DNA-based detection of Leishmania and Crithidia species isolated from humans in cutaneous and post-kala-azar dermal leishmaniasis from Shiraz and Kharameh, southern Iran.

BACKGROUND & OBJECTIVES: Leishmania major and L. tropica are the main pathogens of cutaneous leishmaniasis (CL) in several rural and some urban regions of Iran, respectively. The aim of this study was to detect Leishmania species, and update the distribution data of these species in humans suspected to CL in two endemic foci in southern Iran. METHODS: From March 2016 to March 2017, 276 positive samples from of 350 suspected cases were diagnosed and compared by different diagnostic methods, viz. microscopy, culture, and PCR. In PCR assay, four different gene identifications were performed including minicircle kDNA, and cysteine protease B genes for Leishmania detection, and glyceraldehyde-3-phosphate dehydrogenase, and internal transcribed spacer 1 genes for Crithidia detection. RESULTS: In total, 68% (235/350) and 65.3% (177/271) of patients suspected of leishmaniasis were positive by microscopy and cultivation methods. In PCR assay, L. major, and L. tropica were detected in 86.2% (238/276), and 13.1% (36/276) of CL cases, respectively. Also, dermal L. infantum strain was isolated from 0.7% (2/276) of post-kala-azar dermal leishmaniasis patients. In addition, Crithidia fasciculata was detected in two CL patients chronically infected with L. major. INTERPRETATION & CONCLUSION: It appears that the epidemiology of CL has changed during the last decades and can complicate the control strategy aspects of CL in southern Iran. Therefore, more epidemiological, ecological, and gene polymorphism studies are needed to understand the pathogenic role of these species in human, as a main host of leishmaniasis in Iran.

Morphological discordance of the new trypanosomatid species phylogenetically associated with the genus crithidia.

Three new species of monoxenous parasites from the Neotropical Heteroptera are described on the basis of the ultrastructure of cells in culture, as well as gene sequences of Spliced Leader (SL) RNA, glyceraldehyde phosphate dehydrogenase (GAPDH) and small subunit (SSU) rRNA. The results have highlighted a striking discrepancy between the morphological (dis)similarities and the phylogenetic affinities among the insect trypanosomatids. Although each of the new species is characterized by a distinct set of morphological characters, based on the predominant promastigotes observed in culture, each of them has been provisionally assigned to the genus Leptomonas pending the future revision of this genus. Yet, instead of the phylogenetic affinity with the other members of this polyphyletic genus, the new species are most closely related to Crithidia species. Thus, the extremely long promastigotes of Leptomonas acus sp. n. and the unique morphological features found in Leptomonas bifurcata sp. n. sharply contrast with their respective relatives C. fasciculata and C. deanei both of which are typical choanomastigotes. The results clearly show that the current classification at the genus level is misleading and needs to be revised. The phylogenetic clades potentially representing the candidate new genera of monoxenous trypanosomatids have started to emerge from the presented analyses.

Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species.

Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Gelatin-SDS-PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms. Our survey indicates that the proteolytic profiles of C. deanei and C. desouzai are identical; that C. oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus. This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C. deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.

Systemic lupus erythematosus in mice, spontaneous and induced, is associated with autoimmunity to the C-terminal domain of p53 that recognizes damaged DNA.

The tumor suppressor molecule p53 features a regulatory domain at the C terminus that recognizes damaged DNA. Since damaged DNA might be involved in activating anti-DNA autoantibodies, we tested whether autoimmunity to the C terminus of p53 might mark murine systemic lupus erythematosus (SLE). We now report that MRL / MpJ-Fas(lpr) mice, which spontaneously develop SLE, produce antibodies both to the C terminus of p53 and to a monoclonal antibody (PAb-421) that binds the p53 C terminus. Anti-idiotypic antibodies to PAb-421 (sampled as monoclonal antibodies) could also bind DNA. Thus, the PAb-421 antibody mimics DNA, and the anti-idiotypic antibody to PAb-421 mimics the p53 DNA-binding site. This mimicry was functional; immunization of BALB / c mice to PAb-421 induced anti-DNA antibodies and antibodies to the C terminus of p53, and most of the mice developed an SLE-like disease. Immunization of C57BL / 6 mice to PAb-421 induced antibodies to p53, but not to its C-terminal domain. The C57BL / 6 mice also did not develop anti-DNA antibodies or the SLE-like disease. Thus, network autoimmunity to the domain of p53 that recognizes damaged DNA can be a pathogenic feature in SLE in genetically susceptible strains of mice.

Crithidia luciliae: functional expression of nucleoside and nucleobase transporters in Xenopus laevis oocytes.

