Infrared Fluorescent Imaging as a Potent Tool for In Vitro, Ex Vivo and In Vivo Models of Visceral Leishmaniasis

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. 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. 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 diseaseThis research was supported by Ministerio de Economía y Competitividad (www.mineco.gob.es) grants AGL2010-16078/GAN to RBF and CYTED 214RT0482 to RMR; Instituto de Salud Carlos III (www.isciii.es) grants PI12/00104 to RMR and RICET RD12/0018/0004 to MF; Junta de Castilla y León (www.jcyl.es) grants Gr238 and LE182U13; European Commision (cordis.europa.eu/home_es.html), grant HOMIN - 317057-FP7-PEOPLE-2012-ITN and BIOIMID (http://www.fundacionareces.es) Proyecto de Excelencia Instituto Sanitario “La Princesa” and Fundación Ramón Areces to MF. SK is granted from AECC Foundation (https://www.aecc.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the anuscrip

Deletion Study of DNA Topoisomerase IB from Leishmania donovani: Searching for a Minimal Functional Heterodimer

The substantial differences between trypanosomal and leishmanial DNA topoisomerase IB concerning to their homologues in mammals have provided a new lead in the study of the structural determinants that can be effectively targeted. Leishmania donovani, the causative agent of visceral leishmaniasis, contains an unusual heterodimeric DNA topoisomerase IB. The catalytically active enzyme consists of a large subunit (LdTopIL), which contains the non-conserved N-terminal end and the phylogenetically conserved “core” domain, and of a small subunit (LdTopIS) which harbors the C-terminal region with the characteristic tyrosine residue in the active site. Heterologous co-expression of LdTopIL and LdTopIS genes in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme LdTopIL/S which can be used for structural studies. An approach by combinatorial cloning of deleted genes encoding for truncated versions of both subunits was used in order to find out structural insights involved in enzyme activity or protein-protein interaction. The role played by the non-conserved N-terminal extension of LdTopIL in both relaxation activity and CPT sensitivity has been examined co-expressing the full-length LdTopIS and a fully active LdTopIΔS deletion with several deletions of LdTopIL lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 26 amino acids placed at the N-terminal end and a variable region comprised between Ala548 to end of the C-terminal extension of LdTopIL were enzymatically dispensable. Altogether this combinatorial approach provides important structural insights of the regions involved in relaxation activity and for understanding the atypical structure of this heterodimeric enzyme

Gel-Free Tools for Quick and Simple Screening of Anti-Topoisomerase 1 Compounds

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.This research was funded in part by the Ministerio de Ciencia e Innovación, Spain (PID2021-122558OB-I00, UE) and by Gobierno Vasco, Universidad del País Vasco (GV, IT1701-22; UPV)

Identification and characterization of the regions involved in the nuclear translocation of the heterodimeric leishmanial DNA topoisomerase IB. PLoS One 8

Abstract Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique heterodimeric DNAtopoisomerase IB (LdTopIB). LdTopIB is a heterodimer made up of a large subunit and a small subunit that must interact with each other to build an active enzyme able to solve the topological tensions on the DNA. As LdTopIB is located within the nucleus, one or more nuclear localization signals (NLS) should exist to ensure its nuclear translocation. In this report three novel NLS have been identified through a sequential deletion study of the genes encoding of both subunits fused to that encoding the green fluorescent protein (GFP). NLS1 is a highly basic sequence of 43 amino acids in the C-terminal extension of the large protomer. We found two well-defined sequences in the small protomer: NLS2 is a 10-amino acid motif located in the N-terminal extension of the protein; NLS3 consists of a complex region of 28 amino acids placed in the vicinity of the catalytic Tyr-222 included at the conserved SKINY signature within the C-terminal. Furthermore, by means of yeast cell viability assays, conducted with several LdTopIB chimeras lacking any of the NLS motives, we have revealed that both subunits are transported independently to the nucleus. There was no evidence of LdTopIB accumulation in mitochondria or association to the kinetoplast DNA network. The results rule out the former hypothesis, which attributes nucleocytoplasmic transport of LdTopIB entirely to the large subunit. The LdTopIB is localized to the nucleus only

