ABSTRACT: Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines

Argañaraz, Enrique Roberto

Barbosa, Augusto Mello Simões

Barros, Ana Maria

Nitz, Nadjar

Teixeira, Antonio Raimundo Lima Cruz

Memórias do Instituto Oswaldo Cruz

Integration of kDNA sequences within the genome of the host cell shown
by PCR amplification with primers to the conserved Trypanosoma cruzi
kDNA minicircle sequence was confirmed by Southern hybridization with
specific probes. The cells containing the integrated kDNA sequences
were then perpetuated as transfected macrophage subclonal lines. The
kDNA transfected macrophages expressed membrane antigens that were
recognized by antibodies in a panel of sera from ten patients with
chronic Chagas disease. These antigens barely expressed in the membrane
of uninfected, control macrophage clonal lines were recognized neither
by factors in the control, non-chagasic subjects nor in the chagasic
sera. This finding suggests the presence of an autoimmune antibody in
the chagasic sera that recognizes auto-antigens in the membrane of T.
cruzi kDNA transfected macrophage subclonal lines

Augusto Simões-Barbosa

Ana Maria Barros

Nadjar Nitz

Enrique R. Argañaraz

Antonio R. L. Teixeira

TSpace Repository

Integration of Trypanosoma cruzi kDNA Minicircle Sequence in the Host Genome May Be Associated with Autoimmune Serum Factors in Chagas Disease Patients

