RESUMO
Several persistent pathogens employ antigenic variation to continually evade mammalian host adaptive immune responses. African trypanosomes use variant surface glycoproteins (VSGs) for this purpose, transcribing one telomeric VSG expression-site at a time, and exploiting a reservoir of (sub)telomeric VSG templates to switch the active VSG. It has been known for over fifty years that new VSGs emerge in a predictable order in Trypanosoma brucei, and differential activation frequencies are now known to contribute to the hierarchy. Switching of approximately 0.01% of dividing cells to many new VSGs, in the absence of post-switching competition, suggests that VSGs are deployed in a highly profligate manner, however. Here, we report that switched trypanosomes do indeed compete, in a highly predictable manner that is dependent upon the activated VSG. We induced VSG gene recombination and switching in in vitro culture using CRISPR-Cas9 nuclease to target the active VSG. VSG dynamics, that were independent of host immune selection, were subsequently assessed using RNA-seq. Although trypanosomes activated VSGs from repressed expression-sites at relatively higher frequencies, the population of cells that activated minichromosomal VSGs subsequently displayed a competitive advantage and came to dominate. Furthermore, the advantage appeared to be more pronounced for longer VSGs. Differential growth of switched clones was also associated with wider differences, affecting transcripts involved in nucleolar function, translation, and energy metabolism. We conclude that antigenic variants compete, and that the population of cells that activates minichromosome derived VSGs displays a competitive advantage. Thus, competition among variants impacts antigenic variation dynamics in African trypanosomes and likely prolongs immune evasion with a limited set of antigens.
Assuntos
Trypanosoma brucei brucei , Trypanosoma , Animais , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Trypanosoma brucei brucei/metabolismo , Variação Antigênica/genética , Evasão da Resposta Imune/genética , Glicoproteínas de Membrana/metabolismo , MamíferosRESUMO
With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.
Assuntos
Antiprotozoários/uso terapêutico , Chalcona/metabolismo , Chalcona/farmacologia , Citosol/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Peroxidases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Células Cultivadas , Chalcona/administração & dosagem , Chalcona/análogos & derivados , Citosol/enzimologia , Citosol/parasitologia , Descoberta de Drogas , Humanos , Leishmania/classificação , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Current chemotherapeutics for leishmaniasis have multiple deficiencies, and there is a need for new safe, efficacious, and affordable medicines. This study describes a successful drug repurposing approach that identifies the over-the-counter antihistamine, clemastine fumarate, as a potential antileishmanial drug candidate. The screening for inhibitors of the sphingolipid synthase (inositol phosphorylceramide synthase, IPCS) afforded, following secondary screening against Leishmania major (Lmj) promastigotes, 16 active compounds. Further refinement through the dose response against LmjIPCS and intramacrophage L. major amastigotes identified clemastine fumarate with good activity and selectivity with respect to the host macrophage. On target engagement was supported by diminished sensitivity in a sphingolipid-deficient L. major mutant (ΔLmjLCB2) and altered phospholipid and sphingolipid profiles upon treatment with clemastine fumarate. The drug also induced an enhanced host cell response to infection indicative of polypharmacology. The activity was sustained across a panel of Old and New World Leishmania species, displaying an in vivo activity equivalent to the currently used drug, glucantime, in a mouse model of L. amazonensis infection. Overall, these data validate IPCS as an antileishmanial drug target and indicate that clemastine fumarate is a candidate for repurposing for the treatment of leishmaniasis.
Assuntos
Antiprotozoários , Leishmaniose , Preparações Farmacêuticas , Animais , Antiprotozoários/farmacologia , Clemastina/uso terapêutico , Inositol , Leishmaniose/tratamento farmacológico , CamundongosRESUMO
Cutaneous leishmaniasis (CL) is a major health problem in many countries and its current treatment involves multiple parenteral injections with toxic drugs and requires intensive health services. Previously, the efficacy of a single subcutaneous injection with a slow-release formulation consisting of poly(lactide-co-glycolide) (PLGA) microparticles loaded with an antileishmanial 3-nitro-2-hydroxy-4,6-dimethoxychalcone (CH8) was demonstrated in mice model. In the search for more easily synthesized active chalcone derivatives, and improved microparticle loading, CH8 analogues were synthesized and tested for antileishmanial activity in vitro and in vivo. The 3-nitro-2',4',6'-trimethoxychalcone (NAT22) analogue was chosen for its higher selectivity against intracellular amastigotes (selectivity index = 1489, as compared with 317 for CH8) and more efficient synthesis (89% yield, as compared with 18% for CH8). NAT22 was loaded into PLGA / polyvinylpyrrolidone (PVP) polymeric blend microspheres (NAT22-PLGAk) with average diameter of 1.9 µm. Although NAT22-PLGAk showed similar activity to free NAT22 in killing intracellular parasites in vitro (IC50 ~ 0.2 µm), in vivo studies in Leishmania amazonensis - infected mice demonstrated the significant superior efficacy of NAT22-PLGAk to reduce the parasite load. A single intralesional injection with NAT22-PLGAk was more effective than eight injections with free NAT22. Together, these results show that NAT22-PLGAk is a promising alternative for single-dose localized treatment of CL.
Assuntos
Antiprotozoários/uso terapêutico , Chalconas/uso terapêutico , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/prevenção & controle , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Cutaneous leishmaniasis (CL) is a neglected parasitic disease conventionally treated by multiple injections with systemically toxic drugs. Aiming at a more acceptable therapy, we developed lipid-core nanocapsules (LNCs) entrapping the potent antileishmanial chalcone (CH8) for topical application. Rhodamine-labeled LNC (Rho-LNC-CH8) was produced for imaging studies. LNC-CH8 and Rho-LNC-CH8 had narrow size distributions (polydispersity index <0.10), with similar mean sizes (~180â¯nm) by dynamic light scattering. In vitro, Rho-LNC-CH8 was rapidly internalized by extracellular Leishmania amazonensis parasites macrophages in less than 15â¯min. LNC-CH8 activated macrophage oxidative mechanisms more efficiently than CH8, and was more selectively toxic against the intracellular parasites. In vivo, topically applied Rho-LNC-CH8 efficiently permeated mouse skin. In L. amazonensis-infected mice, LNC-CH8 reduced the parasite load by 86% after three weeks of daily topical treatment, while free CH8 was ineffective. In conclusion, LNC-CH8 has strong potential as a novel topical formulation for CL treatment.