Introduction
Leishmaniasis is a disease complex considered as a public health problem in tropical and subtropical regions in the world, such as Asia, Africa and the Americas. It is caused by obligate protozoan parasites of the genus Leishmania, occurring in 98 countries with 12 million people clinically affected, and 350 million at risk of infection (WHO, 2016). Visceral leishmaniasis (VL) is caused by parasites of the Leishmania donovani and Leishmania infantum species, and it can be fatal if acute and left untreated (Torres-Guerrero et al., Reference Torres-Guerrero, Quintanilla-Cedillo, Ruiz-Esmenjaud and Arenas2017). Regarding the treatment of disease, pentavalent antimonials, liposomal and free amphotericin B, paromomycin, pentamidine and oral miltefosine are used. However, these drugs are toxic and/or present high cost (Alves et al., Reference Alves, Bilbe, Blesson, Goyal, Monnerat, Mowbray, Muthoni, Pécoul, Rijal, Rode, Solomos, Strub-Wourgaft, Wasunna, Wells, Zijlstra, Arana and Alvar2018; Kapil et al., Reference Kapil, Singh and Silakari2018). As a consequence, the prevention of disease should be considered, such as by use of vaccination (Kumar and Samant, Reference Kumar and Samant2016). Although a number of antigens have been tested, and a variable degree of success has been obtained in murine and/or canine models, there is no effective vaccine to protects against human disease (Nico et al., Reference Nico, Claser, Borja-Cabrera, Travassos, Palatnik, Soares, Rodrigues and Palatnik-de-Sousa2010; Martins et al., Reference Martins, Chávez-Fumagalli, Costa, Canavacci, Martins, Lage, Lage, Duarte, Valadares, Magalhães, Ribeiro, Nagem, DaRocha, Régis, Soto, Coelho, Fernandes and Tavares2013; Grimaldi et al., Reference Grimaldi, Teva, Porrozzi, Pinto, Marchevsky, Rocha, Dutra, Bruña-Romero, Fernandes and Gazzinelli2014; Mortazavidehkordi et al., Reference Mortazavidehkordi, Farjadfar, Khanahmad, Najafabadi, Hashemi, Fallah, Najafi, Kia and Hejazi2016; Dias et al., Reference Dias, Martins, Ribeiro, Ramos, Lage, Tavares, Mendonça, Chávez-Fumagalli, Oliveira, Silva, Gomes, Rodrigues, Duarte, Galdino, Menezes-Souza and Coelho2018).
The control of VL requires appropriate diagnosis and adequate treatment, since the precise diagnosis is essential for effective drug regimen for patients (Vijayakumar and Das, Reference Vijayakumar and Das2018). Laboratory strategies are being employed, such as parasitological and immunological evaluations. Parasitological methods include microscopy with the identification of amastigote forms in organ aspirates, such as bone marrow, spleen, liver and/or lymph nodes. However, limitations due to the variable sensitivity, the requirement of technical expertise, the sample's collect be considered an invasive procedure, limit their efficacy (Sakkas et al., Reference Sakkas, Gartzonika and Levidiotou2016). Immunological methods have been used; however, problems related with the sensitivity and/or specificity of the selected antigens are described; thus hampering their use as more appropriate diagnostic tools (Georgiadou et al., Reference Georgiadou, Makaritsis and Dalekos2015; Lima et al., Reference Lima, Costa, Duarte, Menezes-Souza, Salles, Santos, Ramos, Chávez-Fumagalli, Kursancew, Ambrósio, Roatt, Machado-de-Ávila, Gonçalves and Coelho2017).
A commercial kit, Kalazar Detect™ Test (InBios International, Inc., Seattle, Wash, USA), is an immunochromatographic assay applied for the diagnosis of human VL. However, this test cannot discriminate between current, subclinical or past infections, and it is useless for diagnosis of relapses and as a prognostic test (Sundar and Singh, Reference Sundar and Singh2018). As a consequence, the possibility to identify new antigens to be employed in more sensitivity and specific diagnosis should be pointed (Didwania et al., Reference Didwania, Shadab, Sabur and Ali2017). Proteomics is a technology employed for the study and characterization of the information obtained in a cell or organism about the form of protein pathways and networks (Vlahou and Fountoulakis, Reference Vlahou and Fountoulakis2005; Garg et al., Reference Garg, Singh and Ali2018). Using proteomics associated with biological fluids, such as sera samples, can allow for the identification of antigens involved in the development of diseases; thus leading to the identification of biological targets that could be used as diagnostic markers and/or vaccine candidates (Fernandes et al., Reference Fernandes, Coelho, Machado-Coelho, Grimaldi and Gazzinelli2012). Indeed, and due to the high amino acid conservation in Leishmania proteins; the identification of antigenic molecules that stimulate the humoral response in infected hosts could help for the development of more appropriated tests employing these more refined antigens (Jamal et al., Reference Jamal, Shivam, Kumari, Singh, Sardar, Pushpanjali, Narayan, Gupta, Pandey, Das, Ali, Bimal, Das and Singh2017). In this context, in the present study, an immunoproteomics approach performed in L. infantum promastigote and amastigote protein extracts with sera samples of VL patients was developed; with the purpose of identifying antigenic proteins in the parasites to be applied as candidates for the diagnosis of disease. To refine the selection of the candidates, sera samples of Chagas disease patients and healthy subjects living in the endemic region were used to exclude the most cross-reactive spots, aiming to avoid undesired cross-reactivity in the serological assays.
