Leishmania Major

Multi‐locus microsatellite typing (MLMT) has been employed to infer the population structure of Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) sandflies and assign individuals to populations. Phlebotomus papatasi sandflies were collected from 35 sites in 15 countries. A total of 188 P. papatasi individuals were typed using five microsatellite loci, resulting in 113 different genotypes. Unique microsatellite signatures were observed for some of the populations analysed. Comparable results were obtained when the data were analysed with Bayesian model and distance‐based methods. Bayesian statistic‐based analyses split the dataset into two distinct genetic clusters, A and B, with further substructuring within each. Population A consisted of five subpopulations representing large numbers of alleles that were correlated with the geographical origins of the sandflies. Cluster B comprised individuals collected in the Middle East and the northern Mediterranean area. The subpopulations...

Não sei por quantas lutas irei passar... Mas sei que nunca passarei sozinho." Isaias 43:2 A Deus, por ter me concedido a vida e me abençoado em todos os momentos, com saúde, paz, fé e determinação para desenvolver esse trabalho. A todos os meus familiares, em especial meu pai Darci, minha mãe Ivanice, minhas irmãs Fabiana e Franciele e ao meu marido Alex que são a fortaleza da minha vida. Aos meus amigos, colegas de trabalho e orientadora pelo apoio, força, incentivo, companheirismo e amizade. Sem vocês nada disso seria possível! AGRADECIMENTOS A minha orientadora Valeria Marçal Felix de Lima pelo companheirismo, amizade, paciência, dedicação, por me mostrar o caminho da ciência. Muito obrigada pela oportunidade. A minha banca de qualificação Profa. e orientadora Dra. Valéria Marçal Félix de Lima, Profa. Dra. Gisele Fabrino Marques e Profa. Dra. Caris Nunes pelas relevantes considerações sobre o meu trabalho. A minha banca de defesa de dissertação de mestrado, Profa. e orientadora Dra. Valéria Marçal Félix de Lima, Profa. Dra Gisele Fabrino Machado e ao Prof. Dr. Hélio José Montassier por aceitar o convite e pelas sugestões que levaram a melhora do meu trabalho. Aos meus grandes amigos Kathlenn Silva e Breno Almeida por dividir comigo os conhecimentos adquiridos e me ajudar na coleta do material. As

Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host-parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 A resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 A ˚. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3 2 21.

Treatment of cutaneous leishmaniasis (CL) is difficult due to the scarce number of drugs able to eliminate completely the intracellular form of the parasite. In the present study, the aim was to evaluate: i) phytochemical properties of extracts from Libidibia ferrea; ii) antileishmanial activity of extracts from L. ferrea against promastigotes and amastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) guyanensis; iii) the effects of topical treatment using hydrogel containing active extract of L. ferrea on golden hamsters infected with L. (L.) amazonensis. Extracts from leaves, branches and fruits of L. ferrea were obtained with hexane and methanol and were tested by in vitro assays in promastigotes and murine macrophages J774 experimentally infected with amastigotes of Leishmania. Groups of hamsters with CL received topical treatment with a formulation of extract (10%) hydrogels, 50 mg.day-1 for 40 days. In vitro activity of FrMeOH (methanolic extract from frui...

Arginase is an enzyme that converts L-arginine to urea and L-ornithine, an essential substrate for the polyamine pathway supporting Leishmania (Leishmania) amazonensis replication and its survival in the mammalian host. L-arginine is also the substrate of macrophage nitric oxide synthase 2 (NOS2) to produce nitric oxide (NO) that kills the parasite. This competition can define the fate of Leishmania infection. The transcriptomic profiling identified a family of oxidoreductases in L. (L.) amazonensis wild-type (La-WT) and L. (L.) amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. We highlighted the identification of an oxidoreductase that could act as nitric oxide synthase-like (NOS-like), due to the following evidences: conserved domain composition, the participation of NO production during the time course of promastigotes growth and during the axenic amastigotes differentiation, regulation dependence on arginase activity, as well as reduction of NO amount...

is a protozoan parasite that alternates its life cycle between the sand fly and the mammalian host macrophages, involving several environmental changes. The parasite responds to these changes by promoting a rapid metabolic adaptation through cellular signaling modifications that lead to transcriptional and post-transcriptional gene expression regulation and morphological modifications. Molecular approaches such as gene expression regulation, next-generation sequencing (NGS), microRNA (miRNA) expression profiling, Western blot analyses and enzymatic activity profiling, have been used to characterize the infection of murine BALB/c and C57BL/6 macrophages, as well as the human monocytic cell-lineage THP-1, with wild type (WT) or arginase knockout (arg ). These models are being used to elucidate physiological roles of arginine and polyamines pathways and the importance of arginase for the establishment of the infection. In this review, we will describe the main aspects of -host interact...

