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In the mammalian host, the biology of tissue-dwelling Trypanosoma brucei parasites is not completely understood, especially the mechanisms involved in their extravascular colonization. The trypanosome flagellum is an essential organelle in multiple aspects of the parasites' development. The flagellar protein termed FLAgellar Member 8 (FLAM8) acts as a docking platform for a pool of cyclic AMP response protein 3 (CARP3) that is involved in signaling. FLAM8 exhibits a stage-specific distribution suggesting specific functions in the mammalian and vector stages of the parasite. Analyses of knockdown and knockout trypanosomes in their mammalian forms demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and stumpy differentiation. Functional investigations in experimental infections showed that FLAM8-deprived trypanosomes can establish and maintain an infection in the blood circulation and differentiate into insect transmissible forms. However, quantitative bioluminescence imaging and gene expression analysis revealed that FLAM8-null parasites exhibit a significantly impaired dissemination in the extravascular compartment, that is restored by the addition of a single rescue copy of FLAM8. In vitro trans-endothelial migration assays revealed significant defects in trypanosomes lacking FLAM8. FLAM8 is the first flagellar component shown to modulate T. brucei distribution in the host tissues, possibly through sensing functions, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches, especially in the skin.
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Trypanosoma brucei brucei , Tripanossomíase Africana , Animais , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Comunicação Celular , Trypanosoma brucei brucei/metabolismo , Mamíferos , Flagelos/metabolismo , Tripanossomíase Africana/parasitologiaRESUMO
BACKGROUND: The systemic inflammatory syndrome called "cytokine storm" has been described in COVID-19 pathogenesis, contributing to disease severity. The analysis of cytokine and chemokine levels in the blood of 21 SARS-CoV-2 positive patients throughout the phases of the pandemic has been studied to understand immune response dysregulation and identify potential disease biomarkers for new treatments. The present work reports the cytokine and chemokine levels in sera from a small cohort of individuals primarily infected with SARS-CoV-2 during the first wave of the COVID-19 pandemic in Milan (Italy). RESULTS: Among the 27 cytokines and chemokines investigated, a significant higher expression of Interleukin-9 (IL-9), IP-10 (CXCL10), MCP-1 (CCL2) and RANTES (CCL-5) in infected patients compared to uninfected subjects was observed. When the change in cytokine/chemokine levels was monitored over time, from the hospitalization day to discharge, only IL-6 and IP-10 showed a significant decrease. Consistent with these findings, a significant negative correlation was observed between IP-10 and anti-Spike IgG antibodies in infected individuals. In contrast, IL-17 was positively correlated with the production of IgG against SARS-CoV-2. CONCLUSIONS: The cytokine storm and the modulation of cytokine levels by SARS-CoV-2 infection are hallmarks of COVID-19. The current global immunity profile largely stems from widespread vaccination campaigns and previous infection exposures. Consequently, the immunological features and dynamic cytokine profiles of non-vaccinated and primarily-infected subjects reported here provide novel insights into the inflammatory immune landscape in the context of SARS-CoV-2 infection, and offer valuable knowledge for addressing future viral infections and the development of novel treatments.
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COVID-19 , Quimiocinas , Citocinas , SARS-CoV-2 , Humanos , COVID-19/imunologia , COVID-19/sangue , COVID-19/epidemiologia , Itália/epidemiologia , SARS-CoV-2/imunologia , Feminino , Masculino , Pessoa de Meia-Idade , Citocinas/sangue , Idoso , Quimiocinas/sangue , Adulto , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Biomarcadores/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , PandemiasRESUMO
The single flagellum of African trypanosomes is essential in multiple aspects of the parasites' development. The FLAgellar Member 8 protein (FLAM8), localised to the tip of the flagellum in cultured insect forms of Trypanosoma brucei, was identified as a marker of the locking event that controls flagellum length. Here, we investigated whether FLAM8 could also reflect the flagellum maturation state in other parasite cycle stages. We observed that FLAM8 distribution extended along the entire flagellar cytoskeleton in mammalian-infective forms. Then, a rapid FLAM8 concentration to the distal tip occurs during differentiation into early insect forms, illustrating the remodelling of an existing flagellum. In the tsetse cardia, FLAM8 further localises to the entire length of the new flagellum during an asymmetric division. Strikingly, in parasites dividing in the tsetse midgut and in the salivary glands, the amount and distribution of FLAM8 in the new flagellum were seen to predict the daughter cell fate. We propose and discuss how FLAM8 could be considered a meta-marker of the flagellum stage and maturation state in trypanosomes.
