• View in gallery

    Characterization of L6E9 cell infection by four T. cruzi strains. Giemsa staining of L6E9 cells after 72 hours of infection with (A) Y, (B) CL, (C) Brazil, and (D) Tulahuen strains of T. cruzi was performed. All strains studied successfully infected the myoblasts, and at 72 hours post infection (hpi), it was possible to observe amastigote forms in the host cell's cytoplasm as shown in details in A inset. (Bars = 50 μm.) To confirm genetic background of the parasite stocks used for infection, genomic DNA from epimastigote forms was isolated and amplified with Multiplex PCR with primers derived from a hypervariable region of the T. cruzi mini exon. (E) The Y, CL, and Tulahuen strains had a PCR product of 250 bp, indicating that they belong to the T. cruzi (TC) II family, and the Brazil strain had a 200-bp product, indicating that it belongs to TC I family.

  • View in gallery

    Profiles of transcriptomic changes caused by each strain of T. cruzi in host cells. The percentage of genes modulated in L6E9 cells by each T. cruzi strain is shown in A. Genes with fold-change value ³ 1.5 and P value ≤ 0.05 were quantified and plotted in this histogram, in which bars represent a percentage of the valid genes. Bars in white and black represent genes whose expression was decreased or increased exclusively by each strain, respectively. Bars in gray and light gray represent genes that were increased and decreased, respectively, and shared this modulation by at least two strains. The Venn diagrams represent the number of genes significantly (fold change ³ 1.5 and P value < 0.05) (B) increased and (C) decreased in common by all four strains, by three strains, by pairs of strains, and by individual strains of the parasite. The number of genes equally altered by CL + Y and Brazil + Tulahuen strains is indicated below the diagrams. Note the low number of genes altered in the same direction by all strains studied (19 increased and 2 decreased).

  • View in gallery

    Correlation of datasets obtained by microarray analyses of the infection of L6E9 cell line with Y, CL, Brazil, and Tulahuen strains of T. cruzi. The datasets of each strain were plotted against each other in pairs of parasite strains. The histograms show how the transcriptomic changes induced by all strains have a highly significant correlation (all have P values < 0.0001). R2 values were (A) 0.5, (B) 0.51, (C) 0.67, (D) 0.52, (E) 0.47, and (F) 0.53.

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Transcriptomic Signatures of Alterations in a Myoblast Cell Line Infected with Four Distinct Strains of Trypanosoma cruzi

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  • Laboratorio de Ultra-estrutura Celular, Instituto Oswaldo Cruz–Fiocruz, Rio de Janeiro, Brazil; Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York

We examined the extent to which different Trypanosoma cruzi strains induce transcriptomic changes in cultured L6E9 myoblasts 72 hours after infection with Brazil (TC I), Y (TC II), CL (TC II), and Tulahuen (TC II) strains. Expression of 6,289 distinct, fully annotated unigenes was quantified with 27,000 rat oligonucleotide arrays in each of the four replicas of all control and infected RNA samples. Considering changes greater than 1.5-fold and P values < 0.05, the Tulahuen strain was the most disruptive to host transcriptome (17% significantly altered genes), whereas the Y strain altered only 6% of the genes. The significantly altered genes in the infected cells were largely different among the strains, and only 21 genes were similarly changed by all four strains. However, myoblasts infected with different strains showed proportional overall gene-expression alterations. These results indicate that infection with different parasite strains modulates similar but not identical pathways in the host cells.

Introduction

Chagas disease, caused by infection with the flagellate protozoan parasite Trypanosoma cruzi, is a widespread disease in Latin America affecting millions of people.1 Infective trypomastigotes invade peripheral cells and transform into multiplicative amastigote forms. The initial (acute) phase of the disease is characterized by intense tissue parasitism involving the heart, skeletal and smooth muscle cells, liver, fat, and brain that is accompanied by intense focal inflammation and necrosis.2 Some patients can evolve to a chronic phase of the disease that can include cardiac and/or digestive forms. The severity of the chronic phase may be related to the efficiency of the host immune response in resolving the infection during the acute phase,3 but this has never been proven. Moreover, there are several reports of differences of tropism of T. cruzi to host tissue, which is also associated with the pathogenesis of chronic Chagas disease.4,5

Differences in the pathogenesis of the disease among patients may vary according to differences in both hosts and parasite strain.6 Among differences in T. cruzi strains are their resistance to chemotherapy, oxidative stress, and infectivity in the mouse.79 Although previous in vivo and in vitro microarray analyses using cultured cells10,11 and hearts of mice1214 infected with T. cruzi showed that this infection results in profound alterations in the host cells, the degree to which these results are applicable to all T. cruzi strains found in infected individuals has not been explored previously.

