Sequence diversity of the Trypanosoma cruzi complement regulatory protein family.

As a central component of innate immunity, complement activation is a critical mechanism of containment and clearance of microbial pathogens in advance of the development of acquired immunity. Several pathogens restrict complement activation through the acquisition of host proteins that regulate complement activation or through the production of their own complement regulatory molecules (M. K. Liszewski, M. K. Leung, R. Hauhart, R. M. Buller, P. Bertram, X. Wang, A. M. Rosengard, G. J. Kotwal, and J. P. Atkinson, J. Immunol. 176:3725-3734, 2006; J. Lubinski, L. Wang, D. Mastellos, A. Sahu, J. D. Lambris, and H. M. Friedman, J. Exp. Med. 190:1637-1646, 1999). The infectious stage of the protozoan parasite Trypanosoma cruzi produces a surface-anchored complement regulatory protein (CRP) that functions to inhibit alternative and classical pathway complement activation (K. A. Norris, B. Bradt, N. R. Cooper, and M. So, J. Immunol. 147:2240-2247, 1991). This study addresses the genomic complexity of the T. cruzi CRP and its relationship to the T. cruzi supergene family comprising active trans-sialidase (TS) and TS-like proteins. The TS superfamily consists of several functionally distinct subfamilies that share a characteristic sialidase domain at their amino termini. These TS families include active TS, adhesions, CRPs, and proteins of unknown functions (G. A. Cross and G. B. Takle, Annu. Rev. Microbiol. 47:385-411, 1993). A sequence comparison search of GenBank using BLASTP revealed several full-length paralogs of CRP. These proteins share significant homology at their amino termini and a strong spatial conservation of cysteine residues. Alternative pathway complement regulation was confirmed for CRP paralogs with 58% (low) and 83% (high) identity to AAB49414. CRPs are functionally similar to the microbial and mammalian proteins that regulate complement activation. Sequence alignment of mammalian complement control proteins to CRP showed that these sequences are distinct, supporting a convergent evolutionary pathway. Finally, we show that a clonal line of T. cruzi expresses multiple unique copies of CRP that are differentially recognized by patient sera.

DNA-Based immunization with Trypanosoma cruzi complement regulatory protein elicits complement lytic antibodies and confers protection against Trypanosoma cruzi infection.

A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T. cruzi infection. Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T. cruzi trypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruzi DNA vaccine.

Stable transfection of Trypanosoma cruzi epimastigotes with the trypomastigote-specific complement regulatory protein cDNA confers complement resistance.

Trypanosoma cruzi blood stage trypomastigotes are highly resistant to complement-mediated killing in normal serum. A previously described trypomastigote surface glycoprotein was shown to have binding affinity for human complement components C3b and C4b and restrict activation of the complement cascade, thus preventing lysis of the parasites. Insect stage epimastigotes do not produce detectable levels of this 160-kDa complement regulatory protein (CRP) and are highly sensitive to the lytic effects of complement. Epimastigotes were stably transfected with a T. cruzi expression vector carrying the trypomastigote CRP cDNA and produced fully functional recombinant CRP. The recombinant CRP had binding affinity for C3b, and the transfected epimastigotes were protected from complement-mediated lysis. These results demonstrate for the first time that a developmentally regulated gene of T. cruzi trypomastigotes can be expressed in noninfectious epimastigotes and that production of CRP by epimastigotes is sufficient to confer a virulence-associated trait. Furthermore, these studies demonstrate the critical role that trypomastigote CRP plays in the protection of parasites from the deleterious effects of complement, thus establishing the protein as a virulence factor of T. cruzi.

Identification of the gene family encoding the 160-kilodalton Trypanosoma cruzi complement regulatory protein.