Background & objectives: Cutaneous leishmaniasis is an infection caused by protozoan genus Leishmania. Although glucantime is commonly used for the treatment of leishmaniasis, it has some side effects including increased liver enzymes and electrocardiogram changes. In addition, the drug is expensive, the injection is painful, and research shows that resistance of parasite to glucantime is growing in different parts of the world. Therefore, scientists are paying more attention to develop new drugs such as nanosilver solution. The present study is an attempt to evaluate the in vivo topical effects of different concentrations of nanosilver solution in the treatment of leishmaniasis lesions. Methods: In all, 90 female Balb/c mice aged 6-8 wk were infected with 2×10 6 viable stationary-phase promastigotes in the base of tail. Different concentrations (60, 80, 120, 130 and 2000 ppm) nanosilver solution were used in the present study to test the efficacy in the treatment of lesions. Clinical control of the infection trends was conducted weekly for 5 wk by measuring lesion diameter with standard Kulis-Vernieh. Data were analyzed by paired t-test, analysis of variance (ANOVA), and Tukey test. Results: Mean lesion diameter pre-and post-treatment did not significantly differ between different treatment groups (p >0.05). Likewise, a significant difference in splenic parasite load was also not observed between different treatment groups. Interpretation & conclusion: Based on our results, different concentrations of nanosilver are ineffective in reducing mean sizes of lesions.

Background & objectives: Leishmaniasis is a parasitic disease transmitted by sand flies. Many investigations are performed to find an effective and safe treatment for leishmaniasis. In this study, we evaluated the efficacy of herbal extracts of Thymus vulgaris (Thyme) and Achillea millefolium (Yarrow), propolis hydroalcoholic extract and systemic glucantime against cutaneous leishmaniasis in Balb/c mice. Methods: A total of 45 mice were randomised into five groups each including nine mice. They were treated with pure ethanol 70°, systemic glucantime, Achillea millefolium hydroalcoholic extract, Thymus vulgaris hydroalcoholic extract and propolis hydroalcoholic extract for six weeks. The statistical tests including student t-test were used for analysis. Data were analyzed by SPSS software, ver 13.00. Results: Mean of ulcer size reduction were-17.66,-22.57, 43.29, 36.09 and 43.77% for the alcohol, glucantime, yarrow, thyme and propolis groups, respectively. The results were suggestive that Thymus vulgaris, Achillea millefolium and propolis hydroalcoholic extracts were significantly more effective in reduction of ulcer size as compared with glucantime (p = 0.006, 0.002 and 0.008, respectively). Interpretation & conclusion: Our results are suggestive that Thymus vulgaris, Achillea millefolium and propolis extracts are effective for treatment of cutaneous leishmaniasis in mice. Regarding these results, we suggest that efficacy of these extracts alone or in combination are evaluated against human cutaneous leishmaniasis as a randomized clinical trial.

Leishmaniasis is a parasitic disease of global health concern, and an affordable and effective vaccine could be a valuable solution. The aim of this study was to evaluate the immunogenicity of a Leishmania major DNA vaccine based on LACK antigen using calcium phosphate and chitosan adjuvants nano particles. Seventy Balb/c female mice 4–6 weeks old and 20–22 g weight were selected and were divided into 5 groups [Each group 14 mice]. First Group received plasmid LACK vaccine [PCDN3 + LACK], second group received PCDN3 + LACK + Nano adjuvant phosphate Ca, third group received PCDN3 + LACK + Nano adjuvant chitosan, forth group received PBS as negative control and fifth group did not receive vaccine as positive control. The mice were vaccinated two times intramuscular injection with three weeks interval. The mice were challenged with wild type of Leishmania major promastigote via intra dermal injection into the basal of their tail three weeks after the last vaccination. Clinical sign and...