Since host immune response, tissue parasitism, and parasite strains may be important factors in the pathogenesis of chronic Chagas disease, we have used gene-array analysis to compare the alterations in host cells caused by four different stocks of T. cruzi. The present study characterizes the transcriptomic changes in cultured rat myoblasts that result from infection with each strain and highlights common genes that were similarly or differentially modulated by each strain. Analysis reveals host cell changes that might lead to an understanding of previously observed differences in pathogenesis in vivo.

Methods

Cells and parasites.

The L6E9 rat myoblast cell line was maintained in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin at 37°C with 5% CO2 atmosphere.15 Cells were dissociated with trypsin/ ethylenediaminetetracetic acid (EDTA) solution (Gibco), and 106 cells were plated in 100-mm2 cell-culture dishes. After 24 hours of plating, cells were washed with Phosphate Buffered Saline (PBS) containing Ca2+ and Mg2+ (Gibco) and infected with 2 × 106 trypomastigote forms of T. cruzi in DMEM. Parasites of the Y, CL Brener,16 Tulahuen, and Brazil strains were obtained from supernatants of infected L6E9 cultures. Forty-eight hours post-infection, cells were washed twice with Ca2+/Mg2+ PBS to remove free trypomastigotes in the supernatant, and they were re-fed with fresh supplemented DMEM. Total RNA was harvested at 72 hours post-infection using guanidinium thiocyanate-phenol-chloroform extraction (TRIZOL) reagent (Invitrogen, Carlsbad, CA), following the protocol indicated by the manufacturer, when at least 25% of the cultured cells were infected, presented only intracellular amastigotes, and had no release of trypomastigotes, which would lead to re-infection of culture.

T. cruzi genotyping.

The different isolates of T. cruzi used in this work were identified using the method described by Fernandes and others17 according to their phylogenetic lineage. Briefly, genomic DNA from 5 × 108 epimastigote forms of the Y, CL, Brazil, and Tulahuen strains was extracted using the DNeasy kit (Qiagen, Hilden, Germany). Multiplex polymerase chain reaction (PCR) was performed using 150 ng of DNA, and the primers were designed to recognize the mini-exon gene of the parasites using a pool of five nucleotides: three were derived from a hypervariable region of the T. cruzi mini-exon repeat (T. cruzi 1 [TC1], 5¢ ACA CTT TCT GTG GCG CTG ATC G; TC 2, 5¢ TTG CTC GCA CAC TCG GCT GCA T; TC 3, 5¢ CCG CGW ACA ACC CCT MAT AAA AAT G) and an oligonucleotide from a specific region of the Trypanosoma rangelii non-transcribed spacer (TR; 5¢ CCT ATT GTG ATC CCC ATC TTC G). A common downstream oligonucleotide corresponds to sequences present in the most conserved region of the mini-exon gene (ME; 5¢ TAC CAA TAT AGT ACA GAA ACT G). PCR reaction was performed using the Multiplex PCR kit (Qiagen) with the initial denaturing cycle of 95°C (15 seconds) followed by 30 cycles of 94°C (30 seconds, denaturing), 60°C (30 seconds, annealing), and 72°C (30 seconds, extension); a final extension cycle of 72°C lasts for 10 minutes and is followed by a soak cycle (4°C) using a PTC-100 Thermocycler (M.J. Research Inc., Massachusetts, (USA)). PCR fragments were loaded into a 2% agarose/ Tris base, boric acid, EDTA (TBE) gel with 0.008% ethidium bromide, and images were acquired using Kodak 1D Scientific Imaging Systems.

Light microscopy.

Cells (6 × 104) were plated into glass cover slips in 24-well plates. After 24 hours, 106 trypomastigotes were added to cultures in fresh supplemented DMEM, and infection was followed as described above. After 72 hours of infection, cells were washed three times and fixed with glutaraldehyde for Giemsa staining.18 Cover slips were digitally photographed using a Zeiss Axioplan microscope.

Microarray analysis.

