versão On-line ISSN 0717-7712
Parasitol. latinoam. v.63 n.1-2-3-4 Santiago dez. 2008
Parasitol Latinoam 63: 4 -11, 2008 FLAP
Partial purification, immunogenicity and putative new localization of a native Leishmania heat shock protein 70
ELIETE CAVALCANTI DA SILVA*, CYNTHIA D. RAYOL*, PATRICIA M. M. F. MOURA** and PAULO P. ANDRADE***
* Departamento de Histología e Embriología, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recife 50670-901, Brasil.
** Departamento de Parasitología, Instituto de Ciências Biológicas, Universidade de Pernambuco, Recife 50100-010, Brasil.
*** Departamento de Genética, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recife 50670-901, Brasil.
In this work we focused on a recombinant protein, containing approximately 230 aminoacids from the carboxy-terminal extremity of the Leishmania chagasi heat shock protein 70. The heat shock proteins are among the most abundant parasite antigens and conserved proteins in nature, and this family is one of the most immunogenic proteins present within pathogenic organisms. The recombinant protein has been partially purified by electroelution and further precipitation in acetone. The electroelution process did not modify its immunological and antigenic properties, as it continued to be recognized by visceral leishmaniasis positive sera and by the immunological system of rabbits during the immunization, both in ELISA and Western blots. The production of polyclonal sera with an antigen concentration that is far from the maximum dose, strengthens the idea that the proteins of this family are highly antigenic and immunogenic. Our results with these polyclonal sera in the Direct Agglutination Assay allow the conclusion that the Leishmania chagasi native heat shock protein 70 is distributed on the surface of the parasite.
Key words: Leishmania, heat shock proteins, immunogenicity, direct agglutination assay, DAT.
Neste trabalho estudamos urna proteína recombinante (S7) contendo aproximadamente 230 aminoácidos da extremidade carboxi-terminal da proteína de choque térmico de 70 kDa (HSP70) de Leishmania chagasi. As proteínas de choque térmico estâo entre os antígenos parasitarios mais abundantes e mais conservados na natureza. Esta familia pertence a urna das classes de proteínas mais imunogênicas, presentes em organismos patogênicos. Aproteína S7 foi parcialmente purificada por eletroeluição, e em seguida precipitada em acetona. A eletroeluição não modificou suas propriedades imunológicas e antigênicas, pois a proteína continuou a ser reconhecida (tanto no ELISA como no Western blot) por soros positivos para leishmaniose visceral e pelo sistema imunológico de coelhos durante a imunização. Aproducção de soros policlonais com urna concentração antigênica muito inferior a dose máxima, reforca a idéia de que as proteínas desta familia sâo altamente antigênicas e imunogénicas. Nossos resultados com os soros policlonais no ensaio de aglutinação direta (DAT) permitem concluir que a HSP70 nativa de L. chagasi está presente na superficie do parásita.
Leishmania chagasi is the etiological agent of visceral leishmaniasis (VL) in the Americas and other Leishmania species are responsible for the visceral form of the disease in the Old World. The Leishmania are dimorphic organisms that spend part of their life cycle in the alimentary tract of a sandfly vector as flagellated promastigotes and the remainder as obligate intracellular, nonflagellated amastigotes within macrophages in mammalian host1. These changes in the parasite's environment can be responsible for cellular stress and were indeed shown to induce a heat shock response with increased synthesis of a group of proteins known as heat shock proteins (HSPs)2.
The HSPs are among the most abundant parasite antigens and conserved proteins in nature. They can be found in eukaryotic and prokaryotic cells and an increased expression may be observed under stress conditions, such as exposure to high temperatures, chemical agents, oxidative injury, and inflammation3.
