On the introduction of genetically modified Leishmania outside the laboratory.
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Animals↗
Biomedical subjects
S M Beverley
Publications and source records attributed to S M Beverley.
The PGPA gene of Leishmania major mediates antimony (SbIII) resistance by decreasing influx and not by increasing efflux.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism.
Molecular evolutionary relationships within the protozoan order Kinetoplastida were deduced from comparisons of the nuclear small and large subunit ribosomal RNA (rRNA) gene sequences. These studies show that relationships among the trypanosomatid protozoans differ from those previously proposed from studies of organismal characteristics or mitochondrial rRNAs. The genera Leishmania, Endotrypanum, Leptomonas, and Crithidia form a closely related group, which shows progressively more distant relationships to Phytomonas and Blastocrithidia, Trypanosoma cruzi, and lastly Trypanosoma brucei. The rooting of the trypanosomatid tree was accomplished by using Bodo caudatus (family Bodonidae) as an outgroup, a status confirmed by molecular comparisons with other eukaryotes. The nuclear rRNA tree agrees well with data obtained from comparisons of other nuclear genes. Differences with the proposed mitochondrial rRNA tree probably reflect the lack of a suitable outgroup for this tree, as the topologies are otherwise similar. Small subunit rRNA divergences within the trypanosomatids are large, approaching those among plants and animals, which underscores the evolutionary antiquity of the group. Analysis of the distribution of different parasitic life-styles of these species in conjunction with a probable timing of evolutionary divergences suggests that vertebrate parasitism arose multiple times in the trypanosomatids.
Animals↗
Isolation of virulence genes directing surface glycosyl-phosphatidylinositol synthesis by functional complementation of Leishmania.
Trypanosomatid parasites of the genus Leishmania cause a spectrum of widespread tropical diseases. In the vertebrate host they reside within the macrophage phagolysosome; however, the mechanisms employed in this remarkable survival strategy are not well understood. Recent advances in the molecular genetics of these parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant lipophosphoglycan, an abundant glycosyl-phosphatidylinositol-anchored polysaccharide. LPG1, the gene product identified by complementation of the R2D2 mutant, appears to be a glycosyltransferase responsible for the addition of galactofuranosyl residues to the nascent lipophosphoglycan chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism.
Agglutination↗
Shuttle cosmid vectors for the trypanosomatid parasite Leishmania.
We have developed two shuttle cosmid vectors for the trypanosomatid protozoan parasite Leishmania. Cosmids cLHYG and cLNEO contain hyg and neo markers, conferring resistance to hygromycin B and G418, respectively, replicate extrachromosomally after transfection into promastigotes, and bear a unique BamHI cloning site. To ensure the representation of telomeric sequences, which represent about 5% of the Leishmania genome, random insert DNAs were prepared by shearing followed by blunt-end ligation with BamHI adapters. Representative genomic libraries from Leishmania species representing the four major pathogenic complexes were prepared using cosmid cLHYG. The cosmid libraries were efficiently transfected into Leishmania, and individual cosmids were readily recovered by transformation back into Escherichia coli. The relatively small size of the Leishmania genome (50 Mb) combined with the capacity and transfection efficiency of these cosmid libraries (> 1000 Leishmania transfectants/plate) suggests the feasibility of functional genetic complementation in this parasite.
Animals↗
Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.
