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

ABSTRACT: 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.

SUBMITTER: Norris KA

PROVIDER: S-EPMC174602 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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

Norris K A KA Schrimpf J E JE Szabo M J MJ

Infection and immunity 19970201 2

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 anti ...[more]

PMID: 9009282

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Project description: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.

Project description:UNLABELLED:Trypanosoma cruzi is a protozoan parasite of humans and animals, affecting 10 to 20 million people and innumerable animals, primarily in the Americas. Despite being the largest cause of infection-induced heart disease worldwide, even among the neglected tropical diseases (NTDs) T. cruzi is considered one of the least well understood and understudied. The genetic complexity of T. cruzi as well as the limited set of efficient techniques for genome engineering contribute significantly to the relative lack of progress in and understanding of this pathogen. Here, we adapted the CRISPR-Cas9 system for the genetic engineering of T. cruzi, demonstrating rapid and efficient knockout of multiple endogenous genes, including essential genes. We observed that in the absence of a template, repair of the Cas9-induced double-stranded breaks (DSBs) in T. cruzi occurs exclusively by microhomology-mediated end joining (MMEJ) with various-sized deletions. When a template for DNA repair is provided, DSB repair by homologous recombination is achieved at an efficiency several orders of magnitude higher than that in the absence of CRISPR-Cas9-induced DSBs. We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations. Lastly, we show that Cas9 can mediate disruption of its own coding sequence, rescuing a growth defect in stable Cas9-expressing parasites. These results establish a powerful new tool for the analysis of gene functions in T. cruzi, enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome. IMPORTANCE:Trypanosoma cruzi, the causative agent of human Chagas disease, is the leading worldwide cause of infectious myocarditis. Diagnostics for the infection are relatively poor, treatment options are limited and of variable effectiveness, and suitable vaccines are nonexistent. The T. cruzi genome is replete with genes of unknown function and greatly expanded gene families with hundreds of members. The absence of facile genetic engineering tools, including RNA interference, for T. cruzi has prevented elucidation of gene and gene family function and the development of better infection prevention and control measures. In this study, we demonstrate that the CRISPR-Cas9 system is a versatile and powerful tool for genome manipulations in T. cruzi, bringing new opportunities for unraveling the functions of previously uncharacterized genes and how this human pathogen engages its large families of genes encoding surface proteins to interact with human and animal hosts.

Project description:The partial sequence of a gene encoding the COOH terminus of a protein of apparent molecular weight of 160 kD associated with the flagellum of trypomastigotes of Trypanosoma cruzi (FL-160 now renamed to FL-160-1) has been previously reported. The COOH terminus of FL-160-1 has an epitope, defined by 12 amino acids, which molecularly miMics a nervous tissue antigen of 48 kD found in myenteric plexus, sciatic nerve, and a subset of cells in the central nervous system. We now report that FL-160 is a family of highly related genes. The sequence has been determined for the entire open reading frame (ORF) of one of the members of the FL-160 gene family (FL-160-2) and three other partial ORFs. Sequence analysis reveals the various members of the FL-160 gene family to be approximately 80% homologous in the predicted amino acid sequence, but all retain the 12-amino acid molecular mimicry epitope on the COOH terminus. Comparison of the sequence of FL-160-2 to other sequences demonstrates amino acid homology to bacterial sialidase (27%), members of the SA85 gene family (25-30%) and the shed acute-phase antigen/neuraminidase/trans-sialidase gene family (25-30%). Quantitative hybridization at high stringency suggests 750 copies of FL-160 are present in the DNA of each parasite. Reverse transcription and sequence analysis demonstrates that at least five of the members of the FL-160 gene family are transcribed. The NH2 terminus of one of the FL-160 gene products was expressed and antibodies prepared. Antibodies directed to either the COOH or the NH2 terminus of FL-160 bind a 160-kD T. cruzi protein. Both antibodies bind the surface membrane in the flagellar pocket of the trypomastigote. Antibodies to the NH2 terminus bind epineurium and scattered linear densities in sciatic nerve in a pattern distinct from the pattern with antibodies to the COOH terminus. Thus, there are at least two distinct molecular mimicry epitopes on the FL-160 molecule and both mimic epitopes found in nervous tissues. FL-160 may be involved in the generation of autoimmunity to nervous tissues by molecular mimicry, observed in chronic Chagas' disease.

Project description:In our continued effort to search for a Streptococcus suis protein(s) that can serve as a vaccine candidate or a diagnostic reagent, we constructed and screened a gene library with a polyclonal antibody raised against the whole-cell protein of S. suis type 2. A clone that reacted with the antibody was identified and characterized. Analysis revealed that the gene encoding the protein is localized within a 2.0-kbp EcoRI DNA fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 445 amino acid residues with a calculated molecular mass of 46.4 kDa. By in vitro protein synthesis and Western blot experiments, the protein exhibited an electrophoretic mobility of approximately 38 kDa. At the amino acid level the deduced primary sequence shared homology with sequences of unknown function from Streptococcus pneumoniae (89%), Streptococcus mutans (86%), Lactococcus lactis (80%), Listeria monocytogenes (74%), and Clostridium perfringens (64%). Except for strains of serotypes 20, 26, 32, and 33, Southern hybridization analysis revealed the presence of the gene in strains of other S. suis serotypes and demonstrated restriction fragment length differences caused by a point mutation in the EcoRI recognition sequence. We confirmed expression of the 38-kDa protein in the hybridization-positive isolates using specific antiserum against the purified protein. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic and may serve as an antigen of diagnostic importance for the detection of most S. suis infections. Pigs immunized with the recombinant 38-kDa protein mounted antibody responses to the protein and were completely protected against challenge with a strain of a homologous serotype, the wild-type virulent strain of S. suis type 2, suggesting that it may be a good candidate for the development of a vaccine that can be used as protection against S. suis infection. Analysis of the cellular fractions of the bacterium by Western blotting revealed that the protein was present in the surface and cell wall extracts. The functional role of the protein with respect to pathogenesis and whether antibodies against the antigen confer protective immunity against diseases caused by strains of other pathogenic S. suis capsular types remains to be determined.

Project description:Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.

Dataset Information

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

Publications

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

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