HSPs have been classified into families according to their molecular weights. The HSP70 family is one of the most abundant and immunogenic proteins present within pathogenic organisms46. However, HSPs also serve important physiological functions in normal cellular metabolism and the importance of many HSPs is related to their capacity to associate with other proteins in a way that modifies the destiny and function of the latter7. HSP70 is expressed constitutively throughout the life cycle oí Leishmania. However, the protein expression is induced by environmental changes that are inflicted by the switch of host, including alterations in temperature and extracellular pH8. Several authors have described that the members of the HSP70 family are prominent antigens during Leishmania infections9"11. By the other hand, these proteins have been localized intracellularly (e.g. mitochondria, endoplasmic reticulum, cytosol) and surface expression has been observed in human tumor cells1214. Given the strong immune response against HSP70 showed by VL patient15 and the high antibody titers these patients exhibit in a direct agglutination assay (DAT)16 using intact Leishmania promastigotes as antigens, we decided (a) to perform a partial purification of the S7 protein, a 45 kDa recombinant protein (rp) of L. chagasi, corresponding to the C-terminal moiety of HSP70, (b) to study this rp both as antigen and immunogen, and (c) to investigate its putative surface localization in Leishmania using the DAT.
Bacterial strains and culture conditions: Cultures of transformed Escherichia coli XL1-Blue and DH5oc15) containing the plasmid coding for the S7 rp were grown in Luria-Bertani medium supplemented with ampicillin, at 37 °C under agitation. The cultures were used to prepare a total lysate of the transformed bacteria (through sonication with a Thornton Tech sonicator), with a protein concentration of 2 mg/ml, as determined by the Lowry method. Cultures and lysates of wild E. coli DH5a were similarly prepared to be used as controls.
Electrophoresis and electroelution: Four preparative 12% SDS-polyacrylamide gels17 were loaded with the total lysate of the recombinant E. coli DH5a protein concentration of 2 mg/ml). The rp bands were cut out from the gel and electroeluted overnight at 250V in TPJS-glycine buffer containing 0.02% SDS in an electroelution chamber (Biotrap, Schleicher & Schuell). After electroelution, the solution containing the S7 rp was dialyzed against water, precipitated with cold acetone, incubated with 70% ethanol at -70°C for 30 min, centrifugated at 14000 rpm at 4 °C for 15 min, and ressuspended in PBS, pH 7.2, 1/10 of the initial volume. The purity of the partially purified rp was checked on analytical 12% SDS-polyacrylamide gels.
Rabbit immunization: Four NZW female rabbits (obtained at the animal house of the Rural Federal University of Pernambuco, Recife, Brazil) were immunized with 300 |j,g of the S7 rp by using the following schedule: the animals received 3 subcutaneous injections of 100 |j,g of the S7 protein, the first on day 0, the second on the 8th and the third on the 24th day, together with 1000 |LLl of complete Freund adjuvant (Sigma). The serum samples were collected before each immunization. Reactivity of the rabbit serum with the rp was assessed by immunoblot and ELISA with bound antibody detected using protein-A peroxidase (Sigma). Nine weeks after the first immunization, the rabbits were anesthesized and the blood collected by ear puncture.
ELISA: To perform the ELISA test, the partially purified S7 rp, at a concentration of 10 |j,g/ml in carbonate buffer, was used to coat the plates overnight at 4 °C. The plates were then washed in PBS + 0.05% Tween 20 (Sigma), quenched with 2% skeemed milk, washed again in PBS/Tween 20 and incubated for 1 h at 37 °C with immunized rabbit serum diluted at 1:100. After washing, protein A-peroxidase conjugate (Sigma) was added (1:2000 dilution) and incubated for lh at 37 °C. Orthophenylenediamine dihydrochloride (OPD) (Sigma) was added as cromogen and maintained in the dark at room temperature for 30 min. The plate was read at 405 nm in an ELISA reader.
Western blot: The total lysate of the S7 transformed DH5a was separated by SDS-PAGE and transferred to a nitrocellulose membrane18. Nitrocellulose membranes (Schleicher & Schuell) were quenched with PBS + 0.05% Tween 20 containing 2% skeemed milk and incubated overnight at 4 °C. Anti-S7 rabbit serum samples, diluted 1:200,1:400 and 1:1000 in PBS, containing wild E. coli DH5a lysate, were incubated for 30 min at 37 °C. These samples were then incubated with the above mentioned nitrocellulose membranes for lh at room temperature. Immunodetection was performed with protein A-peroxidase (Sigma) 1:5000 diluted in PBS. Proteins were revealed by a solution containing 30% H202, PBS citrate and tetra-methylbenzydine. The reaction was interrupted by washing in running water.
