Silica nanowire conjugated with loop-shaped oligonucleotides: A new structure to silence cysteine proteinase gene in Leishmania tropica.

The main aim of this study was to evaluate the capability of silica nanowire conjugated with loop-shaped oligonucleotides (SNWCLSOs) to silence cysteine proteinase b (Cpb) gene in Leishmania (L) tropica. On the other hand, its toxicity on amastigotes and mouse peritoneal macrophages was evaluated by 5-diphenyl-tetrazolium bromide (MTT) assay. For control, two loop-shaped oligonucleotides (LSO) were considered. LSO1 and LSO2 were 5'-NH2-cccccaaaaaaaaaaaaaaaaaaaaaaaaaggggg-COOH-3' and LSO2: 5'-NH2-cccccttttttttttttttttttttttttttttttttttttttggggg-COOH-3', respectively. After 72 h incubation at 37 °C, AMSNW, LSO1, and LSO2 had no remarkable toxicity on L. tropica amastigote (2 × 10(5)/mL) and mouse peritoneal macrophages (2 × 10(5)/mL). In case of SNWCLSOs, they had high toxicity on L. tropica amastigote, but they had no effect on mouse peritoneal macrophages. At concentrations of 1, 10, and 25 µg/mL, AMSNW, LSO1 and LSO2 had no effect on the gene expression. But, at concentration of 50 and 100 µg/mL, decrease of gene expression was observed. In case of SNWCLSOs, they could dramatically decrease the gene expression. It could be concluded that since SNWCLSOs could silence Cpb gene with no remarkable toxicity, they are good choice for treat cutaneous leishmaniasis in future. As a new agent, it must be checked in vivo.

Detection of Leishmania parasites in the blood of patients with isolated cutaneous leishmaniasis.

BACKGROUND: The consequences of the spread of Leishmania parasites to the blood from lesions in patients with cutaneous leishmaniasis are numerous. To assess the magnitude of this invasion we conducted the present study on patients referred to the American University of Beirut Medical Center for cutaneous leishmaniasis. METHODS: Patients referred for the management of cutaneous leishmaniasis were included in the study. Skin and blood cultures for Leishmania were taken from these patients. RESULTS: One hundred sixty-two patients were proven to have cutaneous leishmaniasis by pathology; 52% were males and 44% females (gender information was missing for 4%). Patient age ranged from 5 months to 70 years. None of the patients had received treatment for Leishmania. We obtained parasite isolates from 85 patients (52.5%), proven by cultures from skin and blood/blood components. Interestingly, the parasite was isolated in the blood and blood components of 50 patients (30.9%). Isoenzyme analysis confirmed the fact that the organisms in blood and skin were the same; from the 28 isolates that were positive in both skin and blood, eight isolates were Leishmania major and two were Leishmania tropica. The remaining isolates, whether positive in both blood and skin or in either of these tissues, skin or blood and its products, were Leishmania infantum sensu lato. CONCLUSIONS: In the current study, the detection rate of parasites in the blood of patients with cutaneous leishmaniasis was high. This illustrates the invasive characteristic of the parasite that has escaped the skin. Testing should be considered in areas other than Lebanon, especially around the Mediterranean basin. Whether these findings support the administration of systemic treatment for cutaneous leishmaniasis or not needs to be confirmed in larger prospective studies.

Characterization of Leishmania (Leishmania) tropica axenic amastigotes.