Materials and methods
Human samples
Sera samples were obtained from VL (n = 30, including 16 males and 14 females with ages ranging from 29 to 63 years) patients, which were collected from the endemic region of disease (Belo Horizonte). Patients were diagnosed by clinical evaluation and demonstration of L. infantum kDNA in spleen or bone marrow aspirates by PCR technique. Sera were also collected from healthy individuals living in an endemic (n = 30, including 18 males and 12 females with ages ranging from 21 to 56 years; Belo Horizonte) or non-endemic (n = 30, including 15 males and 15 females with ages ranging from 19 to 49 years; Poços de Caldas, Minas Gerais, Brazil) region of leishmaniasis. Subjects did not present clinical signal of disease and showed negative serological results. Samples were also obtained from Chagas disease (n = 25, including 15 males and 10 females with ages ranging from 29 to 60 years) patients, with the infection confirmed by hemoculture, Chagatest® recombinant ELISA v.4.0 kit or Chagatest® haemagglutination inhibition kit (Wiener Lab., Rosario, Argentina). Sera from paracoccidioidomycosis (n = 10, six males and four females with ages ranging from 26 to 55 years) patients were also used. The diagnosis was performed by clinical examination and positive Paracoccidioides culture. Samples collected from leprosy (n = 20, with 12 males and eight females, with ages ranging from 24 to 57 years) patients, with the diagnosis confirmed by clinical evaluation, ML Flow rapid test and lesion histopathology, as well as sera from aspergillosis (n = 10, including five males and five females, with ages ranging from 22 to 47 years), tuberculosis (n = 10, six males and four females with ages ranging from 38 to 67 years) or malaria (n = 10, seven males and three females with ages ranging from 20 to 46 years) patients were used in the assays.
Parasites and preparation of Leishmania protein extract
Leishmania infantum (MHOM/BR/1970/BH46) was used. Parasites were cultured at 24 °C in complete Schneider's medium (Sigma), which was supplemented with 20% inactivated foetal bovine serum (FBS; Sigma-Aldrich), 20 mm L-glutamine, 200 U mL−1 penicillin and 100 µg mL−1 streptomycin, at pH 7.4 (Coelho et al., Reference Coelho, Tavares, Carvalho, Chaves, Teixeira, Rodrigues, Charest, Matlashewski, Gazzinelli and Fernandes2003). To obtain the axenic amastigotes, 109 stationary promastigotes were washed three times in sterile phosphate buffer saline (PBS 1×), and incubated in 5 mL FBS for 48 h at 37 °C. Parasites were washed in cold PBS 1×, and their morphology was evaluated after staining by the Giemsa method in an optical microscope (Valadares et al., Reference Valadares, Duarte, Oliveira, Chávez-Fumagalli, Martins, Costa, Leite, Santoro, Régis, Tavares and Coelho2011). The protein extraction and two-dimensional electrophoresis (2DE) were performed following a modified protocol (Lewis et al., Reference Lewis, Hunt, Aveline, Jonscher, Louie, Yeh, Nahreini, Resing and Ahn2000). Briefly, cells from each stage (1010 cells) were washed three times in 40 mm Tris-HCl, pH 7.2, by centrifugation at 5000 × g for 10 min at 4 °C. Pellets were resuspended in lysis buffer solution [7 M urea, 2 M thiourea, 4% cholamidopropyl dimethylammonio-1-propanesulfonate (CHAPS), 40 mm dithiothreitol (DTT), 2% IPG buffer (pH 4–7), 40 mm Tris], and a protease inhibitor cocktail (GE Healthcare, Uppsala, Sweden) was added. Samples were incubated for 1 h at room temperature, with occasional vortexing. Purification was carried out by protein precipitation using a 2D Clean UpKit (GE Healthcare), according to manufacturer instructions. Whole cell extracts were measured by a bidimensional Quant-Kit (GE Healthcare), and aliquots were immediately frozen at −80 °C, until use.