We attempted to generate homozygous dhfr-ts (dihydrofolate reductase-thymidylate synthase) knockouts in virulent Leishmania major, an asexual diploid protozoan parasite. Transfection of a neo (neomycin phosphotransferase) targeting fragment yielded heterozygous replacement lines with high efficiency. However, second transfections with a hyg (hygromycin B phosphotransferase) targeting fragment in the presence of metabolites shown to rescue homozygous knockouts in attenuated Leishmania did not yield the expected dhfr-ts- thymidine auxotrophs obtained previously with attenuated lines. Molecular karyotype, Southern blot, and flow cytometric DNA content analysis of clonal transfectants revealed three classes: (i) genomic tetraploids, containing two wild-type dhfr-ts chromosomes and one neo and one hyg replacement chromosome; (ii) aneuploid trisomic lines with one wild-type dhfr-ts and one neo and one hyg replacement chromosome; (iii) diploids bearing homologous integration of the targeting fragment without replacement. Aneuploid and tetraploid lines predominated. This confirms the common impression that natural populations of Leishmania are often aneuploid. The remarkable ability of these parasites to undergo and tolerate changes in chromosome number suggests a general method for testing whether genes are essential for growth in vitro, as the ability of Leishmania to simultaneously undergo homologous gene replacement while retaining wild-type genes by increasing chromosome number provides a diagnostic and positive experimental result. Our results show that virulent Leishmania require at least one copy of dhfr-ts and argue that DHFR-TS plays an unanticipated role in addition to its role in the de novo synthesis of thymidine. These results also have implications for genetic tests of the organization of Leishmania populations.
Alleles↗
Leishmania mexicana promastigotes induce cytotoxic T lymphocytes in vivo that do not recognize infected macrophages.
The question is addressed whether antigens of Leishmania, a parasite residing in the endosomal compartment of macrophages, can be presented in the context of major histocompatibility complex class I molecules. We used E. coli beta-galactosidase as a model antigen which can be expressed in high levels in L. mexicana promastigotes (L. mexicana-gal). Infection of BALB/c mice with L. mexicana-gal induces beta-galactosidase-specific cytotoxic T cells (CTL), which can be isolated using a beta-galactosidase-expressing mastocytoma line as an antigen-presenting cell. These CTL recognize epitopes of beta-galactosidase in the context of H-2Kd; however, they do not recognize L. mexicana-gal-infected macrophages even after killing of the intracellular amastigotes by drug treatment or macrophage activation by lymphokines, although class I-peptide interaction and the presentation of endogenously produced antigens is normal. It is concluded that parasite antigens can induce a CTL response in vivo but that these CTL cannot recognize infected macrophages because the relevant epitopes cannot gain access to class I molecules. The effect of priming in vivo may be explained by the well-known but ill-understood phenomenon of cross-priming.
Animals↗
Antigens targeted to the Leishmania phagolysosome are processed for CD4+ T cell recognition.
Processing of antigen for recognition by class II-restricted CD4+ T cells occurs within acidic compartments of the antigen-presenting cell. The exact nature of this compartment has yet to be precisely defined, however, but may vary depending upon the cell type studied and the antigen used. The acidic compartments of macrophages are also responsible for the degradation of ingested micro-organisms and play host to others which are adapted to an intracellular existence. To determine whether the phagolysosome (PL) formed in activated macrophages after ingestion of Leishmania parasites is also a site for entry of antigen into the class II presentation pathway, we have used the approach of genetic transformation. Hence, Leishmania were transfected with the genes for the protein antigens ovalbumin (OVA) and beta-galactosidase (beta-gal) and after infection were able to deliver these antigens specifically into the PL. Delivery of antigen to this site resulted in the ability of infected macrophages to present these antigens to antigen-specific CD4+ T cells. After taking into account the absolute levels of antigen uptake by macrophages, a 4-h processing period for OVA delivered by this or a soluble route led to equivalent levels of T cell activation. Unlike macrophages pulsed with soluble OVA, those with PL-targeted OVA still retained the ability to stimulate T cells after a 24-h processing period. This enhanced lifespan of antigen in macrophages corresponded to the kinetics of degradation of the parasite, suggesting slow release of antigen into the processing pathway. beta-gal presentation from the PL was tenfold less efficient under the same conditions. In addition to providing the first information on antigen processing in a protozoan PL, these studies highlight the usefulness of genetically transformed parasites for these types of studies.
Animals↗
Demonstration of circularization of herpes simplex virus DNA following infection using pulsed field gel electrophoresis.
Studies of the configuration of intracellular herpes simplex virus DNA have been limited by the inability of restriction enzyme analyses to distinguish circular DNA from other configurations. To address this issue, we used pulsed-field gel electrophoresis of virus-infected, cycloheximide-treated cells and detected accumulation of viral DNA that failed to migrate out of the sample wells of the gels. This DNA lacked terminal restriction fragments and could be converted to unit-length linear species by gamma-irradiation, demonstrating the circularization of viral DNA following infection.