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Trypanosoma brucei brucei , Trypanosoma , Moscas Tsé-Tsé , Animais , Diferenciação Celular , Flagelos , Estágios do Ciclo de Vida , Proteínas de ProtozoáriosRESUMO
BACKGROUND: The diagnosis of gambiense human African trypanosomiasis (gHAT) typically involves 2 steps: a serological screen, followed by the detection of living trypanosome parasites in the blood or lymph node aspirate. Live parasites can, however, remain undetected in some seropositive individuals, who, we hypothesize, are infected with Trypanosoma brucei gambiense parasites in their extravascular dermis. METHODS: To test this hypothesis, we conducted a prospective observational cohort study in the gHAT focus of Forecariah, Republic of Guinea. Of the 5417 subjects serologically screened for gHAT, 66 were enrolled into our study and underwent a dermatological examination. At enrollment, 11 seronegative, 8 unconfirmed seropositive, and 18 confirmed seropositive individuals had blood samples and skin biopsies taken and examined for trypanosomes by molecular and immunohistological methods. RESULTS: In seropositive individuals, dermatological symptoms were significantly more frequent, relative to seronegative controls. T.b. gambiense parasites were present in the blood of all confirmed cases (n = 18) but not in unconfirmed seropositive individuals (n = 8). However, T. brucei parasites were detected in the extravascular dermis of all unconfirmed seropositive individuals and all confirmed cases. Skin biopsies of all treated cases and most seropositive untreated individuals progressively became negative for trypanosomes 6 and 20 months later. CONCLUSIONS: Our results highlight the skin as a potential reservoir for African trypanosomes, with implications for our understanding of this disease's epidemiology in the context of its planned elimination and underlining the skin as a novel target for gHAT diagnostics.
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Tripanossomíase Africana , Animais , Guiné , Humanos , Estudos Prospectivos , Trypanosoma brucei gambiense , Tripanossomíase Africana/diagnóstico , Tripanossomíase Africana/epidemiologiaRESUMO
African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (µCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions.
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Modelos Animais de Doenças , Trypanosoma brucei brucei , Animais , Camundongos , Trypanosoma brucei brucei/patogenicidade , Imagem Multimodal/métodos , Doenças Negligenciadas/parasitologia , Doenças Negligenciadas/diagnóstico por imagem , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/diagnóstico por imagem , Medições Luminescentes/métodosRESUMO
Visceral leishmaniasis is an emerging neglected tropical disease (NTD) caused by the protozoan Leishmania infantum in the countries bordering the Mediterranean Basin. Currently there is no effective vaccine against this disease, and the therapeutic approach is based on toxic derivatives of Sb(V). Therefore, the discovery of new therapeutic targets and the development of drugs designed to inhibit them comprise an extremely important approach to fighting this disease. DNA topoisomerases (Top) have been identified as promising targets for therapy against leishmaniasis. These enzymes are involved in solving topological problems generated during replication, transcription, and recombination of DNA. Being unlike that of the mammalian host, type IB DNA topoisomerase (TopIB) from Leishmania spp. is a unique bisubunit protein, which makes it very interesting as a selective drug target. In the present investigation, we studied the effect of two TopIB poisons with indenoisoquinoline structure, indotecan and AM13-55, on a murine BALB/c model of infected splenocytes with L. infantum, comparing their effectiveness with that of the clinically tested leishmanicidal drug paromomycin. Both compounds have high selectivity indexes compared with uninfected splenocytes. SDS-KCl-precipitable DNA-protein complexes in Leishmania promastigotes and in vitro cleaving assays confirmed that these drugs are Top poisons. The inhibitory potency of both indenoisoquinolines on L. infantum recombinant TopIB was assessed in vitro, with results showing that indotecan was the most active compound, preventing the relaxation of supercoiled DNA. Experimental infections in susceptible BALB/c mice treated with 2.5 mg/kg body weight/day once every other day for a total of 15 days showed that indotecan cleared more than 80% of the parasite burden of the spleen and liver, indicating promising activity against visceral leishmaniasis.
