Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit visceral muscle contractions in hemimetabolous and holometabolous insects

Female sand flies can acquire protozoan parasites in the genus Leishmania when feeding on an infected vertebrate host. The parasites complete a complex growth cycle in the sand fly gut until they are transmitted by bite to another host. Recently, a myoinhibitory peptide was isolated from Leishmania major promastigotes. This peptide caused significant gut distension and reversible, dose-dependent inhibition of spontaneous hindgut contractions in the enzootic sand fly vector, Phlebotomus papatasi. The current study further characterizes myoinhibitory activity in L. major and other kinetoplastid parasites, using the P. papatasi hindgut and other insect organ preparations. Myoinhibitory activity was greatest in cultured promastigotes and in culture medium in late log-phase and early stationary-phase, coinciding with development of infective Leishmania morphotypes in the sand fly midgut. L. major promastigote lysates inhibited spontaneous contractions of visceral muscle preparations from hemimetabolous (Blattaria and Hemiptera) and holometabolous (Diptera) insects. Inhibition of visceral muscle contractions in three insect orders indicates a conserved mode of action. Myoinhibitory activity was detected also in Leishmania braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced myoinhibition mimics the effect of insect myotropins. Inhibiting host gut contractions protects Leishmania parasites from being excreted after blood meal and peritrophic matrix digestion, allowing development and transmission of infective forms.     

Development of Leishmania (Viannia) braziliensis vianna, 1911 in Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera:Psychodidae:Phlebotominae) under experimental conditions.

The development of Leishmania (Viannia) braziliensis in experimentally infected Lutzomyia intermedia, showed colonization of the hindgut from 48 h after the infective blood-meal, and the migration of flagellates to the foregut, with a massive infection of the cardia at the 5th day post infection. Up to 10 days following the infective blood-meal, very few parasites were seen in the pharynx and cibarium. The role of L. intermedia as a vector of cutaneous leishmaniasis is discussed according to the established criteria.     

In vitro and in vivo behaviour of sympatric Leishmania (V.) braziliensis, L. (V.) peruviana and their hybrids

Leishmania (Viannia) braziliensis is the main cause of highly disfiguring mucocutaneous leishmaniasis (MCL) in South America. The related species L. (V.) peruviana has only been identified in simple cutaneous lesions (CL). Hybrids between L. braziliensis and L. peruviana have been reported although genetic exchange in Leishmania is considered to be rare. Here we compared growth in vitro, adaptive capacity under thermal and oxidative stress and behaviour in a hamster model, of L. braziliensis, L. peruviana, and their putative hybrids. At 24°C, the optimal temperature for in vitro growth, L. braziliensis had the highest growth rate. In in vitro studies hybrid clones presented heterogeneous phenotypes, from slower growth rates, similar to L. peruviana, to higher growth rates, as observed in L. braziliensis. Hamsters infected with hybrid strains, presented the highest parasite densities and aggressive relapses at a later stage of infection. Hybrids generally presented higher plasticity and phenotypic diversity than the putative parental species, with potential eco-epidemiological implications, including an impact on the success of disease control.     

Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana

Leishmania (Viannia) braziliensis and L. (V.) peruviana are two parasite species characterized by a very different pathogenicity in humans despite a high genetic similarity. We hypothesized previously that L. (V.) peruviana would descend from L. (V.) braziliensis and would have acquired its 'peruviana' character during the southward colonization and adaptation of the transmission cycle in the Peruvian Andes. In order to have a first appreciation of the differences in virulence between both species, we evaluated an in vitro and in vivo model for experimental infection. A procedure was adapted to enrich culture forms in infective stages and the purified metacyclics were used to infect macrophage cell lines and golden hamsters. The models were tested with 2 representative strains of L. (V.) braziliensis from cutaneous and mucosal origin respectively and 2 representative strains of L. (V.) peruviana from Northern and Southern Peru respectively. Our models were reproducible and sensitive enough to detect phenotypic differences among strains. We showed in vitro as well as in vivo that the L. (V.) braziliensis was more infective than L. (V.) peruviana. Furthermore, we found that in vitro infectivity patterns of the 4 strains analysed, were in agreement with the geographical structuring of parasite populations demonstrated in our previous studies. Further work is needed to confirm our results with more strains of different geographical origin and their specific clinical outcome. However, our data open new perspectives for understanding the process of speciation in Leishmania and its implications in terms of pathogenicity.     

