Resistance to acute Trypanosoma cruzi infection resulting from immunization of mice with a 90-kilodalton antigen from metacyclic trypomastigotes.

The stage-specific 90-kDa surface antigen of Trypanosoma cruzi metacyclic trypomastigotes was affinity purified and used for immunization studies. Mice immunized with the 90-kDa antigen plus alum as an adjuvant were resistant to challenge with Tulahuen strain metacyclic forms, displaying either reduced or subpatent parasitemias. Mice immunized with the purified antigen without adjuvant or with antigen plus Freund adjuvant or glucan developed high parasitemias, comparable to those of nonimmunized controls, upon metacyclic challenge. Sera of mice immunized with the 90-kDa antigen plus alum showed complement-dependent trypanolytic activity and also greatly inhibited the invasion of Vero cells by metacyclic forms of G and Tulahuen strains in vitro. These sera were found by a competition binding assay to contain antibodies that recognized the same or topographically related sites as the monoclonal antibody 1G7, an antibody previously shown to reduce the infectivity of metacyclic forms in vivo and in vitro. In contrast, 1G7-related antibodies were poorly represented in sera with negative or low trypanolytic activity, such as those from mice immunized with purified antigen without adjuvant.     

Metacyclic neutralizing effect of monoclonal antibody 10D8 directed to the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi.

It was shown in this work that the infectivity of metacyclic forms of Trypanosoma cruzi was affected upon interaction with the monoclonal antibody (10D8), which reacts with a carbohydrate epitope of the 35- and 50-kilodalton (kDa) surface glycoconjugates. The invasion of Vero cells by metacyclic forms of strains Tulahuen and G was inhibited 50 to 67% in the presence of 10D8 (10 micrograms/ml), whereas a nonrelated monoclonal antibody to Plasmodium berghei had no such effect. In mice that were inoculated with metacyclic forms preincubated with 10D8 or that had passively received 10D8 before challenge with metacyclic forms, a considerable decrease in the parasitemia levels was observed. The 35- and 50-kDa antigens were detectable by the galactose oxidase and sodium boro[3H]hydride procedure but not by surface iodination or metabolic labeling with [35S]methionine, suggesting that they may be of glycolipid nature. The finding that the 35- and 50-kDa antigens are major bands recognized by sera of mice immunized with killed metacyclic forms and protected against acute infection, in addition to the results with 10D8, indicate that these glycoconjugates may play an important role in the metacyclic form-host cell association that initiates T. cruzi infection.     

Involvement of the stage-specific 82-kilodalton adhesion molecule of Trypanosoma cruzi metacyclic trypomastigotes in host cell invasion.

This study provides several pieces of evidence indicating that 3F6-Ag, identified by monoclonal antibody (MAb) 3F6 as a stage-specific glycoprotein of approximately 82 kDa on the surface of metacyclic trypomastigotes of different Trypanosoma cruzi strains, promotes the entry of parasites into host cells through a ligand-receptor type interaction. First, invasion of Vero cells by metacyclic trypomastigotes of both CL and Tulahuen strains was significantly inhibited by MAb 3F6 or its Fab fragments. Second, purified 3F6-Ag bound to Vero cells in a dose-dependent and saturable fashion. Third, soluble 3F6-Ag reduced the infection of Vero cells by metacyclic forms of CL and Tulahuen strains by 90 to 97 and 50%, respectively. Unrelated proteins, as well as extracellular matrix components, such as heparan sulfate and collagen, had no effect. Our studies also show that in the Tulahuen strain, 10D8-Ag, a 35/50-kDa glycoprotein identified by MAb 10D8, participates in target cell invasion, confirming previous observations, but the variant form of 10D8-Ag expressed by highly invasive CL strain metacyclic trypomastigotes appears to be irrelevant. Overall, our results indicate that the surface components of T. cruzi metacyclic trypomastigotes involved in the process of host cell penetration are developmentally regulated molecules, such as 3F6-Ag and 10D8-Ag, that have no counterpart in blood- or tissue culture-derived trypomastigotes.     

Polymorphism of the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi metacyclic trypomastigotes.

In this study, we examined the immunochemical properties of the 35- and 50-kDa (35/50-kDa) surface glycoconjugates expressed on the surface of metacyclic trypomastigotes of Trypanosoma cruzi using three different monoclonal antibodies directed to this component. The 35/50-kDa surface antigen was expressed by metacyclic trypomastigotes of 11 different strains of T. cruzi and displayed a significant degree of molecular polymorphism. Results of immunoblotting and complement-mediated lysis were consistent with such diversity. Different monoclonal antibodies reacted with distinct epitopes on the 35/50-kDa antigen, which is resistant to proteases and behaves as an amphiphilic component.     

Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies

Stage-specific Trypanosoma cruzi surface antigens were characterized by using monoclonal antibodies (MAb) which bind specifically to the metacyclic trypomastigotes derived from either the insect vector or acellular cultures. A protein with an apparent molecular weight of 90,000 was detected by the MAb 5E7 on the surface of cultured metacyclics of four strains of T. cruzi: G, CL, Y and Tulahuen. The MAb 1G7, which binds to an epitope of the 90 kDa antigen distinct from that recognized by the MAb 5E7, reacted with metacyclics of the G and Tulahuen strains but not of the Y or CL strain. A polypeptide of approximately 82 kDa, identified by the MAb 3F6, was found in the metacyclics of all four T. cruzi strains. The MAb 3F6 also detected a 75 kDa antigen in the G strain metacyclics. The stage-specific MAb and the polyclonal antibodies from mice protected against acute T. cruzi infection by immunization with killed G metacyclics identified the same set of major surface proteins of G metacyclic trypomastigotes.     

Variation in antigenic determinants specific to the infective stage of Trypanosoma cruzi.

Monoclonal antibodies reactive with the surface antigens of the Peru strain of Trypanosoma cruzi were analyzed by Western blots and immunofluorescence assays to determine their reactivity with three life cycle stages and five strain isolates of T. cruzi. One monoclonal antibody, 7.6, recognized a 68-kilodalton (kDa) polypeptide in Western blots of Peru strain trypomastigotes, epimastigotes, and amastigotes. A 68-kDa polypeptide was also detected by monoclonal antibody 7.6 in trypomastigotes of the CL and Y strains and in the clonal isolates Esmeraldo clone 3 and Silvio X10 clone 1. Positive immunofluorescence results were obtained for all life cycle stages of the five strains that were reacted with monoclonal antibody 7.6, thus indicating that the antigen recognized by monoclonal antibody 7.6 is universally present in all T. cruzi strains tested. In contrast, monoclonal antibody 4.2 reacted with a polypeptide doublet of 90 and 105 kDa in Western blots of Peru strain trypomastigotes, but it did not detect these antigens in epimastigotes or amastigotes. The same polypeptide doublet of 90 and 105 kDa was also detected in Western blots of Y strain trypomastigotes; however, no bands were detected in blots of strain CL or isolate Silvio X10 clone 1 trypomastigotes. In blots of Esmeraldo clone 3 trypomastigotes, a single band of 130 kDa was detected by monoclonal antibody 4.2. In immunofluorescence assays of monoclonal antibody 4.2, positive reactions were obtained only with trypomastigotes of Peru, Y, and Esmeraldo clone 3 strains. Thus, monoclonal antibody 4.2 recognizes a trypomastigote-specific antigen which is not universally present on all strains of T. cruzi.     

Involvement of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells

Upon oral infection, Trypanosoma cruzi metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium, being apparently uniquely specialized for adhesion to mucin and mucosal invasion. Here we investigated the involvement of gp82, the metacyclic-stage-specific surface glycoprotein implicated in host cell entry, in both adhesion to gastric mucin and invasion of the mucosal epithelium upon oral challenge. Metacyclic forms, preincubated with a control monoclonal antibody (MAb) or with MAb 3F6 directed to gp82, were administered orally to BALB/c mice, and parasitemia was monitored. Mice that received parasites treated with MAb 3F6 had greatly reduced parasitemia, displaying at the peak a mean number of blood parasites more than 100-fold lower than that of the control group. MAbs directed to other T. cruzi surface glycoproteins had no such effect. gp82, as either a native or a recombinant molecule, but not the metacyclic trypomastigote surface molecule gp90 or gp35/50, bound to gastric mucin in enzyme-linked immunosorbent assays. MAb 3F6 significantly inhibited the penetration of cultured epithelial HeLa cells by metacyclic forms in the absence or in the presence of gastric mucin. Mucin alone did not affect parasite internalization. Parasite infectivity was not altered by treatment of metacyclic forms with pepsin, to which gp82 was resistant, or with proteinase K, which removed the N-terminal portion of gp82 but preserved its host cell binding site. Taken together, these findings suggest that gp82 mediates the interaction of metacyclic trypomastigotes with gastric mucin and the subsequent penetration of underlying epithelial cells.     

