In Silico Characterization of Calcineurin from Pathogenic Obligate Intracellular Trypanosomatids: Potential New Biological Roles.

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi (Tc) and Leishmania spp. (Lspp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human (Hs) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca2+-binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits TcCaNA1, TcCaNA2, LsppCaNA1, LsppCaNA1_var, and LsppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of HsCaNAα. For regulatory subunits, HsCaNB-1 and LsppCaNB associate preferentially with the nucleus and cytoplasm, and TcCaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.

The terpenic diamine GIB24 inhibits the growth of Trypanosoma cruzi epimastigotes and intracellular amastigotes, with proteomic analysis of drug-resistant epimastigotes.

The effect of N-geranyl-ethane-1,2-diamine dihydochloride (GIB24), a synthetic diamine, was assayed against different developmental forms of the parasitic protozoan Trypanosoma cruzi (strain Dm28c). The compound was effective against culture epimastigote forms (IC50/24h = 5.64 µM; SI = 16.4) and intracellular amastigotes (IC50/24h = 12.89 µM; SI = 7.18), as detected by the MTT methodology and by cell counting, respectively. Incubation of epimastigotes for 6h with 6 µM GIB24 (IC50/24h value) resulted in significant dissipation of the mitochondrial membrane potential, prior to permeabilization of the plasma membrane. Rounded epimastigotes with cell size reduction were observed by scanning electron microscopy. These morpho-physiological changes induced by GIB24 suggest an incidental death process. Treatment of infected Vero cells did not prevent the intracellular amastigotes from completing the intracellular cycle. However, there was a decrease in the number of released parasites, increasing the ratio amastigotes/trypomastigotes. Proteomic analysis of 15 µM GIB24 resistant epimastigotes indicated that the compound acts mainly on mitochondrial components involved in the Krebs cycle and in maintaining the oxidative homeostasis of the parasites. Our data suggest that GIB24 is active against the main morphological forms of T. cruzi.

Hybrids of Cinchona Alkaloids and Bile Acids as Antiparasitic Agents Against Trypanosoma cruzi.

The current chemotherapy of Chagas disease needs to be urgently improved. With this aim, a series of 16 hybrids of Cinchona alkaloids and bile acids were prepared by functionalization at position C-2 of the quinoline nucleus by a radical attack of a norcholane substituent via a Barton-Zard decarboxylation reaction. The antitrypanosomal activity of the hybrids was tested on different stages and strains of T. cruzi. In particular, eight out of 16 hybrids presented an IC50 ≤1 µg/mL against trypomastigotes of the CL Brener strain and/or a selectivity index higher than 10. These promising hybrids yielded similar results when tested on trypomastigotes from the RA strain of T. cruzi (discrete typing unit-DTU-VI). Surprisingly, trypomastigotes of the Y strain (DTU II) were more resistant to benznidazole and to most of the hybrids than those of the CL Brener and RA strains. However, the peracetylated and non-acetylated forms of the cinchonine/chenodeoxycholic bile acid conjugate 4f and 5f were the most trypanocidal hybrids against Y strain trypomastigotes, with IC50 values of 0.5 and 0.65 µg/mL, respectively. More importantly, promising results were observed in invasion assays using the Y strain, where hybrids 5f and 4f induced a significant reduction in intracellular amastigotes and on the release of trypomastigotes from infected cells.

Interleukin-32: An endogenous danger signal or master regulator of intracellular pathogen infections-Focus on leishmaniases.

Interleukin 32 (IL-32) is an intracellular cytokine produced by immune and non immune cells after different stimuli. It contributes to inflammation and control of intracellular pathogens mainly by inducing proinflammatory cytokines and microbicidal molecules. Evidence is rising showing that IL-32 can be considered an endogenous danger signal after tissue injury, amplifying the inflammatory process and acquired immune responses. It seems to be a master regulator of intracellular infectious diseases. In this review, first the general properties of IL-32 are described followed by its role in the immunopathogenesis of inflammatory and infectious diseases. Roles of IL-32 in the control of infectious diseases caused by intracellular pathogens are reported, and later a focus on IL-32 in leishmaniases, diseases caused by an intracellular protozoan, is presented.

Highly potent anti-leishmanial derivatives of hederagenin, a triperpenoid from Sapindus saponaria L.