Isoelectric focusing (IEF) and SDS-PAGE
For the first-dimension electrophoresis, 150 µg of protein extract was added to a volume of 250 µL with a rehydration solution [7 M urea, 2 M thiourea, 2% CHAPS, 40 mm DTT, 2% immobilized pH gradient (IPG-buffer, pH 4–7, trace bromophenol blue)]. Next, samples were applied to IPG strips (13 cm, pH 4–7; GE Healthcare) for passive rehydration overnight at room temperature. After in-gel rehydration for 12 h, isoelectric focusing was performed at 500 V for 1 h, 1.000 V for 1 h, and 8.000 V for 8 h, using a Multiphor II electrophoresis unit and EPS 3500 XL power supply (Amersham, Piscataway, NJ, USA). After IEF, each strip was incubated for 15 min in a solution made up of 10 mL of a 50 mm Tris-HCl buffer pH 8.8, 6 m urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% bromophenol-blue and 125 mm DTT, followed by a second incubation step in the same buffer solution, excluding DTT, which was replaced by 125 mm iodacetamide. IPG strips were transferred to a 12% polyacrylamide and sealed with agarose solution (agarose and bromophenol blue in a Tris-glycine cathode buffer). The protein standard was purchased from Invitrogen (BenchMark™ Protein Ladder). Electrophoresis was performed in a Mini-Protean II system (BioRad) connected to a MultiTemp II cooling bath (Amersham Biosciences), in a Tris/glycine/SDS buffer. Proteins were separated at 200 V, until the dye front had reached the bottom of the gel.
Immunoblottings and protein identification
To select the best spots, immunoblottings were performed using L. infantum promastigote and amastigote protein extracts. For this, they were separated electrophoretically and transferred onto cellulose membranes (Schleicher and Schull, Dassel, Germany) by semi-dry blotting for 2 h at 400 mA. Then, membranes were blocked in 5% (w/v) low-fat dried milk in PBS 1× plus 0.05% Tween 20 for 2 h at room temperature. Next, they were washed seven times (10 min, each) with blocking solution and incubated with sera pools from VL (n = 15) or Chagas disease (n = 10) patients, as well as with sera pool from healthy individuals (n = 15) living in the endemic region of disease. All pools were performed at 1:400 in PBS 1× plus 0.05% Tween 20, and the incubation occurred for 2 h at room temperature. After, membranes were incubated with a peroxidase-conjugated goat anti-human IgG secondary antibody (1:10 000 diluted), for 1 h at room temperature. After having been washed seven times with PBS 1× plus 0.5% Tween 20, blots were revealed by the addition of chloronaphtol, diaminobenzidine and H2O2 30 vol. Bidimensional gels were stained with colloidal Coomassie Brilliant Blue G-250, according described (Neuhoff et al., Reference Neuhoff, Arold, Taube and Ehrhardt1988). The stained gels were scanned using an ImageScanner III (GE Healthcare), and spots mainly recognized by antibodies in sera from VL patients were excised from the gels for protein identification. Three independent preparations were performed using independent parasite cultures, and one representative preparation is shown.
Protein digestion, peptide extraction and spot handling
Spots were excised, and fragments were washed in 25 mm ammonium bicarbonate/50% acetonitrile until completely destained. After drying, gel fragments were placed on ice in a 50 µL protease solution (20 ng mL−1 of a sequence grade-modified trypsin in a 25 mm ammonium bicarbonate) (Promega Biosciences, CA, USA) for 30 min. Excess protease solution was removed and replaced by 25 mm ammonium bicarbonate, when the digestion was performed for 18 h at 37 °C. Peptide extraction was performed twice for 15 min, using 30 µL of 50% acetonitrile/5% formic acid. Trypsin (Promega) digests were concentrated in a Speed-Vac (Savant, USA) to approximately 10 µL and desalted using Zip-Tip (C18 resin; P10, Millipore Corporation, Bedford, MA). Samples were mixed with a matrix (5 mg mL−1 recrystallized α-cyano-4-hydroxycinnamic acid) in a volume of 1 mL (1:1 ratio), and spotted for MALDI-TOF/TOF Ultraflex III (Bruker, Daltonics, Germany).
Protein identification and database search
To determine the MS spectrum of the selected spots, the digests were spotted onto 600 µm Anchorchips (Bruker Daltonics). Spotting was achieved by pipetting, in duplicate, 1 µL of analyte onto the MALDI target plate, then adding 5 mg mL−1 α-cyano-4-hydroxycinnamic acid diluted in 3% TFA/50% acetonitrile, which contained 2 mm ammonium phosphate. The Bruker peptide calibration mixture was spotted down for external calibration. All samples were allowed to air dry at room temperature, and 0.1% TFA was used for on-target washing. All samples were analysed in the positive-ion, reflection mode, through a MALDI-TOF/TOF Ultraflex III mass spectrometer (Bruker, Daltonics, Germany). Each spectrum was produced by accumulating data from 200 consecutive laser shots, with a frequency of 100 Hz, and an m/z range of 1.000–4.000. Instrument calibration was achieved by using peptide calibration standard II (Bruker Daltonics), a mixture of angiotensin I and II, substance P, bombesin, ACTH clip 1–17, ACTH clip 18–39 and somatostatin 28, as the internal standard. Peptide masses were measured as mono-isotopic masses. The MS peaks with the highest intensities were selected for MS/MS fragmentation analyses. The resulting spectra were processed using Flex analysis software, version 2.4 (Bruker Daltonics), with the following settings: peak detection algorithm set at SNAP (Sort Neaten Assign and Place), S/N threshold at 3, precursor and product ion tolerances were set at 0.5 Da, and quality factor threshold at 50. The trypsin autodigestion ion peaks (842.51, 1045.56, 2211.10 and 2225.12 Da) were used as internal standards to validate the external calibration procedure. Matrix, and/or autoproteolytic trypsin fragments, and known contaminants (i.e. keratins) were manually removed. The resulting peptides list was used to search in the NCBI database (http://blast.ncbi.nlm.nih.gov) for the organism option of Leishmania (taxid: 5658). According to the obtained results, and using the peptide sequences identified for each protein, the following parameters were used as selection criteria: total score, query coverage and E value.
