Halictine-2 antimicrobial peptide shows promising anti-parasitic activity against Leishmania spp.

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.

Antileishmanial effect of rapamycin as an alternative approach to control Leishmania tropica infection.

Cutaneous leishmaniosis (CL) is a parasitic disease in animals and human with no satisfactory treatments and vaccination. Rapamycin is a potent inhibitor of mammalian target of rapamycin (mTOR) with various applications. Here, the effect of rapamycin alone or in combination with two other drugs, namely amphotericin B (AmB) and glucantime, was investigated against Leishmania tropica infection. In vitro viability and electron microscopy evaluation of the parasites showed detrimental changes in their appearance and viability. Treatment with clinically relevant dose of rapamycin (10.2 µg/dose) is able to control the parasite load in BALB/c mice infected with L. tropica. Furthermore, the cytokine profiles showed significant polarization towards Th1 immune response. Surprisingly, combination therapy with either AmB or glucantime was not efficient. Rapamycin is showed an effective alternative therapy against leishmaniosis caused by L. tropica.

Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran.

BACKGROUND: Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. METHODS: All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. RESULTS: Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n = 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n = 48, 53.93%) than L. tropica (n = 4, 4.49%) (Mann-Whitney U test: p < 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. CONCLUSION: L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran

ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

PCR and microscopic identification of isolated Leishmania tropica from clinical samples of cutaneous leishmaniasis in human population of Kohat region in Khyber Pakhtunkhwa.

Leishmania tropica was isolated from the clinical patients of cutaneous leishmaniasis in rural community of Kohat district in Khyber Pakhtunkhwa province and was identified through PCR, microscopy, and culture techniques. A total of 113 samples from the clinical patients were examined through PCR, microscopy, and culture which showed 87.61% (99/113), 53.98% (61/113), and 46.90% (53/113) prevalence. During the study, 186 bp Leishmania tropica was identified through PCR. Thus the sensitivity of PCR is very high as compared to the conventional techniques.

Isoenzyme and ultrastructural characterization of Leishmania tropica axenic amastigotes and promastigotes.

Leishmania tropica is one of the main etiological agents of cutaneous leishmaniasis in Iran. For ultrastructural and isoenzyme study, axenic amastigotes were cultured in a brain-heart infusion medium containing 20 % fetal calf serum, pH 4.5, and incubated at 37 °C in 5 % CO(2). Different stages of L. tropica revealed the same isoenzyme profiles after comparing four enzyme systems including phosphoglucomutase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and nucleoside hydrolase II. Different isoenzyme patterns for glucose-phosphate isomerase, glucose-6-phosphate dehydrogenase, nucleoside hydrolase I, and malic enzyme enzymic systems were seen; thus, these isoenzyme systems among the eight systems studied were more efficient in characterizing L. tropica amastigotes. The structure of the axenic amastigotes was essentially similar to that of the promastigotes except for some important characteristics including the flagellum, flagellar pocket, paraxial rod, and the subpellicular microtubules.

Human cutaneous leishmaniasis: ultrastructural interactions between the inflammatory cells and Leishman bodies in the skin lesions.

The ultrastructural interactions between the inflammatory infiltrate and Leishman bodies (LBs) were described in skin lesions from 16 patients with acute cutaneous leishmaniasis. In early stages of the inflammation, the cellular infiltrate consisted of both undifferentiated and differentiated (activated) monocytes (M), macrophages (Mc), multinucleated giant cells (MNGC), plasma cells (PC), lymphocytes (Ly), and fibroblasts (F). In late stages, the infiltrate was in the form of tuberculous granulomas consisted mainly of type I secretory, and type II vesicular epithelioid cells (ECs), in addition to remnant of some inflammatory cells seen in the early stages. The two types of ECs were found only in six patients. The activated M, Mc and MNGC were often parasitized by LBs. The parasites were enclosed within the host cell digestive vacuoles (DVs), or phagolysosomes, together with skin melanosomes which are known to have lysosomal effect. In the DVs, LBs either survived or were killed and expelled from the host cell cytoplasm. This study showed, for the first time, that the melanosomes were apparently involved in killing of the LBs possibly by increasing the fatal effects of the DVs hydrolytic enzymes. Plasma cells were packed with large "Russell's bodies" indicating a high cellular immunoglobulin activity. The large, granular lymphocytes were in close contact to the activated M, possibly to promote delivery of activation signals. The type I secretory ECs contained mucin-like granules with electrondense cores. In late stages of inflammation, the type II vesicular ECs contained lysosomal granules, and were found together with the type I ECs in broken-down tuberculous granulomas. The type I secretory ECs were previously thought to produce a mediator, or "granuloma factor" which recruits undifferentiated mononuclear cells to perpetuate the granulomatous process; while the type II vesicular ECs were thought to appear where the granulomatous process in brought to an end, preceeding the healing by fibrosis.

Visceral and cutaneous leishmaniasis comparative ultrastructure of host-parasite interactions.

