Accepted Manuscript Differentiation of Leishmania species by FT-IR spectroscopy Josafá C. Aguiar, Josane Mittmann, Isabelle Ferreira, Juliana Ferreira-Strixino, Leandro Raniero PII: DOI: Reference:
S1386-1425(15)00018-9 http://dx.doi.org/10.1016/j.saa.2015.01.008 SAA 13176
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
24 June 2014 11 December 2014 5 January 2015
Please cite this article as: J.C. Aguiar, J. Mittmann, I. Ferreira, J. Ferreira-Strixino, L. Raniero, Differentiation of Leishmania species by FT-IR spectroscopy, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.008
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Differentiation of Leishmania species by FT-IR spectroscopy Josafá C. Aguiar1, Josane Mittmann 1, Isabelle Ferreira 1, Juliana Ferreira-Strixino 1, Leandro Raniero 1 1
Laboratório de Nanossensores – Instituto de Pesquisa & Desenvolvimento Universidade do Vale do Paraíba – UNIVAP Shishima Hifumi Avenue, 2911, Urbanova, 12244.000, São José dos Campos-SP, Brazil
E-mail: [email protected]
Abstract Leishmaniasis is a parasitic infectious disease caused by protozoa that belong to the genus Leishmania. It is transmitted by the bite of an infected female Sand fly. The disease is endemic in 88 countries[1] (16 developed countries and 72 developing countries) on four continents. In Brazil, epidemiological data show the disease is present in all Brazilian regions, with the highest incidences in the North and Northeast. There are several methods used to diagnose leishmaniasis, but these procedures have many limitations, are time consuming, have low sensitivity, and are expensive. In this context, Fourier Transform Infrared Spectroscopy (FT-IR) analysis has the potential to provide rapid results and may be adapted for a clinical test with high sensitivity and specificity. In this work, FT-IR was used as a tool to investigate the promastigotes of L. amazonensis, L. chagasi, and L. major species. The spectra were analyzed by cluster analysis and deconvolution procedure base on spectra second derivatives. Results: cluster analysis found four specific regions that are able to identify the Leishmania species. The dendrogram representation clearly indicates the heterogeneity among Leishmania species. The band deconvolution done by the curve fitting in these regions quantitatively differentiated the polysaccharides, amide III, phospholipids, proteins, and nucleic acids. L. chagasi and L. major showed a greater biochemistry similarity and have three bands that were not registered in L. amazonensis. The L. amazonensis presented three specific bands that were not recorded in the other two species. It is evident that the FT-IR method is an indispensable tool to discriminate these parasites. The high sensitivity and specificity of this technique opens up the possibilities for further studies about characterization of other microorganisms.
Keywords: L. amazonensis; L. chagasi; L. major; FT – IR; Cluster Analysis; Biochemistry.
1. Introduction 1.1Leishmania Parasites of the genus Leishmania belong to the Kinetoplastida order and Trypanosomatidae family [2, 3]. These parasites cause the human disease known as leishmaniasis, which can present in different clinical forms, depending on the Leishmania species involved and the relation between the parasite and its host [4]. The transmission occurs during the blood meal of Sand flies from the Phlebotominae family, via injection of promastigotes [5, 6]. The classification of Leishmania includes two subgenera Viannia and Leishmania. The species studied in this article belong to the subgenus Leishmania. Leishmania chagasi and Leishmania amazonensis are found in the New World (Americas), and Leishmania major is found in the Old World
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(Europe, Africa, and Asia) [7-9]. The L. amazonensis and L. major species cause cutaneous leishmaniasis, and L. chagasi causes the most severe form of the disease called visceral leishmaniasis [10]. Epidemiological data show that the highest incidence of leishmaniasis cases in Brazil occurs in the North and Northeast regions. The main factors associated with its increased incidence are the exploration of forest for agricultural activities and ecotourism. The annual average between 2007 and 2011 of cutaneous and visceral leishmaniasis cases were 20,187 and 3,682, respectively. [11-15]. The various approaches to diagnose leishmaniasis include: a) serological detection of antiLeishmania antibodies, though cross-reactivity with other infections such as Chagas disease can occur. b) Indirect immunofluorescence (RIFI) is the test of choice, though its lack of specificity is influenced by cross reactions with other diseases caused by trypanosomes. c) ELISA has better sensitivity and specificity but cross reactions with other diseases caused by trypanosomes may still occur. d) Molecular diagnosis, including PCR to detect DNA, has good sensitivity compared to immunoassays [16-18]. However, PCR is a costly technique, and as such, it is rarely used for diagnosis. Fourier Transform Infrared Spectroscopy (FTIR) analysis has the potential to provide rapid results and may achieve high values of sensitivity and specificity. This technique is able to identify and characterize biological samples by their chemical composition [19-21]. This technique has been applied successfully to identify and characterize microorganisms, such as Salmonella, Bacillus subtilis, Staphylococcus, yeasts, filamentous fungi, etc. [22-26]. FTIR is based on the interaction of infrared radiation with matter [20, 27]. Infrared radiation is absorbed when its frequency (energy) equals that of a vibrational mode. These molecular vibrations are unique to the type and spatial arrangement of the atoms in a sample [20]. Thus, the spectrum is a fingerprint of the illuminated molecule. Furthermore, structural information about important subunits of the molecule, called functional groups, can be obtain.[20, 26, 28]. This article presents the characterization of L. amazonensis, L. major, and L. chagasi by FT-IR, and demonstrates that the species can be identified by analyzing biochemical differences between them.
