Logarithmic Phase Promastigotes Research Articles

Sequencing of hsp70 for discernment of species from the Leishmania (Viannia) guyanensis complex from endemic areas in Colombia

BackgroundColombia is ranked very high among countries with the highest numbers of endemic Leishmania species (n = 9) causing human disease. Although much effort has been devoted to generating simple and specific tools for Leishmania species identification, challenges remain in the discrimination of species belonging to the Leishmania (Viannia) guyanensis complex: L. (V.) guyanensis and L. (V.) panamensis.MethodsA set of seven reference strains of species belonging to the L. (Leishmania) and L. (Viannia) subgenera, clinical strains from human cases of cutaneous leishmaniasis (CL; n = 26) and samples collected from sylvatic mammals and sand flies (n = 7) from endemic areas in Colombia were analyzed in this study. The heat-shock protein 70 gene (hsp70) was amplified by PCR from DNA extracted from logarithmic-phase promastigotes or tissue samples, and the PCR products were sequenced. Sequence alignment was performed against a set of previously published and curated sequences, and phylogenetic analysis based on the maximum-likelihood and Bayesian inference approaches was conducted. Haplotype diversity among strains and species of the L. (V.) guyanensis complex was explored using a median-joining network.ResultsSequencing of the hsp70 gene for L. (Viannia) spp. typing was comparable to species identification using isoenzyme electrophoresis or monoclonal antibodies. Complete species matching was found, except for one sylvatic sample with an identity yet unsolved. Among the L. (V.) panamensis clinical strains, two distinctive phylogenetic clusters were found to correlate with two different zymodemes: L. (V.) panamensis Z2.2 and Z2.3. Analysis of samples from sylvatic environments identified novel records of naturally infected wild mammal and sand fly species.ConclusionsOur results support the adequacy of hsp70 gene sequencing as a single-locus approach for discrimination of L. (Viannia) spp., as well as for exploring the genetic diversity within the L. (V.) guyanensis complex.Graphical

Expression analysis of centrin gene in promastigote and amastigote forms of leishmania infantum iranian isolates: a promising target for live attenuated vaccine development against canine leishmaniasis

BackgroundLeishmania parasites express various essential proteins in different growth phases (logarithmic/stationary) and forms (promastigote/amastigote). Targeting the genes encoding such proteins paves the way for controlling these parasites. Centrin is an essential gene, which its protein product seems to be vital for the proliferation of Leishmania parasites. Herein, this study was contrived to analyze the expression level of the centrin gene in different growth phases and forms of Leishmania infantum (L. infantum) parasites isolated from various endemic areas of canine leishmaniasis (CanL) in Iran.ResultsAll three collected isolates were identified as L. infantum using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time reverse transcription (RT)-PCR revealed a statistically significant up-regulation (3.13-fold) in the logarithmic phase promastigotes compared to stationary ones (p < 0.01), whereas centrin was expressed equally in intracellular amastigotes at different time points during cell culture. Also, our finding revealed a slight up-regulation of the centrin gene (1.22-fold) in the intracellular amastigotes compared to logarithmic phase promastigotes, which was found statistically non-significant (p > 0.05).ConclusionsCentrin gene in Iranian isolates of L. infantum is more expressed in exponential than stationary phases and seems to be considered as a promising target in the development of a genetically modified live attenuated vaccine for CanL control.

In Vitro Activity of Cordia myxa Mucilage Extract Against Leishmania major and L. infantum Promastigotes.