Direct Agglutination Test (DAT): The DAT antigen was prepared using a suspension of L. donovani promastigotes (S-l strain), treated with trypsin, fixed in formaldehyde and then stained with Coomassie brilliant blue (Sigma). The DAT assay was performed with serum samples from the rabbits immunized with the S7 rp, essentially as previously described16. All DAT titers were expressed as a serial dilution (1:25 - 1:800). The rabbit serum samples were diluted in physiological saline containing 1% BSA, and 50 |j,l dispensed in each microtitre well. Two assays were performed, one with addition of P-mer-captoethanol (PME) in the serum dilution and another without P-ME. In each case, serum samples taken from the rabbits prior to the immunization were used as negative controls. The serum samples were diluted in duplicate. 50 |LLl of the parasites' suspension were then added. For each microtiter plate, an additional internal control of the DAT suspension was also added, whereby 50 |j,l of the parasites suspension was incubated only with serum diluent. The microtitre plates were incubated for 16 hours at room temperature. The test was read visually against a white background. The end point titer was taken as the last well where agglutination was seen, considered as the well immediately before the well with a clear, sharp-edged, blue spot identical to the one observed in the negative control well. The DAT titers of the human serum samples were obtained essentially as described elsewhere16.
Immunoadsorption of human serum samples: 60?1 each of total lysates of wild and S7 transformed E.coli DH5a (protein concentration 2 mg/ml) were used to impregnate small nitrocellulose membrane discs (Schleicher & Schuell), which were then transferred do 24-well plates, quenched with PBS + 0.05% Tween 20 containing 2% skeemed milk and incubated overnight at 4 °C. Two positive and 2 negative human serum samples for VL were diluted 1:100,
added to the nitrocellulose discs and incubated at 4° C for 18 hours. After this treatment, these serum samples have been tested with the DAT (serial dilutions were up to 1:409,600).
Partial purification of the S7 rp: The S7 rp was partially purified from the total lysate of the recombinant E.coli DH5a using thick (3 mm) 12% preparative SDS polyacrylamide gels. The rp was localized in the void area of the E. coli DH5a, with a molecular weight of 45 kDa. This void area, containing only discrete natural protein bands, greatly facilitated the electroelution (Figure 1). Starting from a total protein concentration in the total lysate of 2 mg/ml, the electroelution process yield a mean concentration of 0.25 mg/ ml. After acetone precipitation, either the volume of the solution and the protein concentration diminished markedly, yielding a final concentration of 0.5 mg/ml. The partially purified S7 protein was then used in the immunization of rabbits and in an ELISA, in order to identify the humoral immune response against it.
Rabbit immunization: The rabbit humoral immune response against S7 was evaluated either by ELISA (using the electroeluted protein), or by Western blotting the total antigens of the transformed E.coli DH5a. In ELISA (Figure 2), pre-immune (day 0) rabbit serum samples showed no reactivity against the S7 protein. It was also clearly demonstrated that immunization against total antigens of the wild E.coli DH5a does not elicit the production of anti-S7 antibodies, even after boosting. The three animals immunized against S7 showed a humoral response consistent with the schedule: we observed a gradual elevation in the concentration of specific antibodies, specially after the second injection. After 8 weeks, the animals showed an OD > 0.5.
In the Western blot (Figure 3), the anti-S7 serum samples from the three rabbits, containing wild E. coli DH5a lysate, recognized an unique 45 kDa band, compatible with the S7 rp. The negative control serum did not recognize any band.
Antibody recognition: Serially diluted (1:25 -1:800) hiperimmune rabbit sera were used in the DAT, against a suspension of L. donovani promastigotes (Table 1). The control serum was unreactive and the 3 hiperimmune samples showed titers >= 1:400.
The DAT results of the immunoadsorbed positive and negative human VL serum samples showed no difference when compared with the titres prior to the capture of the anti-S7 antibodies. This suggests that the anti-leishmanial HSP70 antibodies are not implicated in the agglutination of the parasites seen in the DAT, in spite of the high titers observed in patients with VL.
