Identification of Virulence Factors in Leishmania infantum Strains by a

Article pubs.acs.org/jpr

Identification of Virulence Factors in Leishmania infantum Strains by a Proteomic Approach Simone da Fonseca Pires,†,# Luiz Carlos Fialho, Jr.,†,# Soraia Oliveira Silva,† Maria Norma Melo,† Carolina Carvalho de Souza,‡ Wagner Luiz Tafuri,‡ Oscar Bruna Romero,§ and Hélida Monteiro de Andrade*,† †

Departamento de Parasitologia, ‡Departamento de Patologia, and §Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-910, Belo Horizonte, Minas Gerais, Brazil S Supporting Information *

ABSTRACT: Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite−host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron−sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase. KEYWORDS: Leishmania infantum, virulence, proteome infected dogs exhibit signs of disease.5 The clinical diversity observed in the distinct hosts that are infected by L. infantum suggests that host and parasite factors could be involved in the clinical variability of the disease, which renders it an excellent model for the study of parasite−host interaction and virulence. Therefore, several research groups have conducted comparative studies between attenuated and virulent parasite forms;6−10 proteomic analysis of the distinct life stages of the parasite and the different Leishmania species has been conducted in an attempt to understand Leishmania pathogenicity and its relationships with the host.11−13 The first contact between the pathogen and the host is dependent on the interaction between the molecules in the cells of both organisms. Thus, the success of the infection depends on the parasite’s ability to escape the host’s protection mechanisms during the host’s attack, allowing the invasion and pathogen proliferation in the target cell. Several molecules that are associated with virulence and host−parasite interaction are proteins; therefore, these

1. INTRODUCTION Visceral Leishmaniasis (VL) is a severe and fatal disease if untreated. There are an estimated 500 000 new cases of VL,1 and in developing nations, the overlap of endemic regions of VL with regions of HIV infection poses a serious threat2 and has become a major challenge to the control of VL.3 The Leishmania infantum (L. chagasi) species is the agent that is responsible for causing the zoonotic VL. This form represents 20% of human VL worldwide (100 000 cases annually), and its incidence is increasing in urban and peri-urban areas of the tropics.4 In the Americas, VL occurs from Mexico to Argentina. Ninety-seven percent of VL human cases are reported from Brazil (http://www.who.int/), where there is an increase of the area of occurrence and number of cases. The human cases range from subclinical infection, in which an unknown number of asymptomatic patients in endemic areas have been observed, to a growing number of HIV coinfection cases and a high mortality rate in children. The disease is severe in dogs and is characterized by chronic and progressive cachexia, hepatosplenomegaly, lymphadenopathy, onychogryphosis, and pancytopenia. However, less than 50% of © XXXX American Chemical Society

Received: September 19, 2013

A

dx.doi.org/10.1021/pr400923g | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

(Cultilab), and 100 μg/mL streptomycin (Cultilab), pH 7.0. The cells were centrifuged at 8000g for 10 min at 4 °C and suspended in RPMI medium (Cultilab) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Cultilab), 0.4 g/L NaHCO3, 4 g/L HEPES, 200 U/mL penicillin (Cultilab), and 100 μg/mL streptomycin (Cultilab), pH 7.4. Approximately 3 × 105 macrophages were plated in 24-well plates containing glass coverslips and incubated at 37 °C in a humidified 5% CO2 incubator for a period of 24 h for attachment. Nonadherent cells were removed by one-step washing with medium, and the subsequent cell infection was performed for approximately 24 h at 37 °C at a multiplicity of infection (MOI) of 10, using fifth passage promastigotes from each L. infantum strain. After infection, cell cultures were washed with medium to eliminate noninternalized parasites, and they were reincubated with fresh media without parasites for 3 days before fixation with methanol. Fixed cultures were stained with Giemsa according to Giaimis et al. 1992,16 and coverslips were mounted onto glass slides for microscopic analysis. Cell infection and the number of amastigotes per cell were evaluated by counting 300 macrophages per coverslip for each parasite population. The data were analyzed using Graph Pad Prism 3.0 software using the mean of experiments that were conducted in triplicate for each strain.

constitute powerful study targets. In this sense, proteomics is the most appropriate analytical technique, and it has been growing rapidly in the postgenomic era. Proteomics is a useful approach to identify Leishmania proteins based on comparative studies. The post-transcriptional regulation of gene expression by polycistronic transcription and trans-splicing in trypanosomatids14,15 allowed detection of small alterations in protein expression and post-translational modification, specifically if two-dimensional electrophoresis (2-DE) and difference gel electrophoresis (DIGE) approaches were used. However, this report is the first description in the literature of the use of this approach to identify virulence factors in L. infantum. To contribute to the host−parasite relationship study, we also conducted a comparative virulence characterization of the two distinct L. infantum strains with differences in pathogenicity. The L. infantum strains, MHOM/BR/1972/BH6 (BH46) and MCAN/BR/2000/BH400 (BH400), were isolated from human and canine cases, respectively. Then, it was our objective in this study to compare the in vitro and in vivo infectivity of both strains by employing a proteomic approach to identify the molecules that are associated with differences in virulence between the strains. The proteomic profiles of both strains were compared by DIGE, and the proteins were identified by matrix-assisted laser desorption ionization time-of-flight/ time-of-flight (MALDI-TOF/TOF) assays. The differential analysis of the expression resulted in the detection of differentially expressed spots, among which 63 were identified (32 up- and 31 downregulated in the more virulent strain). The identified spots corresponded to 36 proteins, of which 16 were virulence factors that have been described in the literature as KMP-11, tryparedoxin, peroxiredoxin, and HSPs. As expected but not yet demonstrated, in L. infantum, virulence appears to be related to the simultaneous increase of several proteins that interact with one another and cause the highest level of parasite invasiveness, proliferation, and host survival. These proteins are excellent targets for therapeutic studies and/or vaccines.

Hamster Infection

Six 4−6 week old female Golden hamsters (Mesocricetus auratus) that weighed 40−60 g were inoculated intraperitoneally with 105 hamster spleen-derived L. infantum amastigotes (MHOM/BR/ 1972/BH6 or MCAN/BR/2000/BH400). Three months post infection the animals were weighed and checked for the presence or absence of ascites and edema prior to being killed in a CO2 chamber. Liver and spleen were collected and weighed. Histopathology

The livers and spleens were submitted to histopathological analysis according to Tafuri et al., 2004.17 The main histological alterations that were analyzed are described as follows: (1) Liver - the presence of a chronic inflammatory disease characterized by an exudate of mononuclear cells in the portal region and intralobular granuloma formation; degenerative hepatocyte lesions (hydropic and esteatosis), hypertrophy and hyperplasia of Kupffer cells and deposition of hemosiderin; (2) Spleen thickening and a chronic inflammatory reaction and in the capsule and trabecule system, depletion of the white pulp; hypertrophy and hyperplasia of macrophages of red pulp (granuloma formation), congestion and hemosiderin deposition in the red pulp. These changes were evaluated semiquantitatively, taking into account the extent of the changes throughout the histological section and they were classified as follows: 1 - none, 2 - discrete (20−30%), 3 - moderate (30−60%) and 4 − severe (>60%).

