Trypanocidal and leishmanicidal activity of six limonoids

Six limonoids [kotschyienone A and B (1, 2), 7-deacetylgedunin (3), 7-deacetyl-7-oxogedunin (4), andirobin (5) and methyl angolensate (6)] were investigated for their trypanocidal and leishmanicidal activities using bloodstream forms of Trypanosoma brucei and promastigotes of Leishmania major. Whereas all compounds showed anti-trypanosomal activity, only compounds 1–4 displayed anti-leishmanial activity. The 50% growth inhibition (GI 50) values for the trypanocidal and leishmanicidal activity of the compounds ranged between 2.5 and 14.9 μM. Kotschyienone A (1) was found to be the most active compound with a minimal inhibition concentration (MIC) value of 10 μM and GI 50 values between 2.5 and 2.9 μM. Only compounds 1 and 3 showed moderate cytotoxicity against HL-60 cells with MIC and GI 50 values of 100 μM and 31.5–46.2 μM, respectively. Compound 1 was also found to show activity against intracellular amastigotes of L. major with a GI 50 value of 1.5 μM. The results suggest that limonoids have potential as drug candidates for the development of new treatments against trypanosomiasis and leishmaniasis

Participação das proteínas ezrina, radixina e moesina (erm proteins) na invasão celular por amastigotas extracelulares de trypanosoma cruzi

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas? disease that affects 6-7 million people worldwide, mostly in the South and Central America. Chagas? disease was responsible for 76% of all deaths caused by Neglected Tropical Diseases in Brazil from 2000 to 2011. In mammalian hosts T. cruzi alternates from extracellular (infective) trypomastigote forms and intracellular (replicative) amastigote forms. Additionally, trypomastigotes can differentiate into amastigotes in the extracellular environment generating infective extracellular amastigotes (EAs). Host cell invasion by these forms is mediated by complex cellular signaling events regulating actin filaments, the main component in EA uptake. ERM proteins (ezrin, radixin and moesin) are key elements linking actin filaments to the plasma membrane, important for the maintenance of cell morphology, cell migration and invasion of intracellular pathogens. The aim of this study was to evaluate the role of ERM proteins in actin cytoskeleton-plasma membrane interplay during host cell invasion by EAs. Our results revealed that depletion of host ezrin and radixin but not moesin inhibited EAs invasion in HeLa cells. Confocal microscopy of HeLa cells transfected with ERM proteins?GFP or -HA revealed recruitment of ERM proteins to EAs invasion sites colocalizing with F-actin. Additionally, invasion assays performed with cells overexpressing ERM proteins revealed increased EAs invasion in ezrin and radixin but not moesin overexpressing cells. Finally, time-lapse live imaging confocal microscopy has shown reduced and delayed actin dynamics in ezrin and radixin depleted HeLa cells when compared to control or moesin groups. Altogether, these findings show distinct roles of ERM proteins in actin filament dynamics and plasma membrane interplay during EAs host cell invasion.Doença de Chagas é causada pelo protozoário Trypanosoma cruzi e afeta 6-7 milhões de pessoas em todo o mundo, principalmente nas Américas do Sul e Central. Entre os anos 2000 e 2011, a doença de Chagas foi responsável por 76% de todas as mortes causadas por doenças tropicais negligenciadas no Brasil. Em hospedeiros mamíferos T. cruzi alterna entre formas tripomastigotas (extracelular e infectivo) e amastigotas (intracelular e replicativo). Além disso, tripomastigotas podem se diferenciar em amastigotas no ambiente extracelular gerando amastigotas extracelulares (AEs) que são capazes de infectar células do hospedeiro. A invasão celular por estas formas é mediada por complexos eventos de sinalização celular que regulam os filamentos de actina, o principal componente do hospedeiro na invasão por AEs. Nesse contexto ERM proteins (ezrina, radixina e moesina) são proteínas chave que ligam os filamentos de actina à membrana plasmática e são importantes para migração celular, manutenção da morfologia e estrutura do tecido além de participar na invasão de patógenos intracelulares. O objetivo deste estudo foi avaliar o papel das ERM proteins durante a invasão da célula hospedeira por AEs. Nossos resultados mostraram que a depleção de ezrina e de radixina, mas não de moesina, inibiu a invasão AEs em células HeLa. Além disso, análise por microscopia confocal de células HeLa trasnsfectadas com ERM proteins-GFP ou -HA revelou recrutamento e co-localização dessas proteínas com F-actina nos sítios de invasão dos AEs. Em ensaios utilizando células HeLa superexpressando ERM proteins observamos que a superexpressão de ezrina e de radixina, mas não de moesina, aumenta a invasão dos parasitas. Finalmente, por microscopia confocal time-lapse em células vivas foi observado retardo entre recrutamento de actina e invasão em células depletadas para ezrina ou radixina em comparação aos grupos de células depletadas para moesina ou sem transdução (controle). Em conjunto, esses resultados mostram papéis distintos das ERM proteins na dinâmica de actina e interação com a membrana plasmática durante a invasão de AEs na célula hospedeira.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Trypanosoma cruzi extracellular amastigotes selectively trigger the PI3K/Akt and Erk pathways during HeLa cell invasion

