Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages.

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.

Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis.

We show here that maintenance of Leishmania infections with Leishmania mexicana complex parasites (Leishmania amazonensis and Leishmania pifanoi) is impaired in the absence of circulating antibody. In these studies, we used mice genetically altered to contain no circulating antibody, with and without functional B cells. This experimental design allowed us to rule out a critical role for B cell antigen presentation in Leishmania pathogenesis. In addition, we show that mice lacking the common gamma chain of Fc receptors (FcgammaRI, FcepsilonRI, and FcgammaRIII) are similarly refractory to infection with these parasites. These observations establish a critical role for antibody in the pathogenesis associated with infection by members of the L. mexicana complex.

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

Biochemical characterization of the protective membrane glycoprotein GP46/M-2 of Leishmania amazonensis.

Biochemical features of the immunologically protective, membrane glycoprotein GP46/M-2 of Leishmania amazonensis have been investigated. The protein appears to have a single carbohydrate side chain of approximately 3 kDa, representing 7% of the mass of the mature GP46/M-2 protein. Experiments removing this carbohydrate side chain from GP46/M-2 indicate that the carbohydrate is not involved in the epitope recognized by the monoclonal antibody, M-2. As this monoclonal antibody recognizes a species-specific epitope, these data suggest that this determinant is defined by the polypeptide portion of the molecule. Studies employing the VSG-lipase as well as anti-CRD antibody clearly indicate that the molecule is anchored to the surface membrane of the promastigote via a phosphatidylinositol-linked lipid anchor. Neither the carbohydrate side chain nor the lipid anchor appear to be responsible for the apparent refractoriness of this protein to protease digestion, suggesting that properties of the polypeptide itself may be responsible. These data are discussed in terms of recent DNA-derived protein sequence of the GP46/M-2.

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.

Evolution of nuclear DNA and the occurrence of sequences related to new small chromosomal DNAs in the trypanosomatid genus Endotrypanum.

Comparisons of nuclear DNA restriction fragment patterns were used to examine the evolutionary relatedness among 17 strains previously identified as Endotrypanum, a trypanosomatid parasite of sloths. Fragments were obtained with 6 restriction enzymes and analyzed by Southern blotting with hybridization probes from three loci. An estimate of the percent nucleotide sequence divergence among strains, delta, was calculated and used to construct molecular evolutionary trees. The 17 isolates fell into four distinct groups, one of which (group D) showed no more relationship to groups A-C than it did to other genera (Leishmania, Crithidia, Leptomonas, Trypanosoma), being too distant to be resolved with this method. These and other data suggest that group D may not actually be Endotrypanum. Molecular karyotype analysis revealed considerable variation among the chromosomes of these strains. One strain (LV88, group B) contained a linear 70-kb chromosome not evident in other isolates. Hybridization probes specific for this chromosome (LV88-70) were developed and revealed that related sequences were present at high levels in group B isolates and low levels in group A isolates, although a complex hybridization pattern was evident. Sequences related to LV88-70 were not present in groups C and D, nor in Leishmania major, showing that this DNA has a disjunct distribution which curiously parallels that of virus-like particles present in these isolates.

Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture.

Inability to culture the disease-producing amastigote form of Leishmania has greatly hampered its study. We have biochemically characterized an axenically cultured amastigote-like form of Leishmania pifanoi. This form closely resembles amastigotes in proteinase, ribonuclease, adenine deaminase and peroxidase activity. It also exhibits comparable rates of growth, transformation, synthesis of DNA, RNA and protein, and metabolism of glucose and linoleic acid. It is distinct from promastigotes in these characteristics. The expression of the genes for beta-tubulin and the P100/11E reductase is developmentally regulated in this axenic form as in amastigotes. These results, combined with previous demonstrations of amastigote morphology and antigenicity in the culture form, confirm that Leishmania amastigotes have been successfully propagated in axenic media. This strain should serve as an excellent model for the study of amastigote biochemistry, pharmacology and immunology, and the molecular genetics of the transformation between amastigote and promastigote forms.

Identification of two distinct cysteine proteinase genes of Leishmania pifanoi axenic amastigotes using the polymerase chain reaction.