Cloning and purification of the parasite proteins
To evaluate the antigenic potential of selected proteins in the immunoproteomics, two antigens, endonuclease III (LINF_090005600) and GTP-binding protein (LINF_250020400) were cloned, expressed, purified and evaluated in ELISA experiments for the serodiagnosis of VL. For this, genes were cloned from L. infantum DNA by using the 5′-TGCTGCTAGCATGAGTAAACACTCCTTT-3′ (forward) and 5′-TCATGGATCCTCACTTTATGCGTCTCTT-3′ (reverse) for endonuclease III, and 5′-TGCTGCTAGCATGCAACAGGCACCCT-3′ (forward) and 5′-TCATGGATCCTCACTCGTCATCGCCCAT-3′ (reverse) for GTP-binding protein. The restriction enzymes used were BamHI and NheI. The DNA fragments were excised from gels, purified and linked into pGEM®-T Vector Systems (Promega, USA), and recombinant plasmids were used to transform Escherichia coli XL1-Blue competent cells. Positive clones were propagated and used for construction of the expression vector. DNA fragments obtained from digestion of pGEM-rENDO and pGEM-rGTP plasmids were ligated into pET28a-TEV, and E. coli BL21 cells (DE3; Agilent Technologies, USA) were transformed with the recombinant plasmids. Gene insertion was confirmed by PCR and sequencing by using the MegaBace 1000 automatic sequencer apparatus (Amersham Biosciences, USA). For the expression and purification of the proteins, cells were induced with IPTG (0.5 µ m) and cultures were shaking at 200 × g per min for 24 h at 12 °C. Cells were ruptured by using six cycles of ultrasonication with cycles of 30 s each (38 MHz), followed by six cycles of freezing and thawing. After, cellular debris was removed by centrifugation, and recombinant proteins were purified onto a HisTrap HP affinity column connected to an AKTA system (GE Healthcare, USA). The eluted fractions containing the rENDO (29.0 kDa) and rGTP (24.0 kDa) proteins were concentrated in Amicon® ultra15 centrifugal filters 10 000 NMWL (Millipore, Germany), and further purified on a Superdex™ 200 gel-filtration column (GE Healthcare Life Sciences, USA). After, recombinant proteins were passed through a polymyxin-agarose column (Sigma) to remove any residual endotoxin content.
ELISA experiments
Previous titration curves were performed to determine the most appropriate antigen concentration and antibody dilution to be used. Falcon flexible microtitre immunoassay plates (Becton Dickinson) were coated with rENDO, rGTP or L. infantum SLA (0.5, 0.25 and 1.0 µg per well, respectively), which were diluted in 100 µL of coating buffer (50 mm carbonate buffer) pH 9.6, for 16 h at 4 °C. Next, free binding sites were blocked using 250 µL of PBS-T (PBS 1× plus Tween 20 0.05%) plus 5% non-fat dry milk (catalogue M7409-1BTL, Sigma-Aldrich, St. Louis, MO, USA), for 1 h at 37 °C. After washing plates five times with PBS-T, they were incubated with 100 µL of human sera (1:400 diluted in PBS-T), for 1 h at 37 °C. Plates were washed seven times in PBS-T, and incubated with anti-human IgG horseradish–peroxidase conjugated antibody (1:20 000 diluted in PBS-T; catalogue SAB3701282; Sigma-Aldrich, USA) for 1 h at 37 °C. After washing plates seven times with PBS-T, reactions were developed by incubation with 100 µL per well of a solution composed by 2 µL H2O2, 2 mg ortho-phenylenediamine and 10 mL citrate-phosphate buffer, at pH 5.0; for 30 min in the dark. Reactions were stopped by adding 25 µL 2 N H2SO4, and optical density (OD) values were read in an ELISA microplate spectrophotometer (Molecular Devices, Spectra Max Plus, Canada), at 492 nm.
Serological follow-up after VL treatment
To evaluate the serological reactivity of rGTP and rENDO proteins using sera samples from treated and untreated VL patients, samples from patients (n = 10, including six males and four females, with ages ranging from 32 to 57 years) were collected before and six months after treatment using pentavalent antimonials (Sanofi Aventis Farmacêutica Ltda., Suzano, São Paulo, Brazil). All patients were submitted to the same therapeutic regimen using the pharmaceutics, at a dose of 20 mg Sb5+ per kg during 30 days, and none of them suffered from any other infections or had any pre-existing disease. When all of them had the treatment completed, they were free of any symptom of disease.