2. Materials and Methods 2.1Parasites The species of parasites used were Leishmania major (MHOM/SU/1973/5-ASKH strain), Leishmania chagasi (MHOM/BR/1974/PP75 strain), and Leishmania amazonensis (MHOM/BR/73 M2269 strain). They were grown in the M199 medium supplemented with 10% Fetal Bovine Serum, Urine 2%, 10 U/ml penicillin, 10 U/ml streptomycin, 0.25% hemin and kept at 26 °C and pH 7.4. The subcultures of promastigotes were replicated weekly. 2.2 Sample preparation For FT-IR analyses, the sample preparation is an important factor, because the composition of the culture medium has a strong influence on the results. To remove the culture medium, 20 ml of the cultures were centrifuged at 3500 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in 15 ml of NaCl solution at 0.2%. This procedure was repeated three times. After each centrifugation, an aliquot was observed under the microscope to verify the integrity of the parasites. Then, IR spectra were collected from five independently grown parasite cultures for each species. 2.3 FT - IR spectroscopy and statistical analysis After the cleaning process to remove media, a drop of 10 µl with approximately 485 parasites was deposited on a calcium fluoride (CaF2) window. The sample was dried by an Eppendorf Concentrator, which allows the thin film formation of the unanalyzed Leishmaniasis. The IR spectra was collected by a microscope (Spotlight Perkin Elmer 400, USA) coupled to FT-IR spectrophotometer (Spectrum 400), which was controlled by a computer with Spotlight 400 software, version 3.6.2. The sample was analyzed in four random regions, totaling 22 points. The spectra were recorded in regions with approximately the same thickness. The band centered at 1650 cm-1 had the highest value of
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absorbance around 0.9 to 1.0 for all spectra. Each spectrum was obtained from an average of 64 spectra per point with spectral resolution of 4 cm-1. The spectral range was from 4000 cm-1 to 750 cm-1. These spectra were analyzed by cluster analysis and deconvolution procedure base on spectra second derivatives. The multivariate statistical analysis was performed by Cluster Analysis [29] using OPUS software version 4.2 with the following parameters: second derivative, smoothing 9 points, Ward's algorithm, and Scaling to 1st range method. Cluster analysis classifies objects into groups which show similarities [30] and is commonly used to rapidly differentiate / classify spectra of microorganisms [31]. Gaussians curve fitting was used in the observed band deconvolutions, thereby quantifying the contribution of specific biochemical species in each region of the spectrum [32]. The biochemical changes, which underlie the separation obtained by cluster analysis, can then be elucidated.
3. Results Fig. 1 shows the average IR spectra for L. major, L. chagasi, and L. amazonensis species. The standard deviations (SD) are represented in gray. The highest variation, observed for L. chagasi, may reflect the development stage of the culture, since they are different parasites, which respond differently in time. This change did not affect the statistical analysis used to separate species.
Figure 1 - Represents the spectra of L. major, L. chagasi, and L. amazonensis with their respective standard deviations. The letters A, B, C, and D represent the main regions.
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The best results of cluster analysis, which allows the differentiation of these three Leishmania species, were found using the spectral regions indicated by A (2890 cm-1 to 3040 cm-1), B (1364 cm-1 to 1472 cm-1), C (1190 cm-1 to 1282 cm-1), and D (1028 cm-1 the 1120 cm-1) in Fig. 1. The A region contains absorption bands of lipids and shows differences in intensities for bands at 2923 cm-1 (asymmetric stretching of CH2) and 2963 cm-1 (C-H asymmetric stretching of CH3), both characteristic of fatty acids.[4, 33, 34]. The B region is related to the absorption bands of lipids and proteins. In this region, two main bands exist: the C = O stretch of COO- bonds associated with amino acids and fatty acids at 1399 cm-1 and scissoring vibrations (δ(CH3) and δ(CH2)) at 1453 cm-1 associated with lipids. [20, 33, 35, 36]. The C region has the absorption bands at 1239 cm-1 of amide III, principal components of proteins. A small difference between the intensity of this band in the spectra was observed. [20, 36]. The D region contains the absorption bands of polysaccharides including a band at 1086 cm-1, which corresponds to P = O symmetric stretching of > PO2- in nucleic acid. A small difference between the intensities of this phosphodiester functional group of DNA/RNA polysaccharide backbone is observed in the spectra.[34] Fig. 2 shows the dendrogram calculated by cluster analysis, where the separation shows clear heterogeneity among Leishmania species. The L. major and L. chagasi have less heterogeneity than L. amazonensis. The heterogeneity between the species is a function of the phylogeny, which is strongly correlated with geographic origin [37]
Figure 2. The dendrogram indicates the difference between the species of Leishmania. Cluster analysis used the second derivatives. The separation regions were between 1028 cm-1 to 1120 cm-1, 1190 cm-1 to 1282 cm-1, 1364 cm-1 to 1472 cm-1, and 2890 cm-1 to 3040 cm-1 .