A developmentally regulated cysteine proteinase associated with an unique lysosomal organelle, the megasome, has been described for the intracellular amastigotes of the Leishmania mexicana complex; this proteinase appears to be important in the survival of the parasite. Degenerate primers encoding the active sites residues have been used to amplify cysteine proteinase cDNA sequences from axenically cultured amastigotes of Leishmania pifanoi, a member of the L. mexicana complex. Based on sequence data, two distinct genes (Lpcys1 and Lpcys2) were identified. Although both genes are preferentially transcribed in the amastigote stage, each is distinct in genomic arrangement and chromosome location, with Lpcys2 showing evidence for the presence of 8-20 tandemly arrayed copies and mRNA levels 10-fold higher than Lpcys1. Related forms of the Lpcys1 and Lpcys2 genes exist in other species of the genus Leishmania, including Leishmania braziliensis, Leishmania major and Leishmania donovani. The protein sequence of an abundant immunoaffinity purified amastigote cysteine proteinase (A-2) is identical to that predicted for the product of Lpcys2; immunofluorescence studies show an intracellular pattern/distribution for the A-2 proteinase consistent with a putative megasomal association. The DNA sequence of a genomic copy of Lpcys2 predicts a C-terminal extension for the proteinase; comparative sequence analyses of the C-terminal extensions found for Trypanosoma cruzi and Trypanosoma brucei reveal the selective conservation of cysteine, as well as proline and glycine residues, suggesting that conservation of folding and secondary structure may be required for biological function.

The biosynthesis, processing, and immunolocalization of Leishmania pifanoi amastigote cysteine proteinases.

Biosynthesis, enzymatic processing, and immunocytochemical localization of an abundant developmentally regulated cysteine proteinase of Leishmania pifanoi, Lpcys2, were investigated employing axenic cultured amastigotes and monoclonal antibodies specifically recognizing either the mature proteinase or the carboxy-terminal extension domain. Pulse labeling and protein sequence data indicated that a 45-kDa precursor is processed to a 40-kDa intermediate, which is further cleaved to generate the 27-kDa mature enzyme and a 15-kDa COOH-terminal domain. Evidence indicates that proteolytic activity is associated with the intermediate form as well as the mature proteinase. Treatment with selected cysteine but not aspartic acid proteinase inhibitors arrested proteolytic processing of Lpcys2 in vivo and inhibited parasite cell division. Electron microscopic immunolocalization of both catalytic and COOH-terminal domains in L. pifanoi and Leishmania amazonensis amastigotes showed intense labeling of megasomes, indicating that cleavage of the COOH-terminal domain probably occurs in the megasome. A low level of the mature proteinase was also associated with the flagellar pocket and plasma membrane; consistent with this observation, low level secretion of Lpcys2 into the culture medium was detected. Lpcys1, a related, less abundant amastigote-specific cysteine proteinase lacking a comparable COOH-terminal domain, was localized to the flagellar pocket and megasomes. Consequently, enzyme sorting to megasomes does not appear to depend upon the COOH-terminal domain; hence this region of Lpcys2 may not be essential for its intracellular targeting.

Cellular trafficking in trypanosomatids: a new target for therapies?

Pathogenic trypanosomatids cause a plethora of diseases marked by the lack of efficient vaccines and therapies. As a consequence, studies are being conducted that are geared towards the understanding of basic mechanisms and various biological aspects of these parasites that might be used as targets for new developments in these areas. One such aspect is the understanding of specific cellular trafficking mechanisms that might be attacked with the intention of disease control. In this paper, we give an overview of our current knowledge of cellular targeting mechanisms in trypanosomatids, with special emphasis on our data related to lysosomal targeting of cysteine proteinases in Leishmania.

Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies.

Five hundred thirty stocks of Leishmania isolated from human and domestic and wild reservoir hosts, representing a wide geographic distribution of endemic foci of American cutaneous (ACL) and visceral leishmaniases (AVL) were characterized and identified at species and/or subspecies levels based on their reactivity to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. This study confirms and extends our preliminary results on the high specificity of some of these monoclonals for the L. braziliensis, L. mexicana, and L. donovani complexes. This study also demonstrates the relative stability of these molecular markers and the general usefulness of the method for parasite identification. Two hundred ninety-two of 420 isolates of ACL were classified as members of the L. braziliensis complex. Two hundred twenty-seven were L. b. braziliensis; these showed the widest geographical distribution (Brazil: Amazonas, Bahia, Ceara, Espirito Santo, Goias, Minas Gerais, Para, Paraiba, Rio de Janeiro, and Sao Paulo; Honduras: Santa Barbara and Yoko; Peru: Ancash, Piura, and Ucayali; and Venezuela: Cojedes, Distrito Federal, Lara, Portuguesa, Vale Hondo, Yaracuy, and Zulia). Forty-one stocks were identified as L. b. guyanensis (from North Brazil: Amazonas, Amapa, Para, and Rondonia). Twenty-one stocks were identified as L. b. panamensis (from Costa Rica: Alajuela, Guanacasten, Limon, Puntarenas, and San Jose; and Honduras: El Paraiso, and Olancho). Out of 128 isolates classified as members of the L. mexicana complex, 74 were differentiated as L. m. amazonensis (from Bolivia; Brazil: Amazonas, Bahia, Ceara, Goias, Maranhao, Mato Grosso do Norte, and Para; Peru: Pasco Forest and Van Humboldt; and Venezuela: Carabobo, Guarico, and Merida). Forty-four stocks were identified as L. m. venezuelensis (from Venezuela: Lara). Six stocks were L. m. mexicana (from Belize; and Mexico: Campeche [corrected] and Quintana Roo, Yucatan). One hundred ten isolates from AVL were identified as L. donovani chagasi (from Brazil: Bahia, Ceara, Maranhao, Minas Gerais, Mato Grosso do Sul, Piaui, Rio de Janeiro, and Sergipe; and Honduras: Valle). The implications of these results with respect to both the clinical and epidemiological data (including the detection of seven unusual characterized stocks) are discussed.