Comparative zymographic analysis of metallopeptidase of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis isolates from Peru

American tegumentary leishmaniasis (ATL) in Peru is mainly associated with Leishmania (Viannia) peruviana and L. (V.) braziliensis. These parasites are genetically related, and their characterization as distinct species is controversial. Despite their genetic similarity, each species is associated with different clinical manifestations of ATL; L. (V.) peruviana causes only cutaneous leishmaniasis, whereas L. (V.) braziliensis can cause both cutaneous and mucocutaneous leishmaniasis. Because the primary cutaneous lesions caused by infection with these species are indistinguishable, it is necessary to develop a suitable method to differentiate them in order to prevent possible metastasis to oropharyngeal mucosa. In the present study, we investigated the proteolytic profile of L. (V.) peruviana and L. (V.) braziliensis isolates from Peru by zymographic analysis in SDS-PAGE copolymerized with gelatin. Enzymes were characterized according to their pH range of activity and sensitivity to distinct peptidase inhibitors. We observed that L. (V.) peruviana isolates displayed three proteolytic bands with molecular masses ranging from 55 to 80kDa, whereas L. (V.) braziliensis isolates showed six proteolytic activities between 55 and 130kDa. Using specific inhibitors, we determined that these proteolytic activities are due to metallopeptidases and present optimal activity between the pH range 5.5 and 10.0. Our results suggest that the expression of metallopeptidases in L. (V.) peruviana and L. (V.) braziliensis isolates from Peru is species-specific.     

Interaction of Leptomonas wallacei with the intestinal tract of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae)

While investigating the distribution of Leptomonas wallacei in the intestine of the insect host Oncopeltus fasciatus, promastigotes and cyst-like forms of L. wallacei were observed only in the midgut ventricles V(3) and V(4) and the hindgut. In video-microscopy, once contact had occurred, the parasites remained attached to the midgut epithelium. Scanning electron microscopy revealed the adhesion of flagellates and cyst-like forms to the midgut wall and to the rectal pads of the hindgut. Using transmission electron microscopy, we observed that adhesion occurred mainly between the flagellum and the perimicrovillar membranes secreted by the midgut epithelium. No modifications were observed either in the parasite or in the epithelial cells. In the hindgut, adhesion to the superficial wax layer of the epithelial cells of the rectal pads was via flagellum. Host cell morphology appeared unaffected by L. wallacei.     

Chitinase secreted by Leishmania functions in the sandfly vector.

Leishmania major parasites ingested with host blood by the sandfly Phlebotomus papatasi multiply confined within the peritrophic membrane. This membrane consists of a chitin framework and a protein carbohydrate matrix and it is secreted around the food by the insect midgut. Histological sections of infected flies show lysis of the chitin layer in the anterior region of the peritrophic membrane that permits the essential forward migration of a concentrated mass of parasites. Both the location and the nature of this disintegration are specific to infected flies. At a later stage the parasites concentrate in the cardiac valve region and subsequently this segment of the fore gut loses its cuticular lining. We have found that chitinase and N-acetylglucosaminidase are secreted by cultured L. major promastigotes, but not by sandfly guts. Hence lysis of the chitin layer of the peritrophic membrane could be catalysed by these enzymes of the parasites. Activity of both enzymes was also observed in other trypanosomatids, including L. donovani, L. infantum, L. braziliensis, Leptomonas seymouri, Crithidia fasciculata and Trypanosoma lewisi.     

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.     