Background and Objective: Leishmaniasis represents a major global health problem with a few effective therapeutic drugs. Gossypium hirsutum has been reported as a traditional herbal medicine to cure cutaneous leishmaniasis. This study was aimed to evaluate the leishmanicidal activities, cytotoxicity, apoptosis and necrosis of G. hirsutum bulb extract and its fractions on Leishmania major promastigotes, amastigote-macrophage model and BALB/C mice and evaluation of cytotoxicity effect of the plant on HepG2, A549, MCF7 and U87. Crude chloroform extract of the plant bulb was fractionated with a column chromatography method using silica gel as stationary phase. Various concentrations of extract and its fractions were evaluated against Leishmania major in comparison with Glucantime ® in a medium containing penicillin/streptomycin and 10% fetal calf serum (FCS) at 24ºC. Optical density (OD) was measured by enzyme-linked immunoabsorbent assay (ELISA) to determine the 50% inhibitory concentration value (IC 50 ) of sample extract and its fractions. For clinical stage, intramacrophage amastigotes were incubated at various concentrations of the extract for 72 h in triplicates. The apoptotic and necrotic values were analyzed by flowcytometry. In order to evaluate in vivo leishmanicidal activity of the extract and fractions, ٤٥ balb/c mice (previously inoculated with ٢×١٠ ٦ promastigotes stationary phase metacyclic promastigotes of L. major) were randomly distributed into nine groups and the lesions diameter and swelling of footpads of the mice were measured weekly for a month. Statistical analyses of data were carried out using ttest, ANOVA, and repeated measure by SPSS software. Result: Following colomn chromatography fractionation of the extract, 12 fractions were achieved. G. hirsutum extract and two effective fractions (F4 and F5) inhibited L major promastigotes and amastigotes proliferation rates in a dose-dependent response at 72h posttreatment. The leishmanical activity as displayed by IC 50 values resulted in 96,3 g/ml and 18,9 µg/ml for promastigotes and amastigotes, respectively.The overall leishmanicidal potency of the crude extract and fractions F4 and F5 was much greater than that of Glucantime ® for both L. major stages. The IC 50 of F4 and F5 were measured at doses of 126,4 µg/ml and 16,3 µg/ml for promastigotes and doses of 154,6 µg/ml and 21,3 µg/ml for amastigotes, respectively. There was no significant cytotoxicity effect as the selectivity index (SI) was over 1000, far beyond ≥10. The mean apoptotic values for L major were superior compared to Glucantime ® . The best antileishmanial activity was shown in balb/c mice that trated with effective fractions and crude extract, respectively. G.hirsutum bulb extract displayed potent antiproliferation cytotoxicity index against A٥٤٩, U٨٧, MCF٧, and HepG٢ cancer cell lines. DPPH and FRAP tests showed a great antioxidant power for the crude extract. The present study demonstrated that G. hirsutum has much stronger antileishmanial effects against L major than that of Glucantime ® . No cytoxicity effects were associated with the extract and induced an excellent apoptotic index, a possible mechanism of parasite death.

The effect of alterations in virulence and transformation by long-term in vitro culture of Leishmania mexicana promastigotes on infectivity and immune responses was investigated. Fresh parasite cultures harvested from Balb/c mice were passaged 20 times in vitro. Infectivity was decreased and was completely avirulent after 20 passages. The qPCR results showed a down-regulation of GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3 genes after passage seven concomitant with a reduced and absence of infectivity by passages seven and 20, respectively. Parasites at passages one and 20 are referred to as virulent and avirulent, respectively. The growth of avirulent and virulent parasite was affected by conditioned media derived from macrophages or monocytes infected with parasites for 2 h. Giemsa staining showed the failure of avirulent but not virulent parasites to transform to the amastigote stage in infected host cells with both virulent and avirulent modulating the expression of CCL-22, Tgad51, Cox2, IL-1, IL-10, TGF-b, TNF-a, Rab7, Rab9 and A2 genes; virulent but not avirulent L. mexicana significantly up-regulated Th2-associated cytokines, but down-regulated Rab7 and Rab9 gene expression. In conclusion, a model for L. mexicana is reported, which is of potential value in studying host-parasite interaction.

Cutaneous Leishmaniasis, caused by Leishmania mexicana complex is associated with unpleasant or disfiguring lesions with no available vaccine. This study aimed to investigate the changes in the gene expression profile of Cytokines (IL-1, IL-6, and TGF-β), and Chemokines (CCL-1, CCL-2, and CCL-5) following infection with avirulent and virulent L. mexicana promastigote using tumour human MonoMac-6, U937 monocyte and macrophage cell lines by qPCR analysis, Furthermore, the effect of their Soluble Leishmania Antigens was (SLA) also investigated. Results showed that the level of cytokine/chemokine by parasite infection was both cell line-and parasite-virulence dependent. Results of this study illustrated that, the gene expression profile IL-1 and IL-6 expression was significantly up regulated in the three cell lines following the avirulent parasite infection. However, the gene expression of TGF-β was up-regulated following the virulent L. mexicana infection in U937 macrophages and MonoMac-6 cell lines. Chemokines CCL-1, CCL-2 and CCL-5 gene expression was down regulated following the infection with L. mexicana in U937 monocytes and macrophages. SLA has also an impact on the gene expression profile of ILs and CCLs in stimulated target cells. For example, the gene expression profile of IL-6 was up regulated after stimulation with avirulent SLA in the three cell line. In conclusion virulent but not avirulent L. mexicana promastigote significantly up regulated T helper cells type 2 mediators which supported the parasite surviving inside the host, and this in vitro model of avirulent L. mexicana parasite has potential role of understanding the parasite pathogenicity.

Leishmania species are recognized to be pathogenic parasites that infect almost 15 million people globally, and up to date there is no vaccine available to stop the spread of Leishmania infection. For Leishmania mexicana to lose its virulence, the parasite culture must undergo in vitro passaging, little is known about the process underlying the reduction of parasite virulence. Thus, the purpose of this work was to look into the expression of lipophosphoglycan (LPG), as a virulence factor, during L. mexicana in vitro culturing and assessing the parasite's capacity to infect target cells. Results illustrated the extreme downregulation in LPG expression on the surface of promastigotes. Since anti-LPG monoclonal antibody staining showed LPG expression was significantly less expressed in passage 20 (P20) compared to its expression in passage 1 (P1). In addition, this was confirmed by flow cytometry analysis, which also showed that LPG was highly expressed in P1 compared to P20. Moreover, an in vitro infectivity assay was performed using Balb/c and C57 macrophages. Both cells were infected with L. mexicana promastigotes from P1 and P20 for 24 hours. The results illustrated the ability of P1, but not P20, to transform into the non-flagellated amastigotes sage inside infected cells. Our results concluded the significant role of LPG protein in L. mexicana virulence and its ability to induce infection.

