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Characterization of Paracoccidioides brasiliensis by FT-IR spectroscopy and nanotechnology
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 152 (2016) 397–403
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Characterization of Paracoccidioides brasiliensis by FT-IR spectroscopy and nanotechnology Isabelle Ferreira a, Juliana Ferreira-Strixino a, Maiara L. Castilho a, Claudia B.L. Campos b, Claudio Tellez a, Leandro Raniero a,⇑ a b
Institute of Research and Development, Universidade do Vale do Paraíba, Univap, Avenida Shishima Hifumi, 2911, Urbanova, 12244-000 São José dos Campos, SP, Brazil Federal University of São Paulo, Rua Talim, 330, 12231-280 São José dos Campos, São Paulo, Brazil
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The development of new approach’s
for identification of P. brasiliensis is needed. PCR associated with a colorimetric methods is safer and cheaper than other methods. Characterize and compare chemical composition of yeast and mycelia forms by FT-IR.
a r t i c l e
i n f o
Article history: Received 15 July 2014 Received in revised form 3 July 2015 Accepted 11 July 2015 Available online 16 July 2015 Keywords: Paracoccidioides brasiliensis Yeast Mycelia Gold nanoprobes FT-IR
⇑ Corresponding author. E-mail address: [email protected] (L. Raniero). http://dx.doi.org/10.1016/j.saa.2015.07.061 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
a b s t r a c t Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, is a dimorphic fungus existing as mycelia in the environment (or at 25 °C in vitro) and as yeast cells in the human host (or at 37 °C in vitro). Because mycological examination of lesions in patients frequently is unable to show the presence of the fungus and serological tests can misdiagnose the disease with other mycosis, the development of new approach’s for molecular identification of P. brasiliensis spurges is needed. This study describes the use of a gold nanoprobe of a known gene sequence of P. brasiliensis as a molecular tool to identify P. brasiliensis by regular polymerase chain reaction (PCR) associated with a colorimetric methods. This approach is suitable for testing in remote areas because it does not require any further step than gene amplification, being safer and cheaper than electrophoresis methods. The proposed test showed a color change of the PCR reaction mixture from red to blue in negative samples, whereas the solution remains red in positive samples. We also performed a Fourier Transform Infrared (FT-IR) Spectroscopy analysis to characterize and compare the chemical composition between yeast and mycelia forms, which revealed biochemical differences between these two forms. The analysis of the spectra showed that differences were distributed in chemical bonds of proteins, lipids and carbohydrates. The most prominent difference between both forms was vibration modes related to 1,3-b-glucan usually found in mycelia and 1,3-a-glucan found in yeasts and also chitin forms. In this work, we introduce FT-IR as a new method suitable to reveal overall differences that biochemically distinguish each form of P. brasiliensis that could be additionally used to discriminate biochemical differences among a single form under distinct environmental conditions. Ó 2015 Elsevier B.V. All rights reserved.
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1. Introduction Paracoccidioides brasiliensis is a thermo-dimorphic fungus that switches between a filamentous hyphae form (under environmental mild temperature or at 25 °C in vitro) and a cellular yeast form (inside the mammalian host or at 37 °C in vitro). This fungus is a pathogenic microorganism that causes a systemic disease named paracoccidioidomycosis (PCM), an important mycosis in Latin America [1–3]. This mycosis is a public health problem due to its morbidity and mortality, especially for agricultural workers. The disease affects mostly male between 30 and 50 years old as a chronic mycosis, but about 5–10% of the patients develop the acute and more severe form of the disease, which affects both genders and all ages indistinctly. The major risk factor for acquisition of infection is the activities related to the management of soil contaminated with fungus [4]. Infection occurs by inhalation of propagules or fragments of mycelia from the environment. Inhaled propagules differentiate to pathogenic yeast in the human lungs, the primary site of the disease, but the fungus can spread to other organs. Many aspects of the biology of this fungus remain unknown. The most prominent difference between both forms is probably the cell wall polysaccharide, being 1,3-ß-glucan usually found in mycelia and 1,3-a-glucan found in yeasts [5], but a plethora of other biochemical differences have already been described between them [6–8]. However, an overall chemical composition for each form is not known and the definition of a chemical signature that distinguish one form from the other, or the same form exposed to different condition (i.e. drug treatment, environmental stresses, etc) could be useful to various applications. Fourier Transform Infrared (FT-IR) Spectroscopy is widely used to characterize the presence of chemical bonds, chemical groups of complex molecules and their interactions (hydrogen bonding by example). For more than a decade, FT-IR has been increasingly applied to characterize or distinguish biological samples [9]. Traditional methods for the diagnosis of PCM require either microscopic identification of P. brasiliensis yeasts in skin lesions, bronchial washings, sputum and biopsies, or serology identification of major antigens, such as GP43, an antigenic glycoprotein known for its diverse function during infection and modulation of immune response, which is based on detection of specific antibodies [6]. However, the mycological examination is frequently unable to show the presence of the fungus, and because symptoms are similar to other fungal or bacterial diseases, such as histoplasmosis or tuberculosis [10–12], paracoccidioidomycosis is often misdiagnosed. The classic ELISA method is usually chosen because it has high sensitivity. However, antibodies-based diagnosis is subject to cross reactions, and paracoccidioidomycosis is especially mistaken with histoplasmosis, leishmaniasis or Chagas disease, which are also endemic in Brazil [13]. The implementation of ELISA is difficult in remote areas. Several molecular techniques to identify and diagnose the paracoccidioidomycosis have being incorporated into clinical laboratories routine in order to increase the effectiveness of current microbiological and immunobiological methods, especially regular PCR or PCR combined with other methodologies. PCR is an important tool for the detection of fungi in patients with negative serological reactions when the concentration of antigen and/or antibody appears low. More recently, new diagnose methods based on the unique properties of metal nanoparticles has been used to improve sensitivity and specificity values of current methods. Particularly, anisotropic nanoparticles of noble metals such as gold and silver have been receiving much attention due to its optical properties in the visible range of electromagnetic spectrum. Their applications are