Dear Editor, Leishmaniasis is an important protozoan disease, which still is a major health problem in endemic countries, such as Iran (1). Despite, recent advances, there is a long way until arriving to the ideal anti-leishmanial agents, with less toxicity, side effects and more potent efficacy. The World Health Organization (WHO) has suggested usage of herbal plants to reach this aim (2). In the Iranian folk medicine, Cordia myxa is an herbal plant, which belongs to the Boraginaceae family and grows in tropical regions, such as Iran (3). Its fruits contain phenolic compounds, which can be a potential therapeutic option for leishmaniasis, similarly to several herbal plants, such as green tea extract (4). The objective of this study was to evaluate the in vitro effects of C. myxa mucilage on Leishmania Major and L. infantum promastigotes. The fruits of C. myxa were collected from a traditional harvesting region for this plant, Ahvaz, Southwest Iran. A total of 50g of C. myxa fruits were boiled slowly, in one liter of hot water, for 30 minutes. The residue was filtrated through Whatman paper No. 33. The mucilage filtrate was freeze-dried. The mucilage extract of C. myxa was initially dissolved in dimethyl sulphoxide (DMSO) (Sigma-Aldrich Corp., St. Louis, MO, USA) and further diluted with the RPMI 1640 medium (GIBCO, Grand Island, NewYork, USA) after a sterilizing the filtrate. To examine the anti-leishmanial activity of the extract, logarithmic phase promastigotes of L. infantum (MCAN/IR/96/LON49) and L. major (MRHO/IR/75/ER) (1×106 cells/ mL) were seeded in a 96-well microtiter plate, in the presence of the serial concentrations (0, 0.61, 1.22, 2.44, 4.88, 9.75, 19.5, 39, 78, and 156 mg/ mL w/v) and then incubated at 24°C, for 72 hours. Anti-leishmanial activity was assayed by light microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) MTT method. Each assay was performed in triplicate, with three independent experiments. The concentration inhibiting parasite growth by 50% (IC50 value) was calculated with a sigmoid dose-response curve. Mucilage extract of C. myxa was active against promastigotes form of L. major and L. infantum, with an IC50 of 26 ± 2.2 mg/mL and an IC50 of 35 ± 2.2 mg/mL, respectively. The survival percentage of L. major and L. infantum promastigotes after 72 hours treatment, with different concentrations of C. myxa mucilage extract, is shown in Table 1. Table 1. Percentage of Survival Leishmania major and L. infantum promastigotes After 72 Hours Treatment With Different Concentrations of Cordia myxa Mucilage Extract This study demonstrated that C. myxa extract had anti-leishmanial effects against L. infantum and L. major promastigotes, under in vitro conditions. Phytochemical studies have demonstrated C. myxa, as a good source of trace elements (such as selenium, copper, zinc, iron and manganese), phenolic, and flavonoid compounds (robinin, datiscoside, rutin, hesperidin, dihydrorobinetin, caffeic acid and chlorogenic acid) (5). Probably, C. myxa has potential anti-leishmanial activity because of phenolic and flavonoid compounds (rutin and caffeic acid) and several trace element content (selenium). Flavonoides bind to the nucleotide binding domains (NBD) of the ATP binding cassette (ABC) transporters, and their role is well established in most medications resistance phenomena, such as anticancer and anti-leishmaniasis drugs. Finally, these cellular events induced an increase of hydrophobic interactions, which leads to inhibition of multiple drug resistance (6). Several studies have focused on the anti-leishmanial activity of selenium compounds. These findings mention the selenium compounds, as novel anti-leishmanial agents for amastigotes and promastigotes, with more potent activity and lower cytotoxicty than miltefosine and edelfosine (IC50 promastigotes = 0.9 - 17 µM and 0.3 - 9 µM for amastigotes) (7, 8). Other benefits of C. myxa can be attributed to less cytotoxicity and cost, compared to current anti-leishmanial drugs, whose main limitation for their administration, particularly in developing countries (9).

Leishmanicidal activity assessment of olive tree extracts

Leishmaniasis, a protozoan parasitic disease that remains a major worldwide health problem with high endemicity in developing countries, is prevalent around the Mediterranean basin. High cost, systemic toxicity, and diminished efficacy due to development of parasite resistance are the serious drawbacks of current treatment options. Thus, identifying new, effective, and safer anti-leishmanial drug(s) is of paramount importance. Here we tested the anti-promastigote and anti-amastigote activity of five natural products, including oleuropein and hydroxytyrosol, present in olive tree leaves and olive mill wastewater. These products are recognized as low-cost starting materials rich in bioactive compounds, particularly biophenols. Oleuropein and hydroxytyrosol exhibited the best inhibitory effect among the natural products tested in both stationary and middle logarithmic phase promastigotes of L. infantum, L. donovani, and L. major. Similarly, oleuropein and hydroxytyrosol demonstrated the highest selectivity index ratio against L. donovani amastigotes that parasitize J774A.1 macrophages. Moreover, oleuropein was tested in vivo in an experimental visceral leishmaniasis model. L. donovani-infected BALB/c mice received intraperitoneal oleuropein a total of 14 times at intervals of every other day. Three days after treatment termination, the spleen parasitic burden was reduced >80%. Of interest, this effect of oleuropein persisted and was even enhanced 6 weeks after the termination of the treatment, as determined by parasite depletion of >95% in liver and spleen. These findings contribute to the potential development of natural products as effective drugs against parasites of the Leishmania genus, with low cost and diminished cytotoxicity.