IL-10 and TGF-beta control the establishment of persistent and transmissible infections produced by Leishmania tropica in C57BL/6 mice

Leishmania tropica is the causative agent of Old World anthroponotic cutaneous leishmaniasis, which is characterized by lesions that take an extended period of time to heal, often resulting in disfiguring scars, and are more refractory to treatment than leishmaniasis caused by Leishmania major. Immunologic studies involving experimental animal models of L. tropica infection are virtually nonexistent. In the current study, infectious-stage L. tropica were used to establish dermal infections in C57BL/6 and BALB/c mice. In both strains, the lesions were slow to develop and showed minimal pathology. They nonetheless contained a stable number of between 10(4) and 10(5) parasites for over 1 year, which were efficiently picked up by a natural sand fly vector, Phlebotomus sergenti. Control of parasite growth depended on the development of a Th1 response, as C57BL/6 mice genetically deficient in Th1 cytokines and BALB/c mice treated with Abs to IFN-gamma harbored significantly more parasites. By contrast, IL-10-deficient mice harbored significantly fewer parasites throughout the infection. To further study the immunologic mechanisms that may prevent efficient clearance of the parasites, IL-10 and TGF-beta signaling were abrogated during the chronic phase of infection in wild-type C57BL/6 mice. Distinct from chronic L. major infection, IL-10 blockade alone had no effect on L. tropica, but required simultaneous treatment with anti-TGF-beta Abs to promote efficient parasite clearance from the infection site. Thus, chronic infection with L. tropica appears to be established via multiple suppressive factors, which together maintain the host as a long-term reservoir of infection for vector sand flies.     

Binding of Leishmania promastigotes to macrophages

Leishmania tropica promastigotes are easily attached to and engulfed by C3H peritoneal macrophages in vitro at 37 degrees C. Different sugars at 0.3-0.5 M inhibited in vitro the attachment of L. tropica promastigotes to C3H peritoneal macrophages with lactose (Gal-beta [1 leads to 4]Glc) being the most efficient. Inhibition of attachment is also affected by pre-treatment of promastigotes with galactose oxidase. Oligosaccharides extending from promastigote and amastigote cell surfaces contain an important proportion of non-reducing galactose as does the carbohydrate-rich factor (EF) excreted by promastigotes of L. tropica and L. donovani. This study suggests that Leishmania, an obligatory intracellular parasite, uses as a means of entering the host cell a cellular mechanism similar to that used in the removal of damaged cells from blood circulation. This mechanism is assumed to take advantage of the exposed sugars, particularly the exposed non-reducing galactose, on the parasite surface during the stage of attachment. Once the parasite is inside the cell, the EF it produces might have a protective function, being inhibitory to some of the host cell lysosomal enzymes.     

The major surface protein of Leishmania promastigotes is a protease

The major surface protein of Leishmania promastigotes is evolutionarily conserved and is found in isolates of L. donovani, L. major, L. tropica, L. mexicana, and L. braziliensis. The data provided in this communication demonstrate that in L. major this integral membrane protein is a protease, which we now designate promastigote surface protease. The enzyme has an alkaline pH optimum and is active both in its detergent-solubilized form and at the surface of living or fixed promastigotes. A water-soluble form of promastigote surface protease is obtained following digestion with the phospholipase C responsible for the release of the variant surface glycoprotein of Trypanosoma brucei. Possible biological functions of promastigote surface protease during the life cycle of Leishmania parasites are discussed.     

Monoclonal antibodies specific for Leishmania tropica. I. Characterization of antigens associated with stage- and species-specific determinants

Four monoclonal antibodies (T-1 through T-4), which were produced to membrane-enriched preparations of Leishmania tropica major promastigotes, reacted specifically with the members of L. tropica complex. The antibodies T-1 and T-4 react exclusively with L. t. major and L. t. minor. The remaining two monoclonal antibodies bind, in addition, to L. t. aethiopica and weakly to L. mexicana amazonensis. No significant cross-reactivity was observed with L. donovani, L. braziliensis braziliensis, and Trypanosoma cruzi. Antibodies T-1, T-2, and T-4 were found to be specific for the promastigote stage (insect) of L. tropica. Antibody T-3 reacts with both the amastigote and promastigote stages of the parasite. All of the monoclonal antibodies react with cell surface components on intact promastigotes. The protein antigens containing the species-specific determinants recognized by each of the four antibodies were identified by radioimmunoprecipitation of solubilized 125I-labeled L. t. major promastigotes. A single 50 kilodalton protein is recognized by clone T-4. T-1 recognized two high m.w. proteins (100 and 200 kilodaltons). These two antigens plus an additional protein of lower m.w. (70 kilodaltons) are also immunoprecipitated by the antibodies T-2 and T-3, demonstrating that species-specific determinants are present on several different cell surface proteins of L. t. major.     