The comparative ultrastructure of host-parasite interactions is described for the first time in patients with visceral (VL) and cutaneous (CL) leishmaniasis. In patients with VL, the parasite invades the bone marrow (BM) macrophages (Mcs) and neutrophils, while in patients with CL, the parasite invades the dermal fibroblasts in addition to Mcs. The skin Mcs seem to have more lethal effects on the parasite than the BM Mcs; this is possibly due to the presence of numerous melanosomes with acid phosphatase activity in the Mcs digestive vacuole. In patients with high level of VL parasitaemia, the parasite may induce the BM reticulocytes to phagocytose both the parasite and mature erythrocytes, i.e. lost recognition. In patients with low level of VLparasitaemia, the parasite may induce the BM Mcs to be haemophagocytic, i.e. temporarily mimick malignant histiocytosis until the course of treatment. In early stages of CL infection, the cellular infiltrate consists of the monocyte-macrophage system, plasma cells, lymphocytes and fibroblasts; while in the late stages, two types of epithelioid cells (ECs) are added to the infiltrate and are involved in the formation of tuberculous granulomas. Type I ECs thought to produce a granuloma factor, while type II ECs possibly precedes healing by fibrosis. However, the severity of host-parasite interactions seems to depend mainly on species of the parasite, the degree of parasitaemia, the type of infected tissue(s), and the variation of host tissue reaction against the parasite from one patient to another.

Life cycle of Leishmania major (Kinetoplastida: Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae).

The development of Leishmania major Yakimoff & Schokhor in the New World sand fly Lutzomyia longipalpis (Lutz & Neiva) was examined by light and electron microscopy. In this unnatural host, parasites differentiated into 10 typical morphological forms, multiplied at three sites, migrated anteriorly and established in the foregut, and attached to gut surfaces. In the blood meal, amastigotes divided and transformed into two successive dividing, stumpy promastigote stages. Elongate nectomonad promastigotes developed from stumpy forms and subsequently rounded up in some flies into paramastigotes and opisthomastigotes. Differentiation into round opisthomastigotes and the apparent fusion of paramastigotes in the blood meal were novel observations in this study. Three nectomonad promastigotes--elongate, short, and metacyclic--were free-swimming in the midgut lumen. Elongate nectomonad promastigotes were highly oriented in the midgut, with their flagella embedded between the epithelial microvilli. Short haptomonad promastigotes were the predominant form attached to the intima of the stomodeal valve, whereas pear-shaped haptomonad promastigotes and paramastigotes colonized surfaces of the esophagus and pharynx. Peripylarian attachment of promastigotes and paramastigotes in the pylorus, ileum, and colon was noted in 21% of flies, suggesting that suprapylarian leishmanias have not lost the ability to colonize the hindgut. L. longipalpis was a successful biological host for L. major, allowing complete development of the parasite.

Expression of a mitochondrial stress protein in the protozoan parasite Leishmania major.

The DNA sequence has been determined of a gene from Leishmania major that shares sequence identity with members of the eukaryotic heat shock protein (hsp) 70 gene family. The deduced open reading frame for translation shares a number of features common to hsp70 stress proteins, including conserved amino acids implicated in ATP binding and a putative calmodulin-binding site. In addition, the protein has an N-terminal sequence characteristic of a mitochondrial targeting signal. Specific antibodies to this protein, generated by the use of recombinant fusion peptides, recognise a 65 kDa molecule of pI 6.7. This molecule is constitutively expressed and localises to the mitochondrion in all stages of the parasite life cycle. These features suggest a role for this protein as a molecular chaperone in Leishmania.

Effect of the anti-microtubule compound tubulozole on Leishmania protozoan parasites in vitro.

For the cis and trans stereoisomers of the synthetic anti-microtubule compound tubulozole, at micromolar concentrations, tubulozole-C is cytotoxic to mammalian cells whereas tubulozole-T is not. The effect of tubulozoles on the parasitic protozoan Leishmania was tested. For the promastigote stage of L. mexicana amazonensis, both isomers inhibited parasite growth. For the amastigote stage of L. mexicana amazonensis and L. major, within murine J774 macrophage line as host cells in vitro, tubulozole-T reduced the infective index. Despite the observation of macrophage cytotoxicity of tubulozole-T, this compound may be a potentially useful and novel anti-leishmanial drug.

Parasitism of epidermal Langerhans cells in experimental cutaneous leishmaniasis with Leishmania major.

Murine epidermal Langerhans cells (LC) have been demonstrated to stimulate a vigorous T cell response to Leishmania major, a cause of human cutaneous leishmaniasis. It was therefore of interest to analyze whether LC can take up viable parasites. Epidermal cells were obtained from mouse ear skin for incubation with L. major and subsequent detection of intracellular parasites by cytochemistry. Freshly isolated LC, but not cultured LC, phagocytosed L. major and the uptake was inhibited by antibodies to the complement receptor type 3. Electron microscopic studies revealed the presence of viable amastigotes within LC. Moreover, with double-labeling techniques, L. major-containing LC could also be detected in infected skin. The results demonstrate that LC can internalize L. major. Since the number of organisms per infected LC remained consistently low, the prime task of LC may not be the promotion of parasite spreading but the presentation of L. major antigen to T cells and, thus, the regulation of the cellular immunity during cutaneous leishmaniasis.

