted fungicidal activity against the CBS 118892 strain, while<italic> trans</italic>
-chalcone exhibited fungistatic activity (<xref ref-type="table" rid="tab2">Table 2</xref>
). Cytotoxicity was evaluated by culturing keratinocytes for 24 h in the presence of the tested drugs and the IC<sub>50</sub>
results are shown in <xref ref-type="table" rid="tab2">Table 2</xref>
.<italic> Trans</italic>
-chalcone and <italic>α</italic>
-solanine exhibited greater cytotoxicity against the mammalian cells than the commercial drug terbinafine but did not compromise interpretation of the results of the coculture assays of<italic> T. rubrum</italic>
on keratinocytes.</p>
</sec>
<sec id="sec3.2"><title>3.2. Coculture of<italic> T. rubrum</italic>
Exposed to the Natural Compounds</title>
<p><xref ref-type="fig" rid="fig1">Figure 1</xref>
illustrates the results obtained for coculture of<italic> T. rubrum</italic>
on keratinocytes and in the presence of the drugs tested. The hyphae were found to be fragmented in the presence of <italic>α</italic>
-solanine. The same was<italic> observed</italic>
for terbinafine, while hyphae were twisted in the presence of<italic> trans</italic>
-chalcone.</p>
</sec>
<sec id="sec3.3"><title>3.3. Modulation of Gene Expression</title>
<sec id="sec3.3.1"><title>3.3.1. Response of<italic> T. rubrum</italic>
to Keratinocytes</title>
<p><xref ref-type="fig" rid="fig2">Figure 2(a)</xref>
shows the effect of keratinocytes on gene expression modulation in<italic> T. rubrum</italic>
. Significant induction of the genes involved in the glyoxylate cycle and ergosterol synthesis and of the genes encoding proteases was observed. In contrast, the<italic> Tru</italic>
MDR2 gene, involved in multiple drug resistance, was repressed.</p>
</sec>
<sec id="sec3.3.2"><title>3.3.2. Response of<italic> T. rubrum</italic>
to Keratinocytes Exposed to the Antifungal Agents</title>
<p>The results of gene expression analysis of the<italic> T. rubrum</italic>
coculture exposed to the antifungal agents are shown in <xref ref-type="fig" rid="fig2">Figure 2(b)</xref>
. The genes involved in the glyoxylate pathway, isocitrate lyase and citrate synthase, were induced in the presence of <italic>α</italic>
-solanine and terbinafine. In contrast,<italic> trans</italic>
-chalcone repressed these target genes. With respect to the genes involved in ergosterol synthesis,<italic> trans</italic>
-chalcone, <italic>α</italic>
-solanine, and terbinafine inhibited the expression of the ERG1 and ERG11 genes. The ERG6 gene was induced by terbinafine, while neither<italic> trans</italic>
-chalcone nor <italic>α</italic>
-solanine modulated the expression of this gene.</p>
<p>Regarding the modulation of the genes encoding proteases, the flavonoid<italic> trans</italic>
-chalcone repressed the expression of serine proteinase (subtilisin). Furthermore, a strong induction of this gene was observed in the presence of terbinafine and, to a lesser extent, in the presence of <italic>α</italic>
-solanine. On the other hand, repression of the MEP4 gene was observed when the fungus was cultured in the presence of terbinafine, <italic>α</italic>
-solanine, and<italic> trans</italic>
-chalcone. Terbinafine induced the expression of the<italic> Tru</italic>
MDR2 gene. In contrast,<italic> trans</italic>
-chalcone and <italic>α</italic>
-solanine repressed the expression of this gene (<xref ref-type="fig" rid="fig2">Figure 2(b)</xref>
).</p>
</sec>
</sec>
</sec>
<sec id="sec4"><title>4. Discussion </title>
<p>Chalcones are an important group of natural compounds that possess a broad range of biological activities, including antifungal activity [<xref rid="B18" ref-type="bibr">18</xref>
–<xref rid="B22" ref-type="bibr">22</xref>