2. EXPERIMENTAL PROCEDURES Ethics Statement

Experiments with hamster and mouse were performed in accordance with guidelines of the Institutional Animal Care and Committee on Ethics of Animal Experimentation (Comitê de É t ica em Experimentação Animal, CETEA) from the Universidade Federal de Minas Gerais, protocol 232/2010 approved on 01/12/2010. Parasite Culture

Promastigotes of L. infantum MHOM/BR/1972/BH6 (BH46) and MCAN/BR/2000/BH400 (BH400) strains were isolated from hamster spleens that were previously infected and grown at 25 °C in α-Mem medium (Cultilab) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Cultilab), 0.4 g/L NaHCO3, 4 g/L HEPES, 200 U/mL penicillin (Cultilab), and 100 μg/mL streptomycin (Cultilab), pH 7.4. Both strains were cultivated under identical conditions (exponential growth phase, temperature, parasite concentration, and medium) until the fifth passage.

Parasite Quantification

Serial Dilution. The number of parasites in the spleen was estimated by a limiting dilution assay. The spleens of six infected animals were aseptically removed, weighed and homogenized in Dulbecco’s modified essential medium (D-MEM, Cultilab) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Cultilab), 200 U/mL penicillin (Cultilab), and 100 μg/mL streptomycin (Cultilab), pH 7.4. The homogenate was then centrifuged at 100g for 5 min at 4 °C to remove large cell debris, and the supernatant was collected and centrifuged at 2000g for an additional 15 min at 4 °C. The resulting pellet was solubilized in 1 mL complete culture medium. The parasite

Macrophage Infections

Macrophages were collected from the peritoneal cavity of female BALB/c mice by washing with cold PBS (phosphate buffered saline), supplemented with 200 U/mL penicillin B



Article

suspension was then serially diluted 10-fold in triplicate in 200 μL aliquots of medium in 96-well plates. Plates were incubated for 4 days at 27 °C, and the wells were inspected for parasite growth. The results were expressed as log parasite titer.18 Immunohistochemical Method for Labeling Amastigote Forms of Leishmania. The immunohistochemical method for the quantitative analysis of the parasite was based on the number of amastigotes that were observed in 20 fields using an optical microscope with a 40× objective, according to Tafuri et al., 2004.17 Real-Time PCR. To quantify parasite burdens, we used primers that were developed by Bretagne et al.19 to amplify a 90-bp fragment of the DNA polymerase gene. This is a single copy-number gene of L. infantum (GenBank accession number AF009147) that was amplified with the following primers: forward, 5′-TGTCGCTTGCAGACCAGATG-3′; reverse, 5′-GCATCGCAGGTGTGAGCAC-3′. Serial dilutions (10×) of a fragment containing 109 to 102 copies were employed in the construction of the calibration curves. PCR was carried out in a final volume of 10 μL containing 1.25 pmol of forward and reverse primers, 1× SYBR GREEN reaction master mix1 (Applied Biosystems, USA), and 1 μL of template DNA. The PCR conditions were as follows: an initial denaturation step at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. To quantify parasite burdens, CT values that were obtained for each test sample were compared to those that were obtained for the corresponding standard curve.20 The hamster β-actin gene was utilized to normalize the DNA concentration. The amplicon with 307 bp was amplified using forward, 5′-CTTCTACAACGAGCTGCGCG-3′, and reverse, 5′-TCATGAGGTAGTCGGTCAGG-3′, primers. The reactions were processed and analyzed in a Step One Sequence Detection System device (SDS, Applied Biosystems, Foster City, CA, USA).

Differentially labeled extracts were pooled, reduced with 2% DTT, complemented with 2% ampholytes (pH 4−7), adjusted to a final volume of 350 μL with sample buffer (7 M urea, 2 M thiourea and 4% CHAPS), and incubated for 20 min on ice in the dark. Samples were then applied to IPG strips (18 cm, pH 4−7 NL; GE Healthcare, USA) for passive rehydration overnight at room temperature. Rehydrated IPG strips were subjected to IEF for 40 000 Vh on an Ettan IPGphor system (GE Healthcare, USA) at 20 °C and a maximum current of 50 μA/strip. Focused IPG strips were equilibrated for 15 min in equilibration solution (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, and 125 mM DTT) and then alkylated for a further 15 min in an equilibration solution containing 13.5 mM iodoacetamide instead of DTT. Strips were transferred onto a 12% SDS-PAGE gel within low-fluorescence glass plates (GE Healthcare, USA), and second dimensional focusing was performed at 10 °C using 20 mA/gel for 1 h, followed by 50 mA/gel, with an Ettan DALT 6 unit (GE Healthcare, USA) until the dye front reached the bottom of the gel. Electrophoresis was performed in Tris/ glycine/SDS buffer and in the dark. Image Analysis. Gels were scanned on a Typhoon FLA 9000 (GE Healthcare, USA) with excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). Images were analyzed using DeCyder 2D software, Version 7.0 (GE Healthcare, USA). After Student’s t test, the spots with p-value 60%). In all spleen fragments, chronic inflammation containing mononuclear cell

Prediction of Protein Interactions

To identify possible interactions between the identified proteins, the analysis of hypothetical interaction was performed using the program String (SearchTool for the Retrieval of Interacting Genes/Proteins) version 9.0-Functional Protein Networks Association (http://string-db.org). The evidence mode was used with an average confidence level of 0.400. The search parameters were as follows: neighborhood, gene fusion, co-occurrence, coexpression, experiments, databases, and text mining. Statistical Methods

Statistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). Data are reported as the mean of the group. Comparisons between different groups were made using the unpaired Student’s t-test or one-way ANOVA. Differences were considered significant at P < 0.05. D



Article

Figure 2. Histopathology of hamsters infected with BH46 and BH400 strains. (A) The spleen and (B) liver histology. The histopathological analysis was performed considering the lesion extension, and the samples were classified based on altered intensity as follows: 1 - without alteration; 2 discrete; 3 - moderate, and 4 - intense. p < 0.05 * BH46 × Control; ** BH400 × Control; # BH400 × BH46.

infiltration in the capsule, splenic cords (Billroth cords), and white and red pulp, were detected mainly in the BH400infected animals. We also observed a moderate hyperplasia and hypertrophy in the red pulp containing parasitized macrophages. Vacuolated macrophages with a cellular aggregate forming granulomas Virchow, as well a discrete increase of the congestion, were observed in the red pulp. T-cell depletion, hyperplasia and hypertrophy ranging from discrete to moderate were detected in the white pulp. Statistically significant differences were detected between the Control and the BH400 strain for the depletion of T-cells (p = 0.0013), hyperplasia and hypertrophy of both red pulp (p = 0.0021) and white pulp (p = 0.0040) (Figure 2A). The microscopic examination of the BH400-infected liver showed portal granulomatous inflammation and intralobular granulomes and indicated the presence of macrophages, lymphocytes, plasmocytes and scarce polymorphonuclear, neutrophils and eosinophils. The hepatocytes showed hydropic degeneration and/or discrete or intense steatosis. The liver also presented capsule thickening that was reactive to the chronic granulomatous process. However, BH46-infected liver did not shown any histological changes. Only one BH46-infected animal presented localized portal inflammation and discrete to intense hydropic degeneration hepatocytes. The alterations of portal inflammation and granulome showed statistically significant differences between the BH46 and BH400 strains, p < 0.0001 and p = 0.0029, respectively (Figure 2B). The quantification of the parasites in the major organs that were affected by L. infantum was determined by immunohistochemistry, serial dilution and qPCR. The immunohistochemistry assays that were conducted in the spleens and livers indicated numbers of parasites that were 30- and 5-fold larger in the BH400-infected organs compared to the BH46-infected organs, respectively (p < 0.0001) (Figure 3A,B). Furthermore, the spleens were individually macerated and serially diluted in 96-well plates. The BH400 parasites were grown until 7a

dilutions; however, the BH46 parasites were grown until the 3a dilution point. The BH400-infected animals showed higher parasitism (107) in the spleen than those infected with BH46 (102) (p < 0.0001) (Figure 3C). The parasite quantification was also conducted by real-time PCR in the spleens of the animals that were infected with BH400 and BH46 strains. The presence of Leishmania DNA was not detected in uninfected Hamsters, as assessed by qPCR. All Hamster samples that were infected with L. infantum were positive. The median amastigote number was greater in the BH400-infected spleens than the organs that were infected by BH46 (p < 0.05) (Figure 3D). These results suggest that the BH400 strain presents more widespread infectivity and division capability, resulting in a higher parasite burden and more severe clinical signals than BH46. Therefore, this virulence characterization encouraged us to look for molecules, by proteomic assay, that can be associated with this phenotype. Proteins Selection and Identification