The sandfly Phlebotomus papatasi is the vector of Leishmania major, the main causative agent of Old World cutaneous leishmaniasis (CL) in Saudi Arabia. Sandflies inject saliva while feeding and the salivary protein PpSP32 was previously shown to be a biomarker for bite exposure. Here we used recombinant PpSP32 to evaluate human exposure to Ph. papatasi bites, and study the association between antibody response to saliva and CL in endemic areas in Saudi Arabia. In this observational study, anti-PpSP32 antibodies, as indicators of exposure to sandfly bites, were measured in sera from healthy individuals and patients from endemic regions in Saudi Arabia with active and cured CL. Ph. papatasi was identified as the primary CL vector in the study area. Anti-PpSP32 antibody levels were significantly higher in CL patients presenting active infections from all geographical regions compared to CL cured and healthy individuals. Furthermore, higher anti-PpSP32 antibody levels correlated with the prevalence and type of CL lesions (nodular vs. papular) observed in patients, especially non-local construction workers. Our findings suggest a possible correlation between the type of immunity generated by the exposure to sandfly bites and disease outcome.

Pythium insidiosum immunomodulatory vaccine (PiV) has been tested in clinical and experimental pythiosis. Previous data showed that P. insidiosum immunogens have the ability to switch the Th2 immune response, normally in place during pythiosis, to a curative Th1 response. Pythiosis cannot be reproduced in experimental rodents with the exception of rabbits, and thus thorough evaluation of PiVś immunomodulatory properties has been limited by the lack of a compatible inbred mouse model. In this study, we took advantage of the murine BALB/c Leishmania infection model, where infected mice produce a Th2 response, to evaluate the PiV Th2 to Th1 immunomodulatory potential. Twenty-one days following challenge with L. major, large cutaneous granulomas developed in control mice, consistent with the expected Th2 response. In contrast, Leishmaniainduced cutaneous lesions in PiV-immunized mice were minimal or absent. Flow cytometry analysis of spleen cells from mice immunized with PiV and

In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease. Leishmanial Ags that elicit proliferative and cytokine responses in PBMCs from cured/exposed/Leishmania patients have been characterized through proteomic approaches, and elongation factor-2 is identified as one of the potent immunostimulatory proteins. In this study, we report the cloning and expression of Leishmania donovani elongation factor-2 protein (LelF-2) and its immunogenicity in PBMCs of cured/exposed Leishmania-infected patients and hamsters (Mesocricetus auratus). Leishmania-infected cured/exposed patients and hamsters exhibited significantly higher proliferative responses to recombinant Lelf-2 (rLelF-2) than those with L. donovani-infected hosts. The soluble L. donovani Ag stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whe...

BACKGROUND Heat shock proteins (HSP) are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen (SLA) was determined. METHODS BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 106 stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks. RESULTS No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the ...

The mechanism by which the salivary gland lysate (SGL) of Lutzomyia longipalpis enables Leishmania infection remains under investigation. One possibility is that saliva promotes cellular recruitment leading to development of skin lesions. In this study, we investigated leukocyte recruitment induced by L. major, L. major + SGL, or SGL alone into the peritoneal cavity of BALB/c mice. The administration of L. major with or without SGL induced neutrophil migration six hours after infection. Interestingly, after seven days, the BALB/c mice still had eosinophils and mononuclear cells in their peritoneal cavities. Flow cytometric analysis showed an increase in the CD4+ CD45RB low T cell subset (effector or memory cells) compared with the CD4+ CD45RB high subset (naive cells). Moreover, the co-injection of L. major with SGL enhanced production of interleukin-10. These results suggest that SGL can facilitate Leishmania infection by modulating leukocyte recruitment and Th2 cytokine production at the inflammatory focus.

Leishmaniasis causes high morbidity and mortality in tropical and subtropical areas. Mast cells can be activated by Leishmania or Leishmania products in vitro and in vivo . Several innate immunity mediators, including some released by mast cells, play roles in the outcome of the disease. In this study, we examined whether pharmacological inactivation of mast cells before infection with L. major interferes with the progressive disease in BALB/c mice. The results show that, when mast cells are degranulated before challenge with L. major , susceptible mice become more resistant to infection, as measured by decrease of lesion size and lower parasite loads. Mast cell degranulation reduced IL-4 production. Moreover, mast cells degranulation enhanced mRNA expression for IFN-γ , inducible nitric oxide, CCL2 and CCL5 in response to infection. Mast cell degranulation also decreased parasite loads in IL-4 KO animals, indicating that mediators other than IL-4 are involved in susceptibility in vivo . Taken together, our results disclose a role for mast cells in the induction of susceptibility to infection. This work contributes to a better understanding of the role of mast cells in Leishmania infection, and suggests a new field of study for strategies to contain the parasite, restricting its dissemination.