based on three fundamental characteristics of the optical response of metallic nanostructures: a high sensitivity to changes in the local vicinity chemistry; the location of the electromagnetic fields of the incident radiation below the diffraction limit and the subsequent generation of high near-field intensity [14]. These properties allow its application in biology, particularly molecular biology. Mirkin et al. (1996) described the use of gold nanoparticles with thiolated oligonucleotides for the colorimetric detection of DNA targets [15]. The non-cross-linking method consists in aggregation of the oligonucleotide-functionalized gold nanoparticles induced by an increasing salt concentration with complementary oligonucleotides [16]. Solution of non-functionalized gold nanoparticles aggregates instantaneously after NaCl addition, which is observed by a color change of the solution from red wine to blue. However, nucleic acid sequences protect gold nanoparticles against aggregation, possibly through electrostatic interactions between the negatively charged phosphate groups of the nucleic acid and the polarized gold nanoparticles [17]. These properties were further exploited and applied in the detection and characterization of gene expression [18,19]. This method has also been successfully applied to detect eukaryotic gene expression without retrotranscription or polymerase chain reaction (PCR) amplification [18]; and in a fast and straight forward assay for Mycobacterium tuberculosis DNA detection in clinical samples [20]. The GP43 gene was first characterized in P. brasiliensis by Cisalpino et al. [21]. This gene encodes a glycosilated protein and a major antigen that react with 100% of sera from patients with PCM [22]. GP43 glycoprotein binds to murine laminin to entail an increased invasiveness and destruction of tissues infected [23], and is abundantly detected in yeast phase [24]. FT-IR spectroscopy has established as a powerful method for the rapid differentiation and identification of microorganisms, and presents a new addition to genetic methods [25,26]. The FTIR analysis allowing the rapid characterization of bacterial isolates and typical marker bands were used to detected and identified bacterial cell components [26,27]. In this work we propose a new molecular method that associates nanotechnology with molecular biology that can contribute to diagnose PCM in remote areas, as well as in epidemiological studies. This method is based on the detection of P. brasiliensis using gold nanoparticles functionalized with DNA in PCR assays in substitution to the further steps of eletrophoresis, which, in turn, avoid the use of ethidium bromide staining and visualization of DNA by UV light. We also propose a new approach to biochemically distinguish both forms of P. brasiliensis by Fourier Transform Infrared Spectroscopy that can be used in various applications in cell and molecular biology to compare general differences between cells that are, for example, exposed to distinct experimental or environmental conditions. 2. Experimental 2.1. Culture of P. brasiliensis The filamentous hypha and yeast forms of the Pb18 isolate were cultivated in liquid Ham’s F12 medium supplemented with 2% glucose either at 25 °C to obtain mycelia or 37 °C to obtain yeast cells, until the middle of the log phase before harvesting. 2.2. FT-IR: sample preparations and spectral conditions Filamentous hypha and yeast forms were washed three times in cold distilled water and fixed in 60% isopropanol for 24 h. After two additional washings, the fungi were resuspended in ultrapure water. Analyses were performed with three independently
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experiments. A drop of each sample was dried in the CaF2 substrate. The samples were analyzed in four different regions and the spectra were collected in the transmission mode, which generated an average of 64 spectra with 4 cm1 resolution. The FT-IR spectrometer (Perkin-Elmer Spectrum 400, USA) was equipped with a microscope (Perkin-Elmer Spotlight 400, USA) coupled to a computer using a Spectrum Image, Spotlight 400 Software, version 3.6.2. 2.3. Polymerase chain reaction (PCR) DNA extraction was performed by mechanical disruption as reported by Cisalpino et al. [21]. The amplification procedure was done in an automatic thermocycler with heating block with linear gradient (Peltier Thermal Cycler MJ96G, Biocycle). The following quantity/reagents were used for PCR: 2 ll of total P. brasiliensis DNA at 200 ng/ll; 45 ll of PCR SuperMix (Invitrogen™) and 2 ll of each primer at 10 mM (Sigma Aldrich), leading to a final volume of 51 ll. Negative controls with no genomic DNA were used to rule out possible contamination of the reagents. The primers sensus 50 -TCGCTTCCTCATGACAGACTT-30 and anti-sensus 50 -TCACCTGCATCCACCATACTT-30 were designed by Primer3 program (version 4.0) using the GP43 gene (GenBank U26160). Primers were purchased from Sigma–Aldrich at 10 mM. Amplification conditions were one cycle of 95 °C (5 min), followed thirty cycles of 95 °C (1 min), 45 °C (1.5 min), 72 °C (2 min), and a final cycle of 5 min at 72 °C. The amplicon size for GP43 was 378 bp, which was confirmed by visualization of ethidium bromide after electrophoresis in 1.5% agarose. 2.4. Nanoprobes detection The gold nanoparticles were prepared using gold chloride reduction by sodium citrate with a molar ratio of 0.026 [28]. The gold nanoparticles were incubated with thiol-modified oligonucleotides (SC4-GGTGCGAGGTC, purchased from Sigma Aldrich) at 10 mM for 16 h. The functionalization of nanoparticles was performed at room temperature under constant stirring. Then all solutions were brought to phosphate buffer (pH 7), sodium phosphate at 10 mM and sodium chloride at 0.1 M, and allowed to stand for 40 h. A volume of 500 ll was centrifuged at 1340 rpm and washed with a phosphate-buffered saline solution three times and dispersed in the same buffer volume. The positive and negative colorimetric tests detections were performed with complementary and non-complementary DNAs mixed to the nanoprobes, which was in the proportion 1:1. In order to denature the DNA, the solution was heated at 95 °C for 7 min and then the temperature was decreased to 25 °C for 25 min. A solution of NaCl was added to achieve the final concentration of 2 M [18,20]. UV–visible spectra were recorded by a Nanodrop ND-1000 spectrophotometer 10 min after the salt addition. 3. Results and discussion 3.1. Nanoprobes of DNA – colorimetric identification GP43, the major antigenic component of P. brasiliensis, is an extracellular glycoprotein with molecular weight of 43 kDa. Considered the main virulence factor of the fungus, it is also the main antigen for serological diagnosis of PCM. This component is found in 100% of sera from patients with PCM, that is, 100% of P. brasiliensis isolates known so far, express this antigen. This characteristic make GP43 a promising molecular target that could be useful for diagnose PCM independent on antibodies-based techniques. GP43 gene was that used as a molecular target to detect P.
