Two new phospholipase D isoforms of Loxosceles laeta: cloning, heterologous expression, functional characterization, and potential biotechnological application.

Toxin phospholipases-D present in the venom of Loxosceles spiders is the principal responsible for local and systemic effects observed in the loxoscelism. In this study, we describe the cloning, expression, functional evaluation, and potential biotechnological application of cDNAs, which code for two new phospholipase D isoforms, LIPLD1 and LIPLD2, of the spider Loxosceles laeta. The recombinant protein rLIPLD1 had hydrolytic activity on sphingomyelin and in vitro hemolytic activity on human red blood cells, whereas rLIPLD2 was inactive. The purified recombinant proteins and the venom are recognized by polyclonal anti-rLIPLD1 and rLIPLD2 sera produced in animals and conferred immunoprotection against the venom. These new isoforms reinforce the importance of the multigene family of phospholipases-D present in Loxosceles spiders. A highly immunogenic inactive isoform such as rLIPLD2 raises important expectation for its use as a potential immunogenic inducer of the immunoprotective response to the toxic action of the venom of Loxosceles laeta.

Tetracycline and penicillin resistant Clostridium perfringens isolated from the fangs and venom glands of Loxosceles laeta: its implications in loxoscelism treatment.

The venom of Loxosceles spiders produces severe dermonecrotic damage, intravascular hemolysis, systemic alterations and risk of death. Clostridium perfringens is present in the microbial flora of the fangs and venom glands of Loxosceles intermedia. Its inoculation with the venom may infect the wound site and exacerbate the dermonecrotic damage. This anaerobic bacterium is widely distributed in nature and capable of damage with similar characteristics and severity to the spider venom. In this study we isolated and characterized species of Clostridium from the fangs and venom glands of Loxosceles laeta, including C. perfringens. The sensitivity patterns of different isolates of C. perfringens were evaluated by minimum inhibitory concentration against penicillin, ampicillin, erythromycin, gentamicin, chloramphenicol, clindamycin and tetracycline, under anaerobic conditions, using the method of microdilution in broth. Strain C. perfringens H28 showed resistance to penicillin, ampicillin, tetracycline and chloramphenicol. Resistance to penicillin and ampicillin was mediated by beta-lactamase. In vivo evaluation of dermonecrosis in rabbits using L. laeta venom co-inoculated with isolate C. perfringens H28 produced an increase in the area of dermonecrotic lesions in the presence of penicillin and tetracycline, but not with gentamicin. Antibiotic therapy Loxosceles poisoning should be re-evaluated, considering the existence of multi-resistant strains of C. perfringens.

Isolation and characterisation of genomic and cDNA clones coding for a serine-, alanine-, and proline-rich protein of Trypanosoma cruzi.

We report here the isolation and characterisation of genomic and cDNA clones encoding a Serine-, Alanine-, and Proline-rich protein (SAP) of Trypanosoma cruzi metacyclic trypomastigotes. The deduced peptides translated from these clones were characterised by a high content of residues of alanine, proline, serine, glycine, valine, and threonine distributed in several repeats: P(2-4), S(2-3), A(2-3), AS, SA, PA, AP, SP, PS, and TP. The repeats are partially homologous to the serine-, alanine-, and proline-containing motifs of Leishmania major and Leishmania mexicana proteophosphoglycans. Genes coding for SAP are part of a polymorphic family whose members are linked to members of gp85/sialidase and mucin-like gene families. This is consistent with the hypothesis that this genetic organisation could be a means by which T. cruzi co-ordinates the expression of major surface proteins.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Characterization of the cell adhesion site of Trypanosoma cruzi metacyclic stage surface glycoprotein gp82.