Differential Surface Deposition of Complement Proteins on Logarithmic and Stationary PhaseLeishmania chagasiPromastigotes

Previous works demonstrated that various species of Leishmania promastigotes exhibit differential sensitivity to complement-mediated lysis (CML) during development. Upon exposure to normal human serum (NHS), cultures of Leishmania chagasi promastigotes recently isolated from infected hamsters (fewer than 5 in vitro passages) are CML-sensitive when in the logarithmic growth phase but become CML-resistant upon transition to the stationary culture phase. Visualization by light and electron microscopy revealed dramatic morphological differences between promastigotes from the 2 culture phases following exposure to NHS. Flow cytometric analysis demonstrated that surface deposition of the complement components C3, C5, and C9 correlated inversely with promastigote CML-resistance. The highest levels of complement protein surface accumulation were observed for logarithmic phase promastigotes, while stationary phase promastigotes adsorbed the least amount of complement proteins. Additionally, fluorescence microscopy revealed that C3 and C5 localized in a fairly uniform pattern to the plasma membrane of promastigotes from logarithmic phase cultures, while the staining of promastigotes from stationary phase cultures was indistinguishable from background. By Western blot analysis, high levels of the complement proteins C3, C5, and C9 were detected in the total lysates of NHS-exposed logarithmic phase L. chagasi promastigotes, relative to NHS-exposed stationary phase promastigotes; this finding indicates that the low levels of C3 and C5 seen on the surface of stationary phase promastigotes were not due to protein uptake/internalization. Together, these data demonstrate the differential deposition of complement proteins on the surfaces of logarithmic and stationary phase L. chagasi promastigotes. The data support a model wherein stationary phase L. chagasi promastigotes resist CML by limiting the deposition of C3 and its derivatives, which, in turn, limit surface levels of complement proteins (including C5 and C9) that form the lytic membrane attack complex.

Olive tree extracts with potential leishmanicidal activity

Nowadays, drugs available for treating leishmaniasis are limited in number and all of them have different disadvantages such as toxicity, resistance and cost. Aiming to identify natural products with leishmanicidal activity, seven natural products derived from olive tree extracts were tested against three different Leishmania species: L. infantum MON-1, L. donovani MON-2 and L.major LV39, the former two causing visceral and the latter cutaneous leishmaniasis. The leishmanicidal activity was evaluated by a quantitative colorimetric assay using the oxidation-reduction Alamar Blue method on stationary or logarithmic phase promastigotes (Table 1).

Molecular genetic analysis of purine nucleobase transport in Leishmania major

Leishmania major and all other parasitic protozoa are unable to synthesize purines de novo and are therefore reliant upon uptake of preformed purines from their hosts via nucleobase and nucleoside transporters. L. major expresses two nucleobase permeases, NT3 that is a high affinity transporter for purine nucleobases and NT4 that is a low affinity transporter for adenine. nt3((-/-)) null mutant promastigotes were unable to replicate in medium containing 10 microM hypoxanthine, guanine, or xanthine and replicated slowly in 10 microM adenine due to residual low affinity uptake of that purine. The NT3 transporter mediated the uptake of the anti-leishmanial drug allopurinol, and the nt3((-/-)) mutants were resistant to killing by this drug. Expression of the NT3 permease was profoundly downregulated at the protein but not the mRNA level in stationary phase compared with logarithmic phase promastigotes. The nt4((-/-)) null mutant was quantitatively impaired in survival within murine bone marrow-derived macrophages. Extensive efforts to generate an nt3((-/-))/nt4((-/-)) dual null mutant were not successful, suggesting that one of the two nucleobase permeases must be retained for robust growth of the parasite. The phenotypes of these null mutants underscore the importance of purine nucleobase transporters in the Leishmania life cycle and pharmacology.