Trypanosoma cruzi trypomastigotes are exquisitely resistant to the lytic effects of vertebrate complement, and this characteristic contributes to the survival of the parasites in the host bloodstream. Trypomastigotes avoid complement-mediated lysis by the production of a surface glycoprotein that inhibits the formation of the alternative and classical C3 convertase, thus preventing activation and amplification of the complement cascade at the parasite surface. We have developed a monoclonal antibody to the 160-kDa T. cruzi complement regulatory protein (CRP) and describe a one-step immunoaffinity purification procedure. The CRP was purified to homogeneity and subjected to amino-terminal peptide sequence analysis. Based on the protein sequence obtained, the CRP was identified as a member of a large family of trypomastigote-specific genes, and a complete cDNA was isolated and sequenced. The complete coding sequence was cloned in Escherichia coli, and antibodies raised against the full-length recombinant protein reacted specifically with a 160-kDa protein in trypomastigote membrane protein preparations as well as with native, purified CRP. Indirect immunofluorescence revealed that the protein is uniformly expressed at the cell surfaces of trypomastigotes.

Ligand-binding renders the 160 kDa Trypanosoma cruzi complement regulatory protein susceptible to proteolytic cleavage.

It has been previously shown that a surface complement regulatory protein (CRP) of Trypanosoma cruzi trypomastigotes binds human complement components C3b and C4b, and inhibits C3 convertase formation, thus contributing to the resistance of the bloodstage parasites to complement-mediated lysis. The blood stage parasites rapidly and spontaneously release a limited set of membrane glycoproteins, including CRP, and the modulation of release of CRP following ligand binding was investigated. Incubation of the parasites with C3b results in the release of CRP at a reduced apparent molecular mass. To determine if proteolytic processing was responsible for the reduction in apparent molecular mass of the released CRP, the proteolytic activity present in trypomastigote membrane preparations was examined. In addition to a well described cysteine protease, a novel 75 kDa protease was identified in tissue culture-derived trypomastigotes and axenically-derived metacyclic trypomastigotes membrane preparations. This protease was inhibited by aprotinin and leupeptin, but not L-trans-epoxysuccinyl-leucylamide-(4-guanidinobutane), N-[N-L-3-transcarboxyirane-2-carbonyl-L-leucinyl]-agmatine (E64). Treatment of the parasites with protease inhibitors did not affect spontaneous shedding of proteins, however, protease inhibitors abrogated the effect of C3b-binding on CRP degradation. These results indicate that binding of C3b to the CRP renders the CRP susceptible to cleavage by a parasite protease, possibly as a means of removing the CRP-C3b from the parasite surface. This process may represent an additional immune evasion mechanism which allows the parasite to avoid both complement-mediated lysis and clearance.

Enhancement of macrophage microbicidal activity: supplemental arginine and citrulline augment nitric oxide production in murine peritoneal macrophages and promote intracellular killing of Trypanosoma cruzi.

The generation of nitric oxide (NO) is largely responsible for the intracellular killing of Trypanosoma cruzi by activated macrophages. The present study was carried out to determine whether the production of NO by activated murine macrophages cultured in physiologic levels of arginine can be augmented by increasing the availability of arginine, the substrate for NO biosynthesis. Increased exogenous arginine or citrulline resulted in a significant increase in NO production and complete clearance of the parasites by activated macrophages. As citrulline fully substituted for arginine in supporting NO production and trypanocidal activity, these results demonstrate the expression of a highly active citrulline-NO cycle in activated macrophages and that levels of arginine in plasma are limiting with respect to both NO production and trypanocidal activity in these cells. The results indicate that increasing plasma substrate levels for both arginine and NO biosynthesis may represent a means of enhancing microbicidal activity in vivo.

Humoral immune response to the Trypanosoma cruzi complement regulatory protein as an indicator of parasitologic clearance in human Chagas' disease.

Immunoprecipitation of the purified 160-kDa complement regulatory protein of Trypanosoma cruzi by Chagas' disease patient sera was examined as a possible correlate of the complement-mediated lysis test and as an indicator of parasite clearance. The results presented demonstrate that assessment of the humoral response to this antigen is a useful indicator of parasite clearance and may be particularly helpful in the assessment of some patients for whom other serological tests produce ambiguous results.

Biochemical analysis of the membrane and soluble forms of the complement regulatory protein of Trypanosoma cruzi.