We used the protocol optimized in our laboratory19 according to the standards of the Microarray Gene Expression Data Society. Briefly, 20 μg Trizol extracted total RNA from each culture dish was reverse transcribed in the presence of fluorescent Alexa Fluor 555-aha-dUTP (green fluorescent emission) or Alexa 647-aha-dUTP (red emission; Invitrogen) to label cDNAs. Differently labeled RNA samples from biological replicas of control (uninfected cells cultured for the same duration) or infected with one strain at a time were co-hybridized (“multiple yellow” strategy20) overnight at 50°C with rat 27k oligonucleotide arrays printed by Duke University (full technical information available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18175). All spots affected by local corruption, with saturated pixels, or with foreground fluorescence less than twice the background fluorescence (where noise may obscure the quantity) were removed from the analysis. The background subtracted signals were normalized iteratively,19 alternating red/green, interblock, lowess and scale intraslide and interslide normalization until the fluctuation of the ratio between the spot median and the corresponding block median of valid spots became less than 5% between successive iteration steps. Normalized expression levels were organized into redundancy groups (each group composed of all spots probing the same gene) and were represented by the weighted average of the values of individual spots. The abundance of host cell transcripts was considered as significantly altered after infection if the absolute fold-change was greater than 1.5-fold and the P value of the heteroscedastic t test (two-sample, unequal variance), applied to the means of the background-subtracted normalized fluorescence values in the four biological replicas of the compared transcriptomes, was greater than 0.05. This composite criterion to identify the significantly altered gene expression minimizes the number of false hits without eliminating too many true hits. The 50% change cut-off (1.5-fold) was selected to be significantly larger than the overall less than 10% interslide technical noise determined for the bacterial controls.

Gene categories.

GenMapp and MappFinder programs (www.genmapp.org; Gladstone Institute, University of California, San Francisco, CA) were used to determine whether or not altered gene expressions differed significantly from chance for the overlapping functional and structural Gene Ontology (GO) categories.

Results

Multiplex PCR was performed to confirm correspondence of the strains of T. cruzi used in this work to the phylogenetic classification as described in the literature. We verified that the Y, CL, and Tulahuen parasites displayed a 250-bp PCR product, indicating that they belong to the T. cruzi II (TC II) group. The Brazil strain, whose effects in host cells and animals have been characterized previously by our group,15,21 belongs to the TC I group, because it displays a 200-bp PCR product (Figure 1E).

Figure 1.
Figure 1.

Characterization of L6E9 cell infection by four T. cruzi strains. Giemsa staining of L6E9 cells after 72 hours of infection with (A) Y, (B) CL, (C) Brazil, and (D) Tulahuen strains of T. cruzi was performed. All strains studied successfully infected the myoblasts, and at 72 hours post infection (hpi), it was possible to observe amastigote forms in the host cell's cytoplasm as shown in details in A inset. (Bars = 50 μm.) To confirm genetic background of the parasite stocks used for infection, genomic DNA from epimastigote forms was isolated and amplified with Multiplex PCR with primers derived from a hypervariable region of the T. cruzi mini exon. (E) The Y, CL, and Tulahuen strains had a PCR product of 250 bp, indicating that they belong to the T. cruzi (TC) II family, and the Brazil strain had a 200-bp product, indicating that it belongs to TC I family.

Citation: The American Society of Tropical Medicine and Hygiene 82, 5; 10.4269/ajtmh.2010.09-0399

Data complying with the Minimum Information about Microarray Experiments (MIAME) were deposited in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18175) as series GSE18175. In this experiment, we quantified 6,289 distinct, well-annotated unigenes in all samples.

The impact of infection with each of the four strains of T. cruzi on the transcriptome changes of the same immortalized cell line was strikingly different (Figure 2). Thus, as shown in Table 5, there were only two (0.03%) genes significantly decreased, and 19 (0.3%) increased by all four T. cruzi strains. However, 4,340 (69%) genes were not significantly altered by any of the strains. The Venn diagrams in Figure 2 illustrate this observation by showing the number of genes equally increased (Figure 2B) or decreased (Figure 2C) by two, three, or all four strains of T. cruzi.

Figure 2.
Figure 2.

Profiles of transcriptomic changes caused by each strain of T. cruzi in host cells. The percentage of genes modulated in L6E9 cells by each T. cruzi strain is shown in A. Genes with fold-change value ³ 1.5 and P value ≤ 0.05 were quantified and plotted in this histogram, in which bars represent a percentage of the valid genes. Bars in white and black represent genes whose expression was decreased or increased exclusively by each strain, respectively. Bars in gray and light gray represent genes that were increased and decreased, respectively, and shared this modulation by at least two strains. The Venn diagrams represent the number of genes significantly (fold change ³ 1.5 and P value < 0.05) (B) increased and (C) decreased in common by all four strains, by three strains, by pairs of strains, and by individual strains of the parasite. The number of genes equally altered by CL + Y and Brazil + Tulahuen strains is indicated below the diagrams. Note the low number of genes altered in the same direction by all strains studied (19 increased and 2 decreased).