To assess differential protein expression between the BH400 and BH46 strains, a 2-D DIGE assay using 18 cm, pH 4−7 strips was conducted. The representative gel images are presented in Figure 4. After in-gel image analysis by DeCyder 2D software, Version 7.2 (GE Healthcare, USA), approximately 1300 spots were located on the gels. The spots were matched and the protein expression difference between the strains was evaluated in the BVA module applying a p-value < 0.05 using one-way ANOVA and Studentś t test. Ninety-six spots with significant expression difference between the strains and present in at least two of the three gels that were analyzed were selected for picking and MS identification. Forty-nine spots were up-regulated and 47 were down-regulated in BH400. Among these, the abundance of 33 (17 up- and 16 downregulated) of the selected spots was too low and could be detected only by the DeCyder software. Thirty-two upregulated and 31 downregulated spots were identified by MS/MS (the peptides sequences were deposited in http://www.peptideatlas. org/PASS/PASS00393) and are circled in green and red, E



Article

Figure 3. Parasite quantification in hamsters infected with BH46 and BH400 strains. Amastigote number determined by immunohistochemical assay of spleens (A) and livers (B). Infected spleens were submitted to serial dilution (C) and real-time PCR (D). *p ≤ 0.0001.

Figure 4. Representative images of 2-D DIGE (SDS-PAGE 12%, strips IPG 18 cm, pH 4−7) of protein extracts from (A) BH46 and (B) BH400 strains and 3D view of the differentially expressed proteins between the L. infantum strains (C). The identified spots that are differentially expressed between the strains are circled: red (right) - down-regulated spots in BH400, and green (left) - upregulated spots in BH400. The numbers correspond to what is shown in Tables 1 and 2. In (C) there is a topographic 3D map of signal intensity was derived from the corresponding peaks F



Article

Table 1. BH400 Upregulated Spot Identificationa spot code

GI

protein name

experimental/theorical (pI/Mr)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

gi|264665718 gi|146078079 gi|401423944 gi|146081643 gi|134073994 gi|146092411 gi|146092411 gi|365927246 gi|365927246 gi|339899227 gi|401415106 gi|146103554 gi|146104107 gi|401421092 gi|401420112 gi|365927246 gi|401420112 gi|758136 gi|146089119 gi|154337338 gi|146086967 gi|145411494 gi|339898641 gi|146086967 gi|146089942 gi|145411494 gi|146087017 gi|146087017 gi|146085415 gi|146093061 gi|401416034 gi|146101120

soluble promastigote surface antigen PSA-34S beta-tubulin metallo-peptidase, Clan MA(E), Family M3 enolase S-adenosylhomocysteine hydrolase putative vacuolar ATP synthase subunit b putative vacuolar ATP synthase subunit b heat-shock protein 70 heat-shock protein 70 putative Rieske iron−sulfur protein precursor putative glutamine synthetase elongation factor 2 14-3-3 protein-like protein conserved hypothetical protein putative phosphomannomutase heat shock protein 70 putative phosphomannomutase heat-shock protein putative 2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase putative proteasome alpha 2 subunit peroxidoxin cytoplasmic tryparedoxin peroxidase putative small GTP-binding protein Rab1 peroxidoxin putative cytochrome c oxidase subunit V cytoplasmic tryparedoxin peroxidase putative endoribonuclease L-PSP (pb5) putative endoribonuclease L-PSP (pb5) putative small myristoylated protein-1 tryparedoxin cofilin-like protein kinetoplastid membrane protein-11

(4.2/53.00 − 4.72/35.55) (4.4/58.00 − 4.74/50.33) (5.77/64.00 − 5.61/77.68) (5.86/54.00 − 5.33/46.63) (5.84/55.00 − 5.66/48.52) (5.90/57.00 − 5.81/55.89) (6.03/57.00 − 5.81/55.89) (5.01/51.00 − 5.31/50.51) (5.12/50.00 − 5.31/50.51) (5.57/44.00 − 5.91/34.04) (6.11/50.00 − 5.71/42.89) (6.24/41.00 − 5.77/94.94) (4.68/38.00 − 4.79/29.78) (5.04/37.00 − 5.37/29.14) (5.18/36.00 − 5.18/28.23) (5.27/37.00 − 6.25/56.63) (5.32/36.00 − 5.18/28.23) (5.50/38.00 − 5.41/71.40) (5.68/36.00 − 5.80/30.72) (5.44/33.00 − 5.43/25.36) (5.91/33.00− 6.43/25.58) (6.24/33.00 − 6.73/22.37) (5.38/31.00 − 5.55/22.46) (5.59/32.00 − 6.43/25.58) (5.64/31.00 − 6.10/22.38) (6.21/29.00 − 6.73/22.37) (5.45/19.00 − 5.87/17.30) (5.83/19.00 − 5.87/17.30) (4.95/16.00 − 5.31/15.15) (5.52/17.00 − 5.24/16.80) (5.44/16.00 − 5.30/15.85) (5.98/11.00 − 5.96/11.28)

abundance vol. ratio p < 0.05 ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑

28.64 2.48 6.82 8.06 9.05 4.77 4.41 15.83 2.01 5.66 5.33 2.32 2.05 2.78 3.96 2.99 1.66 2.51 5.96 4.98 4.24 4.97 2.24 6.58 4.07 9.16 7.85 19.43 3.80 6.13 6.20 1.60

a

The Spot Code refers to the spot identification that was used in Figure 4. Experimental pI and molecular mass-Mr (Da) from DeCyder BVA module; Theorical pI and molecular mass −Mr (Da) from the NCBI database.

respectively, in the representative 2D gel image (Figure 4). The 63 identified spots corresponded to 36 different proteins; some of the identified proteins are present in more than one spot, suggesting the presence of several isoforms and/or posttranslational modifications, which is a common event in trypanosomatides. For example, β-tubulin and heat-shock protein 70 (HSP-70) were identified from five different spots in the BH46 strain and from three spots in the BH400 strain. Twenty unique proteins were up-regulated and 16 were downregulated in BH400. Furthermore, three proteins were identified in both strains: enolase, tryparedoxin, and peroxidoxin. These common proteins corresponded to distinctly localized spots between the gels; the spots that were identified in the down-regulated set always presented increased molecular mass and decreased pI value, which suggests the functional alteration of the protein by post-translational modification. The up- and downregulated proteins that were identified in the BH400 strain for each spot were numbered and are indicated in Tables 1 and 2, respectively. The listed up-regulated proteins had spot intensity ratios ranging from 1.6 to 28.64, and the downregulated proteins had ratios between 1.99 and 16.6. Sixteen of the identified proteins were previously described in virulence studies in Leishmania, confirming the robustness of the data. The extended data analysis (EDA) module was also used to determine the differentially expressed spots using two analytical