A review of the geographic distribution and epidemiology of leishmaniasis in the New World.

A review of the epidemiologic aspects of the New World leishmaniases, including their known geographic distribution, etiologic agents, zoonotic reservoirs, and insect vectors, based on biological and molecular characterization of Leishmania isolates is presented. Data summarized in this paper on parasite taxonomy and geographic distribution come from our studies of greater than 1,000 New World Leishmania isolates identified by species-specific monoclonal antibodies using an indirect radioimmune binding assay and from scientific literature.

Production of a specific monoclonal antibody for the identification of Leishmania (Leishmania) venezuelensis.

We describe a monoclonal antibody (Mab), V1, specific for Leishmania (Leishmania) venezuelensis. Previous Mabs and DNA probes were not specific for this parasite, and so it was not directly possible to distinguish L. (L.) venezuelensis from other Leishmania species. Immunofluorescent staining using Mabs may be performed on very few parasites, whereas other methods of identification usually require far greater numbers of organisms. L. (L.) venezuelensis frequently dies on subculture. Mab V1 can be used to identify this parasite by indirect immunofluorescence and radioimmunoassay.

Mucocutaneous leishmaniasis in Colombia: Leishmania braziliensis subspecies diversity.

It is generally held that with rare exception Leishmania braziliensis braziliensis is the parasite responsible for the metastatic development of mucocutaneous leishmaniasis in the New World. Yet the infrequency of mucocutaneous disease compared with cutaneous manifestations, and the difficulty of isolating parasites from mucocutaneous lesions have restricted the study of the organisms involved. We here report the biologic, isoenzymatic, and monoclonal antibody specificity characteristic of eight Leishmania isolates obtained from the mucosal lesions of the same number of patients. Individually and collectively, the identifying criteria implicate at least two L. braziliensis subspecies as etiologic agents of mucocutaneous leishmaniasis in Colombia and suggest that a spectrum of intrinsically distinguishable organisms may be involved in this disease form.

Characterization and classification of leishmanial parasites from humans, wild mammals, and sand flies in the Amazon region of Brazil.

Ninety-four leishmanial isolates from the Brazilian Amazon Region (Amapá, Amazonas, Pará, and Rondônia) were identified and classified using specific monoclonal antibodies and an indirect radioimmunoassay (serodeme analysis); eighty-two were also characterized by enzyme electrophoresis (zymodeme analysis), the results of which were subjected to a numerical phenetic analysis. Six isolates from humans (3), Didelphis marsupialis (1), Lutzomyia olmeca nociva (1), and Lu, reducta (1) showed reactivity patterns and isoenzyme profiles similar to those obtained with the Leishmania amazonensis reference strains, and were identified as this species. Eighty-six stocks were classified as members of the L. braziliensis complex; of these, 61 were L. guyanensis or variants, which presented three serodeme subtypes, but whose isoenzyme profiles were all similar to the reference strain. A total of 15 isolates were distinguished as L. braziliensis or variants and were classified into five serodeme subtypes. The isolate from Psychodopugus davisi appeared, from the numerical analysis, to be a distinct parasite species. Ten isolates showed reactivity patterns and isoenzyme profiles similar to those obtained with the L. naiffi reference strain. A parasite isolated from Ps. claustrei appeared to be different from all reference strains by both techniques, and was classified as probably being a new species. The importance of these results with respect to the taxonomic status of the New World Leishmania, and their implications for both clinical and epidemiologic data are discussed.

Brazilian Leishmania stocks phenotypically similar to Leishmania major.

