Susceptibility of promastigotes and intracellular amastigotes from distinct Leishmania species to the calpain inhibitor MDL28170

Parasitology Research - Despite the available drug options, leishmaniasis treatment remains unsatisfactory. The repurposing of calpain inhibitors originally developed for human diseases became an...     

Numerical taxonomy of Monascus species based on the electrophoretic analysis of intracellular enzymes

Four isozymes in fifteeen species of Monascus were analyzed using polyacrylamide gel electrophoresis. A numerical analysis was carried out on the basis of similarity values obtained from the electrophoretic mobilities of the enzymes. Non-specific esterase and lactate dehydrogenase showed clearly distinguishable mobilities amont two clusters. From these results obtained, the strains used were divided into two clusters of Group-I and Group-II. Both groups were further subdivided into two subclusters. The type strain, M. ruber VAN TIEGHEM MR MO-10 (IAM 8009) and M. kaoliang MR MO-F2 were found to belong to the Group-I. M. pilosus SATO MR MO-4 (IAM8003) and M. pubigerus SATO MR MO-8 (IAM 8004) were found to belong to the Group-II. Nearly 100% similarity values were found between M. anka SATO MR MO-6 (IAM 8001) and M. rubiginosus SATO IFO 4484, between M. anka var. rubellus SATO MR MO-5 (IAM 8081) and M. purpureus WENT MR MO-9 (IAM 8010), and, moreover, among M. vitreus SATO MR MO-3 (IAM 8007), M. fuliginosus SATO IFO 4483 and M. serorubescens SATO 4487, respectively. Therefore, it was concluded that electrophoretic analysis of enzymes may be useful in presenting a new classification system of Monascus spp.     

Novel method for rapid identification of Nocardia species by detection of preformed enzymes.

The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The beta-naphthylamide-labeled substrate L-pyrrolidonyl-beta-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-alpha-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp.     

Usefulness of a PCR-based method in the detection and species identification of Leishmania from clinical samples

The aim of this study is to assess the usefulness of a simple, low-cost method for the detection and species identification of Leishmania isolated by in vitro culture or detected directly from clinical samples. A total of 110 samples were used in this study. Among these, 21 were human and canine peripheral bloods, 63 skin lesion material samples, eight reference strains and 18 Leishmania culture. Detection of Leishmania DNA with PCR using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene proved sufficiently sensitive at the level of 0.1 parasites per PCR reaction. Furthermore, followed by single-strand conformational polymorphism (SSCP), the PCR-ITS1 allowed the species identification of Leishmania. The inter-specific polymorphism of Leishmania was first validated on reference strains, and then this method was applied on clinical samples and culture. Typing identified all human and canine visceral leishmaniasis samples (21 samples) as L. infantum, 95.23% of the cutaneous leishmaniasis samples as L. major and 3.17% as L. killicki and 1.58% as L. infantum. A scheme of the PCR diagnosis procedure for the detection and identification of Leishmania parasites is proposed in this study.     

Methylglyoxal-catabolizing enzymes of Leishmania donovani promastigotes

Methylglyoxal is a toxic metabolite with growth inhibitory properties against Leishmania donovani promastigotes. We have shown in the present study that both log and stationary phase promastigotes of L. donovani can catabolize methylglyoxal to D-lactate as the major end product. The specific activity of methylglyoxal reductase was found to be the highest of all the catabolic enzymes. In contrast, the anabolic pathway for methylglyoxal could not be detected. Moreover, when control promastigotes or promastigotes in which the glycolytic pathway was inhibited were incubated with glucose, glycerol or dihydroxyacetone phosphate as energy source, neither methylglyoxal nor D-lactate could be detected.     

Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the New and Old World, using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Current PCR presents limitations in terms of sensitivity, which hampers its use for analyzing clinical and biological samples, and specificity, which makes it inappropriate to discriminate between Leishmania and other trypanosomatids. The aim of the study was to improve the sensitivity and specificity of a previously reported hsp70 PCR using alternative PCR primers and RFLPs. Following in silico analysis of available sequences, three new PCR primer sets and restriction digest schemes were tested on a globally representative panel of 114 Leishmania strains, various other infectious agents, and clinical samples. The largest new PCR fragment retained the discriminatory power from RFLP, while two smaller fragments discriminated less species. The detection limit of the new PCRs was between 0.05 and 0.5 parasite genomes, they amplified clinical samples more efficiently, and were Leishmania specific. We succeeded in significantly improving the specificity and sensitivity of the PCRs for hsp70 Leishmania species typing. The improved PCR-RFLP assays can impact diagnosis, treatment, and epidemiological studies of leishmaniasis in any setting worldwide.     

Detection and identification of Leishmania species from clinical specimens by using a real-time PCR assay and sequencing of the cytochrome B gene

Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with >or=99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.     

Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis

The inability to synthesize heme, a well known metabolic defect of trypanosomatid protozoa, accounts for their growth requirement for heme compounds in vitro. We now extend this finding to a pathogen Leishmania mexicana amazonensis, especially the intracellular replicative stage of amastigotes in the macrophage. We measured the level of heme and its biosynthetic enzymes, aminolevulinate dehydratase and porphobilinogen deaminase in the parasites and in infected and non-infected macrophages of J774G8 line. Succinylacetone was used to inhibit heme biosynthesis. Leishmanias transform and grow only in medium containing either heme (usually supplied as hemin) or protoporphyrin IX (the latter is leishmanicidal at high concentrations). We detected 1.2, 8.5 and 25 pmol mg-1 protein of heme in amastigotes, promastigotes and macrophages, respectively. The activities of porphobilinogen deaminase and aminolevulinate dehydratase in macrophages were 70 and 2400 pmol h-1 mg-1 protein, respectively. Leishmania-infected macrophages gave the same results and leishmanias had negligible activities of these enzymes. Succinylacetone at 10(-9)-10(-3) M had no effect on leishmanias, but dose-dependently inhibited the activity of aminolevulinate dehydratase to a negligible level and lowered that of porphobilinogen deaminase in macrophages, resulting in a maximum of 66% reduction in intracellular heme. Amastigotes grew equally well in succinylacetone-treated and control untreated macrophages. The results suggest that L. m. amazonensis, incapable of heme biosynthesis, acquires heme exogenously from the culture medium, i.e., fetal bovine serum, independent of the heme synthesized by the macrophages.     

Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation

The host response to Leishmania infection is regulated by a specific pattern of local cytokine production. We investigated the effect of interleukin (IL)-10 and IL-4 on the leishmanicidal activity of human macrophages (M?). As with L. major, intracellular killing of L. infantum by human M? was obtained following ligation of surface CD23 or cell treatment with Interferon-γ (IFN-γ). This leishmanicidal activity required nitric oxide (NO) generation by activated M?, and it was partially mimicked by cell treatment with chemical NO donors. Addition of recombinant human IL-10 or IL-4 to CD23 mAb or IFN-γ decreased L. infantum and L. major killing by infected M?. IL-10 was more potent than IL-4 in inhibiting the leishmanicidal activity of human M?. Inhibition of Leishmania killing by IL-4 and IL-10 correlated with decreased NO generation from M?, and was reversed when exogenous NO was added to cell cultures. Therefore, IL-10 and IL-4 down-regulate leishmanicidal activity of human M?, in part by inhibiting NO generation by these cells.     

Impairment of the oxidative metabolism of mouse peritoneal macrophages by intracellular Leishmania spp.