Repositório Institucional da Universidade de Brasília

249Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I: 249-252, 1999Integration of Trypanosoma cruzi kDNA MinicircleSequence in the Host Genome May Be Associated withAutoimmune Serum Factors in Chagas Disease PatientsAugusto Simıes-Barbosa, Ana Maria Barros, Nadjar Nitz, Enrique RArgaæaraz, Antonio RL Teixeira+Laboratório Multidisciplinar de Pesquisa em Doença de Chagas, Universidade de Brasília, Caixa Postal 04536,70910-900 Brasília, DF, BrasilIntegration of kDNA sequences within the genome of the host cell shown by PCR amplification withprimers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southernhybridization with specific probes. The cells containing the integrated kDNA sequences were then per-petuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressedmembrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronicChagas disease. These antigens barely expressed in the membrane of uninfected, control macrophageclonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasicsera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizesauto-antigens in the membrane of  T. cruzi kDNA transfected macrophage subclonal lines.Key words: horizontal gene transfer - Trypanosoma cruzi kDNA - Chagas disease - autoimmunityChagas disease is a chronic, consumptive ail-ment of protean manifestations affecting the heart,digestive tract, peripheral and central nervous sys-tems, that may be seen in susceptible hosts infectedwith Trypanosoma cruzi. The acute human infec-tion usually subsides spontaneously. However,chronic Chagas disease is a major public healthproblem because it produces high rates of morbid-ity and mortality.In the absence of correlation between the pres-ence of the parasite and the chronic myocardiallesions, a hallmark of Chagas disease, several au-thors have shown that autoimmunity plays a majorrole in the pathogenesis of the disease (Teixeira1987, Bestetti & Rossi 1997). Policlonal activa-tion of lymphocytes in the acute infection(Minoprio et al. 1986), the existing shared epitopesbetween the parasite and the host heart and neu-ronal tissues (Rizzo et al. 1989, Van Voorkis etal.1991, Cunha-Neto et al. 1995), and the rejec-tion of myocardial cells from normal recipient micethrough immunoreactive isogenic CD4+ and CD8+T cells have suggested autoimmunity plays an im-portant role in the pathogenesis of Chagas disease(Ribeiro-dos-Santos et al. 1992, Higushi et al.1993). However, the origin of immune mechanismsassociated with tissue lesions and clinical mani-festations of the disease is unknown.We have observed that T. cruzi infected rabbitstreated with anti-trypanosomal benznidazole con-tinue to display progressive myocarditis, that is tosay, ablation of the parasitemia did not halt pro-gression of the disease (Teixeira et al. 1990). Thisunderstanding has allowed us to hypothesize thatthe intracellular dividing amastigote form of theparasite may integrate its DNA in the genome ofthe host cell (Teixeira et al. 1991, 1994). Such anintegration of exogenous DNA into the genome ofthe host cell may, as a consequence, produce modi-fication of gene expression. Here we describe re-sults of experiments aimed at the possibility of dis-closing the origin of of autoimmune factors thatcould be associated with wide-ranging manifesta-tions of Chagas disease.MATERIALS AND METHODSCloning transfected macrophages P388D1-IL1- Murine macrophages P388D1-IL1 were infectedwith the Berenice stock trypomastigote forms ofT. cruzi at a 5:1 ratio. The cultures were treatedwith three weekly doses of benznidazole (100 µg/ml). Eradication of the infection was confirmed bythe absence of parasites in the culture medium, andafter successive passages of aliquots of culturestreated in LIT medium and in weanling mice. The+Corresponding author. Fax: +55-61-273.4645. E-mail:limacruz@uol.com.brReceived 9 June 1999Accepted 9 August 1999250 T. cruzi kDNA Minicircle  Augusto Simıes-Barbosa et al.infected-treated macrophages were subjected tolimited dilution cloning on top of a feeder-layer ofmurine peritoneal macrophages disseminated intoeach well of a 96-well plate. Single colony growthwas transferred to the well of a 24-well plate andthen to 25 cm2 culture flasks without feeder-layer.Each colony growth was subjected to PCR analy-sis. Clonal lines yielding expected PCR amplifica-tion products of kDNA were selected for two fur-ther rounds of subcloning. This cloning procedureensured that each kDNA transfected macrophagesubclonal