Standards, and a global similarity analysis between all the spots and between the differentially expressed spots of both strains (Figure 5, panels A and B, respectively) was conducted. In the heat maps, the columns represent the different strains (BH46 and BH400), and the rows indicate the spots. The expression values are shown on a log scale using a normalized colorimetric abundance, ranging from −1 (down-regulated, green) to 1 (upregulated, red). The similarities in the expression patterns of the strains are readily observed, principally at the top of the map, and the larger differences are present in the bottom half of the map (Figure 5A). When we observe the heat map of the differentially expressed proteins, the differences are more visible between the strains (Figure 5B). The analysis was conducted using principal component analysis (PCA), allowing the reduction of multidimensional data and independent strain classification, in which we can observe the separation of two groups of spots (Figure 5C). The PCA score plot shows the separation between the up- (right) and down-regulated (left) spots in the BH400 strain. At PC1 positive values, there are upregulated spots in BH400, while the down-regulated spots are grouped at PC1 negative values. Two identified proteins were selected in the score plot and the location and a 3-D view of these spots in a representative portion of the 2-D DIGE gel image can be observed in Figure 4C. These spots were also selected on score plots (Figure 5C). G



Article

Table 2. BH400 Downregulated Spot Identificationa experimental/theorical (pI/Mr)

spot code

GI

protein name

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

gi|146104321 gi|146104317 gi|146093964 gi|146093964 gi|146081643 gi|146075141 gi|146076586 gi|339897551 gi|339897551 gi|146094146 gi|146102742 gi|146078076 gi|146089026 gi|146104117 gi|146078076 gi|146078076 gi|146078076 gi|339897551 gi|339897551 gi|339897551 gi|146088052 gi|146086967 gi|146086967 gi|146078076 gi|146081837 gi|339897551 gi|146093061 gi|146070754 gi|146093061 gi|146093061 gi|146084262

chaperonin HSP60, mitochondrial precursor chaperonin HSP60 putative heat shock 70-related protein 1, mitochondrial precursor putative heat shock 70-related protein 1, mitochondrial precursor enolase putative eukaryotic initiation factor 4a actin alpha tubulin alpha tubulin conserved hypothetical protein protein disulfide isomerase beta tubulin putative pyruvate dehydrogenase E1 beta subunit putative translation elongation factor 1-beta beta tubulin beta tubulin beta tubulin alpha tubulin alpha tubulin alpha tubulin putative IgE-dependent histamine-releasing factor peroxidoxin peroxidoxin beta tubulin putative ribonucleoprotein p18, mitochondrial precursor alpha tubulin tryparedoxin cyclophilin 2 tryparedoxin tryparedoxin conserved hypothetical protein

(5.07/62.00 (5.26/63.00 (5.53/63.00 (5.62/64.00 (5.64/58.00 (6.24/56.00 (5.64/54.00 (6.00/55.00 (5.43/48.00 (5.46/49.00 (5.58/51.00 (5.56/49.00 (5.44/48.00 (4.60/41.00 (4.83/41.00 (4.93/41.00 (4.93/41.00 (5.23/43.00 (5.18/39.00 (5.18/38.00 (4.29/33.00 (5.20/34.00 (5.42/34.00 (4.83/30.00 (5.83/28.00 (4.91/20.00 (5.38/20.00 (6.74/22.00 (5.26/17.00 (5.13/14.00 (5.56/13.00

− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

5.33/59.66) 5.33/60.85) 6.00/69.31) 6.00/69.31) 5.33/46.63) 5.83/45.35) 5.41/42.30) 5.08/54.94) 5.08/54.94) 5.32/41.15) 5.42/52.76) 4.71/50.39) 5.64/38.44) 4.61/23.22) 4.71/50.39) 4.71/50.39) 4.71/50.39) 5.08/54.94) 5.08/54.94) 5.08/54.94) 4.39/19.60) 6.43/25.58) 6.43/25.58) 4.71/50.39) 6.74/21.64) 5.08/54.94) 5.24/16.80) 6.51/20.46) 5.24/16.80) 5.24/16.80) 5.53/13.38)

abundance vol. ratio p < 0.05 ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

2.35 4.90 7.67 16.66 3.36 6.13 1.99 7.01 8.42 7.40 3.68 4.50 5.43 11.32 2.66 2.78 2.78 4.82 4.39 3.59 5.79 3.28 4.66 2.37 15.86 2.73 5.79 9.60 5.65 5.08 2.46

a

The Spot Code refers to the spot identification that was used in Figure 4. Experimental pI and molecular weight-Mr (Da) from DeCyder BVA module; theorical pI and molecular mass−Mr (Da) from the NCBI database.

Prediction of Protein Interactions

Validation of Differential Protein Expression by Western Blot Assay

Because the focus of our assays is the virulence difference between the strains, we performed an interaction network prediction among the up-regulated and down-regulated proteins using the String 9.0 database (Search Tool for the Retrieval of Interacting Genes/Proteins - http://string-db.org). This approach allows us to explore the relevant interactions that are related to differentially expressed proteins among themselves and other proteins and genes, using a database of known and predicted protein interactions. These interactions can be by direct (physical) and indirect (functional) association (Supplementary Figures S1 and S2, Supporting Information). The nodes are proteins and the annotations are displayed above each node. The virulence associated proteins that were identified in the present study are circled in red, and the edges represent the predicted functional associations. An edge can be represented by seven different colors, which indicates the existence of the seven types of evidence used in predicting the associations. We can observe in S1 and S2 that some interactions of the virulence associated proteins that were identified in the present study (circled in red) occur with other proteins and between themselves, and some proteins do not have any interaction predictions.

The validation of proteomic data was performed using immunoblotting with 2-DE for protein fractionation. AntiKMP-11, EF-1β, and α-tubulin antibodies were used (Figure 6). The selected spot of α-tubulin that was used for assay normalization was distinct from the spot that presented differential expression in 2-D DIGE analysis (Figure 4A and Table 2), and it had a similar abundance in both samples. As indicated in Figure 6A, the anti-α-tubulin signal is strong and similar between the samples. The anti-EF-1β antibody signal revealed different protein isoforms within the strains, as expected; these are more visible and have stronger signals in BH46 when compared to BH400. In membranes that were incubated with anti-KMP-11, we can also verify the presence of weak signals in both strains; however, the intensity of the signal in BH400 is stronger compared to BH46. Semiquantitative data obtained by submitting spots to quantification by ImageJ Software is plotted in Figure 6B. This analysis confirms the visual evaluation of the immunoblotting signals, and the ratio of the signals between the strains was 1.05, 2.9, and 1.7 arbitrary units for α-tubulin, EF-1β, and KMP-11, respectively. These data indicate that EF-1β is down- and KMP-11 is up-regulated in BH400, corroborating the results from our proteomic analysis. H



Article

Figure 5. Heat map and hierarchical clustering of the 2-D DIGE profiles. (A) General protein expression between BH46 and BH400 samples and (B) differentially expressed spots. In A and B, the samples are shown on the vertical axis, and the spots are shown on the horizontal axis. The expression values are shown on a log scale using a normalized colorimetric abundance, ranging from −1 (down-regulated, green) to 1 (up-regulated, red). (C) The principal component analysis that was applied to the data set compressed all differentially expressed spots in up-regulated (right) and down-regulated (left) areas of the Score Plot. Numbers correspond to the described virulence proteins that were used in the immunoblotting assay (46 = EF-1β and 32 = KMP-11).