]. In this respect, Boeck et al. [<xref rid="B23" ref-type="bibr">23</xref>
] showed the chalcones tested in their study were effective against the series of dermatophytes<italic> Microsporum canis</italic>
,<italic> Epidermophyton floccosum</italic>
, and<italic> Microsporum gypseum</italic>
. The chalcone 3-(2-chlorophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one inhibited all clinical strains of<italic> T. rubrum</italic>
isolated from skin infections of immunocompromised patients, with MIC<sub>90</sub>
ranging from 12.5 to 25 <italic>μ</italic>
g/mL. In contrast, the authors observed that none of the chalcones tested was effective against the yeast species<italic> Candida albicans</italic>
,<italic> Saccharomyces cerevisiae</italic>
, or<italic> Cryptococcus neoformans</italic>
, nor against the filamentous fungi<italic> Aspergillus niger</italic>
,<italic> A. fumigates</italic>
, or<italic> A. flavus</italic>
. Furthermore, results from our research group showed that<italic> trans</italic>
-chalcone exerted no antifungal activity against aflatoxigenic<italic> Aspergillus flavus</italic>
strains (MIC<sub>100</sub>
> 1,000 <italic>μ</italic>
g/mL) (unpublished data). Also in the present study,<italic> trans</italic>
-chalcone exhibited marked activity against<italic> T. rubrum</italic>
(MIC = 1.15 <italic>μ</italic>
M), suggesting that this flavonoid exhibits specific activity against dermatophytes, which is supported by previous data.</p>
<p><italic>α</italic>
-Solanine was also effective against<italic> T. rubrum</italic>
(MIC = 17.28 <italic>μ</italic>
g/mL). Much of the biological activity of glycoalkaloids isolated from different<italic> Solanum</italic>
species is due to their ability to disrupt sterol-containing membranes in erythrocytes and fungal protoplasts [<xref rid="B24" ref-type="bibr">24</xref>
]. Other mechanisms of action have been reported for two important glycoalkaloids found in potatoes (<italic>α</italic>
-solanine and chaconine), which exert antifungal activity by inhibiting sporulation and hyphal growth, in addition to causing hyphal distortion. Furthermore, data suggest that these glycoalkaloids can inhibit the biosynthesis of essential compounds or even act as antimetabolites [<xref rid="B25" ref-type="bibr">25</xref>
].</p>
<p>In the present study, the isocitrate lyase and citrate synthase genes involved in the glyoxylate pathway were induced when<italic> T. rubrum</italic>
was cocultured on keratinocytes, suggesting that this pathway is important for the process of infection caused by dermatophytes. In addition to the absence of the glyoxylate cycle in mammals, metabolic pathways are currently being explored as potential antifungal targets since they contribute to metabolic flexibility by permitting microbial cells to survive in nutrient-limited host niches during infection [<xref rid="B26" ref-type="bibr">26</xref>
]. The glyoxylate cycle permits some microorganisms to synthesize glucose from lipids and other alternative carbon sources. Since dermatophytes specifically infect epidermal structures rich in keratin and lipids, the functional characterization of genes involved in the glyoxylate cycle should contribute to the understanding of the virulence of these fungi in the host and also to the discovery of interesting pharmacological targets [<xref rid="B26" ref-type="bibr">26</xref>
]. The importance of three enzymes of the glyoxylate cycle, isocitrate lyase, malate synthase, and citrate synthase for fungal virulence has been demonstrated in<italic> Candida albicans</italic>
[<xref rid="B27" ref-type="bibr">27</xref>
]. In<italic> C. albicans</italic>
, metabolic flexibility is important for adaptation and survival of the fungus in host niches and also for biological parameters of pathogenicity and virulence [<xref rid="B13" ref-type="bibr">13</xref>