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Leishmaniose Visceral/tratamento farmacológico , Tripanossomicidas/uso terapêutico , Animais , Feminino , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Baço/parasitologiaRESUMO
The fatty acids (±)-2-methoxy-6Z-heptadecenoic acid (1), (±)-2-methoxy-6-heptadecynoic acid (2) and (±)-2-methoxyheptadecanoic acid (3) were synthesized and their inhibitory activity against the Leishmania DNA topoisomerase IB enzyme (LdTopIB) determined. Acids 1 and 2 were synthesized from 4-bromo-1-pentanol, the former in ten steps and in 7% overall yield, while the latter in seven steps and in 14% overall yield. Acid 3 was prepared in six steps and in 42% yield from 1-hexadecanol. Acids 1-3 inhibited the LdTopIB enzyme following the order 2 > 1 ⪢ 3, with 2 displaying an EC(50) = 16.6 ± 1.1 µM and 3 not inhibiting the enzyme. Acid 1 preferentially inhibited the LdTopIB enzyme over the human TopIB enzyme. Unsaturation seems to be a prerequisite for effective inhibition, rationalized in terms of weak intermolecular interactions between the active site of LdTopIB and either the double or triple bonds of the fatty acids. Toxicity towards Leishmania donovani promastigotes was also investigated resulting in the same order 2 > 1 > 3, with 2 displaying an EC(50) = 74.0 ± 17.1 µM. Our results indicate that α-methoxylation decreases the toxicity of C(17:1) fatty acids towards L. donovani promastigotes, but improves their selectivity index.
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Erythropoiesis is a complex physiological process by which erythroid progenitors proliferate and differentiate into nonnucleated red blood cells. Several methods can be used to monitor in vitro the differentiation of erythroid precursors, and hence the toxic effects of drugs, chemicals, or pollutants. One of the most commonly available assay of erythropoiesis is the microscopic observation of differentiated cells after benzidine staining, which forms a blue complex with hemoglobin. However, this method is laborious and does not provide accurate results since it heavily relies on the reader's interpretation. Moreover, benzidine is a carcinogen and a highly reactive molecule which forces the reader to microscopically count differentiated and non-differentiated cells within a short time frame (5 min). Here we have developed a simple, inexpensive, in-vitro spectrophotometric assay to measure erythroid differentiation using K562 cell line as a model. Materials needed included 96-well round-bottomed microplates and a microplate reader. Remarkably, carcinogenic benzidine was replaced by its isomeric tetramethyl derivative, the 3,3', 5,5'- tetramethylbenzidine (TMB), which presents several advantages: it is cheap, not mutagenic and a ready-to-use chromogenic substrate. A small volume (50 µl) of TMB added to the samples forms a blue complex in 15 min, and the reaction can be easily stopped and stabilized by the addition of H2SO4. The yellow precipitate is then solubilized, and the absorbance is measured at 450 nm. In addition, the suitability of the assay to determine the effects of compounds on erythroid differentiation was further tested with known inhibitors (artemisinin derivatives) of K562 differentiation. Overall, the reported methodology permits to measure in an accurate and reproducible manner the K562 differentiation and can be used for medium throughput screenings (MTS) of compounds or environmental toxics with potential erythro-toxicity and ability to inhibit erythroid differentiation.
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Eritropoese , Diferenciação Celular , Humanos , Células K562RESUMO
The leishmaniases are vector-borne parasitic diseases affecting humans and animals, with high mortality rates in endemic countries. Infected dogs represent the main reservoir of infection. Disease control is mainly based on chemotherapy, which, at present, shows serious drawbacks both in humans and dogs. Therefore, the discovery or repurposing of new treatments is mandatory. Here, three monovalent ionophores (salinomycin, monensin, nigericin) were tested against promastigotes of Leishmania (L.) infantum, Leishmania tropica, and Leishmania braziliensis, and against amastigotes of L. infantum within human and, for the first time, canine macrophages. All three drugs were leishmanicidal against all Leishmania spp. promastigotes with IC50 values between 7.98 and 0.23 µM. Monensin and nigericin showed IC50 values < 1 µM, whereas salinomycin was the least active compound (IC50 > 4 µM). Notably, the ionophores killed L. infantum amastigotes within human THP-1 cells with IC50 values ranging from 1.67 to 1.93 µM, but they only reduced by 27−37% the parasite burden in L. infantum-infected canine macrophages, showing a host-specific efficacy. Moreover, a selective higher toxicity against canine macrophages was observed. Overall, repurposed ionophores have the potential to be further investigated as anti-Leishmania agents, but different drug options may be required to tackle human or canine leishmaniases.
