Stephen M. Beverley

Stable transfection of the Human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

Molecular Evolution in Drosophila and the Higher Diptera II. A Time Scale for Fly Evolution

In this paper, we examine first the steadiness of the rate of evolutionary change in a larval hemolymph protein, LHP, in numerousDrosophila species. We estimated amino acid sequence divergence from immunological distances measured with the quantitative microcomplement fixation technique. Using tests not depending on knowledge of absolute times of divergence, we estimated the variance of the rate of evolutionary change to be at least 4 times as large as that for a process resembling radioactive decay. Thus, the rate of evolution of this protein is as uniform as that of vertebrate proteins. Our analysis indicates no acceleration of protein evolution in the lineages leading to Hawaiian drosophilines. Second, we give an explicit description of a procedure for calculating the absolute value of the mean rate of evolutionary change in this protein. This procedure is suggested for general use in calculating absolute rates of molecular evolution. The mean rate of evolution of LHP is about 1.2 immunological distance units per million years, which probably coreesponds to a unit evolutionary period of 4 million years; LHP thus evolves at a rate comparable to that of mammalian hemoglobins. Finally, we utilize the calibrated rate of LHP evolution to derive a time scale of evolution in the Drosophilidae and higher Diptera.SummaryIn this paper, we examine first the steadiness of the rate of evolutionary change in a larval hemolymph protein, LHP, in numerousDrosophila species. We estimated amino acid sequence divergence from immunological distances measured with the quantitative microcomplement fixation technique. Using tests not depending on knowledge of absolute times of divergence, we estimated the variance of the rate of evolutionary change to be at least 4 times as large as that for a process resembling radioactive decay. Thus, the rate of evolution of this protein is as uniform as that of vertebrate proteins. Our analysis indicates no acceleration of protein evolution in the lineages leading to Hawaiian drosophilines. Second, we give an explicit description of a procedure for calculating the absolute value of the mean rate of evolutionary change in this protein. This procedure is suggested for general use in calculating absolute rates of molecular evolution. The mean rate of evolution of LHP is about 1.2 immunological distance units per million years, which probably coreesponds to a unit evolutionary period of 4 million years; LHP thus evolves at a rate comparable to that of mammalian hemoglobins. Finally, we utilize the calibrated rate of LHP evolution to derive a time scale of evolution in the Drosophilidae and higher Diptera.

Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites

Infection with Leishmania major induces a protective immune response and long-term resistance to reinfection, which is thought to depend upon persistent parasites. Here we demonstrate that although effector CD4+ T cells are lost in the absence of parasites, central memory CD4+ T cells are maintained. Upon secondary infection, these central memory T cells become tissue-homing effector T cells and mediate protection. Thus, immunity to L. major is mediated by at least two distinct populations of CD4+ T cells: short-lived pathogen-dependent effector cells and long-lived pathogen-independent central memory cells. These data suggest that central memory T cells should be the targets for nonlive vaccines against infectious diseases requiring cell-mediated immunity.

USE OF THE GREEN FLUORESCENT PROTEIN AS A MARKER IN TRANSFECTED LEISHMANIA

We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP+ transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FACS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg- R2D2 mutant of L. donovani. The LPG1(GFP) fusion was localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.

Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania

Approaches which eliminate mRNA expression directly are ideally suited for reverse genetics applications in eukaryotic microbes which are asexual diploids, such as the protozoan parasite Leishmania. RNA interference (RNAi) approaches have been successful in many species, including the related parasite Trypanosoma brucei. For RNAi tests in Leishmania, we developed improved protocols for transient and stable DNA transfection, attaining efficiencies of up to 25 and 3%, respectively. This facilitated RNAi tests at the alpha-tubulin locus, whose inhibition gives a strong lethal phenotype in trypanosomatids. However, transient or stable transfection of DNAs encoding mRNAs for an alpha-tubulin stem-loop construct and GFP to monitor transfection resulted in no effect on parasite morphology, growth or tubulin expression in Leishmania major or L. donovani. Transient transfection of a 24-nucleotide double-stranded alpha-tubulin siRNA also had no effect. Similar results were obtained in studies targeting an introduced GFP gene with a GFP stem-loop construct. These data suggest that typical RNAi strategies may not work effectively in Leishmania, and raise the possibility that Leishmania is naturally deficient for RNAi activity, like Saccharomyces cerevisae. The implications to parasite biology, gene amplification, and genetic analysis are discussed.

