Gene disruption of the DNA topoisomerase IB small subunit induces a non-viable phenotype in the hemoflagellate Leishmania major

<p>Abstract</p> <p>Background</p> <p>The unusual heterodimeric leishmanial DNA topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate <it>Trypanosoma brucei</it>. Unfortunately, this approach is not suitable in <it>Leishmania </it>where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work, we have successfully generated null mutants in the small subunit of the <it>L. major </it>DNA topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively.</p> <p>Results</p> <p>We have successfully replaced both <it>topS </it>loci with two selection markers. However, to achieve the second transfection round, we have had to rescue the null-homozygous with an episomal vector carrying the <it>Leishmania major topS </it>gene. Phenotypic characterization of the <it>L. major </it>rescued strain and a <it>L. major </it>strain, which co-overexpresses both subunits, shows few differences in DNA relaxation and camptothecin cytotoxicity when it was compared to the wild-type strain. Studies on phosphatidylserine externalization show a poor incidence of camptothecin-induced programmed cell death in <it>L. major</it>, but an effective cell-cycle arrest occurs within the first 24 h. S-Phase delay and G₂/M reversible arrest was the main outcome at lower concentrations, but irreversible G₂arrest was detected at higher camptothecin pressure.</p> <p>Conclusion</p> <p>Results obtained in this work evidence the essentiality of the <it>topS </it>gene encoding the <it>L. major </it>DNA topoisomerase IB small subunit. Reversibility of the camptothecin effect points to the existence of effective checkpoint mechanisms in <it>Leishmania </it>parasites.</p

Functional Expression of a DNA-Topoisomerase IB from Cryptosporidium parvum

Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB) in chromosome 7. Heterologous expression of CpTopIB gene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast extracts was detected only when CpTopIB ORF was expressed in a yeast expression system showing time and protein dependence under steady state kinetic conditions. The susceptibility of CpTopIB-transformed yeast to the irreversible inhibitor camptothecin and its water-soluble derivatives (topotecan and SN-38) was assessed

Zoonotic implications of onchocerca species on human health

The genus Onchocerca includes several species associated with ungulates as hosts, although some have been identified in canids, felids, and humans. Onchocerca species have a wide geographical distribution, and the disease they produce, onchocerciasis, is generally seen in adult individuals because of its large prepatency period. In recent years, Onchocerca species infecting animals have been found as subcutaneous nodules or invading the ocular tissues of humans; the species involved are O. lupi, O. dewittei japonica, O. jakutensis, O. gutturosa, and O. cervicalis. These findings generally involve immature adult female worms, with no evidence of being fertile. However, a few cases with fertile O. lupi, O. dewittei japonica, and O. jakutensis worms have been identified recently in humans. These are relevant because they indicate that the parasite's life cycle was completed in the new host-humans. In this work, we discuss the establishment of zoonotic Onchocerca infections in humans, and the possibility of these infections to produce symptoms similar to human onchocerciasis, such as dermatitis, ocular damage, and epilepsy. Zoonotic onchocerciasis is thought to be an emerging human parasitic disease, with the need to take measures such as One Health Strategies, in order to identify and control new cases in humans

Trypanosomatids topoisomerase re-visited. New structural findings and role in drug discovery

AbstractThe Trypanosomatidae family, composed of unicellular parasites, causes severe vector-borne diseases that afflict human populations worldwide. Chagas disease, sleeping sickness, as well as different sorts of leishmaniases are amongst the most important infectious diseases produced by Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively. All these infections are closely related to weak health care services in low-income populations of less developed and least economically developed countries. Search for new therapeutic targets in order to hit these pathogens is of paramount priority, as no effective vaccine is currently in use against any of these parasites. Furthermore, present-day chemotherapy comprises old-fashioned drugs full of important side effects. Besides, they are prone to produce tolerance and resistance as a consequence of their continuous use for decades. DNA topoisomerases (Top) are ubiquitous enzymes responsible for solving the torsional tensions caused during replication and transcription processes, as well as in maintaining genomic stability during DNA recombination. As the inhibition of these enzymes produces cell arrest and triggers cell death, Top inhibitors are among the most effective and most widely used drugs in both cancer and antibacterial therapies. Top relaxation and decatenation activities, which are based on a common nicking–closing cycle involving one or both DNA strands, have been pointed as a promising drug target. Specific inhibitors that bind to the interface of DNA-Top complexes can stabilize Top-mediated transient DNA breaks. In addition, important structural differences have been found between Tops from the Trypanosomatidae family members and Tops from the host. Such dissimilarities make these proteins very interesting for drug design and molecular intervention. The present review is a critical update of the last findings regarding trypanosomatid’s Tops, their new structural features, their involvement both in the physiology and virulence of these parasites, as well as their use as promising targets for drug discovery