Optimum conditions for generating Leishmania (Leishmania) tropica axenic amastigotes (AxA) in culture were determined, pH 5.5/36 degrees C, and the parasites characterized by different techniques, including light microscopy, macrophage infection, stage specific antigen expression and differential display. AxA were morphologically similar to amastigotes and 15.5-fold more infective than stationary phase promastigotes for mouse peritoneal macrophages. Western blotting with promastigote stage specific monoclonal antibodies to either lipophosphoglycan (T2) or a 60 kDa flagella antigen (F3) showed a dramatic decrease in antigen expression when AxA were compared to promastigotes. Similarly F3 gave strong immune fluorescent staining of the promastigote flagellum, but no fluorescence was detected when AxA were examined. Conversely, Western blotting with the amastigote specific monoclonal antibody (T16) showed that this antigen is more highly expressed in AxA than promastigotes. Differential display-PCR was used to identify several parasite genes showing stage specific expression. One gene selectively expressed by AxA was partially sequenced and identified as Leishmania (L.) tropicaamastin. Amastigote specific expression of this gene was further confirmed by reverse transcriptase-PCR (RT-PCR) using AxA and infected macrophages. No amastin expression was observed with promastigotes. Expression of the cysteine protease B (cpb) and protein kinase A catalytic isoform 1 subunit (pkac1) in promastigotes and AxA was also examined by RT-PCR. Pkac1 was strongly expressed by promastigotes, while cpb expression was only seen with AxA or infected macrophages. L. (L.) tropica AxA will prove useful for further studies on parasite differentiation and gene regulation, as well as for drug screening.

Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection.

We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was further simplified by using an oligochromatography dipstick to detect L. infantum and L. donovani products. Because the analytical sensitivity is lower than that of certain other species- and genus-specific PCRs, our assays are especially valuable for use on cultured isolates or directly on cryostabilates. As such, they can be implemented by research and health centers having access to culturing, DNA isolation, and PCR.

The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import.

Leishmania major and Leishmania tropica: II. Effect of an immunomodulator, S(2) complex on the enzymes of the parasites.

S(2) complex has been reported to have a direct antileishmanial effect. The possibility that the direct antileishmanial effect may be due to inhibition of key enzymes involved in glucose metabolism and/ or enzymes associated with virulence was investigated. Cell pellets were prepared from cultures of both axenic amastigotes and promastigotes of Leishmania major (MHOM/IQ/93/MRC6) and L. tropica (MHOM/IQ/93/MRC2). S(2) complex, at various concentrations, was added to the enzyme extracts prepared from the pellets. Results show that in the Embden-Meyerhof pathway, both hexokinase and glucose-phosphate isomerase but not fructophosphokinase were dose dependently inhibited. In the hexose-monophosphate shunt both glucose-6-phosphate dehydrogenase and ribose-5-phosphate isomerase were dose dependently inhibited. Malic dehydrogenase and malic enzyme from the citric-acid cycle were both dose dependently inhibited but succinate dehydrogenase from the same pathway was not inhibited. Both enzymes associated with virulence (protease and acid phosphatase), showed activation rather than inhibition at higher doses of S(2) complex. Thus, the direct antileishmanial effect of S(2) complex may result, partially or entirely, from the inhibition of enzymes that are necessary for the parasites' carbohydrate metabolism.

The molecular characterization of clinical isolates from Indian Kala-azar patients by MLEE and RAPD-PCR.

BACKGROUND: Kala-azar is a serious health problem in India. The situation has worsened further due to the occurrence of cases unresponsive to antimonials. About 30-50% patients do not respond to the prevailing regimen of antimonials. The etiological agent for Indian kala-azar has long been known to be Leishmania donovani. Recently, in a somewhat startling report, it was claimed that L. Tropica causes nearly 25% of current kala-azar cases in India. It was also suggested that this might be in some way related to the unresponsiveness to pentavalent antimonials in the field. MATERIAL/METHODS: Two independent molecular characterization techniques, multilocus enzyme electrophoresis (MLEE) and RAPD-PCR, were employed to analyze 15 clinical isolates from confirmed Indian kala-azar patients collected from the eastern part of the country over a period of nearly 20 years. The collection included six Sb5+-unresponsive and one PKDL case. RESULTS: Our observations strongly suggest that all the clinical isolates, including the antimony Sb5+-unresponsive and PKDL ones, we studied were identical to the WHO reference strain (DD8) for Leishmania donovani by both the above methods and no strain variation might have occurred in two major epidemic and inter-epidemic periods. We also observed that none of the Sb5+-unresponsive stains we analyzed was related to L. Tropica. CONCLUSIONS: We conclude that L. Donovani may be the causal agent for Indian kala-azar and that L. Tropica is most likely not an etiological agent for Indian Kala-azar cases that are unresponsive to antimonials.