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brasiliensis by a PCR-based assay in which a colorimetric detection method was used to develop the result [29,30]. The Fig. 1 shows the GP43 gene amplification and the absorption spectra of gold nanoparticles and gold nanoprobes. The amplified GP43 gene was used as complementary DNA for positive test. The nanoprobe method is based on the physical properties of gold nanoparticles to change their absorption spectrum from 260 nm (or blue color) when nanoprobe is aggregated to 540 nm (or red wine color) when the nanoprobes are dispersed in liquid solution (Fig. 2). In negative samples for P. brasiliensis the gold-labeled nanoprobes specific to the GP43 gene aggregate in the absence of DNA target. This increase in nanoparticle size caused by its aggregation affects the quantum confinement phenomena, turning the absorption to lower wavelengths. Therefore, more light energy passes through the liquid giving a bluish color to reaction mixture. In positive samples, on the other hand, the hybridization of the gold-labeled nanoprobes which then remain dispersed in the developing solution, which achieve a red wine color. In this case, this energy confinement occurs for higher wavelengths energy, allowing light in the red range to pass freely. In Fig. 2 the absorption band 522 nm is the same for nanoprobes dispersed in the liquid shown in Fig. 1. This result was expected because the nanoprobes binding to complementary DNA do not change the colloidal stability. Thus, the positive test kept the same red wine color of the original nanoprobes solution without DNA test. This methodology allowed for the successful detection of GP43 gene. For both tests, the absorption band at 260 nm is a typical absorption of DNA, which mainly is due to the purine and pyrimidine bases. These bases undergo excitations n ? p⁄ and p ? p⁄ transitions [31]. Because PCM does not have a simple identification and the standard method for mycological examination has low sensitivity, the gold-based nanoprobes described here bring a more accurate, quickly and inexpensively method for PCM diagnosis [32]. The non-crosslinking method has been developed to increase the sensitivity and specificity of the test conducted in which provides the discrimination of aggregation states of nanoprobes [10–12,15]. DNA nanoprobes can be an alternative to improve diagnoses in these areas, simple equipment with pre-set amplification program could be made and the results are identified by naked eye without the need of gel electrophoresis with ethidium bromide, which could lead to cancers developing. This is important to minimalize cost with equipment and specialized labor in remote places. 3.2. Comparison between FT-IR spectra of yeast and mycelium The FT-IR spectroscopy is a powerful technique to provide a rapid microanalysis of biological samples from diverse origin. In few minutes it is possible to identify and differentiate microorganisms through comparison of their chemical composition [33]. The Fig. 3 shows the average absorption spectra of P. brasiliensis yeast and mycelium forms. The spectra at higher frequency region than 3000 cm1 show bands that can be assigned as the vibration modes of NH and OH stretching. The region from 3000 to 2800 cm1 enclose bands according to the symmetric and asymmetric stretching modes of CH, CH2 and CH3. In the low frequency from 1700 to 900 cm1 are observed the characteristics bending modes of proteins, lipids, free amino acids, polysaccharides, RNA/DNA, phospholipids radicals. In order to illustrate the biochemical differences between the P. brasiliensis forms, the bands assignments were performed through 2 second derivatives ddrA2 of IR spectra by the frequency, as depicted in Fig. 4. The arrows indicate regions of fluctuations on chemical composition, especially at low frequency, characteristic for glucans modes of vibration.
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Fig. 1. GP43 gene was amplified by PCR and visualized by (1) ethidium bromide staining after electrophoresis in agarose gel and (2) UV absorption in the UV–visible spectra of nanoparticle and nanoprobes.
A
Fig. 2. Absorption spectrum UV–vis of the test; (a) positive test and (b) negative test.
B
Fig. 4. FTIR spectra
Fig. 3. FTIR spectra of mycelium and yeast forms of P. brasiliensis.
d2 A dr2
, of mycelium and yeast forms of P. brasiliensis.