Leishmaniasis is a neglected tropical disease (NTDs), endemic in 88 countries that affect more than 12 million people. Current drugs are limited due to their toxicity, development of biological resistance, length of treatment and high cost. Thus, the search for new effective and less toxic treatments is an urgent need. In this study, we report the synthesis of 3 new amide derivatives of hederagenin (22-24) with yields between 70% and 90%, along with 57 other derivatives of hederagenin (1-21, 25-60) carrying different groups at C-28 previously reported by our group, and the results of their in vitro ability to inhibit the growth of Leishmania infantum. Some derivatives (3, 4, 44, 49 and 52), showed activity at micromolar level and low toxicity against BGM and HepG2 cells. Moreover, the ability of hederagenin derivatives 3 (IC50 = 9.7 µM), 4 (12 µM), 44 (11 µM) and 49 (2 µM), to prevent proliferation of intracellular amastigote forms of L. infantum and their higher selectivity index and low toxicity compared to commercial positive drug control of choice (potassium antimonyl tartrate trihydrate) (IC50 = 80 µM, SI = 0.1), make these compounds promising candidates for the treatment of leishmaniasis.

HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes.

Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10(7) amastigotes--with typical shape and positive for the amastigote marker Ssp4--from 5x10(6) infected Vero cells (48 h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.

Disease resistance in Atlantic salmon (Salmo salar): coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi.

BACKGROUND: Naturally occurring coinfections of pathogens have been reported in salmonids, but their consequences on disease resistance are unclear. We hypothesized that 1) coinfection of Caligus rogercresseyi reduces the resistance of Atlantic salmon to Piscirickettsia salmonis; and 2) coinfection resistance is a heritable trait that does not correlate with resistance to a single infection. METHODOLOGY: In total, 1,634 pedigreed Atlantic salmon were exposed to a single infection (SI) of P. salmonis (primary pathogen) or coinfection with C. rogercresseyi (secondary pathogen). Low and high level of coinfection were evaluated (LC = 44 copepodites per fish; HC = 88 copepodites per fish). Survival and quantitative genetic analyses were performed to determine the resistance to the single infection and coinfections. MAIN FINDINGS: C. rogercresseyi significantly increased the mortality in fish infected with P. salmonis (SI mortality = 251/545; LC mortality = 544/544 and HC mortality = 545/545). Heritability estimates for resistance to P. salmonis were similar and of medium magnitude in all treatments (h2SI = 0.23 ± 0.07; h2LC = 0.17 ± 0.08; h2HC = 0.24 ± 0.07). A large and significant genetic correlation with regard to resistance was observed between coinfection treatments (rg LC-HC = 0.99 ± 0.01) but not between the single and coinfection treatments (rg SI-LC = -0.14 ± 0.33; rg SI-HC = 0.32 ± 0.34). CONCLUSIONS/SIGNIFICANCE: C. rogercresseyi, as a secondary pathogen, reduces the resistance of Atlantic salmon to the pathogen P. salmonis. Resistance to coinfection of Piscirickettsia salmonis and Caligus rogercresseyi in Atlantic salmon is a heritable trait. The absence of a genetic correlation between resistance to a single infection and resistance to coinfection indicates that different genes control these processes. Coinfection of different pathogens and resistance to coinfection needs to be considered in future research on salmon farming, selective breeding and conservation.

A quinoxaline derivative as a potent chemotherapeutic agent, alone or in combination with benznidazole, against Trypanosoma cruzi.

BACKGROUND: Chagas' disease is a condition caused by the protozoan Trypanosoma cruzi that affects millions of people, mainly in Latin America where it is considered endemic. The chemotherapy for Chagas disease remains a problem; the standard treatment currently relies on a single drug, benznidazole, which unfortunately induces several side effects and it is not successful in the cure of most of the chronic patients. In order to improve the drug armamentarium against Chagas' disease, in the present study we describe the synthesis of the compound 3-chloro-7-methoxy-2-(methylsulfonyl) quinoxaline (quinoxaline 4) and its activity, alone or in combination with benznidazole, against Trypanosoma cruzi in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Quinoxaline 4 was found to be strongly active against Trypanosoma cruzi Y strain and more effective against the proliferative forms. The cytotoxicity against LLCMK2 cells provided selective indices above one for all of the parasite forms. The drug induced very low hemolysis, but its anti-protozoan activity was partially inhibited when mouse blood was added in the experiment against trypomastigotes, an effect that was specifically related to blood cells. A synergistic effect between quinoxaline 4 and benznidazole was observed against epimastigotes and trypomastigotes, accompanied by an antagonistic interaction against LLCMK2 cells. Quinoxaline 4 induced several ultrastructural alterations, including formations of vesicular bodies, profiles of reticulum endoplasmic surrounding organelles and disorganization of Golgi complex. These alterations were also companied by cell volume reduction and maintenance of cell membrane integrity of treated-parasites. CONCLUSION/SIGNIFICANCE: Our results demonstrated that quinoxaline 4, alone or in combination with benznidazole, has promising effects against all the main forms of T. cruzi. The compound at low concentrations induced several ultrastructural alterations and led the parasite to an autophagic-like cell death. Taken together these results may support the further development of a combination therapy as an alternative more effective in Chagas' disease treatment.