Cell invasion by Trypanosoma cruzi extracellular amastigotes involves different signaling pathways to induce phagocytosis-like mechanisms. Previous works indicated that PI3K/Akt, Src and Erk might be involved in EA invasion; however, participation of these molecules in this process remains elusive. Here, we observed that EA activated Akt, Erk but not Src. Interference of EA invasion with specific inhibitors corroborated this observation. Our results show that EA is capable of selectively triggering complex signaling pathways. Activation of PI3K/Akt and Erk, kinases related to actin cytoskeleton rearrangement and phagocytosis, reinforces the idea that T. cruzi EA subverts the phagocytic machinery during invasion

Trypanosoma cruzi extracellular amastigotes and host cell signaling: more pieces to the puzzle

Among the different infective stages that Trypanosoma cruzi employs to invade cells, extracellular amastigotes have recently gained attention by our group. This is true primarily because these amastigotes are able to infect cultured cells and animals, establishing a sustainable infective cycle. Extracellular amastigotes are thus an excellent means of adaptation and survival for T. cruzi, whose different infective stages each utilize unique mechanisms for attachment and penetration. Here we discuss some features of host cell invasion by extracellular amastigotes and the associated host cell signaling events that occur as part of the process

Host cell protein LAMP‐2 is the receptor for Trypanosoma cruzi surface molecule gp82 that mediates invasion

Host cell invasion by Trypanosoma cruzi metacyclic trypomastigote (MT) is mediated by MT-specific surface molecule gp82, which binds to a still unidentified receptor, inducing lysosome spreading and exocytosis required for the parasitophorous vacuole formation. We examined the involvement of the major lysosome membrane-associated LAMP proteins in MT invasion. First, human epithelial HeLa cells were incubated with MT in the presence of antibody to LAMP-1 or LAMP-2. Antibody to LAMP-2, but not to LAMP-1, significantly reduced MT invasion. Next, HeLa cells depleted in LAMP-1 or LAMP-2 were generated. Cells deficient in LAMP-2, but not in LAMP-1, were significantly more resistant to MT invasion than wild-type controls. The possibility that LAMP-2 might be the receptor for gp82 was examined by co-immunoprecipitation assays. Protein A/G magnetic beads cross-linked with antibody directed to LAMP-1 or LAMP-2 were incubated with HeLa cell and MT detergent extracts. Gp82 bound to LAMP-2 but not to LAMP-1. Binding of the recombinant gp82 protein to wild-type and LAMP-1-deficient cells, which was dose dependent and saturable, had a similar profile and was much higher as compared with LAMP-2-depleted cells. These data indicate that MT invasion is accomplished through recognition of gp82 by its receptor LAMP-2

Trypanosoma cruzi extracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells

Perfil genético de dois isolados de Trypanosoma evansi do Sul do Brasil com diferentes parasitemias

http://dx.doi.org/10.5007/2175-7925.2014v27n3p73Trypanosoma evansi is the most widespread pathogenic trypanosome, due to its ability to be mechanically transmitted by hematophagous flies. Some studies have shown that T. evansi has a high genetic homology among populations around the world, but they have not described the relationships among infection patterns in the host. In this study, we used molecular techniques to determine the genetic variability of two isolates of T. evansi from southern Brazil showing different infection patterns in a murine model. Genetic variability markers were amplified using the techniques described, as well as the presence of DNA from kinetoplast was also checked. We found a very close genetic profile between the isolates of T. evansi using random amplified polymorphic DNA (RAPD) and markers inter-simple sequence repeats (ISSR). Primers derived from the sequence of Trypanosoma brucei maxicircle encoding the subunit 5 of NADH dehydrogenase (nad5) were used to demonstrate the absence of maxicircles, while immunofluorescence was used to check the lack of DNA from kinetoplast. All methods reveal the absence of kDNA, as occurring in American isolates of T. evansi. Perhaps, differences noticed in the phenotypic patterns of animals that were observed during infection are not associated with the molecular changes, but with the adaptation to different hosts, as described for other trypanosomatids.http://dx.doi.org/10.5007/2175-7925.2014v27n3p73Trypanosoma evansi é o mais difundido dos tripanossomas patogênicos, devido à sua capacidade de ser transmitido mecanicamente por moscas hematófagas. Alguns estudos têm demonstrado que T. evansi possui uma homologia genética entre populações de todo o mundo, mas não descreveram as relações entre os padrões de infecção no hospedeiro. Neste estudo, foram utilizadas técnicas moleculares para determinar as variabilidades genéticas de dois isolados de T. evansi do Sul do Brasil que mostraram diferentes padrões de infecção em modelo murino. Os marcadores de variabilidade genética foram amplificados utilizando as técnicas descritas, bem como a presença de DNA do cinetoplasto também foi verificado. Foi encontrado um perfil genético muito próximo entre os isolados de T. evansi utilizando random amplified polymorphic DNA (RAPD) e marcadores inter-simple sequence repeats (ISSR). Os iniciadores derivados da sequência de maxicírculos de Trypanosoma brucei que codificam a subunidade 5 da NADH desidrogenase (nad5) foram usados para demonstrar a ausência de maxicírculos, enquanto imunofluorescência foi usada para verificar a ausência de DNA do cinetoplasto. Todos os métodos demonstram a ausência de kDNA, como ocorre nos isolados americanos de T. evansi. As diferenças notadas nos padrões fenótipicos dos animais que foram observadas durante a infecção não estão, provavelmente, relacionados com as alterações moleculares, mas à adaptação a diferentes hospedeiros, como descrito para outros tripanosomatídeos

Unique behavior of Trypanosoma cruzi mevalonate kinase: a conserved glycosomal enzyme involved in host cell invasion and signaling

Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion