Amazonensis Strain Research Articles

Genomic analyses reveal moderate levels of ploidy, high heterozygosity and structural variations in a Colombian isolate of Leishmania (Leishmania) amazonensis

Leishmania amazonensis is one of the causative agents of the different forms of cutaneous leishmaniasis present in Latin America. This species has been isolated from humans and animals (canine/feline) in some endemic regions of Colombia. Therefore, L. amazonensis is of great relevance at the clinical and epidemiological levels in medicine and veterinary science. Until now, very few genomes from this species are available. Here, we report the complete genome sequence of a laboratory-adapted L. amazonensis strain isolated from a human patient with clinical manifestation of cutaneous leishmaniasis in Colombia. The genome sequence not only allowed inter and intra-species comparative analyses to be performed with the sequenced genomes of L. amazonensis strains from different geographical regions, but also increased our knowledge about the genomic behavior of this L. amazonensis Colombian strain. This isolate was also characterized in terms of single nucleotide polymorphisms, chromosome and gene copy number variations (CNVs). The results revealed moderate aneuploidy, CNVs in genes involved in the virulence, growth, and survival of the parasite, and in the distributions of the multicopy genes on some chromosomes, as well as a high level of heterozygosity. The data confirmed previous reports that identified unique variations in L. amazonensis, suggesting aneuploidy may not have a high fitness cost and may allow the rapid generation of diversity in Leishmania parasites growing normally.

Mapping Alterations Induced by Long-Term Axenic Cultivation of Leishmania amazonensis Promastigotes With a Multiplatform Metabolomic Fingerprint Approach.

Leishmaniases are widespread neglected diseases with an incidence of 1.6 million new cases and 40 thousand deaths per year. Leishmania parasites may show distinct, species-specific patterns of virulence that lead to different clinical manifestations. It is well known that successive in vitro passages (SIVP) lead to the attenuation of virulence, but neither the metabolism nor the pathways involved in these processes are well understood. Herein, promastigotes of a virulent L. amazonensis strain recently isolated from mice was compared to SIVP derived and attenuated promastigotes, submitted to 10, 40, and 60 axenic passages and named R10, R40, and R60, respectively. In vitro assays and in vivo tests were performed to characterize and confirmed the attenuation profiles. A metabolomic fingerprint comparison of R0, R10, and R60 was performed by means of capillary electrophoresis, liquid and gas chromatography coupled to mass spectrometry. To validate the metabolomic data, qPCR for selected loci, flow cytometry to measure aPS exposure, sensitivity to antimony tartrate and ROS production assays were conducted. The 65 identified metabolites were clustered in biochemical categories and mapped in eight metabolic pathways: ABC transporters; fatty acid biosynthesis; glycine, serine and threonine metabolism; β-alanine metabolism; glutathione metabolism; oxidative phosphorylation; glycerophospholipid metabolism and lysine degradation. The obtained metabolomic data correlated with previous proteomic findings of the SVIP parasites and the gene expression of 13 selected targets. Late SIVP cultures were more sensitive to SbIII produced more ROS and exposed less phosphatidylserine in their surface. The correspondent pathways were connected to build a biochemical map of the most significant alterations involved with the process of attenuation of L. amazonensis. Overall, the reported data pointed out to a very dynamic and continuous metabolic reprogramming process, accompanied by changes in energetic, lipid and redox metabolisms, membrane remodeling and reshaping of parasite-host cells interactions, causing impacts in chemotaxis, host inflammatory responses and infectivity at the early stages of infection.

Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model.

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice

The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis.

Fatty acid and hydrocarbon composition in tropical marine Shewanella amazonensis strain SB2BT

Shewanella amazonensis strain SB2B(T) is an isolate from shallow-water marine sediments derived from the Amazon River delta. This bacterium contained a long-chain polyunsaturated hydrocarbon, all-cis -3,6,9,12,16,19,22,25,28 hentriacontanonaene (C31:9), constituting 1-2% of the total fatty acid methyl ester and hydrocarbon fraction, which was produced dependently of decreased growth temperature. Analysis of its cellular fatty acid composition demonstrated that isopentadecanoic acid was the major fatty acid component and that all the main monounsaturated fatty acids had straight chains with a cis configuration. However, monoenoic cyclopropyl fatty acids, which were previously reported to be present in this bacterium, were not detected by mass spectrometric analysis. The growth temperature affected the content of Δ9-cis -hexadecenoic [16:1(Δ9c)], palmitic, and heptadecanoic acids. These results suggest that C31:9, as well as 16:1(Δ9c) might be involved in adaptation to low temperature in S. amazonensis strain SB2B(T) . Our result suggests that polyunsaturated fatty acid synthase protein complex may be involved in synthesis of C31:9 but not in production of eicosapentaenoic acid.