The contributions of these bands were calculated by spectra deconvolution using Gaussian curves fit as shown in Table 1. The symmetric and asymmetric vibrations assignments m(NH) were
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Table 1 Vibrational modes assignments via Second derivative of yeast and mycelium spectra. Gray cells stand for the calculated areas from spectra deconvolution using Gaussian curves fit.
based on empirical relations proposed by Bellamy and Williams [29] and Krueger and Thompson [39]:
ms ¼ 0:876mas þ 345:5 ms ¼ 0:682mas þ 1023:0 The absorptions bands that is in agreement with this criteria, which has three band pairs of NH2 group at 3433 cm1 mas(NH)(NH2), 3359 cm1 ms(NH)(NH2); 3285 cm1 mas(NH)(NH2), 3214 cm1 ms(NH)(NH2); 3108 cm1 mas(NH)(NH2), 3064 cm1 ms(NH)(NH2); in this way we were able to confirm the presence of primary amines in the amino acid chains of the samples. The attempt of vibrational modes assignments was performed using the chemical composition described in the literature for other microorganisms, but many could not be assigned due to lack of knowledge of P. brasiliensis composition [25,29,34–39]. Chitin is an important polysaccharide cell wall component that is involved in morphogenesis and integrity of the cell wall. Chitin is present in both phases, but yeast form has higher amount (37–
48%) than the mycelial form (7–18%) [3,6,7]. Chitin has been found in three polymorphic forms, b, a and c chitin [40]. a-chitin is the most abundant form and is usually found when hardness is necessary [41]. b-chitin provide toughness, flexibility and motility [42]. The peaks in 3355 cm1, 3199 cm1, 2904 cm1 could be assigned to chitin and are present only in the yeast form. The peaks in 1630 cm1 and 1549 cm1 are typical of b-chitin, and are present in both forms, but is more intense in yeast form. Possibly, at this stage the flexibility and mobility of the wall are necessary for greater interaction with the environment and virulence. According to Firon et al. [41] the cell wall characteristics such as flexibility and toughness are continually changed during the cell cycle, to allow for growth and division. Amide I and amide II bands are the two major bands of proteins in the infrared spectrum. Groups present in proteins which contains Amide I (m(C@O) of the peptide bond) vibrates in the range between 1600 cm1 and 1700 cm1 of the spectrum and amide II (d(NH) of the peptide bond) vibrates in 1510 cm1 and 1580 cm1 region and it is more complex than amide I [8]. The
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mycelial cell wall is richer in proteins (24–41%) while the yeast cell wall has only 7–14% of proteins. The peak in 3197 cm1 could be assigned to NAH str (amide A according Ref. [8]) of proteins there is more intense in micelium phase. The band at 1740 cm1 was associated with lipids [43]. Oleic acid and linoleic acid prevailed in membrane fatty acid composition. The lipid composition of the cell membrane is also distinct in each form. Yeast has twice more lipids than mycelium [44]. There are low rates of galactose in the cell wall of the mycelium [45]. Galactose is recognize in vibrational spectra, specifically in the infrared spectrum by one band at 1150 cm1, this band is more intense in yeast form. The bands around 1038 cm1, 1000 cm1, and 960 cm1 and the peaks centered at 1028 cm1 and 928 cm1 could be assigned as analytical criterium to the presence of 1,3-b-glucan and 1,3-a-glucan, respectively [46]. In both forms of P. brasiliensis, about 40% of the cell wall is composed by glucans, which is a long carbohydrate molecule of D-glucose monomers linked by glycosidic bonds [46,3]. The major polysaccharide found in mycelial’s cell wall is the 1,3-b-glucan, 1,3 a-glucan is the major polysaccharide found in yeast’s cell wall. Only 4–5% of total glucans corresponds to the 1,3-b-glucan in yeast, as observed in the less intense peak corresponding to 1,3-b-glucan (960 cm1) in yeast. The average absorption spectra of P. brasiliensis yeast and mycelium forms have shown different values of absorbance. The absorbance values corresponding to stretching bands assigned as m(CH) of the ACH2 and m(CH)(ACH3) groups were highest in the yeast forms, suggesting an increasing of lipids and protein contents. The absorbance value of the band at 3011 cm1 in yeast spectra can be assigned as m(CH) of unsaturated @CH of lipids or fatty acids. Also, the bending vibrations bands observed at 1435 cm1 (0.19 for mycelium) and at 1442 cm1 (0.27 for yeast), both assigned to the vibrational mode d(HCH) scissoring. This finding reconfirms the high value of lipid content in the yeast form. The quantity of a-helix structure appears to be higher in the mycelium form by the observed values of absorbance at 1659 cm1 (0.83) and 1624 cm1 (0.82) than the absorbance values of the yeast form at 1660 cm1 (0.71) and 1630 cm1 (0.71). The b-chitin also appear to be 6% higher in the mycelium form as was observed from the absorbance values at 1551 cm1 (0.50) than the yeast form at 1546 cm1 (0.44). The sugar content present as galactose, glucose and fructose was 10% higher in yeast form than the mycelium form. Apart from that, the mycelium showed the lowest band intensity at 1746 cm1 with an area value of 0.08 [Abs wn] measured with the OMNIC software [47]. For the yeast sample the calculated area was 6.27 [Abs wn] units. These bands can be assigned as m(C@O) of phospholipids [48]. These results show that the definition of a chemical signature by FT-IR spectroscopy to distinguish both forms of P. brasiliensis is possible. This chemical signature can be further used as a tool to compare the same form of this fungus exposed to different experimental conditions, such as, ie, drug treatment or environmental stresses. 4. Conclusions The mycelia and yeast forms of P. brasiliensis have several differences in the FT-IR, these differences are compatible with the biochemical changes occurring in the transition of mycelium for yeast form. The results suggest difference on chitin concentration as well as chitin structure for each form. The glucan modes also shows variation in its structure, a-glucans was predominate in yeast and b-glucans in mycelium form. The colorimetric identification method was easy to perform, viable and effective in detecting GP43 gene, highly specific for the identification of P. brasiliensis, becoming an alternative to improve diagnoses of this fungus.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Characterization of Paracoccidioides brasiliensis by FT-IR spectroscopy and nanotechnology Isabelle Ferreira a, Juliana Ferreira-Strixino a, Maiara L. Castilho a, Claudia B.L. Campos b, Claudio Tellez a, Leandro Raniero a,⇑ a b
Institute of Research and Development, Universidade do Vale do Paraíba, Univap, Avenida Shishima Hifumi, 2911, Urbanova, 12244-000 São José dos Campos, SP, Brazil Federal University of São Paulo, Rua Talim, 330, 12231-280 São José dos Campos, São Paulo, Brazil
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The development of new approach’s
for identification of P. brasiliensis is needed. PCR associated with a colorimetric methods is safer and cheaper than other methods. Characterize and compare chemical composition of yeast and mycelia forms by FT-IR.