Infection by Trypanosoma cruzi metacyclic forms deficient in gp82 but expressing a related surface molecule, gp30

Trypanosoma cruzi metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium after oral infection. In this study we analyzed the process of infection by T. cruzi isolates deficient in the expression of gp82, the metacyclic stage-specific surface glycoprotein implicated in target cell entry in vitro and in promoting mucosal infection in mice after oral challenge. Mice infected by the oral route with metacyclic forms of gp82-deficient isolate 569 or 588 developed patent parasitemia but at greatly reduced levels compared to those infected with the gp82-expressing isolate CL. Metacyclic forms of both isolates expressed gp30, a surface glycoprotein detectable by monoclonal antibody (MAb) 3F6 directed to gp82. Otherwise, the gp82-deficient isolates displayed a surface profile similar to that of the CL isolate and also entered epithelial HeLa cells in a manner inhibitable by MAb 3F6 and dependent on the parasite signal transduction that involved the activation of protein tyrosine kinase and Ca(2+) mobilization from thapsigargin-sensitive stores. Like gp82, gp30 triggered the host cell Ca(2+) response required for parasite internalization. Purified gp30 and the recombinant gp82 inhibited HeLa cell invasion of metacyclic forms of isolates 569 and 588 by approximately 90 and approximately 70%, respectively. A cell invasion assay performed in the presence of gastric mucin, mimicking the in vivo infection, showed an inhibition of 70 to 75% in the internalization of gp82-deficient isolates but not of the CL isolate. The recombinant gp82 exhibited an adhesive capacity toward gastric mucin much higher than that of gp30. Taken together, our findings indicate that target cell entry of metacyclic trypomastigotes can be mediated either by gp82 or gp30 but that efficient mucosal infection depends on the expression of gp82.     

A Mr 90 000 surface polypeptide of Trypanosoma cruzi as a candidate for a Chagas' disease diagnostic antigen

Trypanosoma cruzi (Peru strain) trypomastigotes and epimastigotes were biosynthetically labeled with [35S]methionine, and the proteins were analyzed by two dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE analysis of the trypomastigotes showed a complex array of polypeptides with distinct clusters at Mr 88 000-92 000, isoelectric point (pI) 5.6-6.0, and Mr 72 000-76 000, pI 5.6-5.8. 2D-PAGE analysis of the epimastigotes did not show the cluster of polypeptides at Mr 90 000. When the trypomastigote lysate was reacted with sera from either mice or humans chronically infected with T. cruzi, 10-50 polypeptides were immunoprecipitated. Five of these polypeptides were recognized by all sera tested. However, of these polypeptides, only three, two of Mr 90 000 and one of Mr 150 000, can be identified by immunoreaction of [35S]methionine-labeled live parasites as surface proteins of T. cruzi trypomastigotes. 125I-iminobiotinylated surface proteins isolated from T. cruzi trypomastigotes were immunoprecipitated with the same series of sera as described above. Chagasic sera immunoprecipitated an antigen of Mr 90 000. The [35S]methionine and 125I-labeled Mr 90 000 polypeptides were not immunoprecipitated with sera from individuals infected with Leishmania donovani, Leishmania braziliensis, Leishmania tropica or Leishmania mexicana. These data indicate that a surface polypeptide of Mr 90000, pI 5.8-5.9 is a viable candidate for a Chagas' disease diagnostic antigen.     

Purification of a Trypanosoma cruzi trypomastigote 60-kilodalton surface glycoprotein that primes and activates murine lymphocytes.

We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of Trypanosoma cruzi trypomastigotes and studied its ability to prime and stimulate the proliferation of murine spleen cells. T. cruzi trypomastigote membrane proteins were separated by preparative isoelectrofocusing. A trypomastigote 60-kDa surface protein with an isoelectric point of 4.2 was enriched by chromatofocusing and was readily purified in native form to homogeneity by gel filtration on a Superose column by use of a fast protein liquid chromatography system. Biotinylated wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin bound to blots containing the purified trypomastigote 60-kDa surface protein, indicating that this protein was glycosylated. The purified trypomastigote 60-kDa glycoprotein was recognized by antibodies produced during human infection, and immunoglobulin G against the purified glycoprotein immunoprecipitated a biotinylated 60-kDa molecule from the surface of trypomastigotes but not epimastigotes. Specific immunoglobulin G against the 60-kDa glycoprotein also increased the uptake of trypomastigotes and promoted parasite killing by macrophages. The purified 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in [3H]thymidine incorporation by spleen cells obtained from CBA mice primed with this glycoprotein, with respect to control values. Furthermore, the 60-kDa glycoprotein did not stimulate unprimed spleen cells, indicating that the lymphoproliferation induced by this glycoprotein was specific and was not due to polyclonal activation. Our findings indicate that this T. cruzi trypomastigote 60-kDa surface glycoprotein primes and activates lymphocytes, which could lead to a beneficial immune response in the host.     

Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi.

The surface antigen makeup of metacyclic trypomastigote forms of strain G of Trypanosoma cruzi, which produce a subpatent infection in mice, differed from those of the virulent strains Y and CL. A 100,000-molecular-weight protein, barely detectable on the Y or CL cell surface, appeared as the main surface antigen of the G metacyclic trypomastigotes. In addition, the G metacyclic forms differed from those of the virulent strains in their susceptibility to complement-mediated immunolysis.     

Biochemical characterization of the 9.3 antigens of human T-cells: simultaneous expression of disulfide-bonded 90-kilodalton dimers and free subunits at the cell surface

Human T-lymphocytes express a heterogeneous family of 90/45-kilodalton (kDa) glycoproteins which bind the 9.3 monoclonal antibody. It was found in previous functional tests carried out with cultures of mononuclear cells or antigen-specific T-cell clones that these glycoproteins have a specific receptor function in early T-cell activation [Gmünder and Lesslauer, Eur. J. Biochem. (1984); Ottenhoff et al., (1985)]; their membrane-biochemical properties are therefore investigated. By screening a number of lines, one continuously growing human T-cell line, HPB-ALL, was identified which expresses the 9.3 antigens in a manner comparable to normal T-cells. Monomeric 45-kDa and dimeric disulfide-bonded 90-kDa forms are precipitated from alkylated surface-iodinated and [35S]methionine-cysteine-labelled cells. The labelled tryptic fragments of surface-iodinated 9.3 antigens have isoelectric points of 4.8 (17-kDa), 4.8 (3-kDa) and 6.0 (17-kDa). By limited proteolysis the 45-kDa monomers are free subunits. The subunits of the 90-kDa dimer appear to be identical. The dimer and the free subunits coexist at the native cell surface and may be in dynamic chemical equilibrium. Human T-cells thus express--in addition to the T-cell antigen receptor--a further disulfide-bonded 90-kDa (homo-) dimeric receptor molecule.     

A recombinant protein based on the Trypanosoma cruzi metacyclic trypomastigote 82-kilodalton antigen that induces and effective immune response to acute infection.

To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection.     

Purification of a Trypanosoma cruzi membrane glycoprotein which elicits lytic antibodies.

Recent studies on the humoral immune response to Trypanosoma cruzi have shown that antibodies which are able to bind living parasites and lyse them in conjunction with complement are associated with host protection. Antibodies which support complement-mediated lysis (CML) of trypomastigotes are elicited as a result of an active infection and not after immunization with killed parasites. In spite of the requirement for immune antibodies, lysis proceeds mainly via the alternative complement pathway. We have purified a 160-kilodalton (kDa) glycoprotein from T. cruzi metacyclic trypomastigotes which appears to be a specific target for lytic antibodies. Rabbit antiserum to the purified 160-kDa protein was prepared, and we have determined that these antibodies will support CML of tissue-culture-derived trypomastigotes. The percentage of killing (65 to 70%) was consistent among three different T. cruzi strains tested. In order to examine the specificity of antibody-dependent CML, antibodies to T. cruzi neuraminidase, an unrelated trypomastigote membrane glycoprotein, were tested in the CML, assays and were not found lytic. Viable trypomastigotes bound anti-160-kDa antibodies uniformly as demonstrated by immunofluorescence, whereas antineuraminidase antibodies were extensively capped. The 160-kDa glycoprotein is specifically produced in infectious trypomastigotes (tissue culture derived and metacyclic) and was not detected in epimastigotes or amastigotes. The identification of the 160-kDa glycoprotein as a specific target for lytic antibodies, as well as its expression only in the infectious stage of the parasite, suggests an important role for this protein in eliciting host immunity.     