Statistical analysis
The results were entered into Microsoft Excel (version 10.0) spreadsheets and analysed using GraphPad Prism™ (version 6.0 for Windows). Receiver operating characteristic (ROC) curves were constructed to obtain the sensitivity (Se), specificity (Sp), area under the curve (AUC) and likelihood ratio (LR); as well as the lower limit of positivity (cut-off). Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by the Bonferroni's post-test for multiple comparisons between the groups. Differences were considered significant at P < 0.05.
Results
Identifying the immunoreactive spots in the L. infantum promastigotes and amastigotes
In our study, L. infantum promastigote protein extracts were reacted with antibodies in sera from VL patients in an immunoproteomics approach. Samples from Chagas disease patients and healthy controls were used to exclude the most cross-reactive spots. Results showed a low reactivity when sera from healthy individuals were used (Fig. 1A). On the other hand, higher reactivity was found when sera from Chagas disease patients were employed in the analysis (Fig. 1B). The majority of the identified spots showed pH between 4.0 and 5.5, and molecular weight varying from 20 to 70 kDa. When sera from VL patients were used, about 200 protein spots were visualized (Fig. 1C). From the selected spots in L. infantum promastigotes (Fig. 1D), a sequencing reaction was performed and 29 valid sequences were identified in this parasite stage. Table 1 shows the identity of the identified proteins, also describing their accession number, molecular weight and isoelectric point.
Two-dimensional profile and immunoproteomic analysis in L. infantum promastigote protein extracts. Bidimensional gels were obtained after separation of promastigote protein extracts of the parasites (150 µg) by using in the first dimension: IEF pH range 4–7, and second dimension: 12% SDS-PAGE. After, they were stained with colloidal Coomassie Brilliant Blue G-250. Immunoblots were developed after incubation of membranes with sera pools from healthy subjects (A), Chagas disease (B) or visceral leishmaniasis (C) patients, all 1:400 diluted in PBS 1× plus 0.05% Tween 20. Bound antibodies were detected with a peroxidase-conjugated goat anti-human IgG secondary antibody (1:10 000 diluted). The x-axis represents the isoelectric point (pI), and the y-axis represents the molecular weight (kDa) indicated by a commercial marker (BenchMark™ protein ladder). The identified spots after their recognition by antibodies in sera of VL patients are marked in the 2DE stained gel (D), and their identities are given in Table 1. Immunoblots are a reliable representation of three independent experiments.
Proteins of L. infantum promastigotes recognized by antibodies in visceral leishmaniasis patients in an immunoproteomic approach
a Spot number in the bidimensional gel.
b Accession numbers according to NCBI.
c Name of the identified protein.
d Predicted isoelectric point (pI).
e Predicted molecular weight (M r, in kDa).
In this same manner, using the L. infantum amastigotes, sera of healthy subjects showed a low reactivity (Fig. 2A), while higher number of spots were recognized by using Chagas disease patients sera were used in the blots (Fig. 2B), with the majority of spots concentrated between 50 and 120 kDa and with pH varying from 4.5 to 5.5. Similarly to found in the promastigote extract, higher number of spots was visualized when VL patients sera were employed in the immunoblottings (Fig. 2C), when over 150 spots were visualized. From the selected spots in L. infantum amastigotes (Fig. 2D), a sequencing reaction was performed and 21 valid sequences were identified in this parasite stage. Table 2 shows the identity of the identified proteins in the amastigote extract, describing their accession number, molecular weight and isoelectric point.
Two-dimensional profile and immunoproteomic analysis in L. infantum amastigote protein extracts. Bidimensional gels were obtained after separation of axenic amastigote protein extracts of the parasites (150 µg) by using in the first dimension: IEF pH range 4–7, and second dimension: 12% SDS-PAGE. After, they were stained with colloidal Coomassie Brilliant Blue G-250. Immunoblots were developed after incubation of membranes with sera pools from healthy subjects (A), Chagas disease (B) or visceral leishmaniasis (C) patients, all 1:400 diluted in PBS 1× plus 0.05% Tween 20. Bound antibodies were detected with a peroxidase-conjugated goat anti-human IgG secondary antibody (1:10 000 diluted). The x-axis represents the isoelectric point (pI), and the y-axis represents the molecular weight (kDa) indicated by a commercial marker (BenchMark™ protein ladder). The identified spots after their recognition by antibodies in sera of VL patients are marked in the 2DE stained gel (D), and their identities are given in Table 2. Immunoblots are a reliable representation of three independent experiments.
Proteins of L. infantum amastigotes recognized by antibodies in visceral leishmaniasis patients in an immunoproteomic approach
a Spot number in the bidimensional gel.
b Accession numbers according to NCBI.
c Name of the identified protein.
d Predicted isoelectric point (pI).
e Predicted molecular weight (M r, in kDa).