a r t i c l e
i n f o
Article history: Received 15 July 2014 Received in revised form 3 July 2015 Accepted 11 July 2015 Available online 16 July 2015 Keywords: Paracoccidioides brasiliensis Yeast Mycelia Gold nanoprobes FT-IR
⇑ Corresponding author. E-mail address: [email protected] (L. Raniero). http://dx.doi.org/10.1016/j.saa.2015.07.061 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
a b s t r a c t Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, is a dimorphic fungus existing as mycelia in the environment (or at 25 °C in vitro) and as yeast cells in the human host (or at 37 °C in vitro). Because mycological examination of lesions in patients frequently is unable to show the presence of the fungus and serological tests can misdiagnose the disease with other mycosis, the development of new approach’s for molecular identification of P. brasiliensis spurges is needed. This study describes the use of a gold nanoprobe of a known gene sequence of P. brasiliensis as a molecular tool to identify P. brasiliensis by regular polymerase chain reaction (PCR) associated with a colorimetric methods. This approach is suitable for testing in remote areas because it does not require any further step than gene amplification, being safer and cheaper than electrophoresis methods. The proposed test showed a color change of the PCR reaction mixture from red to blue in negative samples, whereas the solution remains red in positive samples. We also performed a Fourier Transform Infrared (FT-IR) Spectroscopy analysis to characterize and compare the chemical composition between yeast and mycelia forms, which revealed biochemical differences between these two forms. The analysis of the spectra showed that differences were distributed in chemical bonds of proteins, lipids and carbohydrates. The most prominent difference between both forms was vibration modes related to 1,3-b-glucan usually found in mycelia and 1,3-a-glucan found in yeasts and also chitin forms. In this work, we introduce FT-IR as a new method suitable to reveal overall differences that biochemically distinguish each form of P. brasiliensis that could be additionally used to discriminate biochemical differences among a single form under distinct environmental conditions. Ó 2015 Elsevier B.V. All rights reserved.
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1. Introduction Paracoccidioides brasiliensis is a thermo-dimorphic fungus that switches between a filamentous hyphae form (under environmental mild temperature or at 25 °C in vitro) and a cellular yeast form (inside the mammalian host or at 37 °C in vitro). This fungus is a pathogenic microorganism that causes a systemic disease named paracoccidioidomycosis (PCM), an important mycosis in Latin America [1–3]. This mycosis is a public health problem due to its morbidity and mortality, especially for agricultural workers. The disease affects mostly male between 30 and 50 years old as a chronic mycosis, but about 5–10% of the patients develop the acute and more severe form of the disease, which affects both genders and all ages indistinctly. The major risk factor for acquisition of infection is the activities related to the management of soil contaminated with fungus [4]. Infection occurs by inhalation of propagules or fragments of mycelia from the environment. Inhaled propagules differentiate to pathogenic yeast in the human lungs, the primary site of the disease, but the fungus can spread to other organs. Many aspects of the biology of this fungus remain unknown. The most prominent difference between both forms is probably the cell wall polysaccharide, being 1,3-ß-glucan usually found in mycelia and 1,3-a-glucan found in yeasts [5], but a plethora of other biochemical differences have already been described between them [6–8]. However, an overall chemical composition for each form is not known and the definition of a chemical signature that distinguish one form from the other, or the same form exposed to different condition (i.e. drug treatment, environmental stresses, etc) could be useful to various applications. Fourier Transform Infrared (FT-IR) Spectroscopy is widely used to characterize the presence of chemical bonds, chemical groups of complex molecules and their interactions (hydrogen bonding by example). For more than a decade, FT-IR has been increasingly applied to characterize or distinguish biological samples [9]. Traditional methods for the diagnosis of PCM require either microscopic identification of P. brasiliensis yeasts in skin lesions, bronchial washings, sputum and biopsies, or serology identification of major antigens, such as GP43, an antigenic glycoprotein known for its diverse function during infection and modulation of immune response, which is based on detection of specific antibodies [6]. However, the mycological examination is frequently unable to show the presence of the fungus, and because symptoms are similar to other fungal or bacterial diseases, such as histoplasmosis or tuberculosis [10–12], paracoccidioidomycosis is often misdiagnosed. The classic ELISA method is usually chosen because it has high sensitivity. However, antibodies-based diagnosis is subject to cross reactions, and paracoccidioidomycosis is especially mistaken with histoplasmosis, leishmaniasis or Chagas disease, which are also endemic in Brazil [13]. The implementation of ELISA is difficult in remote areas. Several molecular techniques to identify and diagnose the paracoccidioidomycosis have being incorporated into clinical laboratories routine in order to increase the effectiveness of current microbiological and immunobiological methods, especially regular PCR or PCR combined with other methodologies. PCR is an important tool for the detection of fungi in patients with negative serological reactions when the concentration of antigen and/or antibody appears low. More recently, new diagnose methods based on the unique properties of metal nanoparticles has been used to improve sensitivity and specificity values of current methods. Particularly, anisotropic nanoparticles of noble metals such as gold and silver have been receiving much attention due to its optical properties in the visible range of electromagnetic spectrum. Their applications are