, <xref rid="B28" ref-type="bibr">28</xref>
–<xref rid="B30" ref-type="bibr">30</xref>
].</p>
<p>The ERG1 and ERG11 genes encode, respectively, the squalene epoxidase and lanosterol C-14 <italic>α</italic>
-demethylase, the main target of terbinafine and currently used azolic antifungal agents [<xref rid="B9" ref-type="bibr">9</xref>
, <xref rid="B31" ref-type="bibr">31</xref>
]. In the present study, <italic>α</italic>
-solanine and<italic> trans</italic>
-chalcone inhibited the expression of the ERG1 and ERG11 genes. In fact,<italic> trans</italic>
-chalcone seems to interfere with ergosterol synthesis in the cell, as indicated by previous studies showing a reduction of 74% and 77% in ergosterol levels when a wild-type and a mutant (Δ<italic>Tru</italic>
MDR2) strain of<italic> T. rubrum</italic>
were cultured in the presence of 0.5 MIC of<italic> trans</italic>
-chalcone. At the MIC of<italic> trans</italic>
-chalcone, ergosterol content was reduced by 100% in the two strains [<xref rid="B6" ref-type="bibr">6</xref>
].</p>
<p>Neither<italic> trans</italic>
-chalcone nor <italic>α</italic>
-solanine repressed the expression of the ERG6 gene. This finding might be due to repression of the ERG11 gene which encodes the main enzyme responsible for the first step of ergosterol biosynthesis, impairing activation of the remaining pathway. The ERG6 gene (sterol methyltransferase) encodes an enzyme that participates in the ergosterol pathway, converting zymosterol to fecosterol. However, data suggest that ERG6 is not the main target gene of the pathway, since no differences in vegetative growth were observed in an ERG6 null mutant strain of<italic> Saccharomyces cerevisiae</italic>
and neither meiosis nor sporulation was affected, demonstrating that this gene is not essential for this fungus. However, this gene can affect different phenotypic characteristics such as membrane permeability and fluidity, sensitivity to cycloheximide, the capacity of genetic transformation, and tryptophan uptake [<xref rid="B32" ref-type="bibr">32</xref>
].</p>
<p>Infection with dermatophytes requires installation of the pathogen and the adherence of arthroconidia to the epithelial surface, followed by their germination and penetration of hyphae into the stratum corneum of skin. After adhesion, dermatophytes require nutrients for their development and survival. However, it is known that membrane permeability prevents carbon, nitrogen, phosphorus, and sulfur compounds from being transported into cells, and enzymes able to degrade and transform these macromolecules into smaller molecules are therefore necessary. Different enzymes such as proteases, lipases, elastases, and collagenases are produced during this process [<xref rid="B33" ref-type="bibr">33</xref>
, <xref rid="B34" ref-type="bibr">34</xref>
].</p>
<p>The main families of proteases secreted by fungi are subtilisins (serine proteases) and fungalisins (metalloproteases) [<xref rid="B35" ref-type="bibr">35</xref>
]. Studies on<italic> Microsporum canis</italic>
and<italic> T. rubrum</italic>
have shown that metalloproteases and serine proteinases are potential virulence-related factors in dermatophytes [<xref rid="B36" ref-type="bibr">36</xref>
, <xref rid="B37" ref-type="bibr">37</xref>
]. Zhang et al. [<xref rid="B38" ref-type="bibr">38</xref>
] obtained five metalloprotease mutants by<italic> Agrobacterium tumefaciens</italic>
-mediated transformation. The authors observed a significant reduction in virulence in the MEP4 and MEP5 null mutant strains. The MEP3 mutant strain showed virulence similar to that of the wild-type strain.</p>