Vector competence of some Neotropical sandflies for the Leishmania (Viannia) braziliensis complex

Abstract. To evaluate the vector competence of some Lutzomyia spp. (Diptera: Psychodidae) for Leishmania (Viannia) spp. (Kinetoplastida: Trypanosoma-tidae), experimental infections of anthropophilic sandflies from the Colombian Pacific coast were performed, through membrane feeding and xenodiagnosis on hamsters infected with Le.(V.)braziliensis or Le.(V.)panamensis. Wild-caught or F, generation females of Lutzomyia gomezi, Lu.hartmanni, Lu.panamensis and Lu. trapidoi were allowed to feed on hamster lesions and then maintained at 26^oC and >80% r.h. on a sugar-water diet until dissection on the fifth day post-infection (p.i.).
Despite similar infection rates (range 37–44%) in both Lu.gomezi and Lu.trapidoi , infections were heavier (>100 parasites) in the latter species. Infections of Lu.trapidoi with Le.braziliensis ( n = 21) and Le.panamensis (n = 27) showed parasite migration toward the foregut, with promastigote colonization of the stomodeal valve and appearance of infective forms. In contrast, infections of Lu.gomezi with Le. braziliensis (n = 10) and Le.panamensis (n = 5) were light (<50 parasites) and usually restricted to the pylorus. In Lu.hartmanni , only a few promastigotes were found in the pylorus and midgut of 3/8 specimens infected with Le.braziliensis , and no Le.panamensis developed (n = 19). By day 5 p.i., promastigote colonization of the hind- and midgut by Le.panamensis was observed in 2/4 Lu.panamensis but not Le.braziliensis (n = 3).
It was concluded that Lu.trapidoi is a more efficient vector than Lu.gomezi for both Le.braziliensis and Le.panamensis , and that Lu.hartmanni and Lu.panamensis are of minor importance for Leishmania transmission in this endemic area.     

Size-polymorphism of mini-exon gene-bearing chromosomes among natural populations of Leishmania, subgenus Viannia.

In order to explore genomic plasticity at the level of the mini-exon gene-bearing chromosome in natural populations of Leishmania, the molecular karyotype of 84 Leishmania stocks belonging to subgenus Viannia, originating mostly from Peru and Bolivia, and differing according to eco-geographical and clinical parameters, was resolved and hybridised with a mini-exon probe. The results suggest that size variation of the mini-exon gene-bearing chromosome is frequent and important (up to 245-kb size-difference), and partially involves variation (up to 50%) in copy number of mini-exon genes. There is no significant size-difference between mini-exon-bearing chromosomes of Peruvian and Bolivian populations of cutaneous and mucosal isolates of Leishmania (Viannia) braziliensis, but there is between eco-geographical populations of Leishmania (Viannia) peruviana. Leishmania (V.) peruviana presented a significantly smaller mini-exon-bearing chromosome than the other species of subgenus Viannia. The contrast between the general chromosome size heterogeneity and the homogeneity observed in some Peruvian Andean areas is discussed in terms of selective pressure.     

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.     

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5' extremity of each mRNA, supplying the 5'-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania (Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.     

Cotton rats (Sigmodon hispidus) and black rats (Rattus rattus) as possible reservoirs of Leishmania spp. in Lara State,Venezuela

A total of 519 wild animals belonging to eleven species were collected during a two year study in a cutaneous leishmaniasis endemic area in Venezuela (La Matica, Lara State). The animals were captured in home-made Tomahawk-like traps baited with maize, bananas or other available local fruits, and parasites were isolated from 27 specimens. Two different species were found naturally infected with flagellates, i.e., cotton rats (Sigmodon hispidus) and black rats (Rattus rattus). Characterization of the parasites using PCR, kDNA restriction pattern and hybridization with species-specific probes revealed the presence of Leishmania (L.) mexicana in three of the black rats and Leishmania (V.) braziliensis in two others. The latter species was also identified in the single positive specimen of S. hispidus. The results suggested both species of animals as possible reservoirs of Leishmania sp.     