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More than two years have passed since the viral outbreak that led to the novel infectious respiratory disease COVID-19, caused by the SARS-CoV-2 coronavirus. Since then, the urgency for effective treatments resulted in unprecedented efforts to develop new vaccines and to accelerate the drug discovery pipeline, mainly through the repurposing of well-known compounds with broad antiviral effects. In particular, antiparasitic drugs historically used against human infections due to protozoa or helminth parasites have entered the main stage as a miracle cure in the fight against SARS-CoV-2. Despite having demonstrated promising anti-SARS-CoV-2 activities in vitro, conflicting results have made their translation into clinical practice more difficult than expected. Since many studies involving antiparasitic drugs are currently under investigation, the window of opportunity might be not closed yet. Here, we will review the (controversial) journey of these old antiparasitic drugs to combat the human infection caused by the novel coronavirus SARS-CoV-2.
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Signaling from ciliary microdomains controls developmental processes in metazoans. Trypanosome transmission requires development and migration in the tsetse vector alimentary tract. Flagellar cAMP signaling has been linked to parasite social motility (SoMo) in vitro, yet uncovering control of directed migration in fly organs is challenging. Here we show that the composition of an adenylate cyclase (AC) complex in the flagellar tip microdomain is essential for tsetse salivary gland (SG) colonization and SoMo. Cyclic AMP response protein 3 (CARP3) binds and regulates multiple AC isoforms. CARP3 tip localization depends on the cytoskeletal protein FLAM8. Re-localization of CARP3 away from the tip microdomain is sufficient to abolish SoMo and fly SG colonization. Since intrinsic development is normal in carp3 and flam8 knock-out parasites, AC complex-mediated tip signaling specifically controls parasite migration and thereby transmission. Participation of several developmentally regulated receptor-type AC isoforms may indicate the complexity of the in vivo signals perceived.
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Trypanosoma brucei brucei , Trypanosoma , Moscas Tsé-Tsé , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico , Trypanosoma brucei brucei/metabolismo , Moscas Tsé-Tsé/parasitologiaRESUMO
During a blood meal, female Anopheles mosquitoes are potentially exposed to diverse microbes in addition to the malaria parasite, Plasmodium. Human and animal African trypanosomiases are frequently co-endemic with malaria in Africa. It is not known whether exposure of Anopheles to trypanosomes influences their fitness or ability to transmit Plasmodium. Using cell and molecular biology approaches, we found that Trypanosoma brucei brucei parasites survive for at least 48h after infectious blood meal in the midgut of the major malaria vector, Anopheles coluzzii before being cleared. This transient survival of trypanosomes in the midgut is correlated with a dysbiosis, an alteration in the abundance of the enteric bacterial flora in Anopheles coluzzii. Using a developmental biology approach, we found that the presence of live trypanosomes in mosquito midguts also reduces their reproductive fitness, as it impairs the viability of laid eggs by affecting their hatching. Furthermore, we found that Anopheles exposure to trypanosomes enhances their vector competence for Plasmodium, as it increases their infection prevalence. A transcriptomic analysis revealed that expression of only two Anopheles immune genes are modulated during trypanosome exposure and that the increased susceptibility to Plasmodium was microbiome-dependent, while the reproductive fitness cost was dependent only on the presence of live trypanosomes but was microbiome independent. Taken together, these results demonstrate multiple effects upon Anopheles vector competence for Plasmodium caused by eukaryotic microbes interacting with the host and its microbiome, which may in turn have implications for malaria control strategies in co-endemic areas.
