Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan.

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.     

Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed β-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.     

Sandfly midgut lectin: effect of galactosamine on Leishmania major infections

Galactosamine, which has been shown in vitro to specifically inhibit sandfly midgut lectin activity, was fed to Phlebotomus duboscqi females with blood containing promastigotes of Leishmania major . Non-inhibitory sugar, galactose, was added in controls. For two strains of L. major (LV 561 and Neal-P), galactosamine substantially enhanced the establishment of infection in the sandfly posterior midgut and significantly increased parasite loads after defaecation, but did not affect anterior migration of Leishmania . On day 3 post-infection, most infections in galactosamine-fed sandfly groups (92% of LV 561 and 100% of Neal-P) were found in the ectoperitrophic space of the posterior midgut, whereas most infections in the galactose-fed groups of sandflies (85% in LV 561 and 96% in Neal-P) were restricted to the peritrophic sac. On day 9, however, the proportion of infections colonizing the stomodeal valve was similar in both dietary groups of sandflies for both strains of L. major . The addition of galactosamine prevented the decrease of parasite loads which occurred in controls between days 3 and 6 post-infection. On days 6 and 9, heavy infections were observed almost exclusively in galactosamine-fed females. Differences between groups were more pronounced for the Neal-P strain, which normally developed poorly in sandflies. Morphology of L. major LV 561 was not affected by galactosamine supplement: the lengths of parasite body and flagellum were similar in both sandfly groups. Two hypotheses are considered for the role of sandfly midgut lectin in Leishmania development in the vector midgut. One proposes that sandfly lectin kills Leishmania promastigotes, the other assumes that lectin blocks LPG-mediated binding of promastigotes to sandfly midgut microvilli.     

NKT cells mediate organ-specific resistance against Leishmania major infection

Whereas the acquired T cell-mediated protection against intracellular pathogens such as Leishmania major has been well studied in the past, the cells and mechanisms involved in their innate control are still poorly understood. Here, we investigated the role of natural killer T (NKT) cells in a high dose L. major mouse infection model. In vitro, L. major only weakly stimulated NKT cells and antagonized their response to the prototypic NKT cell ligand alpha-galactosylceramide, indicating that L. major partially escapes the activation of NKT cells. NKT cell deficiency as analyzed by subcutaneous infection of Jalpha281-/- mice (lacking invariant CD1d-restricted NKT cells) and CD1-/- mice (lacking all CD1d-restricted NKT cells) led to a transient increase in skin lesions, but did not impair the clinical cure of the infection, NK cell cytotoxicity, the production of IFN-gamma, the expression of inducible nitric oxide synthase, and the control of the parasites in the lymph node. In the spleen, however, NKT cells were required for NK cell cytotoxicity and early IFN-gamma production, they lowered the parasite burden, and exerted bystander effects on Leishmania antigen-specific T cell responses, most notably after systemic infection. Thus, NKT cells fulfill organ-specific protective functions during infection with L. major, but are not essential for parasite control.     

Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection

In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection.     

Single dose novel Salmonella vaccine enhances resistance against visceralizing L. major and L. donovani infection in susceptible BALB/c mice

Visceral leishmaniasis is a major neglected tropical disease, with an estimated 500,000 new cases and more than 50,000 deaths attributable to this disease every year. Drug therapy is available but costly and resistance against several drug classes has evolved. Despite all efforts, no commercial, let alone affordable, vaccine is available to date. Thus, the development of cost effective, needle-independent vaccines is a high priority. Here, we have continued efforts to develop live vaccine carriers based on recombinant Salmonella. We used an in silico approach to select novel Leishmania parasite antigens from proteomic data sets, with selection criteria based on protein abundance, conservation across Leishmania species and low homology to host species. Five chosen antigens were differentially expressed on the surface or in the cytosol of Salmonella typhimurium SL3261. A two-step procedure was developed to select optimal Salmonella vaccine strains for each antigen, based on bacterial fitness and antigen expression levels. We show that vaccine strains of Salmonella expressing the novel Leishmania antigens LinJ08.1190 and LinJ23.0410 significantly reduced visceralisation of L. major and enhanced systemic resistance against L. donovani in susceptible BALB/c mice. The results show that Salmonella are valid vaccine carriers for inducing resistance against visceral leishmaniasis but that their use may not be suitable for all antigens.     

Protection against Leishmania major infection by oligomannose-coated liposomes

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (Man5-DPPE) have been shown to induce cellular immunity against antigens encapsulated in the liposomes. To assess whether these neoglycolipid-coated liposomes can elicit protective immune response against challenge infection, effects of immunization with soluble leishmanial antigens encapsulated in the liposomes were evaluated using Leishmania major infection in susceptible BALB/c mice. Intraperitoneal immunization of mice with leishmanial antigens in the Man5-DPPE-coated liposomes significantly suppressed footpad swelling in comparison to the control, non-immunized mice, while progression of the disease was observed in mice administered antigens in uncoated liposomes and those administered soluble antigens alone, as seen with control mice. Similarly, the number of parasites decreased substantially in local lymph nodes of mice immunized with the antigen in the Man5-DPPE-coated liposomes. Protection against L. major infection in the immunized mice also coincided with an elevated ratio of antigen-specific IgG2a/IgG1 antibodies, which is a profile of T helper-type 1-like immune response. Taken together, these results indicate the possibility that Man5-DPPE-coated liposome-encapsulated antigens could serve as a vaccine that triggers protection against infectious disease.     