Citation: The American Society of Tropical Medicine and Hygiene 82, 5; 10.4269/ajtmh.2010.09-0399

Table 5

Similar results

Gene nameGene symbolFold change (strains)
BrazilCLTulahuenY
C1q and tumor necrosis factor-related protein 1C1qTNF1–1.90–1.80–2.70–1.80
Matrix metallopeptidase 14MMP14–2.00–1.90–2.00–1.70
Cardiotrophin-like cytokine factor 1Clcf12.202.301.802.30
Cut-like 1 (Drosophila)Cutl17.722.585.361.59
DNA-damage inducible transcript 3Ddit39.303.082.412.64
Excision repair cross-complementing rodent repair deficiency, complementation group 3Ercc32.192.671.592.36
G protein-coupled receptor, family C, group 5, member AGprc5a1.942.531.871.99
GrpE-like 1, mitochondrialGrpel11.601.951.661.91
Pericentriolar material 1Pcm12.704.252.233.70
Proprotein convertase subtilisin/kexin type 7Pcsk76.932.872.382.15
Serologically defined colon cancer antigen 3Sdccag33.362.712.672.50
Solute carrier family 1 (glutamate/neutral amino acid transporter)Slc1a41.592.501.821.68
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteinYwhaq2.842.851.772.56

Analysis of functional classes of genes modulated by the infection with different T. cruzi strains was performed with GenMapp software, and it revealed a very small number of gene functional categories with an abundance that was similarly affected by each pair of parasite strains (Table 7). Below, we describe in more detail the differences that were observed.

Table 7

Gene categories

Gene Ontology (GO) IDGO NameNo. changedNo. measuredNo. in GOPercent changedP value
Y strain
5634Nucleus225571,6433.950000.004
45449Regulation of transcription122648844.550000.010
6355Regulation of transcription, DNA-dependent112377904.640000.012
5509Calcium-ion binding81495385.370000.013
6350Transcription122789204.320000.014
46872Metal-ion binding205931,9133.370000.049
5743Mitochondrial inner membrane4226318.180000.006
19866Organelle inner membrane4297913.790000.026
16757Transferase activity, transferring glycosyl groups54814410.420000.042
43234Protein complex93961,2802.270000.052
16853Isomerase activity4347411.770000.052
CL strain
5829Cytosol4681215.880000.002
5622Intracellular181,3313,7661.350000.004
15031Protein transport51543483.250000.011
6512Ubiquitin cycle31022812.940000.049
3824Catalytic activity81,6115,0730.500000.054
6886Intracellular protein transport31032372.910000.054
16757Transferase activity, transferring glycosyl groups104814420.830000.011
15992Proton transport6246425.000000.023
4197Cysteine-type endopeptidase activity7349120.590000.032
6811Ion transport1811855515.250000.038
3924Gtpase activity5224522.730000.040
16491Oxidoreductase activity2518553913.510000.045
6812Cation transport148637516.280000.046
Brazil strain
8237Metallopeptidase activity3381337.890000.020
4984Olfactory receptor activity9241,03437.500000.000
1584Rhodopsin-like receptor activity9461,39119.570000.001
4872Receptor activity201952,01310.260000.002
7186G-protein–coupled receptor protein-signaling pathway11821,58113.410000.005
43234Protein complex123961,2803.030000.022
7264Small gtpase mediated signal transduction86617912.120000.027
5615Extracellular space42313617.390000.029
6512Ubiquitin cycle11022810.980000.046
5525GTP binding109923710.100000.050
Tulahuen strain
6457Protein folding166813823.530000.001
5488Binding2191,7865,70812.260000.010
3743Translation initiation factor activity7253728.000000.019
6695Cholesterol biosynthesis4111536.360000.021
6955Immune response104625821.739130.033
6629Lipid metabolism2012030416.670000.053
4984Olfactory receptor activity7241,03429.170000.000
1584Rhodopsin-like receptor activity9461,39119.570000.001
16020Membrane637403,7988.510000.003
19866Organelle inner membrane7297924.140000.003
5743Mitochondrial inner membrane5226322.730000.012
4872Receptor activity201952,01310.260000.014
16021Integral to membrane414942,8888.300000.018
5856Cytoskeleton1311433711.400000.021
7186G-protein–coupled receptor protein-signaling pathway10821,58112.200000.029
9058Biosynthesis92849233.170000.036
16874Ligase activity21243291.610000.046
5739Mitochondrion1514929010.070000.048

Y strain.

We observed that the Y strain altered expression of 426 (6%) of the 6,996 quantified host cell genes. Among these genes, 150 were decreased, and 276 were increased after 72 hours of infection. When we identified individual genes whose expression was altered exclusively by the Y strain, we found that 87 genes were increased, such as α-1-integrin (1.76-fold), intercellular adhesion molecule (1.79-fold), presenilin 2 (3.81-fold), transforming growth-factor beta regulated gene (1.73-fold), and vascular cell-adhesion molecule 1 (1.91-fold). Sixty-five genes were decreased, such as cardiac Ca2+ ATPase (−1.6-fold), cadherin (−2.88-fold), and Cyp26b1 (cytochrome P450, family 26, subfamily b, polypeptide 1; −2.83-fold) (Table 1). The Y strain affected the expression of 11 gene categories including calcium and metal binding, regulation of transcription, and complexes of protein (Table 7).

