An anti-Chitinase malaria transmission-blocking single-chain antibody as an effector molecule for creating a Plasmodium falciparum-refractory mosquito

Indirect evidence has suggested the existence of a second chitinase gene, PgCHT2, in the avian malaria parasite Plasmodium gallinaceum. We have now identified PgCHT2 as the orthologue of the P. falciparum chitinase gene PfCHT1, a malaria transmission-blocking target. Computational phylogenetic evidence and biochemical and cell biological functional data support the hypothesis that an avian-related Plasmodium species was the ancestor of both P. falciparum and P. reichenowi, and this single lineage gave rise to another lineage of malaria parasites, including P. vivax, P. knowlesi, P. berghei, P. yoelii, and P. chabaudi. A recombinant PfCHT1/PgCHT2-neutralizing single-chain antibody significantly reduced P. falciparum and P. gallinaceum parasite transmission to mosquitoes. This single-chain antibody is the first anti-P. falciparum effector molecule to be validated for making a malaria transmission-refractory transgenic Anopheles species mosquito. P. gallinaceum is a relevant animal model that facilitates a mechanistic understanding of P. falciparum invasion of the mosquito midgut.     

Transient transfection and expression of firefly luciferase in Giardia lamblia.

We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly luciferase reporter gene. The optimal construct consisted of a GDH/luciferase fusion gene in which the first 18 codons of the GDH gene immediately preceded the luciferase gene; this fusion gene was flanked by the upstream and downstream sequences of the GDH gene. Electroporation of this construct into Giardia yielded luciferase activity that was 3000- to 50,000-fold above background. Removal of either the 5' or 3' GDH flanking sequences from this construct resulted in significantly reduced luciferase activity, and removal of both flanking sequences reduced luciferase activity to background levels. Luciferase activity was proportional to the amount of DNA electroporated and was maximal at 6 hr after electroporation.     

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications.

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.     

Gene transfer into coronary arteries of intact animals with a percutaneous balloon catheter.

Genetic manipulation of the vasculature may offer insights into the pathogenesis of coronary artery disease and may lead to gene therapy for disorders such as restenosis after percutaneous coronary angioplasty. The goal of this study was to develop a percutaneous method for gene transfer into coronary arteries of intact animals. Liposomes were used to facilitate transfection in coronary arteries with a plasmid containing the cDNA encoding luciferase. This reporter was chosen since it is not expressed in mammalian cells, and it can be quantified using a sensitive assay (light production). Mongrel dogs were catheterized, and DNA was delivered to coronary arteries via a porous perfusion balloon system. Luciferase expression was measured 3-5 days after the procedure, when the dogs were killed. Luciferase activity in control arteries (n = 12) was no higher than average background activity. Eight of 12 transfected arteries exhibited gene expression, averaging 4.3 +/- 2.1 pg luciferase (p less than 0.01, transfected versus control arteries). In addition, the ability to transfect DNA into femoral arteries without a transfection vehicle was tested. Five dogs were subjected to surgical transfection attempts in their femoral arteries with either DNA alone or DNA plus liposomes. Luciferase was expressed in all 10 femoral arteries; those treated with DNA alone expressed 35.6 +/- 8 pg luciferase, and those treated with DNA plus liposomes expressed 42.3 +/- 14 pg luciferase (p = 0.70). These results demonstrate the use of a percutaneous catheter to achieve gene transfer and expression in coronary arteries of intact dogs and suggest that the efficiency of intra-arterial gene transfer may be similar whether or not a transfection vehicle is used.     

Transient transfection of the enteric parasite Entamoeba histolytica and expression of firefly luciferase.

Development of DNA-mediated transfection in Entamoeba histolytica will facilitate basic research toward the control of this protozoan parasite. A transient transfection system was established by using the firefly luciferase gene ligated to the 5' and 3' flanking regions of the amebic hgl1 gene. The optimal construct tested encoded an hgl1-luciferase fusion protein and contained 1 kb of 5' flanking sequence with 16 bases of coding sequence from the hgl1 gene ligated in-frame to the luciferase start codon and 2.3 kb of 3' flanking sequence from hgl1 ligated 3' to the luciferase stop codon. Optimal electroporation conditions in strain HM-1:IMSS trophozoites when using this construct were 500 microF and 500 V/cm, which resulted in luciferase activity up to 5000-fold above background 9-12 hr after electroporation. Constructs that contained the luciferase gene without amebic flanking sequences or that contained a simian virus 40 promoter, enhancer, and polyadenylylation signal produced only background levels of luciferase activity. The ability to introduce and express genes in amebae will now permit a genetic analysis of the virulence of this organism, which remains a serious threat to world health.     