The mechanism behind the antileishmanial effect of zinc sulphate. II. Effects on the enzymes of the parasites.

When used in vitro, zinc sulphate has a direct antileishmanial effect. To see if this effect involved the inhibition of the parasites' enzymes, extracts of the promastigotes and axenic amastigotes of Leishmania major (MHOM/IQ/93/MRC6) and L. tropica (MHOM/IQ/93/MRC2) were prepared. Zinc sulphate, at various concentrations, was then added to samples of these extracts before the activities, in the samples, of certain key enzymes of the Embden-Meyerhof pathway, hexose-monophosphate shunt and citric-acid cycle, and of two enzymes associated with virulence (protease and acid phosphatase), were determined. The zinc was found to inhibit every enzyme investigated, usually in a dose-dependent manner. Thus the direct antileishmanial effect of zinc may result, partially or entirely, from the inhibition of enzymes that are necessary for the parasites' carbohydrate metabolism and virulence.

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity.

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.

Members of a unique histidine acid phosphatase family are conserved amongst a group of primitive eukaryotic human pathogens.

Recently, we identified and characterized the genes encoding several distinct members of the histidine-acid phosphatase enzyme family from Leishmania donovani, a primitive protozoan pathogen of humans. These included genes encoding the heavily phosphorylated/glycosylated, tartrate-sensitive, secretory acid phosphatases (Ld SAcP-1 and Ld SAcP-2) and the unique, tartrate-resistant, externally-oriented, surface membrane-bound acid phosphatase (Ld MAcP) of this parasite. It had been previously suggested that these enzymes may play essential roles in the growth, development and survival of this organism. In this report, to further examine this hypothesis, we assessed whether members of the L. donovani histidine-acid phosphatase enzyme family were conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Such phylogenetic conservation would clearly indicate an evolutionary selection for this family of enzymes and strongly suggest and support an important functional role for acid phosphatases to the survival of these parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that homologs of both the Ld SAcPs and Ld MAcP were present in each of the visceral and cutaneous Leishmania species examined (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of enzyme assays showed that all of these organisms expressed both tartrate-sensitive and tartrate-resistant acid phosphatase activities. In addition, homologs of both the Ld SAcPs and Ld MAcP genes and their corresponding enzyme activities were also identified in two Crithidia species (C. fasciculata and C. luciliae) and in Leptomonas seymouri. In contrast, Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens had only very-low levels of such enzyme activities. Cumulatively, results of this study showed that homologs of the Ld SAcPs and Ld MAcP are conserved amongst all pathogenic Leishmania sps. suggesting that they may play significant functional roles in the growth, development and survival of all members of this important group of human pathogens.

A woman and her son with abnormal complicated anthrponotic cutaneous leishmaniasis.

Anthroponotic cutaneous leishmaniasis (ACL.) is known to cause single, self-healing and uncomplicated lesion mainly on the face. This paper presents a son and his mother with positive leishmaniasis IHAT (1/128 and 1/1924 respectively). The two isolates by enzymatic electrophoresis characterization proved to be Leishmania tropica that causes ACL. The son had two large ulcerative ACL on his left hand. The mother had a large ulceration and progressive erosion of the soft tissue (the right cheek and right eye) and the cartilage of the nose disfiguring and debilitating her face. The histpathological examination of the biopsy material obtained from the mother but not the material from her son, showed typical pathological picture of the basal cell carcinoma. No doubt, there are progressive changes in the pathogenesis of L. tropica and that it is one of the predisposing factor of the skin cell carcinoma.

An ecto-ATPase activity present in Leishmania tropica stimulated by dextran sulfate.