or to other endemic areas of the disease in Iran. Spatial distribution of CL cases revealed northeastern and southwestern quarters of the city were the major hot spots of the disease (P<0.05). Hot spot and CL transmission risk analysis across the province indicated that more than 40 villages were located in high and very high risk areas of CL transmission. Due to the existence of hot spots (P<0.05) of CL in successive years in some quarters of Qom city, along with detection of L. major from the patients without a history of travel, there may be potential of local transmission of the disease within the city. Therefore, it is necessary to conduct a comprehensive study concerning the hot spots of CL in Qom city for curtailing the incidence of the disease in the city. The methodology and the results of this study is essential in serving as a yardstick for subsequent similar studies that will be carried out in other endemic areas of CL in Iran and providing an adequate tool for the establishment of a national database of cutaneous leishmaniasis.

Following the epidemic of cutaneous leishmaniasis in Khatam County, Yazd Province, this study was carried out to determine vector, and animal reservoir host(s) and investigate the human infection during 2005-2006. Four rural districts where the disease had higher prevalence were selected. Sticky paper traps were used to collect sand flies during April to November, biweekly. Meanwhile rodents were captured using Sherman traps from August to November. Households and primary schools were visited and examined for human infection in February 2006. The parasite was detected by RAPD-PCR method. The rate of ulcers and scars among the inhabitants was 4.8% and 9.8%, respectively. Three rodent species were captured during the study:Meriones libycus, Rhombomys opimus, andTatera indica. Six sand fly species were also collected and identified; among themPhlebotomus papatasihad the highest frequency.Leishmania majorwas detected as the agent of the disease in the area. It was detected fromR. opimus...

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is a great public health problem in the Old World. Rodents are important reservoir of causative agent of disease is widely distributed in arid and savannah regions. Esfaryen district is one of the most important endemic foci of zoonotic cutaneous leishmaniasis (ZCL) in Northeast of Iran. To identify the reservoir(s) of Leishmania in this focus, a total of 44 rodents were collected using Sherman traps during September to December 2013. They were identified as Rhombomys opimus Lichtenstein (72.7%) and Meriones libycus Lichtenstein (27.3%). Microscopic investigation on blood smear of the animals for amastigote parasites revealed 32 (72.7%) rodents of R. opimus infected with amastigote. L. major infection in these animals was then confirmed by polymerase chain reaction against internal transcribed spacer ribosomal DNA (rDNA) loci of the parasite followed by restriction fragment length polymorphism. Further, sequence analysis...

Leishmaniasis is an important public health disease in many developing countries as well in Iran. The main objective of this study was to investigate on leishmania infection of wild caught sand flies in an endemic focus of disease in Esfarayen district, north east of Iran. Sand flies were collected by sticky papers and mounted in a drop of Puri&#39;s medium for species identification. Polymerase chain reaction techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism were used for identification of DNA of Leishmania parasites within infected sand flies. Among the collected female sand flies, two species of Phlebotomus papatasi and Phlebotomus salehi were found naturally infected with Leishmania major. Furthermore, mixed infection of Leishmania turanica and L. major was observed in one specimen of P. papatasi. Sequence analysis revealed two parasite ITS1 haplotypes including three L. major with accession numbers: KJ425408, KJ425407, KM056403 and one L. turan...

The zinc metalloprotease gp63 (leishmanolysin; promastigote surface protease) is expressed at high density at the surface of Leishmania promastigotes. Efficient non-toxic inhibitors of gp63 do not exist, and its precise role in parasite physiology remains unknown. MARCKS (myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in various cells, including macrophages. We reported previously that MRP is an excellent substrate for gp63. A major cleavage site was identified within the MRP effector domain (ED), a highly basic 24-amino-acid sequence, and the synthetic ED peptide (MRPED) was shown to inhibit MRP hydrolysis. In the present study, MRP cleavage was used as an assay to measure the capacity of various MRP or MARCKS ED peptides to block gp63 activity. On a molar basis, MRPED inhibited gp63 to a greater extent than two previously described gp63 inhibitors, o-phenanthroline and benzyloxycarbonyl-Tyr-Leu-NHOH. MARCK...

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment.

Leishmaniasis is a widely spread zoonotic disease caused by the bite of infected sandflies, particularly in developing countries. Cutaneous leishmaniasis can have a diverse range of presentations, ranging from minor skin nodules to significant mucosal damage. However, nose involvement is infrequent. Our report highlights a 15-year-old female patient with a persistent skin lesion on her nose for three months, which is a rare manifestation of cutaneous leishmaniasis. The lesion started as a raised spot with a brownish-red color and a crust but eventually developed into an ulcer that spread over the entire lobe of the nose and even moved toward the eye. Microscopic examination revealed the presence of Leishmania amastigotes, and a biopsy confirmed a diagnosis of cutaneous leishmaniasis. The patient received daily intravenous sodium stibogluconate doses of 9 mg/kg for 20 days, and three weeks later, there was a significant clinical improvement, with the ulcer beginning to heal and no more amastigotes visible on microscopic examination. It is crucial to keep cutaneous leishmaniasis in mind as a possible diagnosis for patients with skin lesions, even in regions where the condition is not prevalent.