The surface glycoprotein gp82, expressed in the insect-stage metacyclic trypomastigotes of Trypanosoma cruzi, has been implicated in mammalian cell invasion. Here we have characterized the cell adhesion site of gp82 by using recombinant proteins and synthetic peptides based on gp82. The recombinant protein Del-4/8, lacking 65 amino acids of gp82 central domain (at positions 257 to 321), was virtually devoid of cell-binding activity and lacked the ability to inhibit parasite invasion, in contrast to J18, the construct containing the full-length gp82 sequence (amino acids 1 to 516). Constructs with shorter deletions, i.e., Del-4 (deleted from 257 to 271) and Del-8 (deleted from 293 to 321), bound to target cells to a significantly lesser degree than did J18. The sites deleted in recombinant proteins Del-4 and Del-8 contained acidic amino acids critical for cell adhesion. Thus, the cell-binding capacity of protein Del-E/D, lacking the glutamic acid (259/260) and aspartic acid (303/304) pairs, was negligible, as was its capacity to inhibit parasite internalization. Of a set of synthetic peptides spanning the gp82 central domain, a 22-mer hybrid peptide, p4/8, formed by two noncontiguous sequences (at positions 257 to 273 and 302 to 306) and containing the four acidic residues, competed with the binding of J18 protein to target cells and significantly inhibited ( approximately 60%) the penetration of parasites. This peptide, generated by the juxtaposition of sequences that are separated by a hydrophobic stretch in the linear molecule, appears to be mimicking a conformation-dependent cell-binding site of gp82. Experiments of antibody competition with a set of 20-mer overlapping peptides mapped the epitope for 3F6, a monoclonal antibody directed to gp82 that inhibits parasite invasion, to the sequence represented by peptide p3 (244 to 263), which has a partial overlap with the cell adhesion site.

Physical mapping of a 670-kb region of chromosomes XVI and XVII from the human protozoan parasite Trypanosoma cruzi encompassing the genes for two immunodominant antigens.

As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and lambda phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.

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.

Cloning and characterization of a gene for the stage-specific 82-kDa surface antigen of metacyclic trypomastigotes of Trypanosoma cruzi.

We have cloned and sequenced a cDNA clone coding for a metacyclic trypomastigote-specific surface glycoprotein with a molecular mass of 82 kDa (MTS-gp82). By immunoblotting and immunoprecipitation, antibodies against the recombinant protein recognized an 82-kDa protein of metacyclic trypomastigotes, without any detectable reaction towards amastigotes, epimastigotes or tissue culture-derived trypomastigotes. The insert of the MTS-gp82 cDNA clone strongly hybridized with a single 2.2-kb metacyclic trypomastigote mRNA, suggesting that the steady-state levels of mRNAs for MTS-gp82 are developmentally regulated. MTS-gp82 is encoded by a multigene family whose members are distributed in several chromosomes. Sequence analysis revealed 40-56% identity at amino acid level between MTS-gp82 and members of Trypanosoma cruzi gp85/sialidase family (TSA-1, Tt34c1, SA85-1.1). MTS-gp82 showed several amino acid motifs that are characteristic of gp85/sialidase family, such as the Asp box (SxDxGxTW), the subterminal (VTVxNVFLYNR) motif and the putative GPI-anchor sequence. On the basis of its structural features, the MTS-gp82 gene could be included in the T. cruzi gp85/sialidase family, but constituting a distinct group which is preferentially expressed in metacyclic trypomastigotes.

Trypanosoma cruzi: antibody production and T cell response induced by stage-specific surface glycoproteins purified from metacyclic trypomastigotes.

The main surface glycoproteins of metacyclic trypomastigotes of Trypanosoma cruzi, gp90, gp82, and gp35/50, were purified and the immune response elicited by these antigens was analyzed. Balb/c mice immunized with antibody-affinity-purified gp82, plus alum as adjuvant, produced antibodies that recognized both the gp82 and the heterologous gp90 and gp35/50. On the other hand, antisera to gp90 reacted only with the homologous antigen, either by immunoprecipitation or by immunoblotting. Neither sera reacted with unrelated proteins in ELISA. Both antisera lysed 90-100% metacyclic forms in a complement-mediated reaction, a property associated with protection. However, in contrast to gp90, previously shown to induce protective immunity against acute T. cruzi infection, gp82 was not immunoprotective. Lymph node (LN) cells of mice primed with gp82 or gp90, which display 40% amino acid sequence identity at the carboxy terminal domain, were strongly stimulated in vitro by either one of these antigens. Proliferation, inhibitable by anti-CD4 but not by anti-CD8 antibodies, was T. cruzi-specific, no activation being observed with irrelevant antigens. LN cells of mice immunized with unrelated proteins did not proliferate in vitro in the presence of gp82 or gp90. The 35/50-kDa glycoconjugate, which was phenol-extracted, did not elicit any detectable antibody or T cell response.

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.