Table 1

Y Strain

Gene nameGene symbolFold change
Alanyl-tRNA synthetaseAars1.85
Actin, gamma, cytoplasmic 1Actg11.55
Chloride intracellular channel 2Clic21.55
Cyclin-dependent kinase inhibitor 2B (p15; inhibits CDK4)Cdkn2b1.65
Glycogen synthase kinase 3 betaGsk3b1.52
Integrin alpha 1Itga11.76
Intercellular adhesion molecule 1Icam11.79
Lectin, galactose binding, soluble 3Lgals31.52
Presenilin 2Psen23.814
Protein C receptor, endothelialProcr2.04
Pyruvate kinase, musclePkm21.70
Syndecan -proteinSdcbp1.854
Transforming growth-factor beta-regulated gene 1Tbrg11.73
Vascular cell-adhesion molecule 1Vcam11.91
X-linked myotubular myopathy gene 1Mtm11.53
Actin, alpha 1, skeletal muscleActa1–4.84
Acyl-CoA synthetase long-chain family member 3Acsl3–2.63
ATPase, Ca++ transporting, cardiac muscle, slow twitch 2Atp2a2–1.60
Cadherin 15Cdh15–2.88
Calsenilin, presenilin-binding protein, EF hand transcription factorCsen–1.62
Cytochrome P450, family 26, subfamily b, polypeptide 1Cyp26b1–2.83
Guanine monphosphate synthetaseGmps–1.94
Myosin light chain, phosphorylatable, fast skeletal muscleMylpf–1.68
Rho family GTPase 2Rnd2–1.62

CL strain.

When the rat myoblasts were infected with the CL strain, we observed that 763 of 7,133 (11%) genes were significantly altered (53 decreased and 710 increased). The CL strain uniquely modulated the expression of 517 genes (494 were increased and 23 decreased), which corresponded to alterations in 14 gene categories including protein, intracellular protein, proton and ion transport, and ubiquitin cycle (Table 7). Some of the genes that the CL strain modulated were adenylate cyclase 2 (1.56-fold), α-spectrin (1.7-fold), annexin A1 (1.7-fold), caspase 7 (1.76-fold), epsin 2 (1.97), glutamate receptor (3.38), matrix metallopeptidase (MMP) 3 (2.51-fold), and syntaxin (−1.56-fold) (Table 2).

Table 2

CL strain

Gene nameGene symbolFold change
Adenylate cyclase 2Adcy31.56
Alpha-spectrin 2Spna21.70
Angiopoietin-like 2AF1590491.56
Annexin A1Anxa11.56
Casein kinase 1, gamma 1Csnk1g11.95
Caspase 7Casp71.76
Chemokine-like factorCklf1.76
Chondroitin sulfate proteoglycan 6Cspg61.64
DesmuslinDmn1.60
Epsin 2Epn21.97
Glutamate receptor, ionotropic, N-methyl D-aspartate 2BGrin2b3.38
Integrin alpha 5Itga51.84
Interleukin 1 receptor, type IIl1r12.27
Matrix metallopeptidase 10Mmp102.40
Matrix metallopeptidase 3Mmp32.52
Phosphatidylethanolamine N-methyltransferasePemt2.03
Phospholipase D1Pld11.58
Plasminogen activator, urokinase receptorPlaur1.76
Proteasome (prosome, macropain) 26S subunit, non-ATPase, 2Psmd21.99
Chaperonin subunit 4 (delta)Cct4–1.57
Syntaxin 8Stx8–1.56
T-cell immunomodulatory proteinCda08–2.61

Brazil strain.

The Brazil strain induced significant alteration of 7.31% genes of L6E9 by more than 1.5-fold. Among these 494 modulated genes, 117 were decreased, and 377 were increased. Some interesting genes that had their transcription altered by infection with the Brazil strain were cardiomyopathy associated gene (3.37-fold), MMP 1B (2.44-fold), mitogen-activated protein kinase 9 (2.12-fold), fibroblast growth factor (1.94-fold), desmin (1.63-fold), and tropomyosin 4 (−2.45-fold) (Table 3). Ten functional categories of genes were highly modulated in L6E9 by Brazil strain, including metallopeptidase activity, small GTPase-mediated signal transduction, and ubiquitin cycle (Table 7).