In this study we report the effects of sulfated polysaccharides on the ecto-ATPase activity of intact cells of Leishmania tropica. Increasing concentrations of dextran sulfate stimulated progressively the ecto-ATPase activity, but did not modify other ecto-enzymes present on the surface of this parasite, such as 5'nucleotidase, 3'nucleotidase and a membrane-bound acid phosphatase activity. This stimulation was not observed when other sulfated polysaccharides such as chondroitin sulfates and heparin were tested. It depends on size and charge of the dextran sulfated molecule. When the cells were incubated in the presence of dextran sulfate Mr 8,000; 40,000 and 500,000 the stimulation of the ecto-ATPase activity was 11%; 23%; and 63%, respectively, and the stimulation was not observed when desulfated dextran (Mr 40,000) was used. The effects of dextran sulfate also depend on pH of the medium. At pH 7.5, the stimulation was over 60%, whereas at pH 8.5 only 25%. The effects of dextran sulfate 500,000 on the ecto-ATPase activity was totally abolished by spermidine and partially by putrescine, two polyamines synthesized and released by Leishmania.

Mg-dependent ecto-ATPase activity in Leishmania tropica.

ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).

Cutaneous leishmaniasis: iso-enzyme characterisation of Leishmania tropica.

In order to identify and characterise the organisms responsible for Cutaneous Leishmaniasis, parasites were isolated from active lesions, grown in-vitro cultures and identified by iso-enzyme characterisation. Thirteen isolates from different patients were typed as L. tropica. Seven of these isolates were from Afghan refugees encamped in the suburbs of Islamabad, 3 were from patients in Multan, 1 was from a patient from Azad Jammu and Kashmir and 1 was from Besham (Swat, NWFP). The study confirms the presence of anthroponotic Cutaneous Leishamaniasis caused by L. Tropica in Pakistan.

Increased P-type ATPase activity in Leishmania tropica resistant to methotrexate.

In the present report, we show evidence that the membrane from the protozoan parasite Leishmania tropica (LRC-L39), in vitro resistant to 1 mM of methotrexate (MTX), has a significative increased ATPase activity with respect to wild-type line. This ATPase activity is vanadate sensitive, a characteristic of the P-type ATPases included in the ATP-binding casette (ABC) superfamily of transporters, such as P-glycoprotein involved in the multidrug resistance in mammalian cells. Also, this ATPase activity is not modified by MTX, ammonium molybdate and other detergents such as Triton X-100, Brij 58 and lysophosphatidylcholine. However, unlike the P-glycoprotein, we have observed that the ATPase activity is not stimulated by the drugs verapamil and puromycin. This significative ATPase activity could be related to the overexpressed putative P-glycoprotein, with unknown function in these MTX-resistant parasites.

Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) was used to compare promastigote proteinases from two Leishmania species (L. braziliensis and L. major-like). Substrate-SDS-PAGE resolved at least 6 distinct proteinase activities with relative molecular masses between 20 and 65 kDa. The profile of proteinase activity was the same for both species, although qualitative differences were observed. L. major-like expressed a low molecular weight (20 kDa) proteinase with maximum activity at pH 7.0, which was demonstrable from pH 5.0 to pH 8.0. The 20-kDa protease was recovered in the detergent phase of TX-114 and was inhibited by the sulfhydryl group-blocking reagent E-64 (2.5 mM) and the non-specific inhibitor iodoacetic acid (1 mM). Pepstatin (1 microM) and PMSF (2.5 mM) did not inhibit the 20-kDa enzyme. The present study suggests that both Leishmania species studied express hydrophobic cysteine proteases of different molecular weights, since about 2 x 10(7) parasites were analyzed in each lane and the proteolytic activity developed at 37 degrees C for 16 h.

Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) was used to compare promastigote proteinases from two Leishmania species (L. braziliensis and L. major-like). Substrate-SDS-PAGE resolved at least 6 distinct proteinase activities with relative molecular masses between 20 and 65 kDa. The profile of proteinase activity was the same for both species, although qualitative differences were observed. L. major-like expressed a low molecular weight (20 kDa) proteinase with maximum activity at pH 7.0, which was demonstrable from pH 5.0 to pH 8.0. The 20-kDa protease was recovered in the detergent phase of TX-114 and was inhibited by the sulfhydryl group-blocking reagent E-64 (2.5 mM) and the non-specific inhibitor iodoacetic acid (1 mM). Pepstatin (1 microM) and PMSF (2.5 mM) did not inhibit the 20-kDa enzyme. The present study suggests that both Leishmania species studied express hydrophobic cysteine proteases of different molecular weights, since about 2 x 10(7) parasites were analyzed in each lane and the proteolytic activity developed at 37 degrees C for 16 h

Extracellular dephosphorylation in the parasite, Leishmania major.

Leishmania major promastigotes were analyzed for the presence of protein phosphatase activity in intact cells and membrane-enriched fractions. Parasite phosphoproteins, phosphorylated in live cells with [gamma-32P]adenosine 5'-triphosphate (ATP) and an endogenous leishmanial ectokinase, were dephosphorylated by endogenous protein phosphatase-like activity in intact cells and a membrane-rich fractions. An alkaline phosphatase-like activity was also identified using the artificial substrate, p-nitrophenyl phosphate (pNPP). This activity was localized on the extracellular membrane of intact parasites, as well as in the particulate fraction of lysed cells. The phosphatase activity measure using pNPP had inhibition properties and a pH profile between protein phosphatase and general alkaline phosphatases. This study supports the observation that there is extracellular protein phosphorylation/dephosphorylation in L. major which may play a significant role in host cell-parasite recognition and infection.

Extracellular dephosphorylation in the parasite, leishmania major / Desfosforilacion extracelular en el parasito, leishmania major

Leishmania major promastigotes were analyzed for the presence of protein phosphatase activity in intact cells and membrane-enriched fractions. Parasite phosphoprorylated in live cells with {*-32P} adenosine 5'-triphosphate (ATP) and an edogemous leishmanial ectokinase, were dephosphorylated by endogenous protein phosphatase like activity in intact cells and membrane-rich fractions. An alkaline phosphatase-like activity was also identified using the artificial substrate, p-nitrophenyl phosphate (pNPP). This activity was localized on the extracellular membrane of intact parasite, as well as in the particulate fraction of lysed cells. The phosphatase activity measure using pNPP had inhibition properties and a pH profile between protein phosphatases and general alkaline phosphatase. This study supports the observation that there is extracellular protein phosphorylation/dephosphorylation in L.major which may play a significant role in host-cell-parasite recognition and infection

Extracellular phosphorylation in the parasite, Leishmania major.

Intact promastigotes or cell-free extracts of the parasite Leishmania major were labelled with adenosine 5'[gamma-32P]-triphosphate (ATP). This resulted in the identification of eleven phosphoproteins. [gamma-32P]ATP incorporation into endogenous and exogenous substrates was insensitive to most of the commonly used protein kinase inhibitors and activators indicating that the leishmanial enzyme(s) may represent a new class of kinase(s). In addition, exogenous substrate specificity was inconsistent with the preferences of second messenger-dependent protein kinases. Cyclic AMP had differential effects on phosphorylation in intact cells and lysates. The majority of kinase activity could be attributed to an externally oriented membrane-associated protein kinase(s), as no specific cytosolic phosphoproteins were found and intact cells phosphorylated exogenous substrates. Labelled ATP did not cross the membrane and [alpha-32P]ATP was an unsuitable substrate for the phosphorylation activity. The ectokinase activity on live Leishmania exhibited a different substrate preference when compared to the protein kinase activity in the particulate fraction, suggesting that more than one protein kinase may be present in L. major. Three serine-labelled phosphoproteins were specifically released into the medium. The presence of an ecto-kinase and these released phosphoproteins may play a significant role in host-parasite interactions.