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Anopheles/parasitologia , Malária/parasitologia , Plasmodium yoelii/fisiologia , Trypanosoma/fisiologia , Animais , Coinfecção , Interações Hospedeiro-Parasita , Camundongos , Reação em Cadeia da Polimerase , ReproduçãoRESUMO
Trypanosomiases and leishmaniases, caused by a group of related protist parasites, are Neglected Tropical Diseases currently threatening >500 million people worldwide. Reporter proteins have revolutionised the research on infectious diseases and have opened up new advances in the understanding of trypanosomatid-borne diseases in terms of both biology, pathogenesis and drug development. Here, we describe the generation and some applications of a new chimeric triple reporter fusion protein combining the red-shifted firefly luciferase PpyREH9 and the tdTomato red fluorescent protein, fused by the TY1 tag. Expressed in both Trypanosoma brucei brucei and Leishmania major transgenic parasites, this construct was successfully assessed on different state-of-the-art imaging technologies, at different scales ranging from whole organism to cellular level, both in vitro and in vivo in murine models. For T. b. brucei, the usefulness of this triple marker to monitor the entire parasite cycle in both tsetse flies and mice was further demonstrated. This stable reporter allows to qualitatively and quantitatively scrutinize in real-time several crucial aspects of the parasite's development, including the development of African trypanosomes in the dermis of the mammalian host. We briefly discuss developments in bio-imaging technologies and highlight how we could improve our understanding of parasitism by combining the genetic engineering of parasites to the one of the hosting organisms in which they complete their developmental program.
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Leishmania major/genética , Leishmaniose Cutânea/diagnóstico por imagem , Imagem Óptica/métodos , Proteínas Recombinantes de Fusão/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/diagnóstico por imagem , Animais , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Engenharia Genética/métodos , Humanos , Leishmania major/crescimento & desenvolvimento , Leishmania major/metabolismo , Leishmania major/ultraestrutura , Leishmaniose Cutânea/parasitologia , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados , Proteínas Recombinantes de Fusão/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestrutura , Tripanossomíase Africana/parasitologia , Moscas Tsé-Tsé/parasitologia , Proteína Vermelha FluorescenteRESUMO
[This corrects the article DOI: 10.1371/journal.pntd.0003075.].
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The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology.
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Pele/parasitologia , Trypanosoma brucei gambiense/isolamento & purificação , Tripanossomíase Africana/parasitologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tripanossomíase Africana/transmissão , Moscas Tsé-Tsé/parasitologiaRESUMO
The use of genetically engineered pathogens that express fluorescent or luminescent proteins represents a huge stride forward in the understanding of trypanosomatid-borne tropical diseases. Nowadays, such modified microorganisms are being used to screen thousands of compounds under a target-free (phenotypic) approach. In addition, experimental infections with transgenic parasites drastically reduce the number of animals required for preclinical studies, because no animal needs to be put down to assess its parasite load. Finally, the use of fluorescent parasites is contributing to unraveling genetic exchange events between trypanosomatid strains. This phenomenon is important for understanding the mechanism by which traits such as virulence, tissue tropism, and drug resistance are transferred, as well as the emergence of novel strains.
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Diagnóstico por Imagem/métodos , Trypanosoma , Animais , Pesquisa Biomédica , Descoberta de Drogas , Fluorescência , Luminescência , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Parasitos , Trypanosoma/genética , Trypanosoma/metabolismoRESUMO
BACKGROUND: Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years. METHODOLOGY/PRINCIPAL FINDINGS: We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks. CONCLUSIONS/SIGNIFICANCE: The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.
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Modelos Animais de Doenças , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Raios Infravermelhos , Leishmania infantum/genética , Leishmaniose Visceral/tratamento farmacológico , Imagem Óptica/métodos , Animais , Feminino , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Drug discovery programs sponsored by public or private initiatives pursue the same ambitious goal: a crushing defeat of major Neglected Tropical Diseases (NTDs) during this decade. Both target-based and target-free screenings have pros and cons when it comes to finding potential small-molecule leads among chemical libraries consisting of myriads of compounds. Within the target-based strategy, crystals of pathogen recombinant-proteins are being used to obtain three-dimensional (3D) structures in silico for the discovery of structure-based inhibitors. On the other hand, genetically modified parasites expressing easily detectable reporters are in the pipeline of target-free (phenotypic) screenings. Furthermore, lead compounds can be scaled up to in vivo preclinical trials using rodent models of infection monitoring parasite loads by means of cutting-edge bioimaging devices. As such, those preferred are fluorescent and bioluminescent readouts due to their reproducibility and rapidity, which reduces the number of animals used in the trials and allows for an earlier stage detection of the infective process as compared with classical methods. In this review, we focus on the current differences between target-based and phenotypic screenings in Leishmania, as an approach that leads to the discovery of new potential drugs against leishmaniasis.