Novel amidines and analogues as promising agents against intracellular parasites: a systematic review.

Parasitic protozoa comprise diverse aetiological agents responsible for important diseases in humans and animals including sleeping sickness, Chagas disease, leishmaniasis, malaria, toxoplasmosis and others. They are major causes of mortality and morbidity in tropical and subtropical countries, and are also responsible for important economic losses. However, up to now, for most of these parasitic diseases, effective vaccines are lacking and the approved chemotherapeutic compounds present high toxicity, increasing resistance, limited efficacy and require long periods of treatment. Many of these parasitic illnesses predominantly affect low-income populations of developing countries for which new pharmaceutical alternatives are urgently needed. Thus, very low research funding is available. Amidine-containing compounds such as pentamidine are DNA minor groove binders with a broad spectrum of activities against human and veterinary pathogens. Due to their promising microbicidal activity but their rather poor bioavailability and high toxicity, many analogues and derivatives, including pro-drugs, have been synthesized and screened in vitro and in vivo in order to improve their selectivity and pharmacological properties. This review summarizes the knowledge on amidines and analogues with respect to their synthesis, pharmacological profile, mechanistic and biological effects upon a range of intracellular protozoan parasites. The bulk of these data may contribute to the future design and structure optimization of new aromatic dicationic compounds as novel antiparasitic drug candidates.

Triphenylmethane derivatives have high in vitro and in vivo activity against the main causative agents of cutaneous leishmaniasis.

The current standard of care for cutaneous leishmaniasis (CL) is organic antimonial compounds, but the administration of these compounds is complicated by a low therapeutic - toxic index, as well as parenteral administration. Thus, there is an urgent need for the development of new and inexpensive therapies for the treatment of CL. In this study, we evaluate the activity of the triphenylmethane (TPM) class of compounds against three species of Leishmania which are pathogenic in humans. The TPM have a history of safe use in humans, dating back to the use of the original member of this class, gentian violet (GV), from the early 20(th) century. Initially, the in vitro efficacy against Leishmania (Viannia) braziliensis, L. (Leishmania) amazonensis and L. (L.) major of 9 newly synthesized TPM, in addition to GV, was tested. Inhibitory concentrations (IC) IC(50) of 0.025 to 0.84 µM had been found in promastigotes in vitro assays. The four most effective compounds were then tested in amastigote intracellular assays, resulting in IC(50) of 0.10 to 1.59 µM. A high degree of selectivity of antiparasitic activity over toxicity to mammalian cells was observed. Afterwards, GV and TPM 6 were tested in a topical formulation in mice infected with L. (L.) amazonensis leading to elimination of parasite burdens at the site of lesion/infection. These results demonstrated that TPM present significant anti-leishmanial activities and provide a rationale for human clinical trials of GV and other TPM. TPM are inexpensive and safe, thus using them for treatment of CL may have a major impact on public health.

17-AAG kills intracellular Leishmania amazonensis while reducing inflammatory responses in infected macrophages.

BACKGROUND: Leishmaniasis is a neglected endemic disease with a broad spectrum of clinical manifestations. Pentavalent antimonials have been the treatment of choice for the past 70 years and, due to the emergence of resistant cases, the efficacy of these drugs has come under scrutiny. Second-line drugs are less efficacious, cause a range of side effects and can be costly. The formulation of new generations of drugs, especially in developing countries, has become mandatory. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the anti-leishmanial effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, in vitro. This inhibitor is currently in clinical trials for cancer treatment; however, its effects against intracellular Leishmania remain untested. Macrophages infected with L. amazonensis were treated with 17-AAG (25-500 nM) and parasite load was quantified using optical microscopy. Parasite load declined in 17-AAG-treated macrophages in a dose- and time-dependent manner. Intracellular parasite death became irreversible after 4 h of treatment with 17-AAG, and occurred independent of nitric oxide (NO) and superoxide (O(2) (-)) production. Additionally, intracellular parasite viability was severely reduced after 48 h of treatment. Interestingly, treatment with 17-AAG reduced pro-inflammatory mediator production, including TNF-α, IL-6 and MCP-1, yet IL-12 remained unaffected. Electron microscopy revealed morphological alterations, such as double-membrane vacuoles and myelin figures at 24 and 48 h after 17-AAG treatment. CONCLUSIONS/SIGNIFICANCE: The HSP90 inhibitor, 17-AAG, possesses high potency under low dosage and reduces both pro-inflammatory and oxidative molecule production. Therefore, further studies are warranted to investigate this inhibitor's potential in the development of new generations of anti-leishmanials.