line derived from a single cell. The sameprocedure was used to obtain uninfected macroph-age DNA clonal lines.Polymerase chain reaction (PCR) - PCRs wereperformed with Taq DNA polymerase (PharmaciaBiotech) following to the manufacturer’srecomendation. Template DNAs consisted of celllysates (Innis et al. 1990) or genomic DNA from:(i) infected-treated macrophages, (ii) uninfectedcontrol macrophage and (iii) T. cruzi (negative andpositive controls, respectively). Specific primerswere used (SK35/36 and SK34/67) for the con-served region of kDNA (Sturm et al. 1989) and forhighly repetitive sequences (PON1/2 and Tcz1/2)of nuclear DNA of T. cruzi (Moser et al. 1989,Requena et al. 1992).Extraction of DNA and Southern hybridizations- DNA was extracted from uninfected control andfrom transfected macrophage clonal lines. South-ern hybridizations were performed with 20 µg ofDNA digested with restriction enzymes, follow-ing the manufacturer’s recommendation, and thensubmitted to 0.6% agarose gel electroforesis withTBE 1X buffer. The DNA was transferred to apositive charged nylon membrane by capillarity.A kDNA minicircle probe radiolabeled by a ran-dom primer was used in the hybridization assaysin Denhardt’s solution, as described (Sambrook etal.  1989). The membrane was then washed twiceat a maximum estringency of 0.2X SSC, 0.1% SDS,at 65 oC for 30 min, and subjected to autoradiog-raphy.Immunofluorescence assays - Five kDNA-transfected clonal lines (A-to-E) and threeuninfected, control macrophage clonal lines (F,Gand H) were subjected to immunofluorescence as-says to detect cell membrane auto-antigens, throughrecognition by antibodies in chagasic sera. The cellscollected in the exponential growth phase werewashed three times in PBS, pH 7.4. They weretested with a panel of sera from ten patients withchronic Chagas disease, and from ten non-chagasic,control individuals. The panel of chagasic serashowed high anti-T. cruzi antibody titers (1:720 andabove) in ELISA, indirect immunofluorescenceand hemagglutination assays.The cells were fixed in 3.7% paraformaldehydefor 10 min, and permeabilized with 0.5% TritonX-100 in PBS for 15 min at room temperature. Aftertwo washings in PBS, the cells were dispensed ontoglass slides, air-dried and stored at -20°C till use.The immunologic assay consisted of incubation ofaliquots of normal, control macrophage clonallines, and of transfected macrophage clonal lines,fixed onto glass slides, with serial dilutions of non-chagasic and chagasic sera, for 45 min in a cham-ber with humid atmosphere. The cells that werewashed three times in PBS and treated with a 1:160dilution of fluoresceinated rabbit anti-heavy chainhuman IgG, were incubated for 30 min under thesame conditions. The slides were mounted withcoverslips on top of  pH 9.0 buffered  glycerin andobserved under an Axiophot Carl Zeiss fluores-cence microscope with 450-490 excitation and, LP520 barrier filters.Two-dimensional electrophoresis of membraneproteins - Cultured cells were collected by cen-trifugation at 2,000 g for 15 min at 4oC and washedtwice with PBS, pH 7.5. The cell pellet ressus-pended in 3 ml of buffer A (TrisHcl 0,1 M, pH7.5;sacarose 0,25 M, Triton X-100 0.5%, EDTA 20µM, TLCK 20 µM, E-64 20 µM, RAMC 10 µM,PMSF 8 mM) was ground in a power driven tissuehomogeneizer (Tri-R Instruments, NY) with teflonpestle and glass tube adapted to 0.11 to 0.15 mmclearance.  Rupture of the cells was achieved afterfive cycles of stirring at 12,000 rpm for 15 sec,followed by a 5-min rest period, at 4oC. This cellhomogenate was centrifugation at 10,000g for 15min at 4oC. The supernatant was then ultracentri-fuged at 100,000 g for 2 hr to obtain the cell mem-brane proteins. Protein determination showed anaverage of 1300-to-1800 µg/ml. The cell pellet wasressuspended in a solution containing urea 9 M,triton X-100 2% (v/v), DTT 130 mM, PMSF 8 mM,and 2% (v/v) Pharmalyte, pH 3-10.Two-dimensional electrophoresis was per-formed with immobiline, 11 cm drystrip, linear pH3-10, for isoelectric focusing and further separa-tion in ExcelGel SDS gradient (8-to-18%, v/v) fol-lowing the manufacturer’s recommendations(Pharmacia Biotech). A total of 250 µg of mem-brane protein from control (non-transfected) andfrom transfected macrophage clonal lines wasloaded into the gel. Protein spots were detected bysilver staining performed, according to themanufacturer’s protocol.RESULTSThe transfected macrophage clonal lines (A-to-E) template DNA yielded PCR amplificationproducts of expected size