O uso da associação azitromicina e N-metil glucamina no tratamento da leishmaniose cutânea causada por Leishmania (Leishmania) amazonensis em camundongos C57BL6

The first choice treatment for cutaneous Leishmaniasis is N-methyl glucamine: it has high toxicity, requires parenteral administration and cure is not always reached. Azythromycin showed in vitro action and controversial results in humans with the disease. To verify if the association of N-methyl-glucamine - azythromycin is more effective than N-methyl-glucamine alone for the treatment of experimental Leishmaniasis. Twenty-five C57BL/6 mice were inoculated with L. (L.) amazonensis strain and divided into two groups. One group was treated with 400 mgSbV/kg/day of N-methyl glucamine and 200mg/kg/day of azythromycin for 20 days and the other group received the same dose of N-methyl glucamine alone during the same period of time. Clinical and parasitological evaluations were submitted to statistical analyses. There was no statistical difference in clinical analysis, in amastigotes investigation and in cultures. There were significant differences in cultures using limiting dilution, which showed lower efficacy of the association N-methyl glucamine -azythromycin. N-methyl glucamine-azythromycin association was not more effective than N-methyl glucamine alone.

Synthesis and preliminary evaluation of new ursolic and oleanolic acids derivatives as antileishmanial agents

A series of new ursolic and oleanolic acids derivatives was synthesized via ursolic or oleanolic acids, previously extracted from South American Ilex species. These new compounds were tested for in vitro antiparasitic activity on Leishmania amazonensis and Leishmania infantum strains. Some of these compounds showed activity against the promastigote forms of L. amazonensis or L. infantum, with IC50 ranging from 5 to 12 μM. As expected, most of the compounds showed a significant level of cytotoxicity against monocytes (IC50 = 2-50 μM). From a structure-activity relationships point of view, these pharmacological results enlightened mainly the importance of an acetylation at position 3 of the oleanolic acid skeleton in the activity against the L. amazonensis strain, and of a bis-(3-aminopropyl)piperazine moiety on the carboxylic function of ursolic acid against the L. infantum strain.

In vitro Maintenance of Leishmania Amastigotes Directly from Lesions: Advantages and Limitations

Leishmania amazonensis (MHOM/BR/77/LTB0016) amastigotes were obtained from mouse cutaneous lesions and maintained in vitro for 48 hr at pH 4.6, 33 C. These organisms were reproducing, capable of transformation to promastigotes, and did not display the promastigote-specific antigen, GP46. In contrast, 97% of the organisms maintained for 24 hr at 31 C, pH 7.3, were positive for GP46. Thus, short-term cultivation of this L. amazonensis strain under appropriate conditions can provide a high yield of amastigotes for various in vivo and in vitro studies. However, the possible interference of host immunoglobin on the surface of these amastigotes needs to be considered because fluorescent-labeled anti-mouse immunoglobulin was detected on 16% of lesion-derived amastigotes even after 114 hr of cultivation.

Antiprotozoal activity of Jatrogrossidione from Jatropha grossidentata and Jatrophone from Jatropha isabellii

The activity of jatrogrossidione, the main diterpene of Jatropha grossidentata and jatrophone from Jatropha isabellii was determined against Leishmania and Trypanosoma cruzi strains in vitro as well as against Leishmania amazonensis in vivo. Jatrogrossidione showed a strong in vitro leishmanicidal and trypanocidal activity with IC100 of 0.75 and 1.5–5.0 μg/mL, respectively. Under similar conditions, the IC100 of glucantime, ketoconazole and pentamidine towards Leishmania strains were >100, 50–100 and 1 μg/mL, respectively. The IC50 of jatrogrossidione was <0.25 μg/mL against amastigote forms of Leishmania infecting macrophages, with toxicity at concentrations higher than 0.5 μg/mL. BALB/c mice infected with L. amazonensis strain PH 8 were treated 24 h after infection with jatrogrossidione and jatrophone for 13 consecutive days. Jatrophone at 25 mg/kg/day subcutaneously administered was significantly active (p<0.05) against the virulent strain PH 8 of L. amazonesis; it was more active than Glucantime at 112 mg Sbv per kg/day. Subcutaneous administration of jatrophone, however, proved to be too toxic under our assay conditions. Assays of single local treatment on the footpad infection 2 weeks after inoculation of L. amazonensis indicated that jatrogrossidione and jatrophone were inactive at the selected doses.

Antiprotozoal activity of Jatrogrossidione from Jatropha grossidentata and Jatrophone from Jatropha isabellii

The activity of jatrogrossidione, the main diterpene of Jatropha grossidentata and jatrophone from Jatropha isabellii was determined against Leishmania and Trypanosoma cruzi strains in vitro as well as against Leishmania amazonensis in vivo. Jatrogrossidione showed a strong in vitro leishmanicidal and trypanocidal activity with IC100 of 0.75 and 1.5–5.0 μg/mL, respectively. Under similar conditions, the IC100 of glucantime, ketoconazole and pentamidine towards Leishmania strains were >100, 50–100 and 1 μg/mL, respectively. The IC50 of jatrogrossidione was <0.25 μg/mL against amastigote forms of Leishmania infecting macrophages, with toxicity at concentrations higher than 0.5 μg/mL. BALB/c mice infected with L. amazonensis strain PH 8 were treated 24 h after infection with jatrogrossidione and jatrophone for 13 consecutive days. Jatrophone at 25 mg/kg/day subcutaneously administered was significantly active (p<0.05) against the virulent strain PH 8 of L. amazonesis; it was more active than Glucantime at 112 mg Sbv per kg/day. Subcutaneous administration of jatrophone, however, proved to be too toxic under our assay conditions. Assays of single local treatment on the footpad infection 2 weeks after inoculation of L. amazonensis indicated that jatrogrossidione and jatrophone were inactive at the selected doses.