Table.1 shows the contributions of spectral bands from the FT-IR of Leishmania, using second derivative (20d) and Gaussians areas. The statistical parameter used adjusts R – Square = 9,99E-001 was for all regions. Table 1: Assignments of bands found in the spectra of the Leishmania in FT-IR
L. amazonensis
L. chagasi
2ºd
2ºd
GF - %
Assignments*
L. major GF – %
2ºd
GF - %
4
3496 3434 3352 ~3285 3193 3089 3062 3012
3512 3427 3352 3291 3218 3125 3069 3013
---12.7 27 23.7 31.1 1.5 3.4 0.2
3496 3434 3352 3285 3193 3089 3062 3014
3530 3423 3357 3291 3208 3126 3068 2756
---14 19.8 39.5 21.4 2.4 2.6 ---
3496 3434 3352 3285 3193 3089 3062 3014
3509 3433 3341 3286 3223 3169 3068 3015
---8.7 41.4 17.6 20.6 8.4 2.9 0.03
2985 2962
----2959
----23.8
2985 2962
2983 2961
0.14 25.2
2985 2962
3042 2961
----28.7
2925
2926
46.2
2923
2926
46.9
2923
2925
44.5
2897 2874 2854
2900 2870 2854
5.7 16.7 7.3
2897 2874 2854
2899 2872 2853
2.9 18.1 5.8
2897 2874 2854
2899 2873 2853
3.1 18.2 5.3
1467 1453
1468 1456
7.6 25.9
1467 1453
1468 1454
4.5 27.4
1467 1453
1469 1457
6.6 18.4
1443 1413 1401 1380 1367
1442 1417 1396 1379 1368
10 19.2 32.2 3.9 0.8
1443 1414 1401 1387 1368
1441 1413 1401 1387 1368
17.5 11.7 14.2 22.8 1.5
1443 1414 1401 1387 1368
1443 1416 1400 1385 1368
18.4 17.6 18.2 19 1.4
1313 1298 1282
1316 1303 1274
0.9 4.5 27.6
1314
1311
5.7
1314
1312
3.6
1242
1240
60.5
1282 1266 1239
1283 1258 1238
29.4 12.8 46.2
1282 1266 1239
1283 1257 1237
32.1 3.2 60.6
1221
1219
6.5
1172 1153 1119 1101 1086
1172 1154 1119 1104 1086
2.3 8.9 29.6 5.6 25.5
1206 1172 1151 1119 1102 1086
1216 1172 1152 1110 1096 1084
5.6 1.1 7.7 26.2 7.3 13.2
1206 1172 1152 1119 1102 1085
1210 1171 1152 1119 1102 1085
0.2 1.1 6.5 14.4 9.2 22.7
1068
1072
9
1068
1068
6.4
1058
1058
19.2
1057
1059
15.7
1057
1058
23.2
1039
1041
0.9
1034
1040
10.9
1034
1034
7.1
1030
1031
4.7
5
O–H str of hydroxyl groups [28, 35] υ(N–H) of amide A [38] υ (N–H) of amide B [38] NH str vibration (amide A) [19] υsN–H str (amide A) of proteins [33] Fatty acid chains [34] Fatty acid[20, 34] = C–H str of unsaturated fatty acid chains [34] Fatty acid [39] C–H str (asym) of > CH3 in fatty acids [4, 34] C–H str vibration fatty acid of > CH2 [33] C–H str of → C–H aminoacids [20] C–H str (sym) of – CH3 [33] C–H str (sym) of > CH2 in fatty acids[33] C-H def of >CH2[33, 35] δ(CH3) and (CH2) scissoring lipids and proteins [36] CH2 bending vibration [24, 40] υs(CH3)lipids and aromatics [36] C=O str (sym) of COO- [33, 35] δ(CH3) [19] COO- str and CH3 sym bending in proteins and lipids [36] Amide III ,δ(CH) [19, 34] Amide II, (C – N) [39] Amide II [34] Amide III, α – helix [36] Amide III band components of proteins [33] C-O-C and C-O, polysaccharides [20] Tryptophan, υa ( CC) [19] Polysaccharide [34] Polysaccharide [34] Polysaccharide [34] υs(PO2-) nucleic acids [36] P=O str (sym) of >PO2-, in DNA, RNA and phospholipids [20, 35] C-O-C and C-O, polysaccharides [20] Aromatic C – H planar bending vibration [41] C–O–C of the ring of polysaccharides [34] Serine, υs(C – O) [19]
1010 994
1011 996
0.7 1.1
1009 1000
1019 1001
6.5 0.4
1009 1000
1009 998
7.2 ----
969
969
0.9
968
968
1.5
968
968
1.7
tryptophan, υs(CC),δ(CH) [19] C–O–C and C–O, Carbohydrates. Sym str vibration of PO-2 groups in nucleic acids [22, 34] C–C and C–O vibrations in deoxyribose [42]
*str=stretching; sym=symmetric; asym=antisymmetric; ν= stretching vibration; νs=symmetric stretching vibration; νas= antisymmetric stretching vibration; δ= in plane bending vibration. The statistical parameter was adjust R – Square = 9,99E-001 for all Gaussians .
4. Discussion The spectral deconvolution using second derivatives exhibited many differences that might lead to an understanding of the variation of chemical compound of the parasites. Many bands were recorded for all species with differences in relative band areas, but some of them were more specific for a single species. The main components of interest in microorganisms are polysaccharides, nucleic acids, amino acids, lipids, and proteins. Thus, the difference will be discussed using changes in several bands related to the corresponding functional groups. For polysaccharide, the results described in table 1 show that all species presented quantitative differences of polysaccharide in the bands at 1039 cm-1, 1119 cm-1, 1172 cm-1, and 1153 cm-1. L. amazonensis and L. chagasi showed values of 15.4% and 11.8% higher than L. major, in the band at 1119 cm-1, respectively. The band at 1039 cm-1 record for L. chagasi and L. major was 10% and 6.2 % higher than L. amazonensis. Only slight variations were observed in the other two bands. Moreover, the band at 1068 cm-1 was record only for L. chagasi and L. major species. The band at 1221 cm -1 was specific for L. amazonensis. The differences in the aforementioned absorption bands may be explained by specific differences in the surface of the membrane of the Leishmania species. Even though there is a physicochemical similarity due to its phylogenetic proximity, the variations of polysaccharide in these bands help discriminate these parasites. These differences in polysaccharides could be explained by the amount of glycoconjugates, a type of oligosaccharides present in these glycoconjugates. The substitutions in one or more sugars promote high intraspecific and interspecific polimorphism. Within glycoconjugates, lipofosfoglican (LPG) and the glycoproteins (GP63) are more abundant in the parasite membrane and play an important role in survival in the macrophage, invasion, and virulence in the host. Apart from that, glycoconjugates are potential targets for vaccine development [43-48] The most abundant cell-surface molecules of the Leishmania promastigote stage are the glycosylphosphatidylinositol (GPI) protein anchors. The GPI, called membrane anchors, is a glycolipid, connect protein, or carbohydrate polymers to membrane surface.[49, 50] For lipids and proteins, a wide variety of proteins and lipids in the surface of the parasite attached in the membrane by a GPI-anchor varies drastically among species and even between distinct life-cycle stages of the same species. The lipids are structurally essential for formation of phospholipids, which are part of GPI-anchors. Therefore, the quantitative variation of lipids among species also indicates the various types of glycoconjugates present in the external surfaces of parasites.[51] Table 1 shows the quantitative difference of lipids among species and highlights two bands. The band at 1401 cm-1 has greater percentage for L. amazonensis when compared the other two species, and the band 1387 cm-1 for L. chagasi and L. major has a significant percentage when compared to the L. amazonensis. The other bands at 1367 cm-1, 1413 cm-1, and 1467 cm-1 also point to differences among species. In addition to the differences of lipids, a displacement of bands was found. The band at 1380 cm-1 was observed in the L. amazonensis, and in the band at 1387 cm-1 observed in the other two species. The three parasites presented difference in all bands mentioned, because they are biochemically different. [52].