Evaluating the biological functions of identified proteins
Among the proteins recognized by antibodies in VL patients sera in the promastigote extract, seven hypothetical and 22 known antigens were identified. Proteins related to the parasite virulence, such as alpha- and beta-tubulins (Coulson et al., Reference Coulson, Connor, Chen and Ajioka1996; Coelho et al., Reference Coelho, Oliveira, Valadares, Chávez-Fumagalli, Duarte, Lage, Soto, Santoro, Tavares, Fernandes and Coelho2012) and heat-shock protein 70 (Drini et al., Reference Drini, Criscuolo, Lechat, Imamura, Skalický, Rachidi, Lukeš, Dujardin and Späth2016); therapeutic targets, such as RNA-binding protein (Nandan et al., Reference Nandan, Thomas, Nguyen, Moon, Foster and Reiner2017), endonuclease III (Moreira et al., Reference Moreira, Jesus, Soares, Silva, Pinto, Melo, Paes and Pereira2017), GTP-binding protein (Ishemgulova et al., Reference Ishemgulova, Kraeva, Hlaváčová, Zimmer, Butenko, Podešvová, Leštinová, Lukeš, Kostygov, Votýpka, Volf and Yurchenko2017) and pyridoxal kinase (Kumar et al., Reference Kumar, Sharma, Rakesh, Malik, Neelagiri, Neerupudi, Garg and Singh2018); and vaccine candidates, such as paraflagellar rod protein (Carrillo et al., Reference Carrillo, Crusat, Nieto, Chicharro, Thomas, Martínez, Valladares, Cañavate, Requena, López, Alvar and Moreno2008), peroxidoxin (Bayih et al., Reference Bayih, Daifalla and Gedamu2014) and elongation factor (Sabur et al., Reference Sabur, Bhowmick, Chhajer, Ejazi, Didwania, Asad, Bhattacharyya, Sinha and Ali2018) were found. In the amastigote extract, four hypothetical and 17 known proteins were identified. From these, virulence factors, such as leucine-rich repeat protein (Mukherjee et al., Reference Mukherjee, Chakraborty and Chakrabarti2016), diagnosis markers, such as enolase (Duarte et al., Reference Duarte, Lage, Martins, Costa, Salles, Carvalho, Oliveira, Dias, Ribeiro, Chávez-Fumagalli, Machado-de-Ávila, Roatt, Menezes-Souza, Magalhães-Soares and Coelho2017), vaccine candidates, such as eukaryotic initiation factor 4a (Maspi et al., Reference Maspi, Ghaffarifar, Sharifi and Dalimi2015), and drug targets, such as calpain-like cysteine peptidase (Chávez-Fumagalli et al., Reference Chávez-Fumagalli, Schneider, Lage, Machado-de-Ávila and Coelho2017) were recognized.
Testing two recombinant proteins as diagnostic markers for VL
Two proteins that were identified in the promastigote extract, endonuclease III and GTP-binding, were cloned, expressed, purified and their recombinant versions (rENDO and rGTP, respectively) were evaluated in ELISA experiments. The individual OD values obtained against the different antigens are shown (Fig. 3), and ROC curves were constructed to obtain the cut-off values. Results showed that both proteins presented sensitivity and specificity values of 100 and 99.31%, respectively, with AUC of 1.0 and a likelihood ratio of 145; while using L. infantum SLA as a control antigen, sensitivity and specificity values were of 63.31 and 26.67%, respectively, with AUC of 0.67 and likelihood ratio of 38.67 (Table 3).
Evaluation of recombinant antigens for the serodiagnosis of visceral leishmaniasis. ELISA experiments were performed using the rENDO and rGTP proteins and L. infantum SLA, as a control. Sera samples from visceral leishmaniasis (HVL, n = 30) patients and healthy individuals living in non-endemic (HCNEA, n = 30) or endemic (HCEA, n = 30) areas of leishmaniasis were used. To evaluate the cross-reactivity of the antigens, samples from Chagas disease (CD, n = 25), leprosy (HAN, n = 20), aspergillosis (ASP, n = 10), paracoccidioidomycosis (PAR, n = 10), tuberculosis (TB, n = 10) and malaria (MAL, n = 10) patients were used in the assays. The cut-off values were calculated by Receiver Operator Curves (ROC). Results showing the optical density (OD) values of each sample, as well as the mean of sera groups for the rENDO (A), rGTP (B) and SLA (C) antigens, are shown. ROC curves were used to determine ELISA sensitivity (95% CI), specificity (95% CI) and AUC for the diagnostic antigens (D).
Diagnostic performance of the antigens for the serodiagnosis of visceral leishmaniasis
A human serological panel was used in ELISA experiments against rENDO, rGTP and L. infantum SLA. Receiver operating characteristic (ROC) curves were constructed to obtain the sensitivity (Se), specificity (Sp), area under the curve (AUC) and likelihood ratio (LR) values; as well as the lower limit of positivity (cut-off).
IgG antibody levels before and after the VL treatment
With the purpose to evaluate the serological reactivity of the patients before and after treatment, sera were collected and ELISA assays were performed. Results showed a significant decrease in the protein-specific IgG antibody levels, 6 months after the treatment of the patients (Fig. 4). Otherwise, using L. infantum SLA as an antigen, similar antibody levels were found before and after treatment.
Serological reactivity of the recombinant proteins before and after treatment of visceral leishmaniasis patients. Sera samples were collected before and six months after treatment of VL (n = 10), and the anti-protein and anti-parasite IgG levels were investigated. White circles and square shown the optical density (OD) values of the individual samples collected before and after treatment, respectively.