Macrophage interactions with neutrophils regulate Leishmania major infection

Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE(2) and TGF-beta production by macrophages, while parasite killing depended on neutrophil elastase and TNF-alpha production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.     

DNA hybridizations on squash-blotted sandflies to identify both Phlebotomus papatasi and infecting Leishmania major

Epidemiological field studies on leishmaniasis have been hampered by the laborious, and often inefficient, methods used to assess the rates of infection of sandfly vectors (Diptera; Phlebotominae) by species of the causative disease organisms, protozoal parasites of the genus Leishmania (Kinetoplastida; Trypanosomatidae). We report the rapid and accurate identification of both sandfly vector (Phlebotomus (Phlebotomus) papatasi (Scopoli] and infecting Leishmania major Yakimov & Schokov by DNA hybridizations to squash-blotted sandflies. Large numbers of whole (infected) sandflies can be quickly squashed on to nylon hybridization filters and (following standard procedures) the filter-bound DNA can be hybridized sequentially to cloned, multicopy genomic sequences that are specific for species of Leishmania (kinetoplast DNA) or for the sandfly (ribosomal (r) DNA). Our sandfly probe consists of a 3.2 kb fragment of the intergenic 'non-transcribed' spacer of rDNA of P. papatasi that we have detected only in this species: it is present in all six geographically isolated populations tested (from Tunisia through to India) but cannot be detected in the morphologically similar P. (Phlebotomus) duboscqi Neveu-Lemaire, the vector of Leishmania major south of the Sahara; it also cannot be detected in Phlebotomus species of the subgenera Larroussius and Paraphlebotomus that together with P. papatasi are the dominant man-biting sandflies in north African foci of zoonotic cutaneous leishmaniasis, where (as in many arid regions of western Asia) P. papatasi is believed to be the sole vector of L. major.     

Stimulation of nonspecific resistance by thymopentin and its analogs against Leishmania donovani infection in hamsters

Thymopentin and its analogs have been synthesized by the solution phase method of peptide synthesis and evaluated for their prophylactic efficacy against L. donovani infection in hamsters. Thymopentin and some of the analogs were found to stimulate nonspecific resistance of the host against Leishmania donovani infection in hamsters.     

A dhfr-ts- Leishmania major knockout mutant cross-protects against Leishmania amazonensis.

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.     

Lutzomyia longipalpis and the eco-epidemiology of American visceral leishmaniasis, with particular reference to Brazil: a review

An historical review is given of American visceral leishmaniasis (AVL), with particular reference to the eco-epidemiology of the disease in Brazil. Following the first records of AVL in this country, in 1934, the sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) was incriminated as the principal vector. It is now generally accepted, however, that there exist a number of cryptic species under the name of Lu. longipalpis s.l. and that variations in the quantity of the vasodilatory peptide maxadilan in the saliva of flies from different populations of Lu. longipalpis s.l., may account for the variable clinical manifestations of AVL seen in different geographic regions. Distribution of AVL has been shown to extend throughout most of South and Central America, with the domestic dog serving as the principal reservoir of infection for man. However, while one hypothesis suggests that the causative parasite is Leishmania infantum, imported from Europe with the Portuguese and Spanish colonists, the demonstration of a high rate of benign, inapparent infection in foxes in Amazonian Brazil raised an opposing suggestion that the parasite is indigenous to the Americas. Recent reports of similar infections in native marsupials, and possibly rodents, tend to support this view, particularly as Lu. longipalpis is primordially a silvatic sandfly. Although effective control measures in foci of the disease will diminish the number of canine and human infections, the presence of such an enzootic in a variety of native animals will render the total eradication of AVL unlikely.     

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.     

Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Leishmania is a protozoan pathogen which is transmitted to humans through the bite of an infected sandfly. This infection results in a spectrum of diseases throughout the developing world, collectively known as leishmaniasis. During its life cycle, Leishmania differentiates from the promastigote stage in the sandfly vector into the amastigote stage in the mammalian host where it multiplies exclusively in macrophage phagolysosomes. Although differentiation of Leishmania is essential for its survival and pathogenesis in the mammalian host, this process is poorly understood. In higher eukaryotic cells, protein tyrosine phosphorylation plays a central role in cell proliferation, differentiation and overall function. We have therefore investigated the role of protein tyrosine phosphorylation in Leishmania differentiation by undertaking complementary approaches to mediate protein tyrosine dephosphorylation in vivo. In the present study, L. donovani were engineered to express a mammalian protein tyrosine phosphatase, or were treated with inhibitors of protein tyrosine kinases, and the resulting phenotype was examined. Both approaches resulted in a partial differentiation from promastigotes to amastigotes including the expression of the amastigote specific A2 protein, morphological change and increased virulence. These data provide support for the involvement of tyrosine phosphorylation in the differentiation of Leishmania.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.     