Animals↗
Two more independent selectable markers for stable transfection of Leishmania.
Genetic transformation of Leishmania has relied upon two exogenous selectable markers, neo and hyg, encoding resistance to G418 and hygromycin B respectively. There is a need for multiple independent selectable markers, since Leishmania is diploid and experimental sexual crosses are not currently feasible. Here we report on the development of two additional markers: pac, conferring resistance to the glycopeptide antibiotic puromycin, and phleo, conferring resistance to the DNA-binding drug phleomycin. We constructed a set of four analogous shuttle vectors with these four markers, using DNA segments flanking the Leishmania major H region hmtxr gene to provide information required for expression. These constructs (pHM-NEO, pHM-HYG, pHM-PAC and pHM-PHLEO) were successfully transfected into L. major, mostly with efficiencies comparable to those observed with previous DHFR-TS-based neo and hyg-containing constructs. The exception was pHM-PHLEO, which transfected 30-fold less efficiently; this may be related to the nonenzymatic mechanism of resistance encoded by phleo. All four constructs were shown to replicate extra-chromosomally. Stable transfectants bearing all paired combinations of pHM constructs were obtained by a second round of transfection. These data show that the four markers are functionally independent and in conjunction with the Leishmania N-acetylglucosaminyl transferase gene, brings the number of selectable markers available in Leishmania to five.
Animals↗
Coupling of poly(A) site selection and trans-splicing in Leishmania.
Intergenic regions of polycistronic pre-mRNAs of trypanosomatid protozoans are the sites of two processing reactions: polyadenylation of the upstream gene and trans-splicing of the capped miniexon to the downstream gene. Their close proximity and the lack of consensus motifs at poly(A) sites led us to test whether poly(A) site selection is governed by the location of the downstream splice acceptor in the DHFR-TS locus of Leishmania major. Whenever the position of the downstream splice site was altered, the poly(A) site was shifted 400-500 nucleotides upstream of the new splice site. In contrast, when the wild-type poly(A) site was eliminated, the downstream splice site was unaffected, and polyadenylation was maintained 200-500 nucleotides upstream of the splice site. In a second set of experiments, T7 RNA polymerase expressed in Leishmania was used to direct the synthesis of artificial pre-RNAs in vivo whose expression was found to require the presence of a downstream splice acceptor. We conclude that poly(A) site selection in Leishmania is specified by the position of the downstream splice acceptor and propose a scanning model for poly(A) site selection after splice site recognition.
Animals↗
Transfection of Leishmania and Trypanosoma brucei by electroporation.
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Animals↗
Evolution and expression of the Leishmania surface proteinase (gp63) gene locus.
The Leishmania surface proteinase, gp63, is the most abundant, surface-exposed protein on the promastigote form of the parasite. It is the product of a multigene family that, in some Leishmania species, shows marked heterogeneity among its members. The differential expression of structurally distinct gp63 genes shows circumstantial correlation with the differential processing and localization of the protein in the intracellular, amastigote form of Leishmania mexicana. The recent cloning and sequencing of a homologous gene in the monoxenous trypanosomatid, Crithidia fasciculata, provides a reference sequence for comparison with ten Leishmania gp63 genes sequenced to date. The amino terminal and the carboxy terminal regions of the protein sequences suggest different evolutionary histories within some of the genes. The evolutionary significance of the structure, organization, and regulation of the gp63 genes from different Leishmania species is described and speculated upon.
Amino Acid Sequence↗
A member of the aldoketo reductase family confers methotrexate resistance in Leishmania.