Screening by enzyme electrophoresis of isolates of New World Leishmania from different geographic areas revealed a number of stocks with enzyme profiles different from those produced by reference strains of described subspecies of L. mexicana, L. braziliensis, and L. donovani. Analysis by six enzymes (aspartate aminotransferase; alanine aminotransferase; malate dehydrogenase; glucose-6-phosphate dehydrogenase; phosphoglucomutase; and glucose-phosphate isomerase) showed that these stocks have identical enzyme profiles and form a distinct zymodeme grouping. These observations were confirmed using the technique of schizodeme analysis and by comparing the k-DNA fingerprints produced by the restriction enzymes MspI, BspRI and AluI. The stocks were further analyzed by monoclonal antibodies and did not react with any of a large panel of L. mexicana, L. braziliensis, and L. donovani species- and/or subspecies-specific monoclonal antibodies using either an indirect radioimmune binding assay or immunofluorescence. These stocks did, however, react with a panel of monoclonal antibodies specific for L. major (formerly L. tropica major). Furthermore, the stocks could not be differentiated from L. major reference strains by enzyme electrophoresis nor could they be distinguished qualitatively from L. major based on their reactivity patterns using 10 Old World cutaneous species- and subspecies-specific monoclonal antibodies. Kinetoplast DNA restriction enzyme profiles, however, were different between these stocks and L. major reference strains. The implications of these results are discussed including the existence of other L. major-like stocks currently misidentified or uncharacterized.

Characterization of Leishmania colombiensis sp. n (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals, and phlebotomine sand flies in Colombia and Panama.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Identification, using isoenzyme electrophoresis and monoclonal antibodies, of Leishmania isolated from humans and wild animals of Ecuador.

Six strains of Leishmania isolated from wild mammals and humans on the Pacific Coast of Ecuador were identified by isoenzyme electrophoresis and by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. Single isolates from Sciurus vulgaris, Potos flavus, and Tamandua tetradactyla were identified as Leishmania amazonensis. Three other strains, isolated from cutaneous lesions of humans, were identified as Leishmania panamensis.

Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease.

One hundred fourteen Leishmania isolates from patients with different clinical forms of leishmaniasis in the State of Bahia, Brazil, were characterized by indirect radioimmune binding assay using specific monoclonal antibodies (serodeme analysis). Seventy-five of these isolates were also analyzed by enzyme electrophoresis, based on 11 enzyme loci; parasite species were compared, according to their characteristic zymodemes, to those of WHO Leishmania reference strains. All isolates could be classified into one of three species: Leishmania amazonensis (n = 40), L. braziliensis (n = 39) or L. chagasi (n = 35). The most interesting information obtained from this study is the realization that L. amazonensis is capable of producing a wide spectrum of disease in humans. Infection with this parasite was associated with many different clinical presentations, including cutaneous leishmaniasis [CL] (20/49 cases), mucocutaneous leishmaniasis [MCL] (5/13 cases) and, of special note, visceral leishmaniasis [VL] (11/46 cases), as well as four cases of post kalaazar dermal leishmaniasis [PKDL]. In situ tissue parasite characterization, by immunoperoxidase assay and employing anti-L. amazonensis amastigote monoclonal antibodies, confirmed the infection with this species in two cases of CL, one case of DCL, one case of MCL and one case of PKDL. Our results also demonstrate the difficulty of parasite differentiation based on clinical grounds, since at least L. amazonensis infection can be associated with all types of leishmanial diseases, and different Leishmania species may be associated with indistinguishable clinical presentations. Since leishmanial parasites may vary in their biological behavior or in their response to treatment, it is important that their identification be made by reliable methods.

Serodiagnostic assay for visceral leishmaniasis employing monoclonal antibodies.

A highly specific and sensitive competitive serodiagnostic assay for visceral leishmaniasis (VL) was developed using species specific Leishmania donovani monoclonal antibodies. This assay, either RIA or ELISA, is based on the specific inhibition of monoclonal antibody binding to a crude parasite homogenate by serum from patients with VL. 15 monoclonal antibodies were examined. The binding of 13 antibodies was significantly inhibited by VL serum and unaffected by normal serum. 3 species-specific monoclonal antibodies, D-2, D-13 and D-14, which recognize different parasite antigens, were chosen for use in the competitive serodiagnostic assay. In 90% of the positive cases, regardless of geographic origin, VL sera inhibited monoclonal antibody binding to the parasite antigen by more than 30%. No false positive was obtained with sera from Chagas disease, lepromatous leprosy, schistosomiasis, malaria, systemic lupus erythematosus, cutaneous or mucocutaneous leishmaniasis, even at serum dilutions (1:100) which cross-react strongly with Leishmania antigen in direct binding assays. Inhibition by negative control sera from areas endemic for VL and from non-endemic areas was negligible. The assay takes less than 24 h, requires minimum amounts of sera or antigen, and is easily standardized allowing interlaboratory comparison of test data. The competitive serodiagnostic assay will be especially useful in areas where Chagas disease is coendemic and the rapid diagnosis of VL by direct binding serodiagnostic assays presents a problem.