Table 3

Brazil strain

Gene nameGene symbolFold change
H2A histone family, member YH2afy3.70
Cardiomyopathy associated 3Cmya33.37
Protein kinase C, gammaPrkcc2.70
Myocyte enhancer factor 2DMef2d2.43
Mitogen-activated protein kinase 9Mapk92.12
Fibroblast growth factor 21Fgf211.95
ATPase, H+/K+ exchanging, beta polypeptideAtp4b1.90
Phosphatidylinositol 3-kinase, C2 domain containing, gamma polypeptidePik3c2g1.78
PaladinPald1.67
DesminDes1.63
Phosphatidylinositol 3 kinase, regulatory subunit, polypeptide 3Pik3r3–1.53
Coronin, actin-binding protein, 1BCoro1b–1.91
Synaptotagmin XISyt11–1.91
Proteasome (prosome, macropain) 28 subunit, betaPsme2–2.35
Keratin 25DKrt25d–2.38
Tropomyosin 4Tpm4–2.46

Tulahuen strain.

The Tulahuen strain of T. cruzi induced the highest percentage of altered gene expression in the myoblasts (17.35%) with 761 decreased genes and 383 increased genes, of which, 617 and 139, respectively, were uniquely observed in Tulahuen-infected dishes. Additionally, infection with this strain significantly modulated the expression of 18 gene categories, including cholesterol biosynthesis, immune response, lipid metabolism, and receptor activity (Table 7). Some relevant examples of host cell genes significantly modulated during infection were p-cadherin (4.64-fold), cardiac ankyrin repeat kinase (2.54-fold), chymotrypsinogen B (2.64-fold), H2A histone family member Z (3.18-fold), interleukin 11 (1.68-fold), myotrophin (1.51-fold), caspase 9 (−1.75-fold), cytochrome c oxidase, subunit Va (−1.61-fold), heavy and light chain dynein (−1.86- and −1.81-fold, respectively), farnesyltransferase (−1.65-fold), hypoxia-inducible factor 1α subunit (−1.51-fold), janus kinase 3 (−1.64-fold), matrix metallopeptidase 11 (−3.46-fold), and cardiac troponin T2 (−2.89-fold) (Table 4).

Table 4

Tulahuen strain

Gene nameGene symbolFold change
Activin A receptor type II-like 1Acvrl17.60
Cadherin 3, type 1, P-cadherin (placental)Cdh34.65
Cardiac ankyrin repeat kinaseCark2.54
CDK5 regulatory subunit-associated protein 1Cdk5rap12.40
Chymotrypsinogen BCtrb2.64
Glucose 6 phosphatase, catalytic, 3G6pc31.65
Inositol 1,4,5-trisphosphate 3-kinase CItpkc2.37
Interleukin 11Il111.68
Kinesin family member C1Kifc12.04
MyotrophinMtpn1.51
ATPase, Ca++ transporting, plasma membrane 1Atp2b1–2.76
Bcl2 modifying factorBmf–2.93
Calcium channel, voltage-dependent, beta 3 subunitCacnb3–1.88
CalreticulinCalr–2.04
Caspase 9Casp9–1.75
Cytochrome c oxidase, subunit VaCox5a–1.62
Cytokine-induced apoptosis inhibitor 1Ciapin1–1.67
Dynein cytoplasmic 1 heavy chain 1Dync1h1–1.86
Ectonucleoside triphosphate diphosphohydrolase 1Entpd1–2.90
Farnesyltransferase, CAAX box, alphaFnta–1.66
Hypoxia inducible factor 1, alpha subunitHif1a–1.52
Inositol 1,4,5-triphosphate receptor 3Itpr3–3.70
Janus kinase 3Jak3–1.64
Junctional adhesion molecule 3Jam3–2.25
Laminin, beta 2Lamb2–2.21
Matrix metallopeptidase 11Mmp11–3.46
Muscle, skeletal, receptor tyrosine kinaseMusk–2.73
Phospholipase D2Pld2–2.88
Protein kinase C, nuPrkcn–2.57
Synaptojanin 2Synj2–2.35
Troponin T2, cardiacTnnt2–2.89
Tumor protein p53Tp53–1.92

Genes modulated equally by all T. cruzi strains as possible disease biomarkers.