Methotrexate (MTX)-resistant mutants of the parasitic protozoan Leishmania have been used as models for the mechanism and genetic basis of drug resistance in trypanosomatids and other cells. Three resistance mechanisms to MTX, a dihydrofolate reductase inhibitor, have been described in Leishmania: decreased uptake and accumulation of MTX via the folate/MTX transporter, amplification and overexpression of the dihydrofolate reductase-thymidylate synthase gene, and extrachromosomal amplification of H region DNA. We have now identified hmtxr as the H region gene conferring MTX resistance using a transfection-based approach. Data base searches show that the predicted HMTXr protein is related to members of the polyol dehydrogenase/carbonyl reductase family of aldoketo reductases, whose substrates include polyols, quinones, steroids, prostaglandins, fatty acids, and pterins. We therefore propose that HMTXr is also an oxidoreductase and suggest several biochemical mechanisms of resistance in Leishmania that could be exploited in the design of parasite-specific inhibitors.
Amino Acid Sequence↗
Leishmania tarentolae taxonomic relatedness inferred from phylogenetic analysis of the small subunit ribosomal RNA gene.
The sequence of the Leishmania tarentolae SSU rRNA (small subunit or 18S rRNA) gene was completely determined from 2 different strains and used to determine phylogenetic relationships between this organism and other trypanosomatids. Extensive structural similarities were observed between L. tarentolae and mammalian leishmanias the SSU rRNA. Phylogenetic reconstructions, using distance matrix or parsimony methods, showed large evolutionary distances between trypanosomes, either African and American, and L. tarentolae. Further analysis using intergenic rDNA spacer (IGS) sequences as probes in dot blot experiments confirmed the results obtained with the SSU rDNA comparisons. The data presented here clearly indicate that L. tarentolae is closely related to the mammalian parasite Leishmania donovani and highly divergent from trypanosomes.
Animals↗
Thymidine kinase as a negative selectable marker in Leishmania major.
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Animals↗
Stable amplification of a linear extrachromosomal DNA in mycophenolic acid-resistant Leishmania donovani.
Pulsed field gel electrophoretic analysis of chromosomes of MPA100 cells, a strain of Leishmania donovani that possesses an approx. 15-fold amplified IMP dehydrogenase (IMPDH) gene copy number, revealed a new 280-kb extrachromosomal DNA, IMPDH-280, that was not present in wild type parental cells. Southern blots of these pulsed field gels revealed that the vast majority of the amplified impdh genes were localized on IMPDH-280. In addition to the 700-kb wild type chromosome, the impdh probe also recognized a 740-kb chromosome in the MPA100 genome. The pulse time-dependent relative mobility of IMPDH-280 in pulsed field gels, the failure of limited gamma-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to lambda-exonuclease digestion, demonstrated that IMPDH-280 was a linear molecule. IMPDH-280 was also recognized by a telomere probe but not by fragments derived from amplified DNAs found in other drug-resistant Leishmania. IMPDH-280 and the drug resistance phenotype remained stable when MPA100 cells were propagated in the absence of drug for 2 years. The appearance of IMPDH-280 in MPA100 cells represents one of the first examples of an amplification of a linear extrachromosomal DNA element mediating drug resistance in Leishmania and the first instance of a linear DNA amplification that is stable in the absence of selective pressure.
Animals↗
Loss of the GP46/M-2 surface membrane glycoprotein gene family in the Leishmania braziliensis complex.
Immunization with the GP46/M-2 membrane glycoprotein of Leishmania amazonensis has been shown to induce a protective immune response against infection. We have surveyed a variety of trypanosomatid species and genera for the presence and expression of this gene family, information that will be relevant to future vaccine studies against leishmaniasis. Molecular karyotype analysis revealed the presence of GP46/M-2 genes in all members of the Leishmania mexicana complex, Leishmania major, Leishmania donovani, Leishmania tarentolae, and Crithidia fasciculata. In contrast, DNAs from species of the Leishmania braziliensis complex (L. braziliensis, Leishmania guyanensis, and Leishmania panamensis) failed to hybridize to GP46/M-2 probes. Western blot analyses with several polyclonal antisera against the GP46/M-2 protein revealed protein expression in L. major and L. donovani, but not L. panamensis or L. braziliensis. Phylogenetic analysis suggests that a loss of the GP46A gene family occurred following separation of the L. braziliensis complex, prior to speciation events within this complex. These data indicate that GP46/M-2 membrane glycoprotein may not be critical to parasite survival, but may play an ancillary role during the developmental cycle.
Animals↗