When stimulated in vitro with macrophage-activating factor or lipopolysaccharide, mouse peritoneal macrophages acquire the capacity to develop a strong respiratory burst when they are triggered by membrane-active agents. The presence of intracellular parasites of the genus Leishmania (L. enriettii, L. major) significantly inhibited such activity, as measured by chemiluminescence, reduction of cytochrome c and Nitro Blue Tetrazolium, and hexose monophosphate shunt levels. On the contrary, inert intracellular particles such as latex beads strongly increased the macrophage respiratory burst, suggesting that the Leishmania-linked inhibition resulted from a specific parasite effect. Impairment of macrophage oxidative metabolism by intracellular Leishmania spp. was a function of the number of infecting microorganisms and was more pronounced in macrophages infected with living than with dead parasites. Moreover, the metabolic inhibition was less apparent in L. enriettii-infected macrophages that were exposed to both macrophage-activating factor and lipopolysaccharide, i.e., conditions leading to complete parasite destruction. The mechanisms of respiratory burst inhibition by intracellular Leishmania spp. are unclear, but these observations suggest that such effects may contribute significantly to intracellular survival of the microorganisms.     

DNA fingerprints: a tool for identification and determination of the relationships between species and strains of Leishmania.

Using nonradioactive hybridization, the multilocal probes 33.15, F10 and (CAC)5 were shown to recognize multiple minisatellite regions in nuclear DNA of Leishmania, producing on Southern blots complex banding patterns typical of DNA fingerprints. We used the 33.15 probe to study 14 different strains belonging to 6 different species from both the sub-genus Viannia and the Leishmania mexicana complex of the sub-genus Leishmania. Distinct DNA fingerprints were obtained for each strain, permitting their identification. On the other hand, each strain showed little or no clonal variation. The information from the fingerprinting maps could be used for constructing phenograms and cladograms of the species and strains of Leishmania.     

Myd88-dependent in vivo maturation of splenic dendritic cells induced by Leishmania donovani and other Leishmania species

The usual agent of visceral leishmaniasis in the Old World is Leishmania donovani, which typically produces systemic diseases in humans and mice. L. donovani has developed efficient strategies to infect and persist in macrophages from spleen and liver. Dendritic cells (DC) are sentinels of the immune system. Following recognition of evolutionary conserved microbial products, DC undergo a maturation process and activate antigen-specific na?ve T cells. In the present report we provide new insights into how DC detect Leishmania in vivo. We demonstrate that in both C57BL/6 and BALB/c mice, systemic injection of L. donovani induced the migration of splenic DC from marginal zones to T-cell areas. During migration, DC upregulated the expression of major histocompatibility complex II and costimulatory receptors (such as CD40, CD80, and CD86). Leishmania-induced maturation requires live parasites and is not restricted to L. donovani, as L. braziliensis, L. major, and L. mexicana induced a similar process. Using a green fluorescent protein-expressing parasite, we demonstrate that DC undergoing maturation in vivo display no parasite internalization. We also show that L. donovani-induced DC maturation was partially abolished in MyD88-deficient mice. Taken together, our data suggest that Leishmania-induced DC maturation results from direct recognition of Leishmania by DC, and not from DC infection, and that MyD88-dependent receptors are implicated in this process.     

Immunopathogenesis of infection with the visceralizing Leishmania species

Human leishmaniasis is a spectral disease that includes asymptomatic self-resolving infection, localized skin lesions, and progressive visceral leishmaniasis. With some overlap, visceral and cutaneous leishmaniasis are usually caused by different species of Leishmania. This review focuses on host responses to infection with the species that cause visceral leishmaniasis, as they contrast with species causing localized cutaneous leishmaniasis. Data from experimental models document significant differences between host responses to organisms causing these diverse syndromes. The visceralizing Leishmania spp. cause localized organ-specific immune responses that are important determinants of disease outcome. Both the Leishmania species causing cutaneous and those causing visceral leishmaniasis require a Type 1 immune response to undergo cure in mouse models. However, during progressive murine infection with the visceralizing Leishmania sp., the Type 1 response is suppressed at least in part by TGF-beta and IL-10 without type 2 cytokine production. This contrasts with the cutaneous species L. major, in which a Type 2 response suppresses type 1 cytokines and leads to murine disease progression. Population and family studies are beginning to elucidate human genetic determinants predisposing to different outcomes of Leishmania infection. These studies should eventually result in a better understanding of the immunopathogenesis and the spectrum of human leishmaniasis.     