Bardet-Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.

Background: A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes. Methods/Principal Findings: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. Conclusions/Significance: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.

Background: Genome sequencing of Leishmania species that give rise to a range of disease phenotypes in the host has revealed highly conserved gene content and synteny across the genus. Only a small number of genes are differentially distributed between the three species sequenced to date, L. major, L. infantum and L. braziliensis. It is not yet known how many of these genes are expressed in the disease-promoting intracellular amastigotes of these species or whether genes conserved between the species are differentially expressed in the host. Methods/Principal Findings: We have used customised oligonucleotide microarrays to confirm that all of the differentially distributed genes identified by genome comparisons are expressed in intracellular amastigotes, with only a few of these subject to regulation at the RNA level. In the first large-scale study of gene expression in L. braziliensis, we show that only ,9% of the genes analysed are regulated in their RNA expression during the L. braziliensis life cycle, a figure consistent with that observed in other Leishmania species. Comparing amastigote gene expression profiles between species confirms the proposal that Leishmania transcriptomes undergo little regulation but also identifies conserved genes that are regulated differently between species in the host. We have also investigated whether host immune competence influences parasite gene expression, by comparing RNA expression profiles in L. major amastigotes derived from either wild-type (BALB/c) or immunologically compromised (Rag2 2/2 c c 2/2 ) mice. While parasite dissemination from the site of infection is enhanced in the Rag2 2/2 c c 2/2 genetic background, parasite RNA expression profiles are unperturbed. Conclusion/Significance: These findings support the hypothesis that Leishmania amastigotes are pre-adapted for intracellular survival and undergo little dynamic modulation of gene expression at the RNA level. Species-specific parasite factors contributing to virulence and pathogenicity in the host may be limited to the products of a small number of differentially distributed genes or the differential regulation of conserved genes, either of which are subject to translational and/or post-translational controls.

The SHERP genes are found as a tandem pair within the differentially regulated LmcDNA16 locus of Leishmania major. The SHERP gene product (small hydrophilic endoplasmic reticulum-associated protein) is unusual in its small size (6.2 kDa), its acidic pI (4.6) and its exclusive, high-level expression ($ 100 000 copies per cell) in infective non-replicative parasite stages. No homologues have been found to date. Secondary-structure predictions suggest that SHERP contains an amphiphilic α-helix that is presumably involved in proteinprotein interactions. SHERP has been localized to the endoplasmic reticulum as well as to the outer mitochondrial membrane in both wild-type and over-expressing parasites. Given the absence of an N-terminal signal sequence, transmembranespanning domains or detectable post-translational modifications,

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasit...

Background: A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes. Methods/Principal Findings: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. Conclusions/Significance: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.

Leishmania species are intracellular parasites that inhabit a parasitophorous vacuole (PV) within host macrophages and engage with the host endo-membrane network to avoid clearance from the cell. Intracellular Leishmania amastigotes exhibit a high degree of proteolytic/lysosomal activity that may assist degradation of MHC class II molecules and subsequent interruption of antigen presentation. As an aid to further analysis of the endosomal/lysosomal events that could facilitate this process, we have characterised a Leishmania homologue of the late endosomal marker, Rab7, thought to be involved in the terminal steps of endocytosis and lysosomal delivery. The Leishmania major Rab7 (LmRAB7) protein is expressed throughout the life-cycle, shows 73 and 64% identity to Trypanosoma cruzi and Trypanosoma brucei Rab7s (TcRAB7 and TbRAB7), respectively, and includes a kinetoplastidspecific insertion. The recombinant protein binds GTP and polyclonal antibodies raised against this antigen recognise structures in the region of the cell between the nucleus and kinetoplast. By immunoelectron microscopy of axenic amastigotes, Leishmania mexicana Rab7 (LmexRAB7) is found juxtaposed to and overlapping membrane structures labelled for the megasomal marker, cysteine proteinase B, confirming a late-endosomal/lysosomal localisation.

Recent progress in sequencing the genomes of several Leishmania species, causative agents of cutaneous, mucocutaneous and visceral leishmaniasis, is revealing unusual features of potential relevance to parasite virulence and pathogenesis in the host. While the genomes of Leishmania major, Leishmania braziliensis and Leishmania infantum are highly similar in content and organisation, species-specific genes and mechanisms distinguish one from another. In particular, the presence of retrotransposons and the components of a putative RNA interference machinery in L. braziliensis suggest the potential for both greater diversity and more tractable experimentation in this Leishmania Viannia species.