4. DISCUSSION In this study, first we performed comparative in vitro and in vivo virulence characterization in the two L. infantum strains. The results showed that the BH400 strain is more infectious and has a higher parasite number in cells and infected animals than the BH46 strain. BH400-infected hamsters showed greater damage of the analyzed tissue than those that were infected with BH46, confirming the more significant virulence that was induced by BH400. These virulence characterization results encouraged us to determine the molecules that account for this difference in the infectivity of the strains. Thus, we performed a comparative proteomic analysis between the strains to identify the proteins that were associated with virulence. Approximately 1300 spots were detected on 2-D DIGE gels, and only 96 showed significant expression difference between the strains by DeCyder software analysis. Because the parasites were of the same parasite species, we expected that the proteomic profile and expression pattern would be similar, with few differences. Thirty-two up-regulated and 31 down-regulated spots in the BH400 strain were identified by MS/MS, which corresponded to 36 different proteins (20 up- and 16 downregulated) because some identified proteins are present in more than one spot, suggesting the presence of several isoforms and/ or post-translational modifications, which is a common event in trypanosomatides.14,15,22−24 Among the 20 different up-regulated proteins in BH400, 16 had been previously associated with virulence in other studies

using different Leishmania species. Different authors have demonstrated the ability of various molecules, particularly proteins, to interfere with the virulence of several Leishmania species. On the basis of the biology of this protozoan, we expected to detect the increased expression of molecules that are involved in the infection process, prolonging the lifetime of the infected host cell, energy production, stress response, and survival in virulent strains. However, in studies that have been conducted with L. infantum, the simultaneous involvement of proteins that performed all these functions mentioned above had not yet been demonstrated. Accordingly, we will direct our discussion to the simultaneous involvement of the proteins identified here and that have been previously linked to virulence in Leishmania and their interactions. Although somewhat speculative, the discussion is based on experimental data of differential expression and is supported by the scientific literature. Considering our results with what has been reported in the literature, we suggest the hypothesis that the virulence phenotype in L. infatum could be initially a result of the increased expression of kinetoplastid membrane protein-11 (KMP-11) and enolase. These proteins were also detected in an exoproteome study, indicating these proteins could have flexible role in the cell and in immunological studies.25 KMP11, a hydrophobic protein, is involved in the interaction with in the host cell, and its expression is increased in amastigote forms, which has been described to be associated to LPG.26 In I



Article

Clan MA(E) also interacts with another up-regulated protein in BH400, endoribonuclease L-PSP (pb5), which is a class of proteins that act on single-stranded mRNA and are thought to be inhibitors of protein synthesis.33 These nucleases could aid in purine salvage, which is obligatory for Leishmania because they are incapable of de novo purine synthesis.34 Differences in proteins expression involved in proteolysis between the two life stages, amastigote and promastigote, suggest that protein degradation processes might be carried out by different mechanisms between both life stages.35 Increased energy consumption and protein synthesis are also expected in cells that are more invasive and that have high rates of replication. The amplification of precursor synthesis agreed with this requirement, as observed by an increase in Rieske iron−sulfur protein precursor, EF-2, S-adenosylhomocysteine and phosphomannomutase. The Rieske subunit acts by binding an ubiquinol and transferring an electron to the 2Fe-2S cluster, then releasing the electron to cytochrome C, which catalyzes the oxidoreduction of the mobile redox components, generating an electrochemical potential, which is linked to ATP synthesis.36,37 Rieske iron−sulfur protein precursor was also found up-regulated in L. braziliensis antimony resistant, which provides energy for parasite proliferation and contributes to reduce oxidative stress since is a scavenger of peroxides.38 Among the up-regulated molecules is the elongation factor 2 (EF-2), a protein that is associated with the translation process and elongation of the polypeptide chain at ribosomes and that has previously been associated with the virulence phenotype in L. donovani.39 Interactions were predicted to exist between EF2 and ribosomal structural subunits such as the 60S subunit of the L30 and 40S subunit of the S16 ribosomal protein, that are associated with translation.40 The ribosomal proteins mentioned above interact with S-adenosylhomocysteine hydrolase, which is another up-regulated protein that was identified in this study. This hydrolase is a competitive inhibitor of the Sadenosyl-L-methionine-dependent transmethylation reactions and has an important role in methylation control and intracellular S-adenosylhomocysteine regulation. In L. braziliensis and L. donovani, S-adenosylhomocysteine hydrolase was associated with the virulence phenotype, and in this study, it was up-regulated in the more virulent L. infantum strain.41,42 Furthermore, S-adenosylhomocysteine hydrolase also interacts with the ATPase α-subunit that is associated with proton transport through the membrane, a crucial step for ATP synthesis. Moreover, the 60S and 40S subunits of the L30 and S16 ribosomal proteins together with S-adenosylhomocysteine hydrolase also interact with phosphomannomutase, another upregulated protein in the BH400 strain. Phosphomannomutase catalyzes the transformation of mannose-6-phosphate to mannose-1-phosphato, an important step in mannose activation and glycoconjugate biosynthesis in eukaryotes. Phosphomannomutase-deficient L. mexicana loses its virulence capability, suggesting this molecule is a virulence factor and could be used as a promising target for the development of anti-Leishmanial inhibitors.43 Another process that could be involved in prolonging the lifetime of the infected host cell could be promoted by 14-3-3 protein-like protein. These antiapoptotic secreted proteins could be active in prolonging the lifespan of infected host cells.44 Specifically, 14-3-3 protein-like protein was predicted to interact with the 19S proteasome regulatory subunit, metallopeptidase, Clan MP - Family M67, which belongs to a multisubunit complex and is responsible for protein degradation.

Figure 6. Validation of the proteomic analysis by immunoblotting assay. (A) Comparative Western blot from 2-DE gel of BH46 and BH400 strains using anti-α-tubulin, eEF-1β, and KMP-11 antibodies. (B) Intensity measurement of the spots, in arbitrary units, by ImageJ (Wayne Rasband, NIH, USA, http://rsb.info.nih.gov/ij/).