HSPs are highly produced by organisms under several stress conditions, as for example when exposed to temperatures above the optimum for growth19. This work has focused on a rp (S7), containing approximately 230 aminoacids from the C-terminal extremity of L. chagasi HSP70. In L. chagasi, the HSP70 genes constitute a multigenic family, with alíeles differentiating either by small variations in the sequence or by the dependence of promoters to external factors. Heat shock cognates (HSCs) are proteins with high homology to the HSPs and constitutively synthesized by different cells when the organism is not under stress20. It would be more coherent to consider the S7 protein (constitutively expressed in the parasite) as HSC70. Nevertheless, we decided to keep it as HSP, even considering that only the coding region for the protein has been cloned and sequenced, and because of the lack of information concerning its promoter or about differences in aminoacid sequence between the cloned protein and other possible HSP70 of L.chagasi.
The immune response observed in the rabbits, after the immunization with the S7 rp, was very satisfactory and in accordance with the antigenicity and immnunogenicity of the HSPs. Antibodies have been detected in the serum samples of animals between days 8 and 10 after the first injection. The second injection produced a gradually elevated response, as expected when the immune system is exposed for the second time to the same antigen. The injection of 100 |xg of rp was enough to obtain a response of the immune system of the animals. The S7 protein was previously used for immunization of mice, in a concentration of 50 |j,g per injection, with excellent responses observed through serological assays21.
In a field work with dogs of an endemic area, we observed that 5 out of 8 dogs immunized with 400 |Lig of a sonicate extract of E. coli DH5a transformed with the S7 rp in PBS, reacted after 30 days in DTH tests against S7, while those inoculated with Leishvaccin (a mixture of 5 different strains oí Leishmania) did not shown any reaction in DTH against Leishvaccin or HSP70 (Andrade PP, Rayol C, Silva ECS and Moura PMMF, unpublished results). Together with other evidences of the HSP immunogenicity in the literature, these results show that the L. chagasi HSP70 is immunogenic and antigenic for several mammals.
Serological antibodies of control animals, immunized with E.coli DH5a total proteins, had not been able to recognize the Leishmania HSP70. However, E.coli synthesizes a HSP, called DnaK, that shows great homology with the Leishmania HSP70. DnaK is a constitutively express, 70 kDa protein20. A comparative analysis of the sequences of the C-terminal fractions of L. donovanii and E.coli HSP70 show that there are no big differences between these two proteins, even when the carboxi-terminal moiety, its most variable part, is considered: Figure 4 shows that there are only few amino acid exchanges between distinct groups when the sequences of the carboxi-terminal extremities of leishmanial HSP70 and DnaK are compared. Asterisks indicate the exchanges between hydrophilic groups for hydrophobic. In some way, the presence of these amino acid exchanges allows significant differences in the antigenicity of the proteins.
It is difficult to determine potential B-cell epitopes in these sequences. The removal of glutamic acid from the N-terminal region or of alanine from the C-terminal region of the hexapeptide EADDRA reduces the recognition of the leishmanial rp by the visceral leishmaniasis positive sera10. This sequence is specific for L. donovani HSP70 and has not been found in HSP70 of other parasites. However, as previously discussed, epitopes may be distributed among at least 170 aminoacids of the HSP70 C-terminal moiety.
It is indeed surprising that sera from patients with infections induced by taxonomically related parasites, such as T. cruzi, do not recognize the L. chagasi HSP70 protein1523. This specificity has been already observed in other parasitoses, although a constitutive expression of a homologous HSP70 protein in the diverse species of Schistosoma induces a dominant antibody response in the infections. In each case, the parasites antigens are immunologically distinct and do not show cross-reactions24. The same was observed in the case of specific antibodies directed against the Plasmodium falciparum homologous HSP70: these antibodies have not been found in sera from patients infected with Plasmodium vivax. The exception seems to be the Trypanosoma cruzi HSP70, recognized by serum antibodies from some patients with visceral leishmaniasis6. Antibodies against the T. cruzi HSP70, however, do not recognize the human homologous protein, which has 73% homology with its parasitic counterpart, suggesting that the T. cruzi HSP70 do not participate in the process of autoimmunity observed in the patients.