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In this sense, the results showed that L. amazonensis and L. major have in the band 1086 cm-1 differences of 12.3% and 9.5%, respectively, of phospholipids when compared to L. chagasi. These variations of phospholipids among the species contribute to differentiate one species from another. The band at 1101 cm-1 that represent the nucleic acids, exhibited small contributions, but also help in separation. The serine amino acids are part of the composition of one type of phospholipids known as phosphatidylserine. The phosphatidylserine is the membrane component that keeps Leishmania intact and helps in the membrane to perform vital functions such as moving nutrients into Leishmania and pumping waste products out of them .[53] In this sense, the result showed that in the D region, a band at 1030 cm-1 is specific for L. amazonensis of serine. In the C region at 1206 cm-1, the amino acid tryptophan is associated with the virulence of parasite. In the A region, the band at 2897 cm-1 pointed to difference in the amount of amino acids among the species. Therefore, the difference of amino acids found collaborated to differentiate the species. For the fatty acids, the bands that represent the fatty acids region showed small difference among the species. The band at 1453 cm-1 and 1443cm-1 of fatty acids exhibited highest differences. The band at 3012 cm-1 presented very little unsaturated fatty acid. These differences between the Leishmania species with respect to fatty acids is most likely a manifestation of the relative amounts of glycolipids present in the membrane and organelles of the parasite, which can help distinguish the three species. [5456]. The amide III region is another important spectral region to separate the species. In this region, L. amazonensis registered high quantitate amide III in the band at 1242 cm-1, when comparing the L. chagasi and L. major species. L .chagasi and L. major showed differences in the band at 1266 cm-1; this band is specific for these two species. For the amide II region, there was no important difference among the three species. These bands contributed for distinguish these parasites. 5. Conclusion We investigated the potential of FT-IR microspectroscopy for rapid identification of Leishmania. The results demonstrate that infrared absorption is an indispensable tool for differentiating these parasites. The FT-IR spectra of L. amazonensis, L. chagasi, and L. major were analyzed by multivariate statistical analysis (cluster analysis) and the deconvolution procedure based on spectra second derivatives. The cluster analysis demonstrated heterogeneity of 500 when comparing L. amazonensis, L. chagasi, and L. major at the same time; and a heterogeneity of approximately 60 when comparing L. chagasi and L. major. To elucidate the biochemical basis of these differences, the FT-IR spectra of L. amazonensis, L. chagasi, and L. major was analyzed by Gaussian deconvolution. The parasites were discriminated by several spectral bands, attributed to differences in the amount and polimorphism of polysaccharide, fatty acids (phospholipids), nucleic acid, and proteins (amide III). It is evident that the FT-IR method is an indispensable tool to discriminate these parasites. This technique opens up the possibilities for further studies on other species of Leishmania. Acknowledgement The authors gratefully acknowledge the Fundação Valeparaibana de Ensino (FVE) for scholarship.
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[39] J.S.H.-C. Flemming, FTIR - spectroscopy in microbial and material analysis, International Biodeterioration & Biodegradation, 41 (1998) 1-11. [40] A.A. Kamnev, A.V. Tugarova, L.P. Antonyuk, P.A. Tarantilis, L.A. Kulikov, Y.D. Perfiliev, M.G. Polissiou, P.H.E. Gardiner, Instrumental analysis of bacterial cells using vibrational and emission Mossbauer spectroscopic techniques, Anal Chim Acta, 573 (2006) 445452. [41] A. WAHAB, Infrared Absorption Studies on Some New Potential Antimicrobial Diazotization product of 4 – aryl – Thiosemicarbazides, Orient. J. Chem, 27 (2011) 1199-1202. [42] M.K.A. Ahmed, F.; SEALY, E.A, Unique spectral features of DNA infrared bands of some microorganisms, Spectroscopy, 23 (2009) 291 – 297. [43] M.A. Ferguson, The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research, Journal of cell science, 112 ( Pt 17) (1999) 2799-2809. [44] P. Schneider, M.A. Ferguson, M.J. McConville, A. Mehlert, S.W. Homans, C. Bordier, Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease, The Journal of biological chemistry, 265 (1990) 16955-16964. [45] M.J. McConville, J.E. Thomas-Oates, M.A. Ferguson, S.W. Homans, Structure of the lipophosphoglycan from Leishmania major, The Journal of biological chemistry, 265 (1990) 19611-19623. [46] M.J. McConville, J.M. Blackwell, Developmental changes in the glycosylated phosphatidylinositols of Leishmania donovani. Characterization of the promastigote and amastigote glycolipids, The Journal of biological chemistry, 266 (1991) 15170-15179. [47] M.J. McConville, M.A. Ferguson, The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes, The Biochemical journal, 294 ( Pt 2) (1993) 305-324. [48] S.M. Beverley, S.J. Turco, Lipophosphoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania, Trends in microbiology, 6 (1998) 35-40. [49] M. J.J.M. MARR, Biochemistry and Molecular of parasites, Academic Press Inc, 1995. [50] S.J. Turco, A. Descoteaux, The lipophosphoglycan of Leishmania parasites, Annual review of microbiology, 46 (1992) 65-94. [51] H.J. Vial, P. Eldin, A.G. Tielens, J.J. van Hellemond, Phospholipids in parasitic protozoa, Molecular and biochemical parasitology, 126 (2003) 143-154. [52] M.J. McConville, A. Bacic, A family of glycoinositol phospholipids from Leishmania major. Isolation, characterization, and antigenicity, The Journal of biological chemistry, 264 (1989) 757-766. [53] D.E.V.a.J.E. Vance, Biochemistry of Lipids, Lipoproteins and Membranes, 4th edition ed., 2002. [54] S.C. Ilgoutz, M.J. McConville, Function and assembly of the Leishmania surface coat, International journal for parasitology, 31 (2001) 899-908. [55] A. Descoteaux, S.J. Turco, Glycoconjugates in Leishmania infectivity, Biochimica et biophysica acta, 1455 (1999) 341-352. [56] C.W. Roberts, R. McLeod, D.W. Rice, M. Ginger, M.L. Chance, L.J. Goad, Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa, Molecular and biochemical parasitology, 126 (2003) 129-142.