The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts

The abundant cell surface glycolipid lipophosphoglycan (LPG) was implicated in many steps of the Leishmania infectious cycle by biochemical tests. The presence of other abundant surface or secreted glycoconjugates sharing LPG domains, however, has led to uncertainty about the relative contribution of LPG in vivo. Here we used an Leishmania major lpg1- mutant, which lacks LPG alone and shows attenuated virulence, to dissect the role of LPG in the establishment of macrophage infections in vivo. lpg1- was highly susceptible to human complement, had lost the ability to inhibit phagolysosomal fusion transiently, and was oxidant sensitive. Studies of mouse mutants defective in relevant defense mechanisms confirmed the role of LPG in oxidant resistance but called into question the importance of transient inhibition of phagolysosomal fusion for Leishmania macrophage survival. Moreover, the limited lytic activity of mouse complement appears to be an ineffective pathogen defense mechanism in vitro and in vivo, unlike human hosts. In contrast, lpg1- parasites bound C3b and resisted low pH and proteases normally, entered macrophages efficiently and silently, and continued to inhibit host-signaling pathways. These studies illustrate the value of mechanistic approaches focusing on both parasite and host defense pathways in dissecting the specific biological roles of complex virulence factors such as LPG.

Intergenic region typing ( IRT) : A rapid molecular approach to the characterization and evolution of Leishmania

In the New World, Leishmania of the Viannia subgenus cause both cutaneous and mucocutaneous disease. These parasites show considerable intra-species genetic diversity and variation, which complicates taxonomic classification and epidemiology. We have used the variability of the transcribed noncoding regions between the small and large subunit rRNA genes to examine relationships in this group. In a method termed intergenic region typing (IRT), PCR amplification products were obtained for the rapidly evolving 1-1.2-kb internal transcribed spacers (ITS) between the SSU and LSU rRNAs, from 50 parasites isolated from different hosts and geographic areas. Amplified DNAs were digested with 10 different enzymes, and fragment patterns compared after acrylamide gel electrophoresis. High levels of intra- and inter-specific variation were observed, and quantitative similarity comparisons were used to associate different lineages. A complex evolutionary tree was obtained. Some species formed tight clusters (L. equatorensis, L. panamensis, L. guyanensis, L. shawi), while L. braziliensis was highly polymorphic and L. naiffi showed intraspecific distances comparable to the largest obtained within all Viannia. L. colombiensis, L. equatorensis and L. lainsoni clearly represent distinct lineages. Good agreement was obtained with molecular trees based upon isoenzyme or mini-exon repeat sequence comparisons. Overall, IRT appears to be a superb method for epidemiological and taxonomic studies of Leishmania, being sensitive, rapid and quantitative while simultaneously revealing considerable molecular diversity. IRT could also be applied to other nonconserved intergenic regions, including those separating protein-coding genes.

Demonstration of Genetic Exchange During Cyclical Development of Leishmania in the Sand Fly Vector

Genetic exchange has not been shown to be a mechanism underlying the extensive diversity of Leishmania parasites. We report here evidence that the invertebrate stages of Leishmania are capable of having a sexual cycle consistent with a meiotic process like that described for African trypanosomes. Hybrid progeny were generated that bore full genomic complements from both parents, but kinetoplast DNA maxicircles from one parent. Mating occurred only in the sand fly vector, and hybrids were transmitted to the mammalian host by sand fly bite. Genetic exchange likely contributes to phenotypic diversity in natural populations, and analysis of hybrid progeny will be useful for positional cloning of the genes controlling traits such as virulence, tissue tropism, and drug resistance.

Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis.

An RNA virus of a parasite binds to human Toll-like receptor 3 and modulates host immune responses to the parasite. Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus–1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

Migratory dermal dendritic cells act as rapid sensors of protozoan parasites

Dendritic cells (DC), including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC) are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC) is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Gαi protein-coupled receptor–dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.