Visualizing the 3D architecture of multiple erythrocytes infected with Plasmodium at nanoscale by focused ion beam-scanning electron microscopy.

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.

Effects of aqueous leaf extracts of Azadirachta indica A. Juss. (neem) and Melia azedarach L. (Santa Barbara or cinnamon) on the intracellular development of Toxoplasma gondii / Efeito de extrato aquoso de folhas de Azadirachta indica A. Juss. (nim) e Melia azedarach L. (canela ou Santa Bárbara) sobre o desenvolvimento intracelular de Toxoplasma gondii

Melia azedarach (cinnamon) and Azadirachta indica (neem) have a variety of biologically active ingredients against virus, bacteria and protozoan parasites; however, little is known about their action on Toxoplasma gondii intracellular development. Toxoplasma gondii infects all eukaryotic cells, where it establishes and multiplies inside a modified vacuole called the parasitophorous vacuole until the cell ruptures, re-infecting other cells and establishing the infection. There are no efficient chemotherapies for the elimination of T. gondii, minimizing side effects. In this study, we performed in vitro assays with neem and cinnamon aqueous extracts against the intracellular development of T. gondii tachyzoites. After treatment with neem and cinnamon for 24 h, the percentage of infected cells and the number of intracellular parasites drastically decreased. This effect was concentration-dependent. During the incubation of the extracts, progressive morphological and ultrastructure alterations led to intense vesiculation and complete elimination of the parasite from the intracellular medium. However, during the treatment with extracts, no morphological effects were observed in the structure of the host cell. These results suggest that the aqueous extracts of neem and cinnamon were capable of interfering with and eliminating the intracellular development of Toxoplasma gondii.

Melia azedarach (canela) e Azadirachta indica (nim) apresenta grande variedade de ingredientes biologicamente ativos contra vírus, bactérias e protozoários, mas nenhum efeito sobre o desenvolvimento intracelular do Toxoplasma gondii é conhecido. Toxoplasma gondii infecta todos os tipos de células Eucarióticas, onde se estabelece no meio intracelular em vacúolo modificado conhecido como vacúolo parasitóforo. Neste vacúolo ocorre a replicação levando a ruptura da célula hospedeira e reinfecção de novas células, perpetuando a infecção. A quimioterapia utilizada não é capaz de eliminar o parasita além de induzir fortes efeitos colaterais. Neste estudo, nós demonstramos o efeito in vitro de extratos aquosos da canela e nim sobre o desenvolvimento intracelular do taquizoíto do Toxoplasma gondii. Após tratamento de nim e canela por 24 h, a porcentagem de infecção e o número de taquizoítos intracelulares decaiu drasticamente. Este efeito foi concentração-dependente. Durante incubação dos extratos, uma progressiva desorganização morfológica e ultraestrutural levaram a formação de intensa vesiculação e completa destruição do parasita, que passou a uma estrutura amorfa, antes da completa eliminação do meio intracelular. No entanto durante o tratamento com os extratos, efeitos morfológicos não foram observados nas estruturas da célula hospedeira. Estes resultados sugerem que os extratos aquosos de nim e canela foram capazes de interferir e eliminar o desenvolvimento intracelular do Toxoplasma gondii.

Synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial activities of thiosemicarbazides, 4-thiazolidinones and 1,3,4-thiadiazoles.

In this work we reported the synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial activities in vitro of three new compound series obtained from ethyl(5-methyl-1-H-imidazole-4-carboxylate): acylthiosemicarbazide analogues 3a-d, 4-thiazolidinone analogues 4a-d and 1,3,4-thiadiazole analogues 5a-d. All synthesized compounds were characterized by IR, (1)H, (13)C NMR and HRMS. The majority of the tested compounds show excellent anti-T. gondii activity when compared to hydroxyurea and sulfadiazine. In addition it was also shown that most of the compounds in this study have a better performance against intracellular tachyzoites. The results for antimicrobial activity evaluation showed weak antibacterial and antifungal activities for all the tested molecules, when compared with the standard drugs (chloramphenicol and rifampicin for antibacterial activity; nistatin and ketoconazole for antifungal activity).