<p>Another important protease that was found to be modulated in the present study was serine proteinase, which belongs to the subtilisin family and, like metalloproteases, is classified as an endoprotease [<xref rid="B39" ref-type="bibr">39</xref>
, <xref rid="B40" ref-type="bibr">40</xref>
]. Serine proteinases were the proteases most frequently isolated from culture supernatants during the growth of<italic> T. rubrum</italic>
[<xref rid="B41" ref-type="bibr">41</xref>
]. This protease was also detected in another experiment conducted by our group, mimicking skin in minimal culture medium containing keratin and elastin (data not shown). Induction of this protease was observed compared to the control, as well as in the present study in which the result was validated in a keratinocyte model of infection.</p>
<p>Involvement of the<italic> Tru</italic>
MDR2 transporter in the transport of terbinafine, as well as of ethidium bromide compounds and 4NQO, has been demonstrated by Fachin et al. [<xref rid="B16" ref-type="bibr">16</xref>
] in assays testing the susceptibility of the Δ<italic>Tru</italic>
MDR2 mutant to these drugs. Indeed, Zhang et al. [<xref rid="B42" ref-type="bibr">42</xref>
] observed in the microarray experiment that terbinafine induced the transcription of multidrug-resistance (MDR) genes, including DW686642,<italic> Tru</italic>
MDR2, and MDR1 of<italic> T. rubrum</italic>
. In the present study, despite the fact that terbinafine showed antifungal activity and low cytotoxicity against the cells of keratinocytes, terbinafine induced the expression of the<italic> Tru</italic>
MDR2 gene. In contrast, the expression of the<italic> TruMDR2</italic>
gene was repressed in the presence of<italic> trans</italic>
-chalcone and <italic>α</italic>
-solanine. This finding makes these compounds even more interesting since inhibition of the efflux of these drugs by the cell may impair the development of resistance to these inhibitors in<italic> T. rubrum</italic>
. Furthermore, Bitencourt et al. [<xref rid="B6" ref-type="bibr">6</xref>
] observed that the Δ<italic>Tru</italic>
MDR2 mutant strain is more sensitive to<italic> trans</italic>
-chalcone (MIC = 1.9 <italic>μ</italic>
g/mL) than another<italic> T. rubrum</italic>
strain (MYA3108) (MIC = 7.5 <italic>μ</italic>
g/mL). Considering the involvement of the<italic> TruMDR2</italic>
gene both in the mechanism of drug transport [<xref rid="B16" ref-type="bibr">16</xref>
] and in the pathogenicity of<italic> T. rubrum</italic>
[<xref rid="B41" ref-type="bibr">41</xref>
], this gene is an important target to screen for novel antifungal agents.</p>
</sec>
<sec id="sec5"><title>5. Conclusion</title>
<p><italic>Trans</italic>
-chalcone exhibited significant antifungal activity and was moderately cytotoxic to keratinocytes. The interaction between<italic> T. rubrum</italic>
and keratinocytes induced the expression of genes involved in glyoxylate cycle and ergosterol synthesis, as well as genes encoding proteases. However, gene expression analysis demonstrated that<italic> trans</italic>
-chalcone repressed the expression of virulence-related genes (isocitrate lyase, citrate synthase, and MEP4), target genes of commercial antifungal (ERG1 and ERG11), and also the<italic> Tru</italic>
MDR2 transporter involved in multiple drug resistance. These findings render<italic> trans</italic>
-chalcone an interesting compound in future studies, especially its application to resistant strains.</p>
</sec>
</body>
<back><ack><title>Acknowledgments</title>
<p>The authors thank the staff of the Biotechnology Unit, UNAERP, for general support, K. Markendorf for English revision, and Professor Nilce M. Martinez-Rossi for kindly providing the<italic> T. rubrum</italic>