Mice lacking NK cells develop an efficient Th1 response and control cutaneous Leishmania major infection.

NK cells are believed to play a critical role in the development of immunity against Leishmania major. We recently found that transplantation of wild-type bone marrow cells into neonatal tgepsilon 26 mice, which are deficient in T and NK cells, resulted in normal T cell development, but no or poor NK cell development. Using this novel model we analyzed the role of NK cells in the development of Th1 response and control of cutaneous L. major infection. Mice selectively lacking NK cells (NK-T+) developed an efficient Th1-like response, produced significant amounts of IL-12 and IFN-gamma, and controlled cutaneous L. major infection. Administration of neutralizing IL-12 Abs to NK-T+ mice during L. major infection resulted in exacerbation of the disease. These results demonstrate that NK cells are not critical for development of protective immunity against L. major. Furthermore, they indicate that IL-12 can induce development of Th1 response independent of NK cells in NK-T+ mice following L.major infection.     

The human pathogen Leishmania donovani secretes a histidine acid phosphatase activity that is resistant to proteolytic degradation

Promastigotes of all pathogenic Leishmania species secrete acid phosphatase (SAcP) activity during their growth in vitro. It has been suggested that this enzyme may play a role in the survival of the parasite within its sandfly-vector host. To carry out such functions, SAcP would have to be relatively resistant to endogenous sandfly gut-proteases. Therefore, the current study was undertaken to ascertain whether L. donovani SAcP activity was affected by treatment with various proteases. Native L. donovani SAcP was treated with a variety of serine-, thiol-, metallo- and mixed-proteases and subsequently assayed for enzymatic activity. Of the eleven proteases tested, only bromelain and subtilisin treatments caused a pronounced reduction in SAcP activity. Treatment of SAcP with seven out of the remaining nine proteases, resulted in an overall enhancement in SAcP enzymatic activity ranging from approximately 10% (e.g. with trypsin) to > or = 90% (e.g. with ficin). The resistance of the Leishmania SAcP to various proteases may prolong its functional life within the sandfly gut and help to facilitate parasite infection in this host.     

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.     

Cellulase Activity of Leishmania major in the Sandfly Vector and in Culture

ABSTRACT. The secretion of cellulose-degrading enzymes by Leishmania promastigotes in culture and in the sandfly vector was demonstrated. Two types of activity of cellulase enzyme-complexes were measured: endoglucanases, which randomly cleave cellulose chains and cellobioydrolases, which remove cellobiose from the nonreducing end of the molecule. The assays demonstrated that enzymes with these activities were secreted into the culture medium by Leishmania major, L. donovani , and L. braziliensis . These activities were also found in cultures of Sauroleishmania agamae, Leptomonas seymouri, Herpetomonas muscarum, Crithidia fasciculata and Trypanosoma brucei brucei that had a relatively low endoglucanase activity. Both endoglucanase and cellobiohydrolase activities were found in the gut of L. major-infected Phlebotomus papatasi , while gut preparations of uninfected sandflies had only cellobiohydrolase activity. The similar growth of L. major parasites in medium supplemented with either cellulose or glucose suggests these parasites can utilize cellulose.     

The major surface-metalloprotease of the parasitic protozoan, Leishmania, protects against antimicrobial peptide-induced apoptotic killing

Human infection by the vector-borne protozoan Leishmania is responsible for substantial worldwide morbidity and mortality. The surface-metalloprotease (leishmanolysin) of Leishmania is a virulence factor which contributes to a variety of functions including evasion of complement-mediated parasite-killing and host intramacrophage survival. We tested the hypothesis that leishmanolysin serves to protect parasites from the cytolytic effects of various antimicrobial peptides (AMPs) which are important components of the innate immune system. We found that members of the alpha- and theta-defensins, magainins and cathelicidins had substantially higher leishmanicidal activity against leishmanolysin-knock out mutants of L. major. Using the magainin analogue, pexiganan, as a model peptide we show that AMP evasion is due to rapid and extensive peptide degradation by wild-type parasites. Pexiganan-treatment of knock out mutants induced disruption of surface-membrane permeability and expression of features of apoptosis including smaller cell size, loss of mitochondrial membrane potential, exposure of surface phosphatidyl serine as well as induction of caspase 3/7 activity. These results demonstrate leishmanolysin as a virulence factor preventing AMP-mediated apoptotic killing. This study serves as a platform for the dissection of the AMP-mediated death pathways of Leishmania and demonstrates the potential that AMP evasion plays during host infection by this parasite.