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Highlights ► Differentiation of L. amazonensis, L. chagasi and L. major species by FTIR. ► Four spectral regions was able toidentify Leishmania sp. byCluster analysis. ► The polysaccharide region showed greater differences among the Leishmania species.
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S1386-1425(15)00018-9 http://dx.doi.org/10.1016/j.saa.2015.01.008 SAA 13176
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
24 June 2014 11 December 2014 5 January 2015
Please cite this article as: J.C. Aguiar, J. Mittmann, I. Ferreira, J. Ferreira-Strixino, L. Raniero, Differentiation of Leishmania species by FT-IR spectroscopy, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.008
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Differentiation of Leishmania species by FT-IR spectroscopy Josafá C. Aguiar1, Josane Mittmann 1, Isabelle Ferreira 1, Juliana Ferreira-Strixino 1, Leandro Raniero 1 1
Laboratório de Nanossensores – Instituto de Pesquisa & Desenvolvimento Universidade do Vale do Paraíba – UNIVAP Shishima Hifumi Avenue, 2911, Urbanova, 12244.000, São José dos Campos-SP, Brazil
E-mail: [email protected]
Abstract Leishmaniasis is a parasitic infectious disease caused by protozoa that belong to the genus Leishmania. It is transmitted by the bite of an infected female Sand fly. The disease is endemic in 88 countries[1] (16 developed countries and 72 developing countries) on four continents. In Brazil, epidemiological data show the disease is present in all Brazilian regions, with the highest incidences in the North and Northeast. There are several methods used to diagnose leishmaniasis, but these procedures have many limitations, are time consuming, have low sensitivity, and are expensive. In this context, Fourier Transform Infrared Spectroscopy (FT-IR) analysis has the potential to provide rapid results and may be adapted for a clinical test with high sensitivity and specificity. In this work, FT-IR was used as a tool to investigate the promastigotes of L. amazonensis, L. chagasi, and L. major species. The spectra were analyzed by cluster analysis and deconvolution procedure base on spectra second derivatives. Results: cluster analysis found four specific regions that are able to identify the Leishmania species. The dendrogram representation clearly indicates the heterogeneity among Leishmania species. The band deconvolution done by the curve fitting in these regions quantitatively differentiated the polysaccharides, amide III, phospholipids, proteins, and nucleic acids. L. chagasi and L. major showed a greater biochemistry similarity and have three bands that were not registered in L. amazonensis. The L. amazonensis presented three specific bands that were not recorded in the other two species. It is evident that the FT-IR method is an indispensable tool to discriminate these parasites. The high sensitivity and specificity of this technique opens up the possibilities for further studies about characterization of other microorganisms.
Keywords: L. amazonensis; L. chagasi; L. major; FT – IR; Cluster Analysis; Biochemistry.
1. Introduction 1.1Leishmania Parasites of the genus Leishmania belong to the Kinetoplastida order and Trypanosomatidae family [2, 3]. These parasites cause the human disease known as leishmaniasis, which can present in different clinical forms, depending on the Leishmania species involved and the relation between the parasite and its host [4]. The transmission occurs during the blood meal of Sand flies from the Phlebotominae family, via injection of promastigotes [5, 6]. The classification of Leishmania includes two subgenera Viannia and Leishmania. The species studied in this article belong to the subgenus Leishmania. Leishmania chagasi and Leishmania amazonensis are found in the New World (Americas), and Leishmania major is found in the Old World
1
(Europe, Africa, and Asia) [7-9]. The L. amazonensis and L. major species cause cutaneous leishmaniasis, and L. chagasi causes the most severe form of the disease called visceral leishmaniasis [10]. Epidemiological data show that the highest incidence of leishmaniasis cases in Brazil occurs in the North and Northeast regions. The main factors associated with its increased incidence are the exploration of forest for agricultural activities and ecotourism. The annual average between 2007 and 2011 of cutaneous and visceral leishmaniasis cases were 20,187 and 3,682, respectively. [11-15]. The various approaches to diagnose leishmaniasis include: a) serological detection of antiLeishmania antibodies, though cross-reactivity with other infections such as Chagas disease can occur. b) Indirect immunofluorescence (RIFI) is the test of choice, though its lack of specificity is influenced by cross reactions with other diseases caused by trypanosomes. c) ELISA has better sensitivity and specificity but cross reactions with other diseases caused by trypanosomes may still occur. d) Molecular diagnosis, including PCR to detect DNA, has good sensitivity compared to immunoassays [16-18]. However, PCR is a costly technique, and as such, it is rarely used for diagnosis. Fourier Transform Infrared Spectroscopy (FTIR) analysis has the potential to provide rapid results and may achieve high values of sensitivity and specificity. This technique is able to identify and characterize biological samples by their chemical composition [19-21]. This technique has been applied successfully to identify and characterize microorganisms, such as Salmonella, Bacillus subtilis, Staphylococcus, yeasts, filamentous fungi, etc. [22-26]. FTIR is based on the interaction of infrared radiation with matter [20, 27]. Infrared radiation is absorbed when its frequency (energy) equals that of a vibrational mode. These molecular vibrations are unique to the type and spatial arrangement of the atoms in a sample [20]. Thus, the spectrum is a fingerprint of the illuminated molecule. Furthermore, structural information about important subunits of the molecule, called functional groups, can be obtain.[20, 26, 28]. This article presents the characterization of L. amazonensis, L. major, and L. chagasi by FT-IR, and demonstrates that the species can be identified by analyzing biochemical differences between them.