We observed that 13 (0.18%) host cell genes had the same pattern of significant modulation by all four strains of T. cruzi studied. These genes, thus, may represent disease biomarkers that could be useful in detecting the disease independent of the parasite strain. Only two (0.027%) were decreased, MMP-14 (−1.9-fold) and C1q and tumor necrosis factor-related protein 1 (−2.0-fold), and 11 (0.15%) increased, such as solute carrier family 1 (glutamate/neutral amino acid transporter; 1.9-fold), G protein-coupled receptor (2.1-fold), cardiotrophin-like cytokine factor 1 (2.13-fold), pericentriolar material 1 (3.22-fold), and DNA damage-inducible transcript 3 (4.37-fold). Table 5 shows the list of these thirteen genes and their modulation by each strain of the parasite. GenMapp analysis revealed that only nine gene categories were equally expressed by some pairs of strains such as receptor activity (Brazil and Tulahuen) and ubiquitin cycle (Brazil and CL) (Table 7).

Oppositely modulated genes.

Surprisingly, our arrays revealed that some transcripts increased by one specific strain were decreased by another strain. Table 6 contains all the 24 genes that behaved in this manner. Some genes of interest were cytochrome P450, family 2, subfamily d, polypeptide 22 (3-fold by Brazil strain and −2.9-fold by Y strain), neuropathy target esterase-like 1 (2.1-fold by Y strain and −1.8-fold by CL strain), platelet-derived growth-factor receptor, β polypeptide (1.7-fold by CL strain and −1.6-fold by Tulahuen strain), protein kinase D2 (1.5-fold by Brazil strain and −1.6-fold by Y strain), and RNA polymerase 1-1 (1.6-fold by CL strain and −1.6-fold by Tulahuen strain).

Table 6

Opposite results

Gene nameGene symbolFold change (strains)
BrazilCLTulahuenY
ADP-ribosylation factor guanine nucleotide-exchange factor 2Arfgef21.56–1.54
CDC-like kinase 2LOC3658421.52–1.64
Cytochrome P450, family 2, subfamily d, polypeptide 22Cyp2d222.97–2.92
NAD synthetase 1Nadsyn11.56–1.63
Neuropathy target esterase-like 1Ntel1–1.782.11
Platelet-derived growth-factor receptor, beta polypeptidePdgfrb1.68–1.64
Pleckstrin homology, Sec7, and coiled-coil domains 1Pscd11.53–1.53
Preoptic regulatory factor-2PORF-21.62–1.56
Protein kinase D2Prkd21.50–1.62
Regenerating islet-derived 1Reg11.67–2.31
RNA polymerase 1-1Rpo1-11.56–1.56
Son of sevenless homolog 1Sos1–1.531.82
Sulfotransferase family 1A, phenol-preferring, member 1Sult1a11.90–1. 53
Thymoma viral proto-oncogene 2Akt21.54–1.64
Transducer of ErbB-2.1Tob11.58–1.72

Correlations between infections with four strains.

Our studies identifying individual genes that were significantly altered by the four strains of T. cruzi revealed a surprising diversity with only a few genes similarly changed by infection with all strains. However, because this analysis selects only individuals, it does not compare subtle global changes throughout the transcriptome. To compare global trancriptomic alteration patterns in L6E9 cells infected by the four strains used in the current investigation, we compared the entire gene-array datasets obtained from the infection with each strain against each of the other strains. With Origin software (OriginLab, Northampton, MA), we plotted results of these pairs as log2 values of their expression ratios. In all six of the comparisons of expression changes induced by infection with separate T. cruzi strains (Figure 3), the regression coefficients (r2 values) for these linear relations were highly significant, and P values in all cases were less than 0.0001. This finding indicates that although infection with each of the parasite strains leads to only partially overlapping alterations in the genes that are most affected, there is an overall similarity in the pattern of gene-expression alterations resulting from infection with all the strains.

Figure 3.
Figure 3.

Correlation of datasets obtained by microarray analyses of the infection of L6E9 cell line with Y, CL, Brazil, and Tulahuen strains of T. cruzi. The datasets of each strain were plotted against each other in pairs of parasite strains. The histograms show how the transcriptomic changes induced by all strains have a highly significant correlation (all have P values < 0.0001). R2 values were (A) 0.5, (B) 0.51, (C) 0.67, (D) 0.52, (E) 0.47, and (F) 0.53.

Citation: The American Society of Tropical Medicine and Hygiene 82, 5; 10.4269/ajtmh.2010.09-0399

Discussion

Chagas disease represents a spectrum of pathogenesis, varying both in its severity and the organ systems afflicted. Although various host factors, such as competence to launch immune response, may account in part for differences in the pathogenesis of the disease, parasite strain is also an important variable,6 resulting in differences in viability, infectivity, and tissue tropism.79 The present study was undertaken to evaluate the extent to which global gene-expression alteration was similarly altered after in vitro infection of a myoblast cell line with four distinct T. cruzi strains.