Isolation, characterization and analysis of the expression of the Leishmania ribosomal PO protein genes

Two tandemly linked genes are present in the Leishmania infantum genome that code for the acidic ribosomal PO protein. The genes are identical in the coding region, although a striking lack of nucleotide sequence conservation is observed when the boundaries of the coding regions between both genes are compared. The 3′ untranslated regions of the two genes are, moreover, different in size. The deduced amino acid sequence of the L. infantum PO protein ( LiPO) shows a high degree of sequence conservation, including the highly charged conserved C-terminal domain, with the ribosomal PO proteins of other eukaryotic organisms. Northern blot experiments showed that two different size class transcripts are expressed in the gene cluster and that the steady state level of each of the transcripts in logarithmic phase promastigotes is markedly different. The abundance of both transcripts is down-regulated in parasite cultures on reaching stationary phase. Since it seems that the two Leishmania ribosomal PO genes are expressed in a single polycistronic transcript, it is likely that the different levels of PO mRNAs observed in cultured cells is due to a postranscriptional regulatory mechanism.

Genomic organization and expression of two independent gene arrays coding for two antigenic acidic ribosomal proteins of Leishmania.

In the present paper we describe the isolation and characterization of four novel genes of the parasitic protozoan Leishmania infantum. These genes are organized as two independent gene clusters, and they are related by nucleotide sequence to eukaryotic genes encoding acidic ribosomal proteins. Each gene cluster contains two tandemly linked genes coding for identical proteins. Each of the proteins coded by the gene clusters (called LiP and LiP') are highly divergent in sequence, showing the characteristic features of eukaryotic P-proteins from the P2 group. In spite of the sequence conservation of the coding regions of each of the genes in the cluster, the 5'- and 3'-untranslated regions are heterogeneous in sequence. The analysis of the expression of these genes indicates that logarithmic phase promastigotes show increased levels of LiP- and LiP'-specific transcripts compared with stationary phase promastigotes. The steady state RNA levels of the LiP and LiP' genes show a similar dependence of the growth phase of the parasite. Using specific probes for the divergent 3'-untranslated regions of each of the genes, it was found that the abundance of the mature transcripts is different even when the transcripts are derived from the same gene cluster. These findings probably indicate that the 3'-untranslated regions may influence the stability or turnover of the transcripts derived from both LiP and LiP' gene clusters.

The effect of ongoing protein synthesis on the steady state levels of Gp63 RNAs in Leishmania chagasi

G63, the major surface glycoprotein of Leishmania chagasi promastigotes, increases 11-fold in amount as promastigotes grow from logarithmic to stationary phase. Transcripts from three different classes of gp63 genes are differentially expressed during this development (Ramamoorthy, R., Donelson, J. E., Paetz, K. E., Maybodi, M., Roberts, S. P., and Wilson, M. E. (1992) J. Biol. Chem. 267, 1888-1895). We studied the effect of protein synthesis inhibitors on gp63 mRNAs. The steady state level of log class gp63 RNA, expressed primarily in logarithmic phase promastigotes, increased 16.5-fold after incubation in cycloheximide. A similar increase in log gp63 RNAs was caused by inhibitors that block different steps in translation. In contrast, the levels of stationary class gp63 RNA, expressed in stationary phase parasites, and constitutive class gp63 RNA, expressed throughout promastigote growth, increased only 2.3- and 1.5-fold, respectively. The latter was not statistically significant. Nuclear run-on assays showed that the cycloheximide effect was not due to an increased rate of transcription. However, the t1/2 of log RNAs was prolonged 6.5-fold after incubation in cycloheximide, in contrast to a 1.7-fold increase in the t1/2 of ATPase RNA, suggesting that cycloheximide specifically stabilizes log gp63 mRNAs. Thus, a highly labile negative regulatory protein, such as an RNase, may specifically target log gp63 RNAs for degradation.

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO)--a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.

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 electrondensity. 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.