Drug discovery technologies: Caenorhabditis elegans as a model for anthelmintic therapeutics

39 páginas, 3 figuras, 5 tablas.Helminthiasis is one of the gravest problems worldwide. There is a growing concern on less available anthelmintics and the emergence of resistance creating a major threat to human and livestock health resources. Novel and broad-spectrum anthelmintics are urgently needed. The free-living nematode Caenorhabditis elegans could address this issue through automated high-throughput technologies for the screening of large chemical libraries. This review discusses the strong advantages and limitations for using C elegans as a screening method for anthelmintic drug discovery. C elegans is the best model available for the validation of novel effective drugs in treating most, if not all, helminth infections, and for the elucidation the mode of action of anthelmintic candidates. This review also focuses on available technologies in the discovery of anthelmintics published over the last 15 years with particular attention to high-throughput technologies over conventional screens. On the other hand, this review highlights how combinatorial and nanomedicine strategies could prolong the use of anthelmintics and control resistance problems.Consejería de Educación, Junta de Castilla y León, Grant/Award Number: LE020P17; Ministerio de Economía y Competitividad, Grant/Award Numbers: AGL2016‐79813‐C2‐1R, RYC‐2015‐18368; European and Developing Countries Clinical Trials Partnership, Grant/Award Number: RIA2017NCT‐184

Occurrence of Leishmaniasis in Iberian wolves in Northwestern Spain

10 páginas, 2 figuras, 2 tablas.Canine leishmaniasis is an important vector-borne protozoan disease in dogs that is re-sponsible for serious deterioration in their health. In the Iberian Peninsula, as in most countriessurrounding the Mediterranean Sea, canine leishmaniasis is caused byLeishmania infantum(zy-modeme MON-1), a digenetic trypanosomatid that harbors in the parasitophorous vacuoles of hostmacrophages, causing severe lesions that can lead to death if the animals do not receive adequatetreatment. Canine leishmaniasis is highly prevalent in Spain, especially in the Mediterranean coastalregions (Levante, Andalusia and the Balearic Islands), where the population of domestic dogs is veryhigh. However, the presence of this disease has been spreading to other rural and sparsely populatedlatitudes, and cases of leishmaniasis have been reported for years in wildlife in northwestern Spain.This work describes for the first time the presence of wolves that tested positive for leishmaniasis inthe vicinity of the Sierra de la Culebra (Zamora province, northwestern Spain), a protected sanctuaryof this canid species, using PCR amplification ofL. infantumDNA from different non-invasive samplessuch as buccal mucosa and those from both ears and hair. In addition to live animals (21), samplesfrom carcasses of mainly roadkill animals (18) were also included and analyzed using the sametechnique, obtaining a positivity rate of 18 of the 39 wolves sampled (46.1%) regardless of their origin.The authors acknowledge the Centro del Lobo Ibérico “Félix Rodríguez de la Fuente” and the Junta of Castilla y León.Peer reviewe

Presencia de Leishmania infantum en distintas muestras biológicas de lobos ibéricos (Canis lupus signatus) de la provincia de Zamora

Trabajo presentado al: XXII Congreso de Parasitología, SOCEPA. Madrid, Julio. 2022

In vivo toxicity and efficacy of two diamine and one benzimidazole derivatives against the gastrointestinal nematode Haemonchus contortus

Trabajo presentado al: 28th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP). Dublín. Virtual meeting.MINECO (AEI, FEDER, UE): AGL2016-79813-C2-1R/2R and SAF2017-83575-R and Junta de Castilla y León (JCyL) cofinanced by FEDER, UE [LE020p17]. EVG is funded by JCyL and MMV by Ramón y Cajal Programme (RYC-2015-18368).Peer reviewe