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BACKGROUND: The mode of reproduction in Leishmania spp has been argued to be essentially clonal. However, recent data (genetic analysis of populations and co-infections in sand flies) have proposed the existence of a non-obligate sexual cycle in the extracellular stage of the parasite within the sand fly vector. In this article we propose the existence of intraclonal genetic exchange in the natural vector of Leishmania infantum. METHODOLOGY/PRINCIPAL FINDINGS: We have developed transgenic L. infantum lines expressing drug resistance markers linked to green and red fluorescent reporters. We hypothesized whether those cells with identical genotype can recognize each other and mate. Both types of markers were successfully exchanged within the sand fly midgut of the natural vector Phlebotomus perniciosus when individuals from these species were fed with a mixture of parental clones. Using the yellow phenotype and drug resistance markers, we provide evidence for genetic exchange in L. infantum. The hybrid progeny appeared to be triploid based on DNA content analysis. The hybrid clone analyzed was stable throughout the complete parasite life cycle. The progress of infections by the hybrid clone in BALB/c mice caused a reduction in parasite loads in both spleen and liver, and provided weight values similar to those obtained with uninfected mice. Spleen arginase activity was also significantly reduced relative to parental strains. CONCLUSIONS/SIGNIFICANCE: A L. infantum hybrid lineage was obtained from intraclonal genetic exchange within the midgut of the natural vector, suggesting the ability of this parasite to recognize the same genotype and mate. The yellow hybrid progeny is stable throughout the whole parasite life cycle but with a slower virulence, which correlates well with the lower arginase activity detected both in vitro and in vivo infections.
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Leishmania infantum/genética , Leishmania infantum/fisiologia , Phlebotomus/parasitologia , Animais , Sistema Digestório/parasitologia , Feminino , Citometria de Fluxo , Humanos , Hibridização Genética , Leishmaniose Visceral , Estágios do Ciclo de Vida , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The aim of this work is the in vitro and ex vivo assessment of the leishmanicidal activity of camptothecin and three analogues used in cancer therapy: topotecan (Hycantim®), gimatecan (ST1481) and the pro-drug irinotecan (Camptosar®) as well as its active metabolite SN-38 against Leishmania infantum. The activity of camptothecin and its derivatives was studied on extracellular L. infantum infrared-emitting promastigotes and on an ex vivo murine model of infected splenocytes with L. infantum fluorescent amastigotes. In situ formation of SDS/KCl precipitable DNA-protein complexes in Leishmania promastigotes indicated that these drugs are DNA topoisomerase IB poisons. The inhibitory potency of camptothecin derivatives on recombinant L. infantum topoisomerase IB was assessed in vitro showing that gimatecan is the most active compound preventing the relaxation of supercoiled DNA at submicromolar concentrations. Cleavage equilibrium assays in Leishmania topoisomerase IB show that gimatecan changes the equilibrium towards cleavage at much lower concentrations than the other camptothecin derivatives and that this effect persists over time. Gimatecan and camptothecin were the most powerful compounds preventing cell growth of free-living L. infantum promastigotes within the same concentration range. All these compounds killed L. infantum splenocyte-infecting amastigotes within the nanomolar range. The amastigote form showed higher sensitivity to topoisomerase IB poisons (with high therapeutic selectivity indexes) than free-living promastigotes. All the compounds assayed poisoned L. infantum DNA topoisomerase IB leading to a strong leishmanicidal effect. Camptothecin derivatives are suitable for reducing the parasitic burden of ex vivo infected splenocytes. The selectivity index of gimatecan makes it a promising drug against this neglected disease.