when the kDNA prim-ers were used. These template DNA did not yield251Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I, 1999amplification products of expected size when theprimers of  T. cruzi nuclear DNA were used. Thesefindings confirmed the absence of residual T. cruziinfection in the kDNA transfected macrophageclonal lines. The uninfected, control macrophageclonal lines (F, G and H) template DNA did notyield PCR amplification products with either setsof T. cruzi kDNA primers.Southern blot analyses of DNA from trans-fected macrophage clonal lines showed variouspatterns of bands formed by hybridization with theminicircle kDNA probe. The different patterns ofbands formed, varying from 0.8 to 7.4 kb, depend-ing on the restriction enzyme used, suggest thatthe sequences of kDNA minicircles are integratedin the host cell DNA (Fig. 1). None of these bandswere formed when control uninfected macrophagesDNA was hybridized with kDNA minicircle probe.The immunofluorescence assays showed trans-fected macrophage clonal lines membrane antigensto be reactive with antibodies in a panel of tenchagasic sera diluted at 1:200 and below. The pat-Fig. 1: Southern hybridization of EcoRI and of XhoI digests ofDNA from transfected macrophage and from control macroph-age clonal lines with a 32P-dATP kDNA probe. Lanes 1 and 2,EcoRI and of XhoI digests of transfected macrophage DNAshowing 3.4 and 7.0 kb bands, respectively. Notice absence ofbands formed when digests of DNA from control macrophagewere used (Lanes 3 and 4).tern of positive fluorescence that was seen in thefixed and permeabilized cell membrane of the trans-fected macrophage clonal lines A-to-E (Fig. 2) wasnot seen in the uninfected control macrophageclonal lines F, G and H. Furthermore, differencesof membrane protein expression were observed bycomparing the pattern of the two-dimensionalelectrophoresis gel from the uninfected control,and from the transfected macrophage clonal lines,as shown in Fig. 3.Fig. 2: immunofluorescence staining of membrane proteins ofa transfected macrophage clonal line with antibodies in chronicChagas disease patient serum.Fig. 3: two-dimensional electrophoresis of membrane proteins. A and B: spots formed when transfected macrophage  proteinsmigrated at pH 3.0-to-4.5 and 8.0-to-9.5. Notice that proteins spots within circles are not present in C and D, when membraneproteins from control macrophages migrated at same ranges of pH.pH  3.0                        4.5            8.0                       9.536.5 kDa 84 kDaA BC DDISCUSSIONHorizontal transfer of sequences of kDNAminicircle from T. cruzi to the genome of P388D1-IL1 macrophages was obtained by natural infec-tion. The cells transfected with the kDNA parasitewere cloned and the subclonal lines retained theexogenous DNA after 38 months of twice-weeklyserial passages. The kDNA transfected clonal linesremained parasite-free, as demonstrated by PCRamplification products with the T. cruzi kDNAprimers, and absence of amplification productswhen the parasite nuclear DNA primers were used.In order to determine the identity of the inserted1       2         3        4Kb7.03.4252 T. cruzi kDNA Minicircle  Augusto Simıes-Barbosa et al.DNA, we performed Southern blot analyses fromboth uninfected and infected (but parasite-free)macrophage clonal lines. The covalent associationof kDNA with host DNA was confirmed in EcoRIand XhoI digests showing a number of bands ofdifferent sizes over 1.4 kb, hybridized with kDNAminicircles. None of these bands were found inDNA purified from cell-free preparations of theparasite. The different band sizes may representinsertion fragments between parasite and host cellDNA. These results strongly suggest that sequencesof minicircles of kDNA parasite integrated into thehost cell by natural infection.The possibility that T. cruzi kDNA integrationinto the genome of macrophage clonal lines wouldlead to alteration of the host cell expression wassuggested by the immunofluorescence assays.These experiments showed auto-reactive antibod-ies in the chagasic patients’ sera, staining the mem-brane of the transfected macrophage clonal lines.Furthermore, the membrane of the uninfected, con-trol macrophage clonal lines did not show auto-reactive antibody staining. The analysis of two-dimensional electrophoresis gel showed proteinspots formed with the membrane lysates from trans-fected macrophage clonal lines, which were notpresent in the uninfected control macrophage. At-tempts to elute differentially expressed proteins