Lectin-binding properties of different Leishmania species

Carbohydrate cell-surface residues on stationary promastigotes of 19 isolates of Leishmania were studied with a panel of 27 highly purified lectins, which were specific for N-acetyl-D-glucosamine, D-mannose, L-fucose, D-galactose, N-acetyl-D-galactosamine, and sialic acid. The specificity of the cell-surface carbohydrates was analyzed by agglutination and radioiodinated lectin-binding assays. L. (L.) amazonensis and L. (L.) donovani were agglutinated by 12 and 10 of the 27 lectins used, respectively. Artocarpus integrifolia lectin (Jacalin) was incapable of agglutinating the tested species of the donovani complex, and this result was confirmed by radioiodinated Jacalin-binding assays. Jacalin had an average of 3.8 × 10⁶ receptors/L. (L) amazonensis promastigote and bound with an association constant of 5 × 10⁶ M ⁻¹. Received: 14 September 1998 / Accepted: 27 January 1999     

In silico prediction of the glycosomal enzymes of Leishmania major and trypanosomes

In total, 37080 protein sequences of the three trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi, were used to predict the trypanosomatid glycosomal proteome. All protein sequences were analyzed for the presence of either a C-terminal (PTS1) or an N-terminal (PTS2) peroxisomal targeting sequence. For L. major 191 potential PTS1-containing proteins and 68 potential PTS2-containing proteins with homologues in T. brucei and T. cruzi were identified. About 50% of them were hypothetical proteins to which no function was attributed. From those proteins with known function it appears that the predicted glycosomal proteome of L. major strongly resembles that of T. brucei and T. cruzi with respect to enzyme content. Glycosomes are not only involved in glycolysis, but are predicted to carry out also gluconeogenesis, reactions of the hexose-monophosphate pathway, purine salvage and pyrimidine biosynthesis, beta-oxidation of fatty acids, fatty acid elongation and the biosynthesis of ether lipids. In addition, they seem to catalyze several reactions of isoprenoid synthesis and are involved in oxidant stress protection.     

New primers for the detection Leishmania species by multiplex polymerase chain reaction

Parasitology Research - Leishmaniasis is caused by protozoa of the Leishmania genus, which is divided into subgenus Viannia and Leishmania. In humans, the course of infection largely depends on the...     

Molecular karyotype of species and subspecies of Leishmania

The DNA karyotypes of three species and several subspecies of New World Leishmania were found to be distinct. The karyotypes were more similar among closely related isolates than among more distantly related groups. Two classes of chromosomal DNA differences were detected among stocks; ± 50 kb size differences among DNAs, some of which were shown to contain homologous sequences, and DNAs having no obvious corresponding chromosomal DNA in other isolates. A total of 14–24 chromosomal DNA bands were resolved, depending on the isolate, but densitometric analyses suggest that these isolates contain 26–33 distinct DNA molecules. These molecules total about 2.5 × 10⁷ bp, a substantial fraction of the genomic DNA. The chromosomal DNA locations of gene sequences homologous to - and β-tubulin, ribosomal RNA, thymidylate synthetase-dihydrofolate reductase, and the H-region sequence were determined. The homologous sequences were located on chromosomal DNAs of similar, but not identical sizes among different stocks. We also found species- and some subspecies-specific β-tubulin chromosomal loci. We conclude that the DNA karyotype is useful for stock identification, taxonomy, and gene localization in Leishmania. Its potential for identifying the species and subspecies in natural infections appears less useful unless applied in conjunction with specific hybridization probes.