strain CBS. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grants 2012/03845-9 and 2014/23481-3). CAPES and FAPESP fellowships were granted to Tatiana Takahasi Komoto and Tamires Aparecida Bitencourt (Process no. 2012/02920-7), respectively.</p>
</ack>
<sec sec-type="conflict"><title>Conflict of Interests</title>
<p>The authors declare that they have no conflict of interests.</p>
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<floats-group><fig id="fig1" orientation="portrait" position="float"><label>Figure 1</label>
<caption><p>Coculture of<italic> Trichophyton rubrum</italic>
in HaCaT cells exposed to antifungal agents. The<italic> T. rubrum</italic>
solution (1 × 10<sup>7</sup>
conidia/mL) was preincubated in Sabouraud medium for 7 h, 2.5 × 10<sup>5</sup>
keratinocytes/mL were added, and the culture was exposed to the antifungal agents for 24 h. (a) Control; (b) 0.0162 <italic>μ</italic>
M terbinafine; (c) 4.32 <italic>μ</italic>
M <italic>α</italic>
-solanine; (d) 0.288 <italic>μ</italic>
M<italic> trans</italic>
-chalcone.</p>
</caption>
<graphic xlink:href="ECAM2015-180535.001"></graphic>
</fig>
<fig id="fig2" orientation="portrait" position="float"><label>Figure 2</label>
<caption><p>(a) Modulation of gene expression in<italic> Trichophyton rubrum</italic>
cocultured with HaCaT cells for 24 h. The reference used was the conidia solution. (b) Modulation of gene expression in<italic> T. rubrum</italic>
cocultured with HaCaT cells exposed to 0.0162 <italic>μ</italic>
M terbinafine, 4.32 <italic>μ</italic>
M <italic>α</italic>
-solanine, and 0.288 <italic>μ</italic>
M<italic> trans</italic>
-chalcone for 24 h. The reference used was the coculture without antifungals.</p>
</caption>
<graphic xlink:href="ECAM2015-180535.002"></graphic>
</fig>
<table-wrap id="tab1" orientation="portrait" position="float"><label>Table 1</label>
<caption><p>Primers used for RT- PCR.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Gene</th>
<th align="left" rowspan="1" colspan="1">Sequence</th>
<th align="center" rowspan="1" colspan="1">Size (bp) </th>
<th align="center" rowspan="1" colspan="1">Reference</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1">Isocitrate lyase </td>
<td align="left" rowspan="1" colspan="1">F: 5′-ACAACCTCTCGCCTTCATTC-3′ <break></break>
R: 5′-GGTCAGATATCAGGGCAGTTG-3′</td>
<td align="center" rowspan="1" colspan="1">144</td>
<td align="center" rowspan="1" colspan="1">This paper </td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Citrate synthase</td>
<td align="left" rowspan="1" colspan="1">F: 5′-GAGGAGGGTATTCGCTTCCG-3′ <break></break>
R: 5′-CGAACTTGTTGCTCGGTTGG-3′</td>
<td align="center" rowspan="1" colspan="1">143</td>
<td align="center" rowspan="1" colspan="1">This paper </td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Metalloprotease MEP4 </td>
<td align="left" rowspan="1" colspan="1">F: 5′-GCATGGACTTATGCTTGCGG-3′ <break></break>
R: 5′-TGGATATCTGGGGAAGGCGA-3′</td>
<td align="center" rowspan="1" colspan="1">131</td>
<td align="center" rowspan="1" colspan="1">This paper </td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Serine proteinase </td>
<td align="left" rowspan="1" colspan="1">F: 5′-GCTGGCTCCAATCTACTCATAC-3′ <break></break>
R: 5′-CGCTGTATCCCTTCATCTTGT-3′</td>
<td align="center" rowspan="1" colspan="1">105</td>
<td align="center" rowspan="1" colspan="1">This paper </td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">ERG1</td>
<td align="left" rowspan="1" colspan="1">F: 5′-GTGAAGATACCTTTCCCTAGCG-3′ <break></break>
R: 5′-TTATGGTAGAAACGGCCTTGG-3′</td>
<td align="center" rowspan="1" colspan="1">148</td>
<td align="center" rowspan="1" colspan="1">This paper </td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">ERG6</td>
<td align="left" rowspan="1" colspan="1">F: 5′-CTCTGGCAAGACACGAACAC-3′ <break></break>
R: 5′-CCTTGCAGCCGGTGAAGG-3′</td>