based on three fundamental characteristics of the optical response of metallic nanostructures: a high sensitivity to changes in the local vicinity chemistry; the location of the electromagnetic fields of the incident radiation below the diffraction limit and the subsequent generation of high near-field intensity [14]. These properties allow its application in biology, particularly molecular biology. Mirkin et al. (1996) described the use of gold nanoparticles with thiolated oligonucleotides for the colorimetric detection of DNA targets [15]. The non-cross-linking method consists in aggregation of the oligonucleotide-functionalized gold nanoparticles induced by an increasing salt concentration with complementary oligonucleotides [16]. Solution of non-functionalized gold nanoparticles aggregates instantaneously after NaCl addition, which is observed by a color change of the solution from red wine to blue. However, nucleic acid sequences protect gold nanoparticles against aggregation, possibly through electrostatic interactions between the negatively charged phosphate groups of the nucleic acid and the polarized gold nanoparticles [17]. These properties were further exploited and applied in the detection and characterization of gene expression [18,19]. This method has also been successfully applied to detect eukaryotic gene expression without retrotranscription or polymerase chain reaction (PCR) amplification [18]; and in a fast and straight forward assay for Mycobacterium tuberculosis DNA detection in clinical samples [20]. The GP43 gene was first characterized in P. brasiliensis by Cisalpino et al. [21]. This gene encodes a glycosilated protein and a major antigen that react with 100% of sera from patients with PCM [22]. GP43 glycoprotein binds to murine laminin to entail an increased invasiveness and destruction of tissues infected [23], and is abundantly detected in yeast phase [24]. FT-IR spectroscopy has established as a powerful method for the rapid differentiation and identification of microorganisms, and presents a new addition to genetic methods [25,26]. The FTIR analysis allowing the rapid characterization of bacterial isolates and typical marker bands were used to detected and identified bacterial cell components [26,27]. In this work we propose a new molecular method that associates nanotechnology with molecular biology that can contribute to diagnose PCM in remote areas, as well as in epidemiological studies. This method is based on the detection of P. brasiliensis using gold nanoparticles functionalized with DNA in PCR assays in substitution to the further steps of eletrophoresis, which, in turn, avoid the use of ethidium bromide staining and visualization of DNA by UV light. We also propose a new approach to biochemically distinguish both forms of P. brasiliensis by Fourier Transform Infrared Spectroscopy that can be used in various applications in cell and molecular biology to compare general differences between cells that are, for example, exposed to distinct experimental or environmental conditions. 2. Experimental 2.1. Culture of P. brasiliensis The filamentous hypha and yeast forms of the Pb18 isolate were cultivated in liquid Ham’s F12 medium supplemented with 2% glucose either at 25 °C to obtain mycelia or 37 °C to obtain yeast cells, until the middle of the log phase before harvesting. 2.2. FT-IR: sample preparations and spectral conditions Filamentous hypha and yeast forms were washed three times in cold distilled water and fixed in 60% isopropanol for 24 h. After two additional washings, the fungi were resuspended in ultrapure water. Analyses were performed with three independently
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experiments. A drop of each sample was dried in the CaF2 substrate. The samples were analyzed in four different regions and the spectra were collected in the transmission mode, which generated an average of 64 spectra with 4 cm1 resolution. The FT-IR spectrometer (Perkin-Elmer Spectrum 400, USA) was equipped with a microscope (Perkin-Elmer Spotlight 400, USA) coupled to a computer using a Spectrum Image, Spotlight 400 Software, version 3.6.2. 2.3. Polymerase chain reaction (PCR) DNA extraction was performed by mechanical disruption as reported by Cisalpino et al. [21]. The amplification procedure was done in an automatic thermocycler with heating block with linear gradient (Peltier Thermal Cycler MJ96G, Biocycle). The following quantity/reagents were used for PCR: 2 ll of total P. brasiliensis DNA at 200 ng/ll; 45 ll of PCR SuperMix (Invitrogen™) and 2 ll of each primer at 10 mM (Sigma Aldrich), leading to a final volume of 51 ll. Negative controls with no genomic DNA were used to rule out possible contamination of the reagents. The primers sensus 50 -TCGCTTCCTCATGACAGACTT-30 and anti-sensus 50 -TCACCTGCATCCACCATACTT-30 were designed by Primer3 program (version 4.0) using the GP43 gene (GenBank U26160). Primers were purchased from Sigma–Aldrich at 10 mM. Amplification conditions were one cycle of 95 °C (5 min), followed thirty cycles of 95 °C (1 min), 45 °C (1.5 min), 72 °C (2 min), and a final cycle of 5 min at 72 °C. The amplicon size for GP43 was 378 bp, which was confirmed by visualization of ethidium bromide after electrophoresis in 1.5% agarose. 2.4. Nanoprobes detection The gold nanoparticles were prepared using gold chloride reduction by sodium citrate with a molar ratio of 0.026 [28]. The gold nanoparticles were incubated with thiol-modified oligonucleotides (SC4-GGTGCGAGGTC, purchased from Sigma Aldrich) at 10 mM for 16 h. The functionalization of nanoparticles was performed at room temperature under constant stirring. Then all solutions were brought to phosphate buffer (pH 7), sodium phosphate at 10 mM and sodium chloride at 0.1 M, and allowed to stand for 40 h. A volume of 500 ll was centrifuged at 1340 rpm and washed with a phosphate-buffered saline solution three times and dispersed in the same buffer volume. The positive and negative colorimetric tests detections were performed with complementary and non-complementary DNAs mixed to the nanoprobes, which was in the proportion 1:1. In order to denature the DNA, the solution was heated at 95 °C for 7 min and then the temperature was decreased to 25 °C for 25 min. A solution of NaCl was added to achieve the final concentration of 2 M [18,20]. UV–visible spectra were recorded by a Nanodrop ND-1000 spectrophotometer 10 min after the salt addition. 3. Results and discussion 3.1. Nanoprobes of DNA – colorimetric identification GP43, the major antigenic component of P. brasiliensis, is an extracellular glycoprotein with molecular weight of 43 kDa. Considered the main virulence factor of the fungus, it is also the main antigen for serological diagnosis of PCM. This component is found in 100% of sera from patients with PCM, that is, 100% of P. brasiliensis isolates known so far, express this antigen. This characteristic make GP43 a promising molecular target that could be useful for diagnose PCM independent on antibodies-based techniques. GP43 gene was that used as a molecular target to detect P.