Glycophosphatidylinositol-anchored proteins in metacyclic trypomastigotes of Trypanosoma cruzi

We searched for the presence of glycophosphatidylinositol (GPI)-anchored proteins in epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi, by treatment of parasite lysates with the GPI-specific phospholipase C of Trypanosoma brucei. Upon treatment, several proteins (70-90 kDa) in metacyclics, but none in epimastigotes, reacted with antibodies to the cross-reacting determinant (CRD), an epitope revealed on the variant surface glycoproteins of T. brucei following removal of the diacylglycerol moiety from their GPI-anchor. Since these T. cruzi metacyclic proteins also lost their original amphiphilicity, as judged by Triton X-114 phase separation, it is very likely that they are linked to the membrane by GPI. One of these proteins is the 90 kDa protein, the major surface protein of G and Tulahuen strains, recognized by the monoclonal antibody 1G7. A variable portion of the 90 kDa molecules was resistant to solubilization by T. brucei lipase. The reasons for this are not clear but susceptibility appeared to increase with the age of the T. cruzi culture. Enzymes that solubilize GPI-anchored proteins were detected in epimastigotes and metacyclics, but the enzymatic activity in these forms was smaller than the activity detected in the same cell numbers of trypomastigotes of T. cruzi originated from infected mammalian cells or from T. brucei bloodstream forms. A preliminary characterization of these activities indicates that at least two classes of enzymes, one of them inhibited by o-phenanthroline, are present in epimastigotes and metacyclics. None of the reagents tested fully inhibited the phospholipases.     

A surface antigen of Trypanosoma cruzi involved in cell invasion (Tc-85) is heterogeneous in expression and molecular constitution

A monoclonal antibody (McAb), H1A10, causes 46-96% inhibition of the invasion of tissue culture cells by trypomastigotes from different strains and clones of Trypanosoma cruzi. Higher inhibition was observed when axenic medium-derived metacyclic trypomastigotes (72-96%) were employed instead of tissue culture-derived trypomastigotes (46-60%). In contrast to the metacyclic population, in which all individuals reacted with the McAb, part of the tissue-cultured trypomastigote population did not bind the antibody. The molecular masses and isoelectric points of the glycoproteins recognized by the H1A10 McAb differed when tissue culture trypomastigotes of the Y strain and YuYu strain were analyzed. Variability between the strains was also observed when the antigens were metabolically labelled in the presence of tunicamycin. These results suggest that the differences described are probably not due solely to the carbohydrate portion of the molecule.     

The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope.

Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific. Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes. The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R. tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium. In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli. DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide. A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with [35S]methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide. Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A). Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene.     

Monoclonal antibodies to 52-kilodalton protein of Salmonella typhi.

Ten monoclonal antibodies (6 immunoglobulin G1 kappa [IgG1 kappa] and 4 IgG2b kappa) from six hybrid clones specific for Salmonella typhi antigen were produced by immunizing BALB/cJ mice with affinity-purified S. typhi proteins (Bp). The latter were prepared by passing crude S. typhi Bp through an affinity column made from Sepharose conjugated to IgG antibodies against partially purified S. typhi Bp. The eluent was subsequently used as the immunogen for the production of monoclonal antibody. All 10 monoclonal antibodies reacted specifically with a 52-kilodalton (kDa) protein of S. typhi and were species specific. The presence of IgM antibody to the 52-kDa antigen in the sera of a majority of patients with acute typhoid fever suggested that this 52-kDa protein is also a good immunogen for humans. The potential usefulness of this antigen in the early diagnosis of typhoid fever is discussed.     

The major surface protein of Leishmania promastigotes is a fibronectin-like molecule

The major surface glycoprotein of Leishmania chagasi promastigotes showed crossreactivity with fibronectin (Fn), a large glycoprotein that is a major constituent of the extracellular matrix of most mononuclear cells. Polyclonal and monoclonal antibodies against Fn precipitated two molecules of 63-58 kDa from the lysates of both 125I and [35S]methionine-labeled promastigotes. In addition, a monoclonal antibody against a 15-kDa fragment of Fn containing the Arg-Gly-Asp-Ser (RGDS) sequence and several polyclonal monospecific mouse antibodies against a synthetic RGDS peptide also recognized the above two molecules. The attachment of Leishmania promastigotes to mouse peritoneal macrophages in vitro was partially inhibited when promastigotes were treated with F(ab')2 fragment of an anti-Fn IgG. Identical results were obtained by saturating the Fn receptors on macrophages using different peptides containing the RGDS sequence. Moreover, antigen preparations rich in glycoprotein 63 could efficiently promote the attachment and spreading of 3T3 mouse fibroblasts to surfaces coated with the antigen. These results clearly suggest that the gp63 of L. chagasi promastigotes is an Fn-like molecule that shares certain biological and molecular characteristics with Fn.