The expression of purine-specific nucleoside and base transporters of Crithidia luciliae has been demonstrated in Xenopus laevis oocytes. Poly(A)+-mRNA from C. luciliae, cultured in either purine-replete or purine-starved conditions, was microinjected into X. laevis oocytes. For "purine-replete" mRNA, expression of adenosine and hypoxanthine uptake in microinjected X. laevis oocytes was increased on average 9- and 3-fold above water-injected controls, respectively. Expression of adenosine and hypoxanthine uptake in oocytes microinjected with "purine-starved" mRNA was 8 and 3-fold above water-injected controls, respectively. Substrate competition indicated an adenosine/deoxyadenosine transporter and a separate base transporter specific for hypoxanthine. In contrast to C. luciliae in vivo, where the level of activity of adenosine and hypoxanthine transport was regulated by the level of purines in the medium, the heterologous expression of these transporters (from both purine replete and deplete cultures) in X. laevis oocytes was independent of the extracellular purine concentration. These results may suggest that the presence of specific transporter message is independent of the extracellular purine content, indicating that the regulation of activation and expression of these transporters in C. luciliae may not be under transcriptional control.

Monophyly of endosymbiont containing trypanosomatids: phylogeny versus taxonomy.

To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp. TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H. roitmani. The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA. The size of minicircles in both isolates is 4.2 kilobases. The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic. Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree. These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.

African trypanosomes have differentially expressed genes encoding homologues of the Leishmania GP63 surface protease.

The genomes of various Leishmania parasites contain tandemly arrayed genes encoding an abundant 63-kDa surface glycoprotein called GP63. Leishmania GP63s are metalloproteases that play an important role in the invasion and survival of the parasites within the macrophage, and their presence on the Leishmania surface has been correlated with resistance to complement-mediated lysis. Here we report the identification of GP63-like genes in African trypanosomes. The predicted trypanosome and Leishmania GP63s share a metalloprotease catalytic site motif of HEXXH as well as 19 cysteines and 10 prolines, implying a conservation of enzymatic activity and secondary/tertiary structure. The trypanosome GP63 genes are transcribed equally in procyclic and bloodstream trypanosomes, but their mRNAs accumulate to a 50-fold higher steady state level in bloodstream trypanosomes, where the ratio of mRNAs for GP63 and variant surface glycoprotein is about 1:150. Transcription of the GP63 genes is sensitive to alpha-amanitin, indicating that they are transcribed by a different polymerase than the variant surface glycoprotein genes. These results lead to a reconsideration of the potential functions of GP63, inasmuch as African trypanosomes are not known to interact with macrophages and do not have an intracellular stage during their life cycle.

Drosophila topoisomerase II-DNA interactions are affected by DNA structure.

The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.

A rapidly rearranging retrotransposon within the miniexon gene locus of Crithidia fasciculata.

The tandemly arrayed miniexon genes of the trypanosomatid Crithidia fasciculata are interrupted at specific sites by multiple copies of an inserted element. The element, termed Crithidia retrotransposable element 1 (CRE1), is flanked by 29-base-pair target site duplications and contains a long 3'-terminal poly(dA) stretch. A single 1,140-codon reading frame is similar in sequence to the integrase and reverse transcriptase regions of retroviral pol polyproteins. Cloned lines derived from a stock of C. fasciculata have unique arrangements of CRE1s. In different cloned lines, CRE1s, in association with miniexon genes, are located on multiple chromosomes. By examining the arrangement of CRE1s in subclones, we estimate that the element rearranges at a rate of ca. 1% per generation. These results indicate that the C. fasciculata miniexon locus is the target for a novel retrotransposon.

A photochemical method to map ethidium bromide binding sites on DNA: application to a bent DNA fragment.

It is shown that, when irradiated in the visible, ethidium bromide (EB) engages in direct photochemistry with its DNA binding site. At the photochemical end point, an average of one single-strand break is produced per bound EB molecule in a reaction which also bleaches the dye chromophore. Using high-resolution electrophoresis, we have mapped the distribution of EB photocleavage sites on DNA, at one-base resolution. It is argued that because the photocleavage is stoichiometric, the resulting pattern is similar to, if not identical with, the local distribution of EB binding affinity. When interpreted in the context of the extensive thermodynamic and structural data which are available for EB, a binding distribution of that kind can be used to infer details of DNA structure variation within the underlying helix. As a first application of the method, we have used EB to probe the structure of a 265 bp fragment of DNA, which had been described as being bent as the result of a periodic array of oligo(A) segments [Kitchin et al. (1986) J. Biol. Chem. 261, 11302]. The EB mapping data provide evidence that the oligo(A) elements in this fragment assume a local secondary structure which is different than that assumed by isolated ApA nearest neighbors and that the ends of the oligo(A) elements comprise a junctional domain with EB binding properties which differ from those of the oligo(A) element or of random-sequence DNA.

[Various elements of the structure of mini-ring kinetoplast DNA of Crithidia fasciculata]. / Nekotorye élementy struktury mini-kol'tsevoi kinetoplastnoi DNK Crithidia fasciculata.