The microarray analyses described in this study were performed on the myoblast cell line L6E9 during infection with four reference strains of T. cruzi, each with well-characterized rates of in vivo and in vitro infectivity, resistance to chemotherapy, and pathogenesis in vivo. We used the Y and CL strains as representatives of the TC II group of T. cruzi, known to be found in central and eastern Brazil, which is commonly associated with the “mega” syndromes (cardiomegaly, megacolon, and megaesophagous).22 The Tulahuen strain was chosen to represent the TC I group, however, the Multiplex PCR performed with genomic DNA of parasites of this strain revealed that it was actually a strain belonging to the TC II family. The Brazil strain was selected, because it has been shown to cause a dilated cardiomyopathy associated with a reduction in fractional shortening and myocardial wall thinning in mice.13 Genotyping revealed that the Brazil strain belongs to the TC I group, the predominant group in Venezuela and central Brazil, which is usually associated with electrocardiographic (ECG) abnormalities.22

We have compared the datasets obtained from the microarray analyses in two ways, resulting in different but complementary conclusions regarding the pathogenesis of T. cruzi infection in vitro and perhaps, applicable to in vivo infection as well. First, through identification of the genes that had altered expression after infection, we identified a large number of gene expression changes in the infected cells, but only a very small number of these were common to infection with each parasite strain. We concluded from this analysis that each strain induces a particular fingerprint of pathology. One such fingerprint may include genes encoding cell-junction proteins, which was evidenced by our finding that both Tulahuen and Y strains down-regulated several cellular junction genes such as junction plakoglobin, junctional adhesion molecule 3, and adipocyte-specific adhesion molecule as well as cadherin 15 (Y strain). T. cruzi infection was also shown to alter adhesion molecules of the host such as connexin4323 and cadherin-catenin.11,24 These findings highlight an evolving concept that many types of cardiomyopathy target expression or involve mutations in molecular components of the intercalated disk (see reference 25 for review and reference 26 for changes in sepsis). Thus, as pointed out in a recent review,27 cardiomyopathies, including chronic chagasic cardiomyopathy, may be considered to be junctionopathies.

An additional conclusion from the small number of genes found to be commonly regulated in infections with all strains (e.g., cut-like 1, DNA-damage inducible transcript 3, proprotein convertase subtilisin/kexin type 7) may provide a subset of biomarkers that could be potentially useful for diagnosis of acute T. cruzi infection and possibly, also reliably determine both indeterminate phase and chronic disease.

The second type of analysis that we performed on the gene-expression datasets was to compare overall transcriptomic changes in myoblasts infected with each T. cruzi strain through regression analysis of relative expression levels of each gene. For this, we compared each of the six pair-wise combinations of parasite strains, in each case finding a highly significant positive slope. This analysis emphasizes the concept that although Chagas disease shows a spectrum of manifestations, it does indeed represent a syndrome of common phenotypic alterations. The shallow slopes of the regression lines indicate that the changes induced by the parasite strains, although similar, are on average very low in amplitude; this emphasizes again the potential utility of the few commonly altered biomarkers.

The significant alteration in few overlapping genes among myoblasts infected with all strains poses a formidable challenge to the development of disease biomarkers that can be used to detect disease endemic areas where endogenous infective strains differ, although the few genes that we have detected with strong and significant expression changes offer such a possibility. Nevertheless, the overall transcriptomic signature of the disease that is revealed in the strong correlations between subtle expression alteration of all genes may provide a source whereby additional biomarkers may be discovered as common principal components of the acute response.

Acknowledgments:

The authors thank Vicki L. Braunstein (AECOM) and Angela Santos (LUC–Fiocruz) for the technical support with maintenance of the T. cruzi strains and Nadia Nehme and Dr. Octavio Fernandes (Fiocruz) and Aisha Cordero (AECOM) for the assistance with T. cruzi genotyping. We also thank Ethan Mackenzie (AECOM) for the help with GenMapp analysis. D.A. was supported in part by a grant from the Fogarty International Center-NIH D43 W007129 (HBT) and grants from The US National Institutes of Health AI-076248(HBT), HL-73732(HBT, DCS) and from CNPq, CNPq, PAPES IV-FIOCRUZ.

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Author Notes

*Address correspondence to Daniel Adesse, Avenida Brasil 4365, Pavilhão Carlos Chagas, Rio de Janeiro, Rio de Janeiro, Brazil. E-mail: adesse@ioc.fiocruz.br

Authors' addresses: Daniel Adesse, Luciana Ribeiro Garzoni, and Maria de Nazareth Meirelles, Laboratório de Ultraestrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil. Dumitru A. Iacobas, Sanda Iacobas, and David C. Spray, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY. Herbert B. Tanowitz, Department of Pathology, Albert Einstein College of Medicine, Bronx, NY.

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