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brasiliensis by a PCR-based assay in which a colorimetric detection method was used to develop the result [29,30]. The Fig. 1 shows the GP43 gene amplification and the absorption spectra of gold nanoparticles and gold nanoprobes. The amplified GP43 gene was used as complementary DNA for positive test. The nanoprobe method is based on the physical properties of gold nanoparticles to change their absorption spectrum from 260 nm (or blue color) when nanoprobe is aggregated to 540 nm (or red wine color) when the nanoprobes are dispersed in liquid solution (Fig. 2). In negative samples for P. brasiliensis the gold-labeled nanoprobes specific to the GP43 gene aggregate in the absence of DNA target. This increase in nanoparticle size caused by its aggregation affects the quantum confinement phenomena, turning the absorption to lower wavelengths. Therefore, more light energy passes through the liquid giving a bluish color to reaction mixture. In positive samples, on the other hand, the hybridization of the gold-labeled nanoprobes which then remain dispersed in the developing solution, which achieve a red wine color. In this case, this energy confinement occurs for higher wavelengths energy, allowing light in the red range to pass freely. In Fig. 2 the absorption band 522 nm is the same for nanoprobes dispersed in the liquid shown in Fig. 1. This result was expected because the nanoprobes binding to complementary DNA do not change the colloidal stability. Thus, the positive test kept the same red wine color of the original nanoprobes solution without DNA test. This methodology allowed for the successful detection of GP43 gene. For both tests, the absorption band at 260 nm is a typical absorption of DNA, which mainly is due to the purine and pyrimidine bases. These bases undergo excitations n ? p⁄ and p ? p⁄ transitions [31]. Because PCM does not have a simple identification and the standard method for mycological examination has low sensitivity, the gold-based nanoprobes described here bring a more accurate, quickly and inexpensively method for PCM diagnosis [32]. The non-crosslinking method has been developed to increase the sensitivity and specificity of the test conducted in which provides the discrimination of aggregation states of nanoprobes [10–12,15]. DNA nanoprobes can be an alternative to improve diagnoses in these areas, simple equipment with pre-set amplification program could be made and the results are identified by naked eye without the need of gel electrophoresis with ethidium bromide, which could lead to cancers developing. This is important to minimalize cost with equipment and specialized labor in remote places. 3.2. Comparison between FT-IR spectra of yeast and mycelium The FT-IR spectroscopy is a powerful technique to provide a rapid microanalysis of biological samples from diverse origin. In few minutes it is possible to identify and differentiate microorganisms through comparison of their chemical composition [33]. The Fig. 3 shows the average absorption spectra of P. brasiliensis yeast and mycelium forms. The spectra at higher frequency region than 3000 cm1 show bands that can be assigned as the vibration modes of NH and OH stretching. The region from 3000 to 2800 cm1 enclose bands according to the symmetric and asymmetric stretching modes of CH, CH2 and CH3. In the low frequency from 1700 to 900 cm1 are observed the characteristics bending modes of proteins, lipids, free amino acids, polysaccharides, RNA/DNA, phospholipids radicals. In order to illustrate the biochemical differences between the P. brasiliensis forms, the bands assignments were performed through 2 second derivatives ddrA2 of IR spectra by the frequency, as depicted in Fig. 4. The arrows indicate regions of fluctuations on chemical composition, especially at low frequency, characteristic for glucans modes of vibration.
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Fig. 1. GP43 gene was amplified by PCR and visualized by (1) ethidium bromide staining after electrophoresis in agarose gel and (2) UV absorption in the UV–visible spectra of nanoparticle and nanoprobes.
A
Fig. 2. Absorption spectrum UV–vis of the test; (a) positive test and (b) negative test.
B
Fig. 4. FTIR spectra
Fig. 3. FTIR spectra of mycelium and yeast forms of P. brasiliensis.
d2 A dr2
, of mycelium and yeast forms of P. brasiliensis.
