The genomic fingerprinting of the coding region of the β-tubulin gene in Leishmania identification

Concepción

Antimicrob Agents Chemother

et al. 2008

ER-119884 and E5700, novel arylquinuclidine derivatives developed as cholesterol-lowering agents, were potent in vitro growth inhibitors of both proliferative stages of Leishmania amazonensis, the main causative agent of cutaneous leishmaniasis in South America, with the 50% inhibitory concentrations (IC 50 s) being in the low-nanomolar to subnanomolar range. The compounds were very potent noncompetitive inhibitors of native L. amazonensis squalene synthase (SQS), with inhibition constants also being in the nanomolar to subnanomolar range. Growth inhibition was strictly associated with the depletion of the parasite's main endogenous sterols and the concomitant accumulation of exogenous cholesterol. Using electron microscopy, we identified the intracellular structures affected by the compounds. A large number of lipid inclusions displaying different shapes and electron densities were observed after treatment with both SQS inhibitors, and these inclusions were associated with an intense disorganization of the membrane that surrounds the cell body and flagellum, as well as the endoplasmic reticulum and the Golgi complex. Cells treated with ER-119884 but not those treated with E5700 had an altered cytoskeleton organization due to an abnormal distribution of tubulin, and many were arrested at cytokinesis. A prominent contractile vacuole and a phenotype typical of programmed cell death were frequently found in drug-treated cells. The selectivity of the drugs was demonstrated with the JC-1 mitochondrial fluorescent label and by trypan blue exclusion tests with macrophages, which showed that the IC 50 s against the host cells were 4 to 5 orders of magnitude greater that those against the intracellular parasites. Taken together, our results show that ER-119884 and E5700 are unusually potent and selective inhibitors of the growth of Leishmania amazonensis, probably because of their inhibitory effects on de novo sterol biosynthesis at the level of SQS, but some of our observations indicate that ER-119884 may also interfere with other cellular processes.

Section: Methodsmentioning

confidence: 99%

In Vitro Activities of ER-119884 and E5700, Two Potent Squalene Synthase Inhibitors, against Leishmania amazonensis : Antiproliferative, Biochemical, and Ultrastructural Effects

Concepción

Antimicrob Agents Chemother

et al. 2008

“…Molecular diversity within Leishmania species has been analyzed by several methods (Macedo et al 1992;Fernandes et al 1994Fernandes et al , 1999Luis et al 1998;Mauricio et al 1999) and comparison of the sequences of ribosomal RNA (rRNA) genes has been valuable in phylogenetic studies (Sogin et al 1986;Ramirez and Guevara 1987;Herna´ndez et al 1990;Briones et al 1992;Fernandes et al 1993;Landweber and Gilbert 1994;Maslov et al 1994;Uliana et al 1994;Cupolillo et al 1995;Scho¨nian et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana

Berzunza-Cruz¹,

Cabrera

Crippa-Rossi³

et al. 2002

Parasitology Research

Leishmania mexicana causes a wide spectrum of clinical diseases. In spite of the variety of clinical forms, no data exist regarding genetic polymorphism of L. mexicana. We analyzed the polymorphism of the internal transcribed spacer (ITS) and the small subunit rRNA genes of 3 reference strains and 24 Mexican isolates of L. mexicana, by means of polymerase chain reaction and subsequent digestion by restriction enzymes. All strains of L. mexicana had invariant patterns for both the ITS and the small subunit of rRNA genes. Leishmania amazonensis and Leishmania venezuelensis displayed polymorphism only in the ITS. The high degree of identity of this region was confirmed by sequencing DNA from three L. mexicana isolates. There was almost complete identity of the sequence for the ITS region of L. venezuelensis and that of strains of Leishmania major, suggesting that these species may be more closely related than previously thought.

“…With the advances in molecular techniques, a number of molecular markers and PCR protocols for the detection or identification of Leishmania on different taxonomical levels (genus, complex, and species) have been reported (11,43). Target sequences for characterization include either nuclear DNA, such as the small subunit rRNA (SSU rRNA) gene (40), a repetitive genomic sequence (30), the miniexon (spliced leader) gene repeat (18), the beta-tubulin gene region (22), the gp63 gene locus (41), and internal transcribed spacer (ITS) regions (9); microsatellite DNA (36,37); or kinetoplast DNA, such as minicircle sequences (34).…”

mentioning

confidence: 99%

Identification and Differentiation ofLeishmaniaSpecies in Clinical Samples by PCR Amplification of the Miniexon Sequence and Subsequent Restriction Fragment Length Polymorphism Analysis

et al. 2003

We recently developed a new PCR-restriction fragment length polymorphism (RFLP)-based assay using the miniexon sequence from the genus Leishmania. Here we report the application of this new genotyping method to naturally infected clinical samples for the differentiation of New and Old World Leishmania species. Of the newly developed assay and four currently applied diagnostic tests (i.e., in vitro cultivation, serology, and two other molecular assays using either the small subunit-internal transcribed spacer sequence or a repetitive genomic sequence), the miniexon assay showed the highest sensitivity, 89.7%, compared to 70.6, 57.1, 51.7, and 79.3%, respectively. Species differentiation was robust and reliable compared with that by two other Leishmania genotyping techniques. The assay provides a valuable tool for the identification of Leishmania directly from clinical samples and enables determination of the infecting species by a facile technique with high discrimination power. Since Leishmania causes a broad spectrum of diseases distinguished by different parasite and host factors, detection and characterization of the infecting species is crucial for the confirmation of a diagnosis as well as the establishment of the clinical prognosis and the initiation of an adequate therapeutic approach. The miniexon PCR-RFLP assay will facilitate such determination and might improve diagnosis and treatment of leishmaniasis.