L. donovani, the expression of KMP-11 was decreased along with parasite virulence as a function of the time of the subculture.27 KMP-11 was also reported in L. amazonensis to be able to exacerbate the infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The authors showed that the use of anti-KMP-11 antibodies inhibited the increase in parasite load.28,29 It was also observed in multiple independent experiments that the isolation of a Sb(III) resistant L. infantum cell line always correlated with a marked decrease in the KMP-11 protein but not mRNA levels. To the authors, these data suggest that in the Sb(III) resistant mutant the stability of the KMP-11 may be compromised resulting in an increased turnover rate of this protein. It is conceivable that alteration in the posttranslational modifications of KMP-11 in drug resistant cell lines may accelerate degradation of KMP-11.30 Conversely, the up-regulation of enolase, which was described in L. mexicana, L. donovani, and L. major as a virulence factor,31 would perform versatile functions as a chaperone, DNA-binding protein acting in transcription, plasminogen receptor, cell migration, and metallo-protease activating protein.32 Furthermore, there could be an increase of the proteinases that are involved in the infection process. The proteinases are enzymes that hydrolyze peptide bonds and thus have the potential to degrade proteins and peptides that participate in the infection process. In our analysis, metallo-peptidase Clan MA(E), Family M3 was up-regulated in BH400, and it was predicted to interact with vacuolar-type proton translocating pyrophosphatase1. In L. amazonensis, a soluble pyrophosphatase (LaVSP1) was localized to acidocalcisomes and preferentially expressed in metacyclic promastigotes. Metallo-peptidase J



Article

It was also predicted to interact with the α-2 subunit of the proteasome that degrades labeled proteins by ubiquitin or by other mechanisms.45 Additionally, during the infection, the host cell produces a large amount of reactive oxygen intermediates and nitric oxide (NO and ROI), creating unfavorable environments for intracellular pathogens. Next, we could expect an increase in the secretion of certain proteins that are responsible for protecting the parasite against the stress response, such as tryparedoxin peroxidase and peroxidoxin. The following proteins were up-regulated in both L. infantum strains: cytoplasmic tryparedoxin peroxidase and peroxidoxin; both proteins were associated with the Leishmania virulence phenotype.46 Another group described the markedly increased expression of tryparedoxin after exposure of L. donovani to H2O2, which did not cause any cell death in this parasite. In addition, tryparedoxin transfectants demonstrated increased virulence, causing higher parasite burden in macrophages.47 The interaction between tryparedoxin peroxidase and peroxidoxin, which has cellular detoxification functions and is involved in signaling, proliferation, and differentiation, was also reported by Castro et al.48 to be crucial for parasite survival in oxidative environments. Corroborating this idea, a proteomic analyses between L. braziliensis and L. chagasi antimony resistant had shown up-regulation of tryparedoxin peroxidase and peroxiredoxin, suggesting that an increased metabolism of peroxides and higher antioxidant defense may play a significant role in the resistance of parasites to antimonials.38 Accordingly, we hypothesize that although both proteins were up-regulated in the two strains, their role in L. infantum virulence is significantthe spot localization on the gels was distinct, presenting distinct pI and MW and consequently different functions. This phenomenon was observed by previous authors who suggested these spots could represent different variants of tryparedoxin peroxidase, due to post-translational modifications, since differences between the calculated and experimental pI of identified spots were found.38 Another hypothesis proposed by Danishvar et al.49 in a study using gentamicin attenuated line of L. infantum (H-Line) was these enzymes are encoded by multiple paralogous genes, encoding very similar proteins that differ in predicted size and pI, and, given the number of protein spots that were identified as tryparedoxin peroxidase, it seems likely that multiple gene products are each expressed as multiple isoforms.49 Furthermore, increased survival could be related to the increase of chaperones and endoribonuclease L-PSP (PB5). HSP-70, a chaperone, is more abundant in cells that are stressed by elevated temperatures, protects proteins that have been denatured by heat, nascent peptides, and blocks the folding of proteins that must remain unfolded until they have been translocated across membranes. In L. infantum, the absence of HSP70-II caused a major alteration in growth as the promastigotes reached the stationary phase. Leishmania exosomes containing HSP-70 and -90, EF-1α, aldolase, and GP63 were identified in the cytosolic fraction of L. donovaniinfected macrophages. Leishmania secretes virulence factors into the host cytoplasm, where they interact with host signaling molecules to subvert host immune responses.50 The presence of HSPs in exosomes most likely ensures the correct folding of the exosomal proteins and consequently their action as virulence factors. HSP-70 was also associated in antimony resistance mechanisms and cells development in a proteome study using L. braziliensis and L. chagasi antimony resistant.38

Another virulence associated molecule is the small GTPbinding protein Rab1 that is known to be involved in vesicular transport. Although these transport vesicle regulatory proteins may normally regulate vesicle trafficking in Leishmania, ectopically following secretion, they may have the potential to affect vesicle trafficking in infected cells.40 Surprisingly, both strains had three common proteins (enolase, peroxidoxin, and tryparedoxin). These proteins were located at distinct points of the gel, and the group of downregulated proteins always presented an increased molecular mass and decreased pI. A proteome can be modified by co- or post-translational modifications, which can change some protein properties such as charge, hydrophobicity, conformation, and/or stability, and consequently, its function. Some of these modifications are ubiquitination, oxidation, glycosylation, phosphorylation, methylation, and acetylation, among others. The protein sequence is very important in determining the post-translational change that can occur at a specific residues, and some protein motifs serve as signals for certain posttranslational modifications. However, this is modulated by a protein’s context, the cellular compartment, certain pathways, and the features of the surrounding amino acid sequence.51 Many post-translationally modified proteins exist as isoforms. Therefore, proteins can exist in modified and unmodified states and can carry different numbers of one or more types of modifications, leading to the presence of a large number of protein products for one gene. Post-translational modifications such as phosphate, sulfate, or carboxyl groups (for example, sialic acid) directly confer negative charges onto proteins, resulting in multiple isoforms after 2-D (PAGE) separation.52 These multiple spots can vary between different samples such as disease and healthy states, different tissues, cells submitted to distinct treatments, cell development, and, based on our data, parasite strains with different levels of virulence. The mechanism for this could be associated with the distinct functionality of the modified proteins, or the fact that some modifications, such as ubiquitin addition, can target the proteins for degradation, which is a hypothesis that needs to be further investigated. Another unexpected result was the presence of a protein associated with the virulence phenotype in Leishmania that was down-regulated in BH400. Elongation factor-1α (EF-1 α) has been shown to inhibit macrophage activation.53 EF-1α is exported to the host cytoplasm by the secretion of exosomes that could subvert host immune responses. 51 In an exoproteome study from logarithmic and stationary L. infantum promastigotes, EF-1α was the most abundant protein in vesicles provided from logarithmic parasite.25 Our hypothesis is that the turnover of these proteins is extremely high, and excess machinery is disposed of via secretion in addition to the reported processes of ubiquitination and proteasome-mediated degradation. On the other hand, it is important to consider the possibility of proteolysis in EF-1α, as substantiated in both promastigotes and amastigotes stages.54 It was also demonstrated that eEF-1α was up-regulated in the L. infantum amastigote membranous preparation; this protein has been implicated in intramacrophage survival of amastigotes in L. donovani.55 In the present study, we identified some proteins that were described as virulence factors in other Leishmania species, as well as other proteins that were predicted to be their “partner”. We would like to emphasize that based on what was indicated by the interaction network, it was observed that proteins described as virulence factors and that were identified in this study sometimes interact with each other, for example, enolase and K