Attempts to confirm the presence of a surface leishmanial HSP have been done with DAT. Hyperimmune sera produced against the S7 rp showed sometimes agglutination reactions even more intense when in presence of P- ME. However, the agglutination observed for the negative serum was completely abolished by the addition of P-ME. To correctly evaluate these results, it is necessary to keep in mind the particularities of the DAT and those of agglutination reactions in general.
The agglutination process is rather complex and depends, among other factors, on the reaction of antibodies against epitopes on the surface of the cell. The linkage between the cells is made by an antibody, where an idiotipic extremity is bound to one parasite and another extremity is potentially bound to another parasite. The antigens that provoke agglutination are usually sparsely distributed on the surface of the cells, otherwise both extremities of the antibody would be in average bound to the same parasite.
On the other hand, agglutinating antibodies can be either of the IgG class or of the IgM class. In our study, antibodies originated from sacrificed rabbits 10 days after the third and last immunization, lead to the conclusion that the humoral immune response was essentially of the IgG class. In serum samples from human natural infection, it has been observed that the agglutinating fraction is IgG26.
The addition of P-ME, however, does not affect the specific agglutination and eliminates non-specific agglutinins. The precise role of ? -ME in this assay is not known. Non-specific agglutinins could be split by alcohol, or unfoldings of the surface of the parasite could be provoked by reduction of dissulphid bonds between adjacent cisteines in surface glycoconjugates. In fact, P-ME exposes new sugars on the surface of 'Leishmania1'1'. The addition of P-ME to samples of human sera also increased both the specificity and sensitivity of the DAT for the diagnosis of visceral leishmaniasis18.
The reduction in non-specific reactions demonstrates that there are agglutinins in the rabbits sera. The maintainance of the specific reaction after addition of P-ME, however, shows that surface epitopes are being recognized by non-IgM antibodies. These results allow the conclusion that the L. chagasi HSP70 native protein is distributed on the surface of the parasite.
HSP70 proteins have been located in eucaryotic cells in diverse cellular compartments, as the nucleus, mitochondrion, chloroplasts and endoplasmic reticulum22,28. These proteins have been found associated with a number of viral and cellular proteins in an ATP-dependent form. The presence of HSP on the surface of cells, however, is not generally recognized. A member of the GroEL family, a 60 kDa HSP ofLegionella pneumophila and Helicobacter pylori, was detected in the periplasmic space and on the surface of these intracellular bacteria, related to the level of stress in these organisms29,30.
The S7 rp, belonging to the HSP70 family, was detected on the surface of L. donovani promastigotes. What functions would the HSP70 fulfill on the surface of the parasite? The chaperone role of HSP70 could justify its transient presence on the surface of the cell, when a membrane protein is joined by the HSP70 during the passage through the cytoplasm (or the endoplasmic reticulum) to the surface. In this case, this HSP, that would be adsorbed to the surfaces through its linkage with chaperoned molecules, would have to return to the interior of the cell or at least to be excreted. This hypothesis justifies the observed localization of the HSP70, even in the absence of stress. An alternative hypothesis is based on the antigenic role of HSP70 in several parasitoses: they apparently function as molecules for the evasion of the immune system, purposedly directed, presented and excreted on the surface. In any case, the presence of HSP70 on the surface of the parasite is always transient.
Acknowledgements: We thank Dr. A. El-Harith (Lelystad, The Netherlands) for gifts of the DAT antigen suspension and Prof. Luiz B. Carvalho for allowing the use of laboratory facilities at LIKA (Laboratorio de Imunopatologia Keizo Asami, Federal University of Pernambuco, Recife, Brazil).
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Correspondence to: Eliete Cavalcanti da Silva
Departamento de Histología e Embriología - CCS Universidade Federal de Pernambuco.
Av. Prof. Moraes Regó, s/n, Cidade. Universitária Recife 50670-901, Brazil.
Telephone: +55-81-21268515, Fax:+55-81-21268516. e-mail: firstname.lastname@example.org