Discussion
Visceral leishmaniasis is a neglected disease in which the outcome of infection can be fatal if acute and left untreated (Burza et al., Reference Burza, Croft and Boelaert2018). Diagnostic tests are still related to invasive aspiration or by means of immunochromatographic evaluations. Advancements in immunoproteomics employing bidimensional electrophoresis and mass spectrometry have enabled the identification of new markers for disease diagnosis and prognosis, as well as to they use in therapeutic monitoring and the understanding of the infected hosts’ immune response (Kumari et al., Reference Kumari, Kumar, Samant, Sundar, Singh and Dube2008; Ejazi et al., Reference Ejazi, Bhattacharyya, Choudhury, Ghosh, Sabur, Pandey, Das, Das, Rahaman, Goswami and Ali2018). In this study, an immunoproteomics approach was performed using VL patients sera, as well as from Chagas disease patients and healthy endemic control; those employed to exclude the most cross-reactive spots. From this approach, 29 and 21 valid sequences were identified by MALDI-TOF/TOF and mass spectrometry in the promastigote and amastigote stages of the parasites. On the basis of results obtained, these antigens can be expected to be antigenic in the human VL. Some of them, such as tubulins and heat shock proteins are housekeeping in Leishmania (Coelho et al., Reference Coelho, Oliveira, Valadares, Chávez-Fumagalli, Duarte, Lage, Soto, Santoro, Tavares, Fernandes and Coelho2012); while others, such as paraflagellar rod protein, elongation factor and enolase present immunological role in the disease in mammalian hosts (Carrillo et al., Reference Carrillo, Crusat, Nieto, Chicharro, Thomas, Martínez, Valladares, Cañavate, Requena, López, Alvar and Moreno2008; Santos et al., Reference Santos, Martins, Lage, Costa, Salles, Carvalho, Dias, Ribeiro, Chávez-Fumagalli, Machado-de-Ávila, Roatt, Magalhães-Soares, Menezes-Souza, Coelho and Duarte2017; Sabur et al., Reference Sabur, Bhowmick, Chhajer, Ejazi, Didwania, Asad, Bhattacharyya, Sinha and Ali2018). Not less important, a number of hypothetical proteins were identified by antibodies in VL patients sera, when both proteins extracts were tested; thus demonstrating the possibility to also evaluate these antigens as diagnostic markers and/or vaccine candidates against disease (Lage et al., Reference Lage, Martins, Duarte, Costa, Garde, Dimer, Kursancew, Chávez-Fumagalli, Magalhães-Soares, Menezes-Souza, Roatt, Machado-de-Ávila, Soto, Tavares and Coelho2016; Ribeiro et al., Reference Ribeiro, Dias, Novais, Lage, Tavares, Mendonça, Oliveira, Chávez-Fumagalli, Roatt, Duarte, Menezes-Souza, Ludolf, Tavares, Oliveira and Coelho2018; Chávez-Fumagalli et al., Reference Chávez-Fumagalli, Lage, Tavares, Mendonça, Dias, Ribeiro, Ludolf, Costa, Coelho and Coelho2019).
In our study, we have chosen two parasite proteins that were recognized in the promastigote extract, aiming to validate our data generated in the immunoproteomics approach, by using ELISA experiments and a large serological panel. Results showed that the recombinant proteins, rENDO and rGTP, showed high sensitivity and specificity values to identify VL samples. Additionally, they were not recognized by antibodies in sera from patients with paracoccidioidomycosis, leprosy, aspergillosis, tuberculosis or malaria; as well as by sera from non-endemic or endemic healthy controls. This fact opens the possibility to test these candidates in future studies for the serodiagnosis of human disease.
GTP-binding proteins are considered as virulence factors in Leishmania, being involved in the drug resistance against L. amazonensis species (Lang et al., Reference Lang, Chastellier, Frehel, Hellio, Metezeau, Leão and Antoine1994; Ishemgulova et al., Reference Ishemgulova, Kraeva, Hlaváčová, Zimmer, Butenko, Podešvová, Leštinová, Lukeš, Kostygov, Votýpka, Volf and Yurchenko2017). These molecules participate in secretion pathways and/or environmental response of parasites, as well as in the communication with the host cell during the infection (Ishemgulova et al., Reference Ishemgulova, Kraeva, Hlaváčová, Zimmer, Butenko, Podešvová, Leštinová, Lukeš, Kostygov, Votýpka, Volf and Yurchenko2017). Recently, Magalhães et al. (Reference Magalhães, Duarte, Mattos, Martins, Lage, Chávez-Fumagalli, Lage, Menezes-Souza, Régis, Alves, Soto, Tavares, Nagen and Coelho2014) applied a proteomic approach in L. amazonensis to analyse the variation of the protein expression profile, when parasites were in vitro cultured for a 150-day period. Results showed that 37 proteins presented a significant decrease in their expression content, whereas 19 proteins showed a significant increase in their content during the cultivation. The authors associated some of these proteins as diagnosis markers, vaccine candidates and/or drug targets on leishmaniasis. One of them, the GTP-binding protein, was showed to decrease its expression content in the order of 3.13-fold during the cultivation. It and others that also decrease their expression were considered as parasites’ infectivity factors, in which a significant reduction in their in vitro and in vivo infectivity was found after 150 days of cultivation (Magalhães et al., Reference Magalhães, Duarte, Mattos, Martins, Lage, Chávez-Fumagalli, Lage, Menezes-Souza, Régis, Alves, Soto, Tavares, Nagen and Coelho2014).