A developmentally regulated, 160-kDa trypomastigote surface glycoprotein was previously shown to bind the third component of complement and to inhibit activation of the alternative complement pathway, thus providing the parasites a means of avoiding the lytic effects of complement. We now show that this complement regulatory protein (CRP) binds human C4b, a component of the classical pathway C3 convertase, and may therefore also act to restrict classical complement activation. Characterization of the extent of carbohydrate modification of the protein revealed extensive N-linked glycosylation and no apparent O-linked sugars. The CRP purified from parasites treated with an inhibitor of N-linked glycosylation exhibited a decreased binding affinity for C3b compared with that of the fully glycosylated protein. We have previously shown that the protein was anchored to the membrane via a glycosyl phosphatidylinositol linkage and was spontaneously shed from the parasite surface. The spontaneous release of CRP from the parasite surface may augment the protection of the parasites from complement-mediated lysis by the removal of complement-CRP complexes. The majority of the shed CRP had an apparent molecular mass of 160 kDa and lacked the glycolipid anchor, whereas the membrane form was recovered with the glycolipid anchor attached and had an apparent molecular mass of 185 kDa. Both the membrane form (185 kDa) and the soluble form (160 kDa) retained binding affinity for C3b. Evidence is presented to indicate that the conversion of the 185-kDa membrane form to the 160-kDa form is the result of cleavage by an endogenous phospholipase C.

Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor.

Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement-mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway. We have purified a developmentally regulated, trypomastigote glycoprotein, gp160, and shown that it has complement regulatory activity. The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor. Using anti-gp160 antibodies we have isolated a bacteriophage lgt11 clone expressing a portion of the gp160 gene that shares significant DNA sequence homology with the human DAF gene. These results provide functional, biochemical, and genetic evidence that the T. cruzi gp160 is a member of the C3/C4 binding family of complement regulatory proteins, and that gp160 may provide the infectious trypomastigotes with a means of evading the destructive effects of complement.

Generation of a cytotoxic T-lymphocyte response using a Salmonella antigen-delivery system.

We have constructed a general-use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S. typhimurium strain. Mice immunized with CS-expressing Salmonella recombinants mount a CS-specific cytotoxic T-lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen-delivery system.

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.

Colorimetric enzymatic determination of serum total carbon dioxide, as applied to the Vickers Multichannel 300 discrete analyzer.

Phosphoenolpyruvate carbosylase (EC 4.1.1.31) has been used in developing a simple, inexpensive colorimetric assay for serum total carbon dioxide in an open system. The oxaloacetate formed by the action of the enzyme on bicarbonate and phosphoenolpyruvate is measured by use of the diazonium salt of Fast Violet B. Interferences from billirubin, pyruvate, and drugs are negligible. Acetoacetate interference is significant only in highly ketotic samples, and a serum blank corrects for it. Serum protein interference is equivalent to 3.3 plus or minus 1.25 mmol of CO2 per liter and hence is sufficiently constant to be corrected for by use of a serum standard or serum blank. The method has been applied to the Vickers M-300 and D-300 systems and within-batch standard deviations of plus or minus 0.1 to plus or minus 0.6 mmol/liter have been observed. Excellent correlation with orthodox techniques has been obtained.

Heterogeneous distribution of enzymes among plasma-membrane fragments sedimenting with the microsomal fraction of rat liver.

Plasma-membrane fragments recovered in the microsomal fraction of rat liver homogenates were shown to be heterogeneous in density. It was demonstrated that 5'-nucleotidase, the most commonly used plasma-membrane marker, is concentrated in the lightest subfraction. Two of the published procedures for the isolation of plasma-membrane fragments from the microsomal fraction (Touster et al., 1970; Hinton et al., 1971) are shown to give products which are not representative of all the plasma-membrane fragments of microsomal size, and it is argued that a third procedure (House & Weidemann, 1970) is likely to give a similar product.

Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes.

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl(2). Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg(2+), there being at least a 12-fold difference between the activity in the presence of Mg(2+) and of EDTA. There is, however, a difference in the response of the enzymes to Mg(2+) and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl(2) and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl(2) for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.