To investigate the role of clathrin-mediated traYcking during the Leishmania lifecycle, open reading frames encoding clathrin heavy chain and the -adaptins, major components of the adaptor complexes, have been analysed both in silico and experimentally. The Leishmania genome encodes three -adaptins, which arose at a time predating speciation of these divergent trypanosomatids. Unlike Trypanosoma brucei, both clathrin heavy chain and -adaptin1 are constitutively expressed throughout the Leishmania life cycle. Clathrin relocalises in amastigotes relative to promastigotes, consistent with developmental alterations to the morphology of the endo-membrane system.

Leishmania hydrophilic acylated surface protein B (HASPB) family are only expressed in infective parasites (both extra-and intracellular stages) and, together with the peripheral membrane protein SHERP (small hydrophilic endoplasmic reticulum-associated protein), are essential for parasite differentiation (metacyclogenesis) in the sand fly vector. HASPB is a 'nonclassically' secreted protein, requiring N-terminal acylation for trafficking to and exposure on the plasma membrane. Here, we use live cell imaging methods to further explore this pathway to the membrane and flagellum. Unlike HASPB trafficking in transfected mammalian cells, we find no evidence for a phosphorylation-regulated recycling pathway in metacyclic parasites. Once at the plasma membrane, HASPB18-GFP (green fluorescent protein) can undergo bidirectional movement within the inner leaflet of the membrane and on the flagellum. Transfer of fluorescent protein between the flagellum and the plasma membrane is compromised, however, suggesting the presence of a diffusion barrier at the base of the Leishmania flagellum. Full-length HASPB is released from the metacyclic parasite surface on to macrophages during phagocytosis but while expression is maintained in intracellular amastigotes, HASPB cannot be detected on the external surface in these cells. Thus HASPB may be a dual function protein that is shed by the infective metacyclic but retained internally once Leishmania are taken up by macrophages.

The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of &lt;1 μM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16–66 μM and low toxicity to murine macrophages. These results suggest that targeting ...

Zoonotic cutaneous leishmaniasis (ZCL) is an expanding disease and public health problem in Iran. In the current study, natural Leishmania infection rate and seasonal fluctuation of the infection in Rhombomys opimus population of a hyperendemic focus of ZCL in Iran was investigated. The study was conducted from October 2006 to October 2008 in Esfahan Province, central part of Iran. An extensive sampling of rodents using Sherman traps was done in different seasons. Nested PCR assay was used for detection and identification of Leishmania species and the results were confirmed using PCR-RFLP. Leishmania infection rate was 58.6% (34 of 58) using nested PCR. 44.8% of the gerbils were infected only with L. turanica and 1.7% with L. gerbilli alone. A mixed natural infection with L. major and L. turanica was seen in 12.1% of the rodents. L. major infection alone was not seen in R. opimus population in the study area. The highest and lowest Leishmania infection rates were observed in fall an...

Local tissue acidosis affects anti-tumor immunity. In contrast, data on tissue pH levels in infected tissues and their impact on antimicrobial activity is sparse. In this study, we assessed the pH levels in cutaneous Leishmania lesions. Leishmania major-infected skin tissue displayed pH levels of 6.7 indicating that lesional pH is acidic. Next, we tested the effect of low extracellular pH on the ability of macrophages to produce leishmanicidal NO and to fight the protozoan parasite Leishmania major. Extracellular acidification led to a marked decrease in both NO production and leishmanicidal activity of lipopolysaccharide (LPS) and interferon g (IFN-g)-coactivated macrophages. This was not directly caused by a disruption of NOS2 expression, a shortage of reducing equivalents (NAPDH) or substrate (L-arginine), but by a direct, pH-mediated inhibition of NOS2 enzyme activity. Normalization of intracellular pH significantly increased NO production and antiparasitic activity of macrophages even in an acidic microenvironment. Overall, these findings indicate that low local tissue pH can curtail NO production and leishmanicidal activity of macrophages.

Project: This study investigated the in vitro and in vivo effectiveness of biogenic selenium nanoparticles (Se NPs), biosynthesized by Bacillus sp. MSh-1, against Leishmania major (MRHO/IR/75/ER). Procedure: The 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity effects of the biogenic Se NPs against both promastigote and amastigote forms of L. major. In a separate in vivo experiment, we also determined the preventive and therapeutic effects of biogenic Se NPs in BALB/c mice following subcutaneous infected with L. major. Results: The MTT assays showed that the highest toxicity occurred after 72 h against both promastigote and amastigote forms of L. major. The cytotoxicity of Se NPs was higher at all incubation times (24, 48, and 72 h) against the promastigote than the amastigote form (p < 0.05). The 50% inhibitory concentrations (IC 50 ) of the Se NPs were 1.62 ± 0.6 and 4.4 ± 0.6 g ml -1 against the promastigote and amastigote forms, respectively, after a 72-h incubation period. Apoptosis assays showed DNA fragmentation in promastigotes treated with Se NPs. In an animal challenge, prophylactic doses of biogenic Se NPs delayed the development of localized cutaneous lesions. Moreover, daily administration of Se NPs (5 or 10 mg kg -1 day -1 ) in similarly infected BALB/c mice that had not received prophylactic doses of Se NPs also abolished the localized lesions after 14 days. Conclusion: Based on these in vitro and in vivo studies, biogenic Se NPs can be considered as a novel therapeutic agent for treatment of the localized lesions typical of cutaneous leishmaniasis.