Miltefosine and nifuratel combination: a promising therapy for the treatment of leishmania donovani visceral leishmaniasis

[EN] Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel-a nitrofurantoin used against vaginal infections-as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine-the only oral drug currently used against leishmaniasis-with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.SIThis research was partially funded by MINECO; SAF2017-83575-R and PID 2020-119031RB-100 to R.M.R. E.M.-F. is contracted with EU PRIMA (PCI2022-132925); G.G. is recipient of FPI scholarship (PRE 2021 096909) supported by AE

Butyric acid-based feed additives help protect broiler chickens from Salmonella Enteritidis infection

[EN] Sodium butyrate is a sodium salt of a volatile short-chain fatty acid (butyric acid) used to prevent Salmonella Enteritidis infection in birds. Three groups of fifty 1-d-old broilers each were fed the following diets: T0 = standard broiler diet (control); T1 = standard broiler diet supplemented with 0.92 g/kg of an additive with free sodium butyrate (Gustor XXI B92); and T2 = standard broiler diet supplemented with 0.92 g/kg of an additive with sodium butyrate partially protected with vegetable fats (Gustor XXI BP70). Twenty percent of the birds were orally infected with Salmonella Enteritidis at d 5 posthatching and fecal Salmonella shedding was assessed at d 6, 9, 13, 20, 27, 34, and 41 of the trial. At d 42, all birds were slaughtered and 20 of them dissected: crop, cecum, liver, and spleen were sampled for bacteriological analyses. Both butyrate-based additives showed a significant reduction (P < 0.05) of Salmonella Enteritidis infection in birds from d 27 onward. However, the partially protected butyrate additive was more effective at the late phase of infection. Partially protected butyrate treatment successfully decreased infection not only in the crop and cecum but also in the liver. There were no differences in the spleen. These results suggest that sodium butyrate partially protected with vegetable fats offers a unique balance of free and protected active substances effective all along the gastrointestinal tract because it is slowly released during digestion.S

Further Investigations of Nitroheterocyclic Compounds as Potential Antikinetoplastid Drug Candidates

[EN] Due to the lack of specific vaccines, management of the trypanosomatid-caused neglected tropical diseases (sleeping sickness, Chagas disease and leishmaniasis) relies exclusively on pharmacological treatments. Current drugs against them are scarce, old and exhibit disadvantages, such as adverse effects, parenteral administration, chemical instability and high costs which are often unaffordable for endemic low-income countries. Discoveries of new pharmacological entities for the treatment of these diseases are scarce, since most of the big pharmaceutical companies find this market unattractive. In order to fill the pipeline of compounds and replace existing ones, highly translatable drug screening platforms have been developed in the last two decades. Thousands of molecules have been tested, including nitroheterocyclic compounds, such as benznidazole and nifurtimox, which had already provided potent and effective effects against Chagas disease. More recently, fexinidazole has been added as a new drug against African trypanosomiasis. Despite the success of nitroheterocycles, they had been discarded from drug discovery campaigns due to their mutagenic potential, but now they represent a promising source of inspiration for oral drugs that can replace those currently on the market. The examples provided by the trypanocidal activity of fexinidazole and the promising efficacy of the derivative DNDi-0690 against leishmaniasis seem to open a new window of opportunity for these compounds that were discovered in the 1960s. In this review, we show the current uses of nitroheterocycles and the novel derived molecules that are being synthesized against these neglected diseasesSIThis collaborative research was partially funded by MINECO; SAF2017-83575-R to RM

Antileishmanial activity of terpenylquinones on Leishmania infantum and their effects on Leishmania topoisomerase IB