Article

(4) Dye, C. The logic of visceral Leishmaniasis control. Am. J. Trop. Med. Hyg. 1996, 55, 125−130. (5) Berrahal, F.; Mary, C.; Roze, M.; Berenger, A.; Escoffier, K.; Lamouroux, D. Canine Leishmaniasis: identification of asymptomatic carriers by polymerase chain reaction and immunoblotting. Am. J. Trop. Med. Hyg. 1996, 55, 273−277. (6) Azizi, H.; Hassani, K.; Taslimi, Y.; Shateri-Najafabadi, H.; Papadopoulou, B.; Rafati, S. Searching for virulence factors in the nonpathogenic parasite to humans Leishmania tarentolae. Parasitology 2009, 136, 723−735. (7) Zhang, W. W.; Matlashewski, G. Screening Leishmania donovanispecific genes required for visceral infection. Mol. Microbiol. 2010, 77 (2), 1−13. (8) Brittingham, A.; Miller, M. A.; Donelson, J. E.; Wilson, M. E. Regulation of GP63 mRNA stability in promastigotes of virulent and attenuated Leishmania chagasi. Mol. Biochem. Parasitol. 2001, 112, 51− 59. (9) Yao, C.; Chen, Y.; Sudan, B.; Donelson, J. E.; Wilson, M. E. Leishmania chagasi: Homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model. Exp. Parasitol. 2008, 118, 129−133. (10) Yao, C.; Li, Y.; Donelson, J. E.; Wilson, M. E. Proteomic examination of Leishmania chagasi plasma membrane proteins: contrast between avirulent and virulent (metacyclic) parasite forms. Proteomics Clin. Appl. 2010, 4, 4−16. (11) Morales, M. A.; Watanabe, R.; Dacher, M.; Chafey, P.; Osorio, Y.; Fortéa, J.; Scott, D. A.; Beverley, S. M.; Ommen, G.; Clos, J.; Hem, S.; Lenormand, P.; Rousselle, J. C.; Namane, A.; Späth, G. F. Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage. Proc. Natl. Acad. Sci., U. S. A. 2010, 107, 8381−8386. (12) Costa, M. M.; Andrade, H. M.; Bartholomeu, D. C.; Pires, S. F.; Chapeaurouge, A. D.; Gazzinelli, R. T. Analysis of Leishmania chagasi by 2-D Difference Gel Eletrophoresis (2-D DIGE) and Immunoproteomic: Identification of Novel Candidate Antigens for Diagnostic Tests and Vaccine. J. Proteome Res. 2011, 10, 2172−2184. (13) Pescher, P.; Blisnick, T.; Bastin, P.; Späth, G. Quantitative proteome profiling informs on phenotypictraits that adapt Leishmania donovani for axenic and intracellular proliferation. Cell. Microbiol. 2011, 13, 978−991. (14) Clayton, C.; Shapira, M. Post-transcriptional regulation of gene expression in trypanosomes and leishmanias. Mol. Biochem. Parasitol. 2007, 156, 93−101. (15) Clayton, C. E. Life without transcriptional control? From fly to man and back again. EMBO J. 2002, 21, 1881−1888. (16) Giaimis, J.; Lombard, Y.; Makaya-Kumba, M.; Fonteneau, P.; Poindron, P. A new and simple method for studying the binding and ingestion steps in the phagocytosis of yeasts. J. Immunol. Methods 1992, 154 (2), 185−193. (17) Tafuri, W. L.; Santos, R. L.; Arantes, R. M.; Gonçalves, R.; De Melo, M. N.; Michalick, M. S.; Tafuri, W. L. An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues. J. Immunol. Methods. 2004, 292, 17−23. (18) Afonso, L. C.; Scott, P. Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis. Infect. Immun. 1993, 61, 2952−2959. (19) Bretagne, S.; Durand, R.; Olivi, M.; Garin, J. F.; Sulahian, A.; Rivollet, D.; Vidaud, M.; Deniau, M. Real-time PCR as a new tool for quantifying Leishmania infantum in liver in infected mice. Clin. Diagn. Lab. Immunol. 2001, 8, 828−831. (20) Bruna-Romero, O.; Hafalla, C. R.; González-Aseguinolaza, G.; Sano, G. I.; Tsuji, M.; Zavala, F. Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR. Int. J. Parasitol. 2001, 31, 1499−1502. (21) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using

S-adenosylhomocysteine hydrolase, and tryparedoxin and peroxiredoxin. We know that a protein cannot act alone in a system; cellular functions are complex and are typically the result of the coordinated action of several proteins acting in molecular assemblies or pathways to achieve a particular task. The interaction network schemes are believed to contribute to our understanding of the relationships between proteins and phenotypes. Furthermore, events such as post-translational modifications clearly indicate that there is an increased molecular diversity that could contribute to phenotypic complexity. Again we emphasize that there is no experimental validation that these proteins are indeed directly related to virulence in L. infantum. It would be very informative and, perhaps, even conclusive if overexpression of either protein, in each one or both strains, could lead to a notable increase in infectivity. This is what we intend to do next. The variability of the protein expression between different L. infantum strains remains an unexplored area in Leishmania biology. Additional studies will allow us to understand the various aspects of the parasite and the differences in pathogenicity between strains of the same species more thoroughly, thereby generating hypotheses that can be tested in future studies as the role of these proteins in the cell and in the host−parasite interaction become better understood.

■

ASSOCIATED CONTENT

* Supporting Information S

One large table and two figures: Supplementary Table 1: Identification of differentially expressed protein spots between BH400 and BH46 L. infantum strains. Supplementary Figure S1: Interaction network of the up-regulated proteins in BH400. Supplementary Figure S2: Interaction network of the downregulated proteins in BH400. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +55(31) 3409-3010. Author Contributions #

S.d.F.P. and L.C.F.J. have equally contributed to the work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was funded by Fundaçaõ de Amparo as Pesquisa do Estado de Minas Gerais (FAPEMIG- Grant 00321-10), ́ Superio Coordenaçaõ de Aperfeiçoamento de Pessoal de Nivel (CAPES- Grant 2682/2010), Conselho Nacional de Desenvolví e Tecnológico (CNPq- Grant 472214/2010-1) mento Cientifico and Pro-reitoria de Pesquisa da Universidade Federal de Minas Gerais (PRPq/UFMG).

■

REFERENCES

(1) Desjeux, P. Leishmaniasis: current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis. 2004, 27, 305−318. (2) Alvar, J.; Aparicio, P.; Aseffa, A.; Den Boer, M.; Cañavate, C.; Dedet, J. P.; Gradoni, L.; Ter Horst, R.; López-Vélez, R.; Moreno, J. The relationship between leishmaniasis and AIDS: the second 10 years. Clin. Microbiol. Rev. 2008, 21 (2), 334−359. (3) Hailu, A.; Berhe, N. The performance of direct agglutination tests (DAT) in the diagnosis of visceral leishmaniasis among Ethiopian patients with HIV co-infection. Ann Trop Med Parasitol. 2002, 96 (1), 25−30. L