Recent evidence has shown that the characteristics of the Leishmania replication and repair machinery can be used as targets for the development of new therapeutic strategies. In this context, the evaluation of DNA repair enzymes as biological targets can open the way to the identification of potential targets for the development of novel diagnostic and therapeutic strategies (Rajão et al., Reference Rajão, Furtado, Alves, Passos-Silva, Moura, Schamber-Reis, Kunrath-Lima, Zuma, Vieira-da-Rocha, Garcia, Mendes, Pena, Macedo, Franco, Souza-Pinto, Medeiros, Cruz, Motta, Teixeira and Machado2014). One of the proteins identified here, endonuclease III, is a DNA repair glycosylase, previously known for its repair activity on oxidative pyrimidine damage (Mishra et al., Reference Mishra, Khan, Jha, Kumar, Das, Das and Sinha2018). In this study, the diagnostic potential of this enzyme was successfully evaluated, then speculate about the possible employment of this recombinant antigen for the serodiagnosis of disease.
Different strategies have been tested to provide understanding about the hosts’ immune response against infection by different pathogens, such as Leishmania, such as by proteomic studies (Chambers et al., Reference Chambers, Lawrie, Cash and Murray2000; Sundar and Singh, Reference Sundar and Singh2018). Previous work published used the immunoproteomics tool to identify antigenic proteins in L. braziliensis protein extracts by antibodies in sera from tegumentary leishmaniasis (TL) patients. With the purpose to reduce the cross-reactivity of the identified proteins, spots highly recognized by antibodies in sera from Chagas disease patients and healthy individuals living in an endemic region were excluded. Results showed that 20 proteins were identified in the protein extracts, and five of them were cloned, expressed, purified and tested in ELISA experiments for the diagnosis of TL, with serological results showing high sensitivity and specificity values (Duarte et al., Reference Duarte, Pimenta, Menezes-Souza, Magalhães, Diniz, Costa, Chávez-Fumagalli, Lage, Bartholomeu, Alves, Fernandes, Soto, Tavares, Gonçalves, Rocha and Coelho2015).
Here, the experimental strategy was also based on the exclusion of the most reactive spots, since sera samples from Chagas disease patients and healthy controls were used. This fact is based on the high cross-reactivity found when serodiagnosis tests are applied in such patients, and could justify the low number of identified proteins. In addition, several antigens recognized by antibodies in parasite extracts were recognized as multiple spots or proteolytic fragments. Although their degradation cannot be discarded; extracts were prepared in the presence of a cocktail of protease inhibitors. As a consequence, these findings can be associated with the presence of isoforms or post-translational modifications, known to occur in Leishmania parasites (Brotherton et al., Reference Brotherton, Racine, Foucher, Drummelsmith, Papadopoulou and Ouellette2010; Coelho et al., Reference Coelho, Oliveira, Valadares, Chávez-Fumagalli, Duarte, Lage, Soto, Santoro, Tavares, Fernandes and Coelho2012; Moreira et al., Reference Moreira, Pescher, Laurent, Lenormand, Späth and Murta2015).
The serological evaluation in cured patients should also be considered, since the purpose of diagnostic tools is to develop a test that can be used in endemic regions to detect active disease. Results obtained in this study can be considered promising, since lower serological reactivity specific to rGTP and rENDO proteins was found in the cured and treated VL patients. Conversely, rA2 and SLA did not show such discriminative results, and new experiments must be performed to confirm the lack of their diagnostic role in human disease. To the best of our knowledge, the present study is the first in which these proteins were effective to discriminate patients after treatment had been completed; thus suggesting the possibility of using them as serological markers for VL, as well as to correlate the presence of low levels of specific antibodies with the clinic cure of patients. As limiting factor of the work, the small number of patients evaluated before and after treatment should be considered and certainly new studies are necessary to be performed.
Taking into account, results indicated patterns of protein recognition by antibodies in sera from L. infantum-infected patients by an immunoproteomics approach, and suggested that rENDO and rGTP could be evaluated as diagnostic markers for human VL, as well as in the monitoring of human disease treatment. Additional studies using these antigens, as well as others identified in the amastigote forms, should be performed to validate our findings about the use of these proteins as diagnostic markers for human VL.
Author ORCIDs
Eduardo A. F. Coelho, 0000-0002-6681-9014.
Financial support
The authors would like thank CAPES, CNPq and FAPEMIG for scholarships. This work was supported by grants from CNPq (APQ-408408/2016-2 and APQ-408675/2018-7).
Conflict of interest
None.
Ethical standards
The study was approved by the Human Research Ethics Committee from Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil (protocol number CAAE-32343114.9.0000.5149).