2. Materials and Methods 2.1Parasites The species of parasites used were Leishmania major (MHOM/SU/1973/5-ASKH strain), Leishmania chagasi (MHOM/BR/1974/PP75 strain), and Leishmania amazonensis (MHOM/BR/73 M2269 strain). They were grown in the M199 medium supplemented with 10% Fetal Bovine Serum, Urine 2%, 10 U/ml penicillin, 10 U/ml streptomycin, 0.25% hemin and kept at 26 °C and pH 7.4. The subcultures of promastigotes were replicated weekly. 2.2 Sample preparation For FT-IR analyses, the sample preparation is an important factor, because the composition of the culture medium has a strong influence on the results. To remove the culture medium, 20 ml of the cultures were centrifuged at 3500 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in 15 ml of NaCl solution at 0.2%. This procedure was repeated three times. After each centrifugation, an aliquot was observed under the microscope to verify the integrity of the parasites. Then, IR spectra were collected from five independently grown parasite cultures for each species. 2.3 FT - IR spectroscopy and statistical analysis After the cleaning process to remove media, a drop of 10 µl with approximately 485 parasites was deposited on a calcium fluoride (CaF2) window. The sample was dried by an Eppendorf Concentrator, which allows the thin film formation of the unanalyzed Leishmaniasis. The IR spectra was collected by a microscope (Spotlight Perkin Elmer 400, USA) coupled to FT-IR spectrophotometer (Spectrum 400), which was controlled by a computer with Spotlight 400 software, version 3.6.2. The sample was analyzed in four random regions, totaling 22 points. The spectra were recorded in regions with approximately the same thickness. The band centered at 1650 cm-1 had the highest value of
2
absorbance around 0.9 to 1.0 for all spectra. Each spectrum was obtained from an average of 64 spectra per point with spectral resolution of 4 cm-1. The spectral range was from 4000 cm-1 to 750 cm-1. These spectra were analyzed by cluster analysis and deconvolution procedure base on spectra second derivatives. The multivariate statistical analysis was performed by Cluster Analysis [29] using OPUS software version 4.2 with the following parameters: second derivative, smoothing 9 points, Ward's algorithm, and Scaling to 1st range method. Cluster analysis classifies objects into groups which show similarities [30] and is commonly used to rapidly differentiate / classify spectra of microorganisms [31]. Gaussians curve fitting was used in the observed band deconvolutions, thereby quantifying the contribution of specific biochemical species in each region of the spectrum [32]. The biochemical changes, which underlie the separation obtained by cluster analysis, can then be elucidated.
3. Results Fig. 1 shows the average IR spectra for L. major, L. chagasi, and L. amazonensis species. The standard deviations (SD) are represented in gray. The highest variation, observed for L. chagasi, may reflect the development stage of the culture, since they are different parasites, which respond differently in time. This change did not affect the statistical analysis used to separate species.
Figure 1 - Represents the spectra of L. major, L. chagasi, and L. amazonensis with their respective standard deviations. The letters A, B, C, and D represent the main regions.
3
The best results of cluster analysis, which allows the differentiation of these three Leishmania species, were found using the spectral regions indicated by A (2890 cm-1 to 3040 cm-1), B (1364 cm-1 to 1472 cm-1), C (1190 cm-1 to 1282 cm-1), and D (1028 cm-1 the 1120 cm-1) in Fig. 1. The A region contains absorption bands of lipids and shows differences in intensities for bands at 2923 cm-1 (asymmetric stretching of CH2) and 2963 cm-1 (C-H asymmetric stretching of CH3), both characteristic of fatty acids.[4, 33, 34]. The B region is related to the absorption bands of lipids and proteins. In this region, two main bands exist: the C = O stretch of COO- bonds associated with amino acids and fatty acids at 1399 cm-1 and scissoring vibrations (δ(CH3) and δ(CH2)) at 1453 cm-1 associated with lipids. [20, 33, 35, 36]. The C region has the absorption bands at 1239 cm-1 of amide III, principal components of proteins. A small difference between the intensity of this band in the spectra was observed. [20, 36]. The D region contains the absorption bands of polysaccharides including a band at 1086 cm-1, which corresponds to P = O symmetric stretching of > PO2- in nucleic acid. A small difference between the intensities of this phosphodiester functional group of DNA/RNA polysaccharide backbone is observed in the spectra.[34] Fig. 2 shows the dendrogram calculated by cluster analysis, where the separation shows clear heterogeneity among Leishmania species. The L. major and L. chagasi have less heterogeneity than L. amazonensis. The heterogeneity between the species is a function of the phylogeny, which is strongly correlated with geographic origin [37]
Figure 2. The dendrogram indicates the difference between the species of Leishmania. Cluster analysis used the second derivatives. The separation regions were between 1028 cm-1 to 1120 cm-1, 1190 cm-1 to 1282 cm-1, 1364 cm-1 to 1472 cm-1, and 2890 cm-1 to 3040 cm-1 .