Article

Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9, 255−262. (22) Teixeira, S. M. R.; Otsu, K.; Hill, K. L.; Kirchhoff, L. V.; Donelson, J. E. Expression of a marker for intracellular Trypanosoma cruzi amastigotes in extracellular spheromastigotes. Mol. Biochem. Parasitol. 1999, 98, 265−270. (23) Di Noia, J. M.; D’orso, I.; Sanchez, D. O.; Frasch, A. C. AU-rich elements in the 3′-untranslated region of a new mucin-type gene family of Trypanosoma cruzi confers mRNA instability and modulates translation efficiency. J. Biol. Chem. 2000, 275, 10218−10227. (24) D’orso, I.; Frasch, A. C. TcUBP-1, a developmentally regulated U-rich RNA-binding protein involved in selective mRNA destabilization in trypanosomes. J. Biol. Chem. 2001, 276, 34801−34809. (25) Santarém, N.; Racine, G.; Silvestre, R.; Cordeiro-da-Silva, A.; Ouellette, M. Exoproteome dynamics in Leishmania infantum. J. Proteomics 2013, 12 (84), 106−118. (26) Jardim, A.; Funk, V.; Caprioli, R. M.; Olafson, R. W. Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein. Biochem. J. 1995, 305, 307−313. (27) Mukhopadhyay, S.; Sen, P.; Majumder, H. K.; Roy, S. Reduced expression of lipophosphoglycan (LPG) and kinetoplastid membrane protein (KMP)-11 in Leishmania donovani promastigotes in axenic culture. J. Parasitol. 1998, 84, 644−647. (28) Lacerda, D. I.; Cysne-Finkelstein, L.; Nunes, M. P.; De-Luca, P. M.; Genestra, M.; Da, S.; Leon, L. L.; Berrêdo-Pinho, M.; MendonçaLima, L.; Matos, D. C.; Medeiros, M. A.; Mendonça, S. C. Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages. Mem. Inst. Oswaldo Cruz. 2012, 107, 238−245. (29) Matos, D. C.; Faccioli, L. A.; Cysne-Finkelstein, L.; Luca, P. M.; Corte-Real, S.; Armoa, G. R. Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis. Mem Inst Oswaldo Cruz. 2010, 105, 341−347. (30) El Fadili, K.; Drummelsmith, J.; Roy, G.; Jardim, A.; Ouellette, M. Down regulation of KMP-11 in Leishmania infantum axenic antimony resistant amastigotes as revealed by a proteomic screen. Exp. Parasitol. 2009, 123 (1), 51−57. (31) Swenerton, R. K.; Zhang, S.; Sajid, M.; Medzihradszky, K. F.; Craik, C. S.; Kelly, B. L.; Mckerrow, J. H. The oligopeptidase B of Leishmania regulates parasite enolase and immune evasion. J. Biol. Chem. 2011, 286, 429−40. (32) Vanegas, G.; Quiñones, W.; Concepción, L.; Avilán, L. Enolase as a plasminogen binding protein in Leishmania mexicana. Parasitol Res. 2007, 101, 1511−1516. (33) Ruhland, A.; Leal, N.; Kima, P. E. Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis. Cell Microbiol. 2007, 9, 84−96. (34) Rappu, P.; Shin, B. S.; Zalkin, H.; Mantsala, P. A Role for a Highly Conserved Protein of Unknown Function in Regulation of Bacillus subtilis purA by the Purine Repressor. J. Bacteriol. 1999, 181, 3810−3815. (35) Brotherton, M. C; Racine, G.; Ouameur, A. A; Leprohon, P.; Papadopoulou, B.; Ouellette, M. Analysis of membrane-enriched and high molecular weight proteins in Leishmania infantum promastigotes and axenic amastigotes. J. Proteome Res. 2012, 11 (8), 3974−3985. (36) Priest, J. W.; Hajduk, S. L. In Vitro Import of the Rieske IronSulfur Protein by Trypanosome Mitochondria. J. Biol. Chem. 1996, 271, 20060−20069. (37) Harnisch, U.; Weiss, H.; Sebald, W. The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing. Eur. J. Biochem. 1985, 149, 95−99. (38) Matrangolo, F. S.; Liarte, D. B.; Andrade, L. C.; de Melo, M. F.; Andrade, J. M.; Ferreira, R. F.; Santiago, A. S.; Pirovani, C. P.; SilvaPereira, R. A.; Murta, S. M. Comparative proteomic analysis of antimony-resistant and -susceptible Leishmania braziliensis and

Leishmania infantum chagasi lines. Mol. Biochem. Parasitol. 2013, 190 (2), 63−75. (39) Kushawaha, R.; Gupta, R.; Amogh, A.; Sahasrabuddhe, S. Elongation factor-2, a Th1 stimulatory protein of Leishmania donovani, generates strong IFN-γ and IL-12 response in cured Leishmaniainfected patients/hamsters and protects hamsters against Leishmania challenge. J Immunol. 2011, 187, 6417−6427. (40) Bargis-Surgey, P.; Lavergne, J. P.; Reboud, J. P. Interaction of elongation factor eEF-2 with ribosomal P proteins. Eur. J. Biochem. 1999, 262, 606−611. (41) Mottram, J. C.; Coombs, G. H.; Alexander, J. Cysteine peptidases as virulence factors of Leishmania. Curr. Opin. Microbiol. 2004, 7, 375−381. (42) Walker, J.; Vasquez, J. J.; Gomez, M. A. Identification of developmentally-regulated proteins in Leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes. Mol. Biochem. Parasitol. 2006, 147, 64−73. (43) Kedzierski, L.; Malby, R.; Smith, B.; Handman, E. Structure of Leishmania Mexicana Phosphomannomutase Highlights Similarities with Human Isoforms. J. Mol. Biol. 2006, 363, 215−227. (44) Silverman, J. M.; Chan, S. K.; Robinson, D. P.; Dwyer, D. M. Proteomic analysis of the secretome of Leishmania donovani. Genome Biol. 2008, 9, 1−21. (45) Malygin, A. A.; Shaulo, D. D.; Karpova, G. Proteins S7, S10, S16 and S19 of the human 40S ribosomal subunit are most resistant to dissociation by salt. Biochem. Biophys. Acta 2000, 1494, 213−216. (46) Romao, S.; Castro, H.; Sousa, C.; Carvalho, S.; Tomás, A. The cytosolic tryparedoxin of Leishmania infantum is essential for parasite survival. Int. J. Parasitol. 2009, 39, 1−9. (47) Iyer, J. P.; Kaprakkaden, A.; Choudhary, M. L.; Shaha, C. Crucial role of cytosolic tryparedoxin peroxidase in Leishmania donovani survival, drug response and virulence. Mol. Microbiol. 2008, 68, 372− 391. (48) Castro, H.; Sousa, C.; Santos, M.; Tomas, A. M. Complementary antioxidant defense by cytoplasmic and Mitochondrial peroxiredoxins in Leishmania infantum. Free Radical Biol. Med. 2002, 33, 1552−1562. (49) Daneshvar, H.; Wyllie, S.; Phillips, S.; Hagan, P.; Burchmore, R. Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiolredox metabolism. J. Proteomics 2012, 75 (5), 1463−1471. (50) Silverman, J. M.; Clos, J.; de’Oliveira, C. C.; Shirvani, O.; Fang, Y.; Wang, C.; Foster, L. J.; Reiner, N. E. An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages. J. Cell Sci. 2010, 123, 842−852. (51) Jensen, O. N. Interpreting the protein language using proteomics. Nat. Rev., Mol. Cell Biol. 2006, 7, 391−403. (52) Hughes, G. J.; Frutiger, S.; Paquet, N.; Ravier, F.; Pasquali, C.; Sanchez, J. C.; James, R.; Tissot, J. D.; Bjellqvist, B.; Hochstrasser, D. Plasma protein map: an update by microsequencing. Electrophoresis 1992, 13, 707−714. (53) Nandan, D.; Yi, T.; Lopez, M.; Lai, C.; Reiner, N. E. Leishmania EF-1 alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. J. Biol. Chem. 2002, 277, 50190−50197. (54) Brotherton, M. C.; Racine, G.; Foucher, A. L.; Drummelsmith, J.; Papadopoulou, B.; Ouellette, M. Analysis of stage-specific expression of basic proteins in Leishmania infantum. J. Proteome Res. 2010, 9 (8), 3842−3853. (55) Lynn, M. A.; Marr, A. K.; McMaster, W. R. Differential quantitative proteomic profiling of Leishmania infantum and Leishmania mexicana density gradient separated membranous fractions. J. Proteomics 2013, 82, 179−192.

M


Identification of Virulence Factors in Leishmania infantum Strains by a

Recommend Documents