Receptor-mediated gene transfer into macrophages.

Gene transfer systems targeting various receptors have been developed to introduce functional genes into cells in culture and into intact animals. A synthetic molecular conjugate, consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor, was constructed and complexed to expression plasmids containing either the Photinus pyralis luciferase or Escherichia coli beta-galactosidase (lacZ) reporter genes. The DNA complexes were used to transfect murine macrophages isolated from peritoneal exudates in vitro. Luciferase and beta-galactosidase activity was found in transfected cells in culture, whereas complexes consisting of an irrelevant plasmid bound to mannosylated polylysine or the expression plasmid bound to galactosylated polylysine resulted in no detectable transgene expression. Gene transfer was inhibited by the addition of excess mannosylated bovine serum albumin to the culture medium before transfection. Reporter genes were also transferred into macrophages residing in the spleen and liver of adult animals using this system. Luciferase activity was maximal at 4 days after transfection and decreased to lower levels by 16 days. Transgene expression conformed to the distribution of cells that had nonspecific esterase, a cytochemical marker for macrophages. Thus, this system can be used to introduce functional genes into macrophages and may be an approach to the treatment of storage diseases that affect the reticuloendothelial system.     

Targeted gene transfer to human hematopoietic progenitor cell lines through the c-kit receptor

In this report, we describe a novel gene therapy approach for hematopoietic stem/progenitor cells using a specific receptor-mediated gene transfection procedure to target c-kit+ cell lines. The vector consists of plasmid DNA containing a luciferase reporter gene that is condensed by electrostatic forces with polylysine (PL) covalently linked to streptavidin (binds biotinylated ligand) and PL covalently linked to adenovirus (AD; to achieve endosomal lysis) with the final addition of biotinylated steel factor (SLF-biotin). Targeted transfection of growth factor-dependent hematopoietic progenitor cell lines that express c-kit showed specific luciferase gene expression over cell lines that did not express c-kit. This effect was dependent on the dose of SLF-biotin and was competed by excess SLF or with monoclonal antibodies that recognize c-kit and block the binding of SLF to its receptor. Maximum transfection efficiency (> 90%) requires a 2- hour incubation period of the vector with the cells, and maximum gene expression occurred 30 hours later. Removal of the endosomalytic agent, AD, from the vector resulted in the loss of gene expression. Vector targeting was versatile and could be changed by the addition of other biotinylated ligands. In principle, this vector should be broadly applicable to deliver genes to hematopoietic stem/progenitor cells in vitro and in vivo.     

Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells.

Most current gene transfer methods function satisfactorily in specialized systems involving established cell lines but are often not applicable with nonadherent, primary hematopoietic cells, which are notoriously difficult to transfect. To approach this problem, we have investigated an alternative method of gene transfer, "transferrinfection," in which DNA complexed to transferrin-polycation conjugates is introduced into cells by receptor-mediated endocytosis [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We show here that transferrin-polylysine and transferrin-protamine, when complexed to plasmid DNA containing a luciferase reporter gene, is efficiently bound and moved into avian erythroblasts by endocytosis. Successful transfer and expression of the luciferase reporter gene depends on specific interaction of the transferrin-polylysine-DNA complex with the transferrin receptor and occurs in a significant fraction (greater than 95%) of the cells. Gene transfer efficiency by transferrinfection is lower than with an optimized DEAE-dextran transfection method but reaches similar efficiencies when the cells are treated with chloroquine. Because the procedure in the absence of chloroquine is completely nontoxic to cells, a constant expression level of transferred genes may be maintained by repeated additions of transferrin-polylysine-DNA complex. In addition, the usefulness of transferrinfection for gene transfer into primary hematopoietic cells is demonstrated.     

Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies

Monoclonal antibodies (MoAb) were produced against both salivary gland sporozoites (SGS) and oocyst sporozoites (OS) of Plasmodium gallinaceum, an avian malaria parasite. By indirect immunofluorescence, all of the MoAbs reacted with both SGS and OS of P. gallinaceum and two of the MoAbs cross-reacted weakly with P. berghei sporozoites. None of the MoAbs reacted with sporozoites of six additional species of mammalian plasmodia. In Western blot analysis of extracts of either SGS or OS of P. gallinaceum, these MoAbs identified two polypeptides with molecular weights of approximately 76,000 and 64,000 D. The results of a MoAb inhibition of binding assay and a two-site one-antibody immunoradiometric assay indicate that the circumsporozoite protein of P. gallinaceum, like those of mammalian malaria parasites, contains a repetitive immunodominant epitope. Two of the anti-P. gallinaceum MoAbs were tested in a sporozoite neutralization assay and decreased, but did not abolish, the infectivity of sporozoites for chickens, indicating that the polypeptide of P. gallinaceum identified by immunoblot is probably the protective antigen.     

A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum

Genomic information is rapidly accumulating for the human malaria pathogen, Plasmodium falciparum. Our ability to perform genetic manipulations to understand Plasmodium gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of P. falciparum. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes BSD, encoding blasticidin S deaminase of Aspergillus terreus, and NEO, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (HRPIII) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and BSD or NEO expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the P. falciparum genome.     

Identification of a conserved Plasmodium falciparum var gene implicated in malaria in pregnancy

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is a highly polymorphic class of variant surface antigens encoded by var genes that play an important role in malaria pathogenesis. This report describes the unexpected finding that 1 of the var genes encoding a PfEMP1 variant that binds to the host receptor chondroitin sulfate A (CSA) and is implicated in malaria in pregnancy is well conserved among P. falciparum isolates worldwide. The N-terminal domains of this PfEMP1 variant are especially highly conserved, whereas the functional CSA binding domain is more variable. Analysis of var gene expression in placental parasites from primigravid women in Malawi did not support a role for this conserved gene in placental infection but identified a second commonly occurring var gene. These results indicate the need for reevaluation of previous assumptions of a minimal overlap between var gene repertoires from different parasite isolates.     

Expression and assembly of functional bacterial luciferase in plants

The luxA and luxB structural genes of Vibrio harveyi luciferase [alkanal,reduced FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] were introduced into a plant expression vector and transferred into tobacco and carrot cells by Agrobacterium-mediated or direct DNA transformation. Simultaneous expression of the luxA and luxB genes was monitored by protein immunoblot analysis. Luciferase-mediated light emission provided evidence for the assembly of the two protein subunits into a functional dimeric enzyme in plant protoplasts, in transformed calli, and in leaves of transformed plants. Bacterial luciferase may provide a useful marker-gene system for the quantitative assay of coordinate gene expression in transgenic plants.     

Inhibitor RNA blocks the protein translation mediated by hepatitis C virus internal ribosome entry site in vivo

AIM:To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA(IRNA) on gene expression mediated by HCV IRES in vivo.METHODS:By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5′ untranslated region (5′UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5′UTR-Iuc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons, pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-β Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope.RESULTS: Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48h after transfection, the expression level of reportor gene descreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24h after transfection and the highest inhibitory activity was 80% by 72h, and the inhibitory activity was not increased until 7d after transfection.CONCLUSION:IRNA can inhibit HCV IRES mediated gene expression in vivo.     

恶性疟原虫己糖转运体编码基因的体外扩增、克隆及表达

[目的 ]体外扩增、克隆和表达恶性疟原虫海南分离株的己糖转运体 (PfHT1)基因 ,为研究其保护性免疫创造条件。 [方法 ]恶性疟原虫FCC1/HN株的体外培养 ;碱裂解法提取基因组DNA ;PfHT1基因的PCR扩增、克隆 ;脂质体介导法转染HEPG2细胞株及真核表达。 [结果 ]从恶性疟原虫海南分离株基因组DNA中扩增出特异性的编码PfHT1的基因序列 ,片段大小为 15 16bp ;成功构建 pN3 HT1真核表达重组质粒并在肝癌细胞HEPG2中稳定表达。 [结论 ]体外成功扩增、克隆恶性疟原虫PfHT1编码序列 ;重组质粒转染成功并获稳定表达融合蛋白的 pN3 HT1/HEPG2细胞株     

A DNA delivery system containing listeriolys in Oresults in enhanced hepatocytedirected gene expression

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The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC(50) (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.