Table.1 shows the contributions of spectral bands from the FT-IR of Leishmania, using second derivative (20d) and Gaussians areas. The statistical parameter used adjusts R – Square = 9,99E-001 was for all regions. Table 1: Assignments of bands found in the spectra of the Leishmania in FT-IR
L. amazonensis
L. chagasi
2ºd
2ºd
GF - %
Assignments*
L. major GF – %
2ºd
GF - %
4
3496 3434 3352 ~3285 3193 3089 3062 3012
3512 3427 3352 3291 3218 3125 3069 3013
---12.7 27 23.7 31.1 1.5 3.4 0.2
3496 3434 3352 3285 3193 3089 3062 3014
3530 3423 3357 3291 3208 3126 3068 2756
---14 19.8 39.5 21.4 2.4 2.6 ---
3496 3434 3352 3285 3193 3089 3062 3014
3509 3433 3341 3286 3223 3169 3068 3015
---8.7 41.4 17.6 20.6 8.4 2.9 0.03
2985 2962
----2959
----23.8
2985 2962
2983 2961
0.14 25.2
2985 2962
3042 2961
----28.7
2925
2926
46.2
2923
2926
46.9
2923
2925
44.5
2897 2874 2854
2900 2870 2854
5.7 16.7 7.3
2897 2874 2854
2899 2872 2853
2.9 18.1 5.8
2897 2874 2854
2899 2873 2853
3.1 18.2 5.3
1467 1453
1468 1456
7.6 25.9
1467 1453
1468 1454
4.5 27.4
1467 1453
1469 1457
6.6 18.4
1443 1413 1401 1380 1367
1442 1417 1396 1379 1368
10 19.2 32.2 3.9 0.8
1443 1414 1401 1387 1368
1441 1413 1401 1387 1368
17.5 11.7 14.2 22.8 1.5
1443 1414 1401 1387 1368
1443 1416 1400 1385 1368
18.4 17.6 18.2 19 1.4
1313 1298 1282
1316 1303 1274
0.9 4.5 27.6
1314
1311
5.7
1314
1312
3.6
1242
1240
60.5
1282 1266 1239
1283 1258 1238
29.4 12.8 46.2
1282 1266 1239
1283 1257 1237
32.1 3.2 60.6
1221
1219
6.5
1172 1153 1119 1101 1086
1172 1154 1119 1104 1086
2.3 8.9 29.6 5.6 25.5
1206 1172 1151 1119 1102 1086
1216 1172 1152 1110 1096 1084
5.6 1.1 7.7 26.2 7.3 13.2
1206 1172 1152 1119 1102 1085
1210 1171 1152 1119 1102 1085
0.2 1.1 6.5 14.4 9.2 22.7
1068
1072
9
1068
1068
6.4
1058
1058
19.2
1057
1059
15.7
1057
1058
23.2
1039
1041
0.9
1034
1040
10.9
1034
1034
7.1
1030
1031
4.7
5
O–H str of hydroxyl groups [28, 35] υ(N–H) of amide A [38] υ (N–H) of amide B [38] NH str vibration (amide A) [19] υsN–H str (amide A) of proteins [33] Fatty acid chains [34] Fatty acid[20, 34] = C–H str of unsaturated fatty acid chains [34] Fatty acid [39] C–H str (asym) of > CH3 in fatty acids [4, 34] C–H str vibration fatty acid of > CH2 [33] C–H str of → C–H aminoacids [20] C–H str (sym) of – CH3 [33] C–H str (sym) of > CH2 in fatty acids[33] C-H def of >CH2[33, 35] δ(CH3) and (CH2) scissoring lipids and proteins [36] CH2 bending vibration [24, 40] υs(CH3)lipids and aromatics [36] C=O str (sym) of COO- [33, 35] δ(CH3) [19] COO- str and CH3 sym bending in proteins and lipids [36] Amide III ,δ(CH) [19, 34] Amide II, (C – N) [39] Amide II [34] Amide III, α – helix [36] Amide III band components of proteins [33] C-O-C and C-O, polysaccharides [20] Tryptophan, υa ( CC) [19] Polysaccharide [34] Polysaccharide [34] Polysaccharide [34] υs(PO2-) nucleic acids [36] P=O str (sym) of >PO2-, in DNA, RNA and phospholipids [20, 35] C-O-C and C-O, polysaccharides [20] Aromatic C – H planar bending vibration [41] C–O–C of the ring of polysaccharides [34] Serine, υs(C – O) [19]
1010 994
1011 996
0.7 1.1
1009 1000
1019 1001
6.5 0.4
1009 1000
1009 998
7.2 ----
969
969
0.9
968
968
1.5
968
968
1.7
tryptophan, υs(CC),δ(CH) [19] C–O–C and C–O, Carbohydrates. Sym str vibration of PO-2 groups in nucleic acids [22, 34] C–C and C–O vibrations in deoxyribose [42]
*str=stretching; sym=symmetric; asym=antisymmetric; ν= stretching vibration; νs=symmetric stretching vibration; νas= antisymmetric stretching vibration; δ= in plane bending vibration. The statistical parameter was adjust R – Square = 9,99E-001 for all Gaussians .
4. Discussion The spectral deconvolution using second derivatives exhibited many differences that might lead to an understanding of the variation of chemical compound of the parasites. Many bands were recorded for all species with differences in relative band areas, but some of them were more specific for a single species. The main components of interest in microorganisms are polysaccharides, nucleic acids, amino acids, lipids, and proteins. Thus, the difference will be discussed using changes in several bands related to the corresponding functional groups. For polysaccharide, the results described in table 1 show that all species presented quantitative differences of polysaccharide in the bands at 1039 cm-1, 1119 cm-1, 1172 cm-1, and 1153 cm-1. L. amazonensis and L. chagasi showed values of 15.4% and 11.8% higher than L. major, in the band at 1119 cm-1, respectively. The band at 1039 cm-1 record for L. chagasi and L. major was 10% and 6.2 % higher than L. amazonensis. Only slight variations were observed in the other two bands. Moreover, the band at 1068 cm-1 was record only for L. chagasi and L. major species. The band at 1221 cm -1 was specific for L. amazonensis. The differences in the aforementioned absorption bands may be explained by specific differences in the surface of the membrane of the Leishmania species. Even though there is a physicochemical similarity due to its phylogenetic proximity, the variations of polysaccharide in these bands help discriminate these parasites. These differences in polysaccharides could be explained by the amount of glycoconjugates, a type of oligosaccharides present in these glycoconjugates. The substitutions in one or more sugars promote high intraspecific and interspecific polimorphism. Within glycoconjugates, lipofosfoglican (LPG) and the glycoproteins (GP63) are more abundant in the parasite membrane and play an important role in survival in the macrophage, invasion, and virulence in the host. Apart from that, glycoconjugates are potential targets for vaccine development [43-48] The most abundant cell-surface molecules of the Leishmania promastigote stage are the glycosylphosphatidylinositol (GPI) protein anchors. The GPI, called membrane anchors, is a glycolipid, connect protein, or carbohydrate polymers to membrane surface.[49, 50] For lipids and proteins, a wide variety of proteins and lipids in the surface of the parasite attached in the membrane by a GPI-anchor varies drastically among species and even between distinct life-cycle stages of the same species. The lipids are structurally essential for formation of phospholipids, which are part of GPI-anchors. Therefore, the quantitative variation of lipids among species also indicates the various types of glycoconjugates present in the external surfaces of parasites.[51] Table 1 shows the quantitative difference of lipids among species and highlights two bands. The band at 1401 cm-1 has greater percentage for L. amazonensis when compared the other two species, and the band 1387 cm-1 for L. chagasi and L. major has a significant percentage when compared to the L. amazonensis. The other bands at 1367 cm-1, 1413 cm-1, and 1467 cm-1 also point to differences among species. In addition to the differences of lipids, a displacement of bands was found. The band at 1380 cm-1 was observed in the L. amazonensis, and in the band at 1387 cm-1 observed in the other two species. The three parasites presented difference in all bands mentioned, because they are biochemically different. [52].
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In this sense, the results showed that L. amazonensis and L. major have in the band 1086 cm-1 differences of 12.3% and 9.5%, respectively, of phospholipids when compared to L. chagasi. These variations of phospholipids among the species contribute to differentiate one species from another. The band at 1101 cm-1 that represent the nucleic acids, exhibited small contributions, but also help in separation. The serine amino acids are part of the composition of one type of phospholipids known as phosphatidylserine. The phosphatidylserine is the membrane component that keeps Leishmania intact and helps in the membrane to perform vital functions such as moving nutrients into Leishmania and pumping waste products out of them .[53] In this sense, the result showed that in the D region, a band at 1030 cm-1 is specific for L. amazonensis of serine. In the C region at 1206 cm-1, the amino acid tryptophan is associated with the virulence of parasite. In the A region, the band at 2897 cm-1 pointed to difference in the amount of amino acids among the species. Therefore, the difference of amino acids found collaborated to differentiate the species. For the fatty acids, the bands that represent the fatty acids region showed small difference among the species. The band at 1453 cm-1 and 1443cm-1 of fatty acids exhibited highest differences. The band at 3012 cm-1 presented very little unsaturated fatty acid. These differences between the Leishmania species with respect to fatty acids is most likely a manifestation of the relative amounts of glycolipids present in the membrane and organelles of the parasite, which can help distinguish the three species. [5456]. The amide III region is another important spectral region to separate the species. In this region, L. amazonensis registered high quantitate amide III in the band at 1242 cm-1, when comparing the L. chagasi and L. major species. L .chagasi and L. major showed differences in the band at 1266 cm-1; this band is specific for these two species. For the amide II region, there was no important difference among the three species. These bands contributed for distinguish these parasites. 5. Conclusion We investigated the potential of FT-IR microspectroscopy for rapid identification of Leishmania. The results demonstrate that infrared absorption is an indispensable tool for differentiating these parasites. The FT-IR spectra of L. amazonensis, L. chagasi, and L. major were analyzed by multivariate statistical analysis (cluster analysis) and the deconvolution procedure based on spectra second derivatives. The cluster analysis demonstrated heterogeneity of 500 when comparing L. amazonensis, L. chagasi, and L. major at the same time; and a heterogeneity of approximately 60 when comparing L. chagasi and L. major. To elucidate the biochemical basis of these differences, the FT-IR spectra of L. amazonensis, L. chagasi, and L. major was analyzed by Gaussian deconvolution. The parasites were discriminated by several spectral bands, attributed to differences in the amount and polimorphism of polysaccharide, fatty acids (phospholipids), nucleic acid, and proteins (amide III). It is evident that the FT-IR method is an indispensable tool to discriminate these parasites. This technique opens up the possibilities for further studies on other species of Leishmania. Acknowledgement The authors gratefully acknowledge the Fundação Valeparaibana de Ensino (FVE) for scholarship.
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Highlights ► Differentiation of L. amazonensis, L. chagasi and L. major species by FTIR. ► Four spectral regions was able toidentify Leishmania sp. byCluster analysis. ► The polysaccharide region showed greater differences among the Leishmania species.
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