inthe gel resulting from lysate of the transfected cellline yielded minunte amounts only and, therefore,did not permit sequencing.These data suggest that the kDNA integrationevent may alter gene expression of the host cells.We believe that genes differentially expressed inthe transfected macrophage, which are not triggeredin the uninfected control macrophage clonal lines,will be identified by differential display of  mRNA.Further studies will be undertaken to determinewhose proteins are up or down regulated. The iden-tification of gene products that are differentiallyexpressed in the kDNA transfected host cells maysuggest some of the important autoantigens asso-ciated with the origin of autoimmunity that has beendescribed in human and experimental Chagas dis-ease.REFERENCESBestetti RB, Rossi MA 1997. A rational approach formortality risk stratification in Chagas heart disease.Intern J Cardiol 58: 199-209.Cunha-Neto E, Duranti M, Gruber A, Zingales B, DeMessias I, Stolf N, Bellotti G, Patarroyo ME, PilleggiF, Kalil J 1995. Autoimmunity in Chagas diseasecardiopathy: Biological relevance of a cardiac myo-sin specific epitope crossreactive to animmunodominant Trypanosoma cruzi antigen. ProcNatl Acad Sci USA 92: 3541-3545.Higushi M de L, Gutierrez PS, Aiello VD, Palomino S,Bocchi E, Kalil J, Belloti G, Pileggi F 1993. Immu-nohistochemical characterization of infiltrating cellin human chronic chagasic myocarditis: comparisionwith myocardial rejection process. Virchows Arch APathol Anat Histopathol 423: 157-160.Innis MA, Gelfand DH, Sninsky JJ, White TJ 1990. PCRProtocols, A Guide to Methods and Applications,Chapter 18,  p.146-152. Academic Press, New York.Minoprio P, Eisen H, Fomi L,  D’Imperio Lima MR,Joskowicz M,  Coutinho A 1986. Polyclonal lym-phocyte response to murine T. cruzi infection I.Quantitation of both T and B-cell responses. ScandJ Immunol 24: 661-668.Moser DR, Kirchhoff LV, Donelson JE 1989. Detectionof Trypanosoma cruzi by DNA amplification usingthe polymerase chain reaction. J Clin Microbiol 27:1477-1482.Requena JM, Jimenez-Ruiz A,  Soto RM, Lopez MC,Alonso C 1992. Characterization of a highly repeatedinterspersed DNA sequence of Trypanosoma cruzi:its potential use in diagnosis and strain classifica-tion. Mol Biochem Parasitol 51: 271-280.Ribeiro-dos-Santos R, Rossi MA, Laus JL, Silva JS,Savino W, Mengel J 1992. Anti CD-4 abrogates re-jection and reestablishes long-term tolerance tosyngenic newborn hearts grafted in mice chronicallyinfected with Trypanosoma cruzi. J Exp Med 175:29-39.Rizzo LV, Cunha-Neto E, Teixeira ARL 1989. Autoim-munity in Chagas disease: specific inhibition of re-activity of CD4+T cells against myosin in micechronically infected with Trypanosoma cruzi. InfectImmun 57: 2640-2644.Sambrook J, Fritsh EF, Maniatis T 1989. MolecularCloning. A Laboratory Manual, Cold Spring Har-bor, New York.Sturm NR, Degrave W, Morel C, Simpson L 1989. Sen-sitive detection and schizodeme classification ofTrypanosoma cruzi cells by amplification of kineto-plast minicircle DNA sequences: use in  diagnosisof Chagas disease. Mol Biochem Parasitol 33: 205-214.Teixeira ARL 1987. Doença de Chagas e OutrasDoenças por Tripanossomos, Universidade deBrasília, Brasília, 161 pp.Teixeira ARL, Argañaraz ER, Freitas Jr LM, LacavaZGM, Santana JM, Luna H 1994. Possible integra-tion of  Trypanosoma cruzi  kDNA  minicircles intothe host cell genome by infections. Mut Res 305:197-209.Teixeira ARL, Cordoba JC, Souto-Maior Solorzano E1990. Chagas disease: lymphoma growth in rabbitstreated with benznidozole. Am J Trop Med Hyg 43:146-158.Teixeira ARL, Lacava ZL, Santana JM, Luna H 1991.Inserção de DNA de Trypanosoma cruzi no genomada célula hospedeira de mamíferos por meio deinfecção. Rev Soc Bras Med Trop 24: 55-58.Van Voorkis WC, Schlekewy L, Trong HL 1991. Mo-lecular mimicry by Trypanosoma cruzi: the F1-160epitope that mimics mammalian nerve  can bemapped to a 12-amino acid peptide. Proc Natl AcadSci USA 88: 5993-5997.

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in chagas disease patients

University of Toronto Research Repository

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines

Simões-Barbosa, Augusto

Argañaraz, Enrique R

Teixeira, Antonio RL

Scientific Electronic Library Online - Brazil

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

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Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in chagas disease patients

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