<td align="center" rowspan="1" colspan="1">126</td>
<td align="center" rowspan="1" colspan="1">[<xref rid="B6" ref-type="bibr">6</xref>
]</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">ERG 11</td>
<td align="left" rowspan="1" colspan="1">F: 5′-CACTTCCTTGCCCTGTAGAGATC-3′ <break></break>
R: 5′-GGAGTTTTCAATGTCAGCAAGGTTT-3′</td>
<td align="center" rowspan="1" colspan="1">78</td>
<td align="center" rowspan="1" colspan="1">[<xref rid="B15" ref-type="bibr">15</xref>
]</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><italic>Tru</italic>
MDR2</td>
<td align="left" rowspan="1" colspan="1">F: 5′-GCACTGATCTGCAGCTCGACC-3′ <break></break>
R: 5′-CCAACGTCATCCTCCCAGAC-3′</td>
<td align="center" rowspan="1" colspan="1">91</td>
<td align="center" rowspan="1" colspan="1">[<xref rid="B16" ref-type="bibr">16</xref>
]</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Beta-tubulin</td>
<td align="left" rowspan="1" colspan="1">F: 5′-AACATGATGGCTGCCACTGA-3′ <break></break>
R: 5′-AAGATGGCAGAGCAGGTAAGGT-3′</td>
<td align="center" rowspan="1" colspan="1">253</td>
<td align="center" rowspan="1" colspan="1">[<xref rid="B17" ref-type="bibr">17</xref>
]</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="tab2" orientation="portrait" position="float"><label>Table 2</label>
<caption><p>Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the antifungal agents against <italic>Trichophyton rubrum</italic>
strain CBS 118892 (<italic>µ</italic>
M). IC<sub>50</sub>
(<italic>µ</italic>
M) of the antifungal agents in a human keratinocyte cell line (HaCaT).</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Compound</th>
<th align="center" rowspan="1" colspan="1">MIC<break></break>
</th>
<th align="center" rowspan="1" colspan="1">MFC<break></break>
</th>
<th align="center" rowspan="1" colspan="1">IC<sub>50</sub>
</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1"><italic>Trans</italic>
-chalcone</td>
<td align="center" rowspan="1" colspan="1">1.15</td>
<td align="center" rowspan="1" colspan="1">2.30</td>
<td align="center" rowspan="1" colspan="1">44.18</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><italic>α</italic>
-Solanine</td>
<td align="center" rowspan="1" colspan="1">17.28</td>
<td align="center" rowspan="1" colspan="1">17.28</td>
<td align="center" rowspan="1" colspan="1">61.60</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Terbinafine</td>
<td align="center" rowspan="1" colspan="1">0.065</td>
<td align="center" rowspan="1" colspan="1">0.065</td>
<td align="center" rowspan="1" colspan="1">315.7</td>
</tr>
</tbody>
</table>
</table-wrap>
</floats-group>
</pmc>
<affiliations><list><country><li>Brésil</li>
</country>
<region><li>État de São Paulo</li>
</region>
</list>
<tree><country name="Brésil"><region name="État de São Paulo"><name sortKey="Komoto, Tatiana Takahasi" sort="Komoto, Tatiana Takahasi" uniqKey="Komoto T" first="Tatiana Takahasi" last="Komoto">Tatiana Takahasi Komoto</name>
</region>
<name sortKey="Beleboni, Rene Oliveira" sort="Beleboni, Rene Oliveira" uniqKey="Beleboni R" first="Rene Oliveira" last="Beleboni">Rene Oliveira Beleboni</name>
<name sortKey="Bitencourt, Tamires Aparecida" sort="Bitencourt, Tamires Aparecida" uniqKey="Bitencourt T" first="Tamires Aparecida" last="Bitencourt">Tamires Aparecida Bitencourt</name>
<name sortKey="Fachin, Ana Lucia" sort="Fachin, Ana Lucia" uniqKey="Fachin A" first="Ana Lúcia" last="Fachin">Ana Lúcia Fachin</name>
<name sortKey="Marins, Mozart" sort="Marins, Mozart" uniqKey="Marins M" first="Mozart" last="Marins">Mozart Marins</name>
<name sortKey="Silva, Gabriel" sort="Silva, Gabriel" uniqKey="Silva G" first="Gabriel" last="Silva">Gabriel Silva</name>
</country>
</tree>
</affiliations>
</record>
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