[EN] Leishmania is the aethiological agent responsible for the visceral leishmaniasis, a serious parasite-borne disease widely spread all over the World. The emergence of resistant strains makes classical treatments less effective; therefore, new and better drugs are necessary. Naphthoquinones are interesting compounds for which many pharmacological properties have been described, including leishmanicidal activity. This work shows the antileishmanial effect of two series of terpenyl-1,4-naphthoquinones (NQ) and 1,4-anthraquinones (AQ) obtained from natural terpenoids, such as myrcene and myrceocommunic acid. They were evaluated both in vitro and ex vivo against the transgenic iRFP-Leishmania infantum strain and also tested on liver HepG2 cells to determine their selectivity indexes. The results indicated that NQ derivatives showed better antileishmanial activity than AQ analogues, and among them, compounds with a diacetylated hydroquinone moiety provided better results than their corresponding quinones. Regarding the terpenic precursor, compounds obtained from the monoterpenoid myrcene displayed good antiparasitic efficiency and low cytotoxicity for mammalian cells, whereas those derived from the diterpenoid showed better antileishmanial activity without selectivity. In order to explore their mechanism of action, all the compounds have been tested as potential inhibitors of Leishmania type IB DNA topoisomerases, but only some compounds that displayed the quinone ring were able to inhibit the recombinant enzyme in vitro. This fact together with the docking studies performed on LTopIB suggested the existence of another mechanism of action, alternative or complementary to LTopIB inhibition. In silico druglikeness and ADME evaluation of the best leishmanicidal compounds has shown good predictable druggabilitySIFinancial support came from Spanish MINECO (CTQ2015-68175-R, AGL2016-79813-C2-1-R, AGL2016-79813-C2-2-R and SAF2017-83575-R), ISCIII-RICET Network (RD12/0018/0002) and Consejería de Educación de la Junta de Castilla y León (LE020P17) co-financed by the Fondo Social Europeo of the European Union (FEDER-EU). P. G. J. acknowledges funding by Fundación Salamanca Ciudad de Cultura y Saberes (’‘Programme for attracting scientific talent to Salamanca’‘

Axenic interspecies and intraclonal hybrid formation in Leishmania: Successful crossings between visceral and cutaneous strains

[EN] Neglected Tropical Diseases (NTDs) represent a serious threat to humans, especially for those living in poor or developing countries. Leishmanianiosis is considered a zoonotic NTD transmitted by the bite of female phlebotomine sandflies, and is manifested mainly as a visceral form (caused by L. infantum and L. donovani) and a cutaneous form (caused by many species including L. major, L. tropica and L. braziliensis). Although it is now known that sexual reproduction occurs in these parasites, more studies are necessary to characterize the ability of Leishmania to generate hybrids, which may represent an important mechanism for virulence, drug resistance or adaptation to the host immune system. Therefore, several experiments were conducted to generate either intraclonal or interspecies hybrids in vitro. Results demonstrated that hybrids can be formed even with outcrosses between parasites causing visceral and cutaneous forms of the disease. Characterization of hybrids in terms of ploidy, kDNA content, growth rate and infection capacity provide important information about sexual reproduction in these parasites.SI: C.G.C (LE255-16) and B.D.A (LE208-17) are recipients of Junta de Castilla y Leo´n (JCyL) and European Social Found (ESF)’s Fellowships Scheme for Doctoral Training Programs. This research was funded by MINECO; SAF2017- 83575-R to RMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Antiparasitic effect of synthetic aromathecins on Leishmania infantum

[EN} Background Canine leishmaniasis is a zoonotic disease caused by Leishmania infantum, being the dogs one of the major reservoirs of human visceral leishmaniasis. DNA topology is a consolidated target for drug discovery. In this regard, topoisomerase IB – one of the enzymes controlling DNA topology – has been poisoned by hundreds of compounds that increase DNA fragility and cell death. Aromathecins are novel molecules with a multiheterocyclic ring scaffold that have higher stability than camptothecins. Results Aromathecins showed strong activity against both forms of L. infantum parasites, free-living promastigotes and intra-macrophagic amastigotes harbored in ex vivo splenic explant cultures obtained from infected BALB/c mice. However, they prevented the relaxation activity of leishmanial topoisomerase IB weakly, which suggests that the inhibition of topoisomerase IB partially explains the antileishmanial effect of these compounds. The effect of aromathecins was also studied against a strain resistant to camptothecin, and results suggested that the trafficking of these compounds is not through the ABCG6 transporter. Conclusions Aromathecins are promising novel compounds against canine leishmaniasis that can circumvent potential resistances based on drug efflux pumps.SIThis research had the financial support of: Ministerio de Economía y Competitividad (MINECO, AEI, FEDER, UE) [MINECO: AGL2016–79813-C2-1R, SAF2017–83575-R]. Junta de Castilla y León cofinanced by FEDER UE [LE020P17, Grant UIC108]. The funding body does not participate in the design of the study; collection, analysis and interpretation of data and in writing the manuscript