The nucleotide sequence of 1.4 kbp SmaI-fragment of minicircle DNA from kinetoplasts of Crithidia fasciculata has been determined and some sequence elements characterized. The sequence contains several oligo(dT)blocks located on the same strand in phase with a period of DNA helix turn, thus representing a "bent helix". Both sides of the bent helix region are flanked by sequences capable of forming a cloverleaf structure. There are also two direct 150 bp repeats located 180 degrees apart on the circular map of the molecule. Each repeat contains the sites of H-strand and L-strand replication origin. The specific stem-loop secondary structure may be folded by the nucleotide sequence within the origins region. The alignment of the sequence determined with two other C. fasciculata minicircle sequences spanning over the bent helix and the adjacent regions has indicated the presence of several conserved sequence blocks, one of them representing the sequence of the bend. The divergence of three sequences occurred mainly by small insertions-deletions. Several open reading frames were found, the largest of which being capable of coding for the approximately 200 amino acids polypeptide.

The terminus of SV40 DNA replication and transcription contains a sharp sequence-directed curve.

We have examined nucleosome positioning on two DNA segments containing sharp sequence-directed curvatures. A 223 bp DNA from Crithidia fasciculata was cloned into two sites in pBR325 separated by 28%. These sites were found to selectively reconstitute nucleosomes 5- to 7-fold more effectively than the adjoining straight DNA. The terminus of replication and termini of transcription of SV40 DNA are contained within a region of approximately 200 bp centrally located in a 1216 bp fragment. Visualization of this fragment by electron microscopy revealed a sharp curve of approximately 200 degrees in the terminal region. Reconstitution of histone protein with this fragment revealed a 2- to 5-fold higher probability of assembling nucleosomes in the terminal region over the adjacent DNA.

Curved helix segments can uniquely orient the topology of supertwisted DNA.

To show that DNA containing sharp sequence-directed curvature can preferentially establish ends of supertwisted domains, a highly curved DNA from Crithidia fasciculata was cloned into two sites separated by 28% in pBR325. When this construct (pJGC2) was examined by electron microscopy, 63% of the supercoiled molecules were branched with three or more arms, and the remaining molecules appeared as linear interwound rods. The distance between the tips of two of the arms for the branched molecules measured within 2% of 28% of the DNA contour for 32% of the pJGC2 molecules, as contrasted with only 6.6% for the poorly branched pBR325 DNA. When one of the curved segments in pJGC2 was replaced by a highly curved fragment from SV40, similar results were obtained.

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. II. Effects of variations in ligand structure on the in situ detection of mercurated probes.

In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond could be stabilized by an extra polar interaction between ligand and nucleic acid. This was achieved by reversing the net charge of the ligand. Such ligands were synthesized by reacting aliphatic diamines to the carboxyl groups of Tnp-glutathione using a water soluble carbodiimide. Gel chromatographic analysis of mercurated polynucleotide-ligand complexes showed that the stability of the mercury-sulfhydryl bond is increased by the reversal of the net charge of the ligand. In situ hybridized mercurated mouse satellite DNA to mouse liver nuclei and mercurated kinetoplast cRNA hybridized to Crithidia fasciculata were immunocytochemically detected after the introduction of these positively charged ligands. The described method is applicable for RNA and DNA probes. It has a sensitivity comparable to other non-autoradiographic methods, is relatively simple to perform and can be carried out with ordinary laboratory chemicals.

Tubulin heterogeneity in the trypanosome Crithidia fasciculata.

The interphase cell of Crithidia fasciculata has three discrete tubulin populations: the subpellicular microtubules, the axonemal microtubules, and the nonpolymerized cytoplasmic pool protein. These three tubulin populations were independently and selectively purified, yielding, in each case, microtubule protein capable of self-assembly. All three preparations polymerized to form ribbons and sheets rather than the more usual microtubular structures. Analyses of the tubulin by two-dimensional polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping indicated that the beta-tubulin complex remained constant regardless of source but that some heterogeneity was present in the alpha subunit. Cytoplasmic pool alpha tubulins (alpha 1/alpha 2) were the only alpha isotypes in the cytoplasm and also formed most of the alpha tubulin species in the pellicular fraction. Flagellar alpha tubulin (alpha 3) was the sole alpha isotype in the flagella; it appeared in small amounts in the pellicular fraction but was completely absent from the cytoplasm. In vitro translation products from polyadenylated RNA from C. fasciculata were also examined by two-dimensional polyacrylamide gel electrophoresis and possessed a protein corresponding to alpha 1/alpha 2 tubulin but lacked any alpha 3 tubulin. The alpha 3 polypeptide arose from a post-translational modification of a precursor polypeptide not identifiable by two-dimensional polyacrylamide gel electrophoresis as alpha 3. Peptide mapping data indicated that cytoplasmic alpha tubulin is the most likely precursor. These results demonstrate alpha-tubulin heterogeneity in this organism and also how close the relationship between flagellar and cytoskeletal tubulins can be among lower eucaryotes.