Snake venom can affect the growth of Trypanosoma cruzi and Leishmania spp. As new classes of therapeutic drugs against protozoan parasites could be derived from snake venom, alterations in the ultrastructure and growth of the epimastigotes, trypomastigotes and amastigotes of T. cruzi, as well as the promastigotes of Leishmania major, were analyzed after treatment with crude venom from Bothrops jararaca. Parasite growth (epimastigotes and promastigotes) of venom treated cultures showed a negative correlation between cell growth and venom concentration. No growth occurred at a dose of 100 lg/ml of venom, while 50% growth inhibition was obtained in the range 0.1-0.3 lg/ml. Ultrastructural observations of treated bloodstream trypomastigotes, intracellular amastigotes, as well as axenic cultures of epimastigotes and promastigotes, demonstrated mitochondrial swelling and kinetoplast disorganization. Our data show that B. jararaca venom effectively inhibited the growth of T. cruzi and L. major parasites. Growth inhibition was probably related to mitochondrial impairment.

Introduction: Visceral Leishmaniasis (VL) also known as Kala-azar is a fatal neglected tropical disease (NTD) with a 95% fatality rate among untreated cases. Although there is little information available about the disease's prevalence in Isiolo County, it is extremely endemic in a number of villages in Merti Sub-County. Case Presentation: In this case presentation, a family of three children resulted to traditional therapy when the children contracted Kala-azar. All the three children exhibited signs of fever, loss of appetite, swollen abdomen, vomiting, and general weakness. Discussion: Low levels of Visceral Leishmaniasis knowledge, ignorance characterized by cultural beliefs, and the use of local therapies are among socio-cultural challenges impeding implementation of effective Kala-azar control strategies in Isiolo County. Conclusion: This case highlights the importance of focused community awareness for prompt and efficient diagnosis and treatment to lower Kala-azar prevalence, monitoring the burden and spread of the disease as well as lowering transmission.

BACKGROUND Leishmaniasis, a neglected disease caused by the parasite Leishmania, is treated with drugs associated with high toxicity and limited efficacy, in addition to constant reports of the emergence of resistant parasites. In this context, snake serums emerge as good candidates since they are natural sources with the potential to yield novel drugs. OBJECTIVES We aimed to show the antileishmanial effects of γCdcPLI, a phospholipase A 2 inhibitor from Crotalus durissus collilineatus snake serum, against Leishmania (Leishmania) amazonensis. METHODS Promastigotes forms were exposed to γCdcPLI, and we assessed the parasite viability and cell cycle, as well as invasion and proliferation assays. FINDINGS Despite the low cytotoxicity effect on macrophages, our data indicate that γCdcPLI has a direct effect on parasites promoting an arrest in the G1 phase and reduction in the G2/M phase at the highest dose tested. Moreover, this PLA 2 inhibitor reduced the parasite infectivity when promastigotes were pre-treated. Also, we demonstrated that the γCdcPLI treatment modulated the host cell environment impairing early and late steps of the parasitism. MAIN CONCLUSIONS γCdcPLI is an interesting tool for the discovery of new essential targets on the parasite, as well as an alternative compound to improve the effectiveness of the leishmaniasis treatment.

shawi causes cutaneous lesions in humans. Parasite antigens conferring signiWcant protection against American tegumentar leishmaniosis (ATL) might be important for the development of eVective vaccine. Therefore, this work evaluates the protective eVect of antigenic fractions released by L. shawi. Antigens released by promastigotes to culture medium were concentrated and isolated by SDS-PAGE. The three main fractions LsPass1 (>75 kDa), LsPass2 (75-50 kDa) and LsPass3 (<50 kDa) were electro-eluted according with their molecular mass. Immunized BALB/c mice were challenged with L. shawi promastigotes and the course of infection monitored during 5 weeks. LsPass1-challenged mice showed no protection, however, a strong degree of protection associated to smaller lesions and high expression of IFN-and TNF-by CD4 + T, CD8 + T and double negative CD4CD8 cells was achieved in LsPass3-challenged mice. Furthermore, LsPass2-challenged mice showed an intermediated degree of protection associated to high levels of IFN-, IL-4 and IL-10 mRNA. In spite of increased expression of IFNand TNF-, high amounts of IL-4 and IL-10 mRNA were also detected in LsPass3-challenged mice indicating a possible contribution of these cytokines for the persistence of a residual number of parasites that may be important in inducing long-lasting immunity. Therefore, LsPass3 seems to be an interesting alternative that should be considered in the development of an eVective vaccine against ATL.