A lipophosphoglycan-independent development of Leishmania in permissive sand flies

Leishmaniases are serious parasitic diseases the etiological organisms of which are transmitted by insect vectors, phlebotominae sand flies. Two sand fly species, Phlebotomus papatasi and P. sergenti, display remarkable specificity for Leishmania parasites they transmit in nature, but many others are broadly permissive to the development of different Leishmania species. Previous studies have suggested that in 'specific' vectors the successful parasite development is mediated by parasite surface glycoconjugates and sand fly lectins, however we show here that interactions involving 'permissive' sand flies utilize another molecules. We did find that the abundant surface glycoconjugate lipophosphoglycan, essential for attachment of Leishmania major in the specific vector P. papatasi, was not required for parasite adherence or survival in the permissive vectors P. arabicus and Lutzomyia longipalpis. Attachment in several permissive sand fly species instead correlated with the presence of midgut glycoproteins bearing terminal N-acetyl-galactosamine and with the occurrence of a lectin-like activity on Leishmania surface. This new binding modality has important implications for parasite transmission and evolution. It may contribute to the successful spreading of Leishmania due to their adaptation into new vectors, namely transmission of L. infantum by Lutzomyia longipalpis; this event led to the establishment of L. infantum/chagasi in Latin America.     

Leishmania major and Leishmania tropica: I the in vitro effects of an immunomodulator, S2-Complex

This study was undertaken to try to determine the possible anti-leishmanial activity of S2-Complex, an organic complex of copper chloride, ascorbic acid, and nicotinamide. The promastigotes, axenic amastigotes, and intracellular amastigotes of both Leishmania major and Leishmania tropica were incubated with different concentrations of S2-Complex. The EC50 for each form was calculated. Results show that all forms of the parasites were dose dependently inhibited by S2-Complex. The promastigotes of both parasites were the most resistant with highest EC50 followed by axenic amastigotes. While intracellular amastigotes were the most sensitive with the lowest EC50.These results indicate that S2-Complex has a direct anti-leishmanial effect. When mice were treated with S2-Complex or BCG for four days before harvesting the macrophages, and the macrophages infected with both L. major and L. tropica, they showed increased phagocytosis and increased parasite killing. The results of S2-Complex were not statistically different from the immunomodulating agent BCG. These results indicate that S2-Complex has an immunomodulating effect in addition to the direct anti-leishmanial effect.     

Phlebotomus (Adlerius) halepensis vector competence for Leishmania major and Le. tropica

In Eurasia, phlebotomine sandflies of the subgenus Adlerius (Diptera: Psychodidae) comprise about 20 known species. Some are suspected vectors of visceral leishmaniasis (VL) and at least one species has been implicated as a vector of cutaneous leishmaniasis (CL). We tested Phlebotomus (Adlerius) halepensis Theodor (Jordan strain) for CL vector competence, compared with three standard vectors: Phlebotomus (Phlebotomus) duboscqi N-L. from Senegal, Phlebotomus (Paraphlebotomus) sergenti Parrot from Turkey and the Neotropical Lutzomyia longipalpis (L. & N) (Jacobina strain). Sandfly females were membrane-fed on amastigote suspensions of Leishmania major Y. & S. and Le. tropica (Wright) (Kinetoplastida: Trypanosomatidae) and examined for parasite development 3, 6 and 10 days post-infection. Phlebotomus halepensis showed high susceptibility to both leishmanias, supporting typical suprapylarian parasite development similar to the other vectors. Phlebotomus halepensis infection rates were approximately 90% for Le. major and approximately 80% for Le. tropica, with high parasite densities. Development of infections was relatively fast, colonizing the thoracic midgut by 6 days post-bloodmeal in every case and reaching the stomodeal valve in >80% of flies. In late-stage infections, 10 days post-bloodmeal, nearly all P. halepensis females had cardia and stomodeal valve filled with very high numbers of parasites and some Le. tropica-infected females had promastigotes in the pharynx and proboscis. Host choice experiments in the laboratory showed that P. halepensis females fed readily on rat or rabbit and preferred the human forearm. In view of its vector competence and partial anthropophily, we infer that P. halepensis is a potential vector of cutaneous as well as visceral leishmaniases.