The contributions of these bands were calculated by spectra deconvolution using Gaussian curves fit as shown in Table 1. The symmetric and asymmetric vibrations assignments m(NH) were
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Table 1 Vibrational modes assignments via Second derivative of yeast and mycelium spectra. Gray cells stand for the calculated areas from spectra deconvolution using Gaussian curves fit.
based on empirical relations proposed by Bellamy and Williams [29] and Krueger and Thompson [39]:
ms ¼ 0:876mas þ 345:5 ms ¼ 0:682mas þ 1023:0 The absorptions bands that is in agreement with this criteria, which has three band pairs of NH2 group at 3433 cm1 mas(NH)(NH2), 3359 cm1 ms(NH)(NH2); 3285 cm1 mas(NH)(NH2), 3214 cm1 ms(NH)(NH2); 3108 cm1 mas(NH)(NH2), 3064 cm1 ms(NH)(NH2); in this way we were able to confirm the presence of primary amines in the amino acid chains of the samples. The attempt of vibrational modes assignments was performed using the chemical composition described in the literature for other microorganisms, but many could not be assigned due to lack of knowledge of P. brasiliensis composition [25,29,34–39]. Chitin is an important polysaccharide cell wall component that is involved in morphogenesis and integrity of the cell wall. Chitin is present in both phases, but yeast form has higher amount (37–
48%) than the mycelial form (7–18%) [3,6,7]. Chitin has been found in three polymorphic forms, b, a and c chitin [40]. a-chitin is the most abundant form and is usually found when hardness is necessary [41]. b-chitin provide toughness, flexibility and motility [42]. The peaks in 3355 cm1, 3199 cm1, 2904 cm1 could be assigned to chitin and are present only in the yeast form. The peaks in 1630 cm1 and 1549 cm1 are typical of b-chitin, and are present in both forms, but is more intense in yeast form. Possibly, at this stage the flexibility and mobility of the wall are necessary for greater interaction with the environment and virulence. According to Firon et al. [41] the cell wall characteristics such as flexibility and toughness are continually changed during the cell cycle, to allow for growth and division. Amide I and amide II bands are the two major bands of proteins in the infrared spectrum. Groups present in proteins which contains Amide I (m(C@O) of the peptide bond) vibrates in the range between 1600 cm1 and 1700 cm1 of the spectrum and amide II (d(NH) of the peptide bond) vibrates in 1510 cm1 and 1580 cm1 region and it is more complex than amide I [8]. The
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mycelial cell wall is richer in proteins (24–41%) while the yeast cell wall has only 7–14% of proteins. The peak in 3197 cm1 could be assigned to NAH str (amide A according Ref. [8]) of proteins there is more intense in micelium phase. The band at 1740 cm1 was associated with lipids [43]. Oleic acid and linoleic acid prevailed in membrane fatty acid composition. The lipid composition of the cell membrane is also distinct in each form. Yeast has twice more lipids than mycelium [44]. There are low rates of galactose in the cell wall of the mycelium [45]. Galactose is recognize in vibrational spectra, specifically in the infrared spectrum by one band at 1150 cm1, this band is more intense in yeast form. The bands around 1038 cm1, 1000 cm1, and 960 cm1 and the peaks centered at 1028 cm1 and 928 cm1 could be assigned as analytical criterium to the presence of 1,3-b-glucan and 1,3-a-glucan, respectively [46]. In both forms of P. brasiliensis, about 40% of the cell wall is composed by glucans, which is a long carbohydrate molecule of D-glucose monomers linked by glycosidic bonds [46,3]. The major polysaccharide found in mycelial’s cell wall is the 1,3-b-glucan, 1,3 a-glucan is the major polysaccharide found in yeast’s cell wall. Only 4–5% of total glucans corresponds to the 1,3-b-glucan in yeast, as observed in the less intense peak corresponding to 1,3-b-glucan (960 cm1) in yeast. The average absorption spectra of P. brasiliensis yeast and mycelium forms have shown different values of absorbance. The absorbance values corresponding to stretching bands assigned as m(CH) of the ACH2 and m(CH)(ACH3) groups were highest in the yeast forms, suggesting an increasing of lipids and protein contents. The absorbance value of the band at 3011 cm1 in yeast spectra can be assigned as m(CH) of unsaturated @CH of lipids or fatty acids. Also, the bending vibrations bands observed at 1435 cm1 (0.19 for mycelium) and at 1442 cm1 (0.27 for yeast), both assigned to the vibrational mode d(HCH) scissoring. This finding reconfirms the high value of lipid content in the yeast form. The quantity of a-helix structure appears to be higher in the mycelium form by the observed values of absorbance at 1659 cm1 (0.83) and 1624 cm1 (0.82) than the absorbance values of the yeast form at 1660 cm1 (0.71) and 1630 cm1 (0.71). The b-chitin also appear to be 6% higher in the mycelium form as was observed from the absorbance values at 1551 cm1 (0.50) than the yeast form at 1546 cm1 (0.44). The sugar content present as galactose, glucose and fructose was 10% higher in yeast form than the mycelium form. Apart from that, the mycelium showed the lowest band intensity at 1746 cm1 with an area value of 0.08 [Abs wn] measured with the OMNIC software [47]. For the yeast sample the calculated area was 6.27 [Abs wn] units. These bands can be assigned as m(C@O) of phospholipids [48]. These results show that the definition of a chemical signature by FT-IR spectroscopy to distinguish both forms of P. brasiliensis is possible. This chemical signature can be further used as a tool to compare the same form of this fungus exposed to different experimental conditions, such as, ie, drug treatment or environmental stresses. 4. Conclusions The mycelia and yeast forms of P. brasiliensis have several differences in the FT-IR, these differences are compatible with the biochemical changes occurring in the transition of mycelium for yeast form. The results suggest difference on chitin concentration as well as chitin structure for each form. The glucan modes also shows variation in its structure, a-glucans was predominate in yeast and b-glucans in mycelium form. The colorimetric identification method was easy to perform, viable and effective in detecting GP43 gene, highly specific for the identification of P. brasiliensis, becoming an alternative to improve diagnoses of this fungus.
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