Real-time quantitative elemental analysis and mapping: microchemical imaging in cell physiology.

Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X-ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics-based microcomputer to acquire spectrally filtered X-ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post-acquisition image map with regard to specimen drift. Both high-resolution quantitative energy-dispersive X-ray images of freeze-dried cyrosections and low-dose quantitative bright-field images of frozen-hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on-line in real time. In addition, the unified design of the software program provides for off-line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer-aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure-function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.

The comparative fine structure and surface glycoconjugate expression of three life stages of Leishmania major.

The cellular ultrastructure and surface glycoconjugate expression of three life stages of Leishmania major were compared. Noninfective logarithmic phase promastigotes (LP) are immature cells bearing a thin cell coat, short flagellum, small and empty flagellar pocket, and a loose cytoplasm filled with profiles of ER and large Golgi complex. LP also contain subpopulations of maturing cells containing less ER and Golgi and synthesizing cytoplasmic granules of different size, number, and electron-density. Infective or metacyclic promastigotes (MP) are fully differentiated nondividing forms with a thickened, prominent cell coat, long flagellum, distended flagellar pocket filled with secretory material, and few cytoplasmic organelles other than abundant electron-dense granules. Tissue amastigotes also contain electron-dense cytoplasmic granules, their flagellar pockets are also enlarged and contain secretory material, but they lack a discernable cell coat. Immunogold labeling of GP63 on the cell surface was extensive only on amastigotes. Promastigote GP63 appeared to be masked by the presence of a densely packed lipophosphoglycan (LPG) coat which was extensively labeled on the entire surface of MP and LP. An elongated, developmentally modified form of LPG was abundantly labeled only on MP. LPG was poorly labeled on amastigotes, arguing that the promastigote cell coat is a stage-specific structure which is lost during intracellular transformation.

[Effect of high temperature on promastigotes of 2 species of Leishmania]. / Effetto delle alte temperature su promastigoti di due specie di Leishmania.

A morphological and chemical transformation of the protozoon Leishmania results during the shift from sand fly vector to mammalian host. This is due to the increase in temperature from about 26 degrees to about 35 degrees C. We evaluated the modifications induced by 37 degrees C on stationary phase promastigotes of two different Leishmania species (L. major and L. infantum). L. infantum changed to amastigote-like forms in a greater and quicker way than L. major; this fact could relate with the more elevated spreading capacity of this species in the human body.

Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major.

Leishmania major promastigotes contain electron-dense vacuoles. The elemental composition of these vacuoles and of the cytoplasm was measured by electron probe X-ray microanalysis, using rapid cryopreservation techniques to prevent alterations in composition due to diffusion. The electron-dense vacuoles are rich in P, presumably present as polyphosphate (poly P). Mg is present at about 9 times its cytoplasmic level. There is sufficient Mg to largely neutralize most of the negative charge of the Poly P. The electron-dense vacuoles also contain appreciable amounts of Ca and Zn, which are not detectable in the cytoplasm, as well as Na, K, and Cl, the latter two at concentrations below that of the cytoplasm. These results suggest that the vacuolar membranes have at least one cation transport system. Incubation of the promastigotes for 1 h in the absence of phosphate in the presence or absence of glucose did not cause significant changes in the vacuolar contents of P, Mg, or Zn, but changes in K and Cl content were observed in both the electron-dense vacuoles and in the cytoplasm.

Cell surface nanoanatomy of Leishmania major as revealed by fracture-flip. A surface meshwork of 44 nm fusiform filaments identifies infective developmental stage promastigotes.

Fracture-flip (Anderson-Forsman and Pinto da Silva, J. Cell Sci. 90, 531-541; 1988) was used to reveal the nanoanatomy of the surface of Leishmania major promastigotes. Over the cell surface of infective metacyclic promastigotes we identify a meshwork of 44 nm long, fusiform filaments. These filaments are not seen in noninfective stages of the parasite. Replica-staining immunocytochemistry with monoclonal antibody against infective metacyclic lipophosphoglycan shows a uniform distribution of protein A-colloidal gold complexes over the cell surface. Thin sections show that acquisition of the high molecular weight lipophosphoglycan is reflected in a thicker glycocalyx. Conventional freeze-fracture shows that in infective metacyclic promastigotes there is a reversal of the partition of intramembrane particles--an additional morphological marker for the infective developmental stage. We hypothesize that the fusiform filaments represent metacyclic developmental lipophosphoglycan.

[Ultrastructural study of Leishmania tropica in the amastigote phase]. / Estudio ultraestructural de la Leishmania trópica en fase amastigota.

A case is reported of cutaneous leishmaniasis with an ultrastructural study of the amastigote form, emphasizing and discussing the presence of two flagella inwards in the same parasite, a fact which is indicative of the great activity of the parasite in host lesions.