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Among neglected tropical diseases, leishmaniasis is one of the leading causes, not only of deaths but also of disability-adjusted life years. This disease, caused by protozoan parasites of the genus Leishmania, triggers different clinical manifestations, with cutaneous, mucocutaneous, and visceral forms. As existing treatments for this parasitosis are not sufficiently effective or safe for the patient, in this work, different sesquiterpenes isolated from the red alga Laurencia johnstonii have been studied for this purpose. The different compounds were tested in vitro against the promastigote and amastigote forms of Leishmania amazonensis. Different assays were also performed, including the measurement of mitochondrial potential, determination of ROS accumulation, and chromatin condensation, among others, focused on the detection of the cell death process known in this type of organism as apoptosis-like. Five compounds were identified that displayed leishmanicidal activity: laurequinone, laurinterol, debromolaurinterol, isolaurinterol, and aplysin, showing IC50 values against promastigotes of 1.87, 34.45, 12.48, 10.09, and 54.13 µM, respectively. Laurequinone was the most potent compound tested and was shown to be more effective than the reference drug miltefosine against promastigotes. Different death mechanism studies carried out showed that laurequinone appears to induce programmed cell death or apoptosis in the parasite studied. The obtained results underline the potential of this sesquiterpene as a novel anti-kinetoplastid therapeutic agent.

Current therapies of leishmaniasis and Chagas disease, two of the most widespread neglected tropical diseases, have limited efficacy and toxic side effects. In this regard, natural products play an important role in overcoming the current need for new antiparasitic agents. The present study reports the leishmanicidal and trypanocidal activities of twenty-four known silyl-ether derivatives of withaferin A. Eleven compounds from this series (4, 7, 8, 10, 12, 15, 17, 18, 20, 22 and 25) showed a potent dose-dependent inhibitory effect on the proliferation of Leishmania amazonensis promastigotes and Trypanosoma cruzi epimastigotes respectively, even higher than the references drugs, miltefosine and benznidazole. Among them, the most promising compound, derivative 10, exhibited approximately 34-fold higher leishmanicidal activity and 49-fold higher trypanocidal activity compared to the reference drugs, as well as lower cytotoxicity. Moreover, compounds 4, 7, 10, 12 and 15 were more active than the reference drugs against the amastigote forms of L. amazonensis, presenting a high selectivity index. Assays performed to study the ATP levels, mitochondrial membrane potential, plasma membrane permeability, chromatin condensation, reactive oxygen species and autophagy indicated that these withaferin A-silyl analogs appear to induce events characteristic of apoptosis-like and also autophagy leading to programmed cell death. These findings support the therapeutic potential of withaferin A-related steroids as anti-Leishmania and Trypanosoma agents.

Statins are effective sterol lowering agents with high amoebicidal activity. Nevertheless, due to their poor aqueous solubility, they remain underused especially in eye drop formulation. The aim of the present study is to develop Pitavastatin loaded nanoparticles suitable for ophthalmic administration and designed for the management of Acanthamoeba Keratitis. These nanocarriers are aimed to solve both the ophthalmic route-associated problems and the limited aqueous drug solubility issues of Pitavastatin. Nanoparticles were obtained by a nanoprecipitation-solvent displacement method and their amoebicidal activity was evaluated against four strains of Acanthamoeba: A. castellanii Neff, A. polyphaga, A. griffini and A. quina. In Acanthamoeba polyphaga, the effect of the present nanoparticles was investigated with respect to the microtubule distribution and several programmed cell death features. Nanoparticles were able to eliminate all the tested strains and Acanthamoeba polyphaga was determined to be the most resistance strain. Nanoparticles induced chromatin condensation, autophagic vacuoles and mitochondria dysfunction.

Background The in vitro activity against Leishmania spp. of a novel group of compounds, phenalenone derivatives, is described in this study. Previous studies have shown that some phenalenones present leishmanicidal activity, and induce a decrease in the mitochondrial membrane potential in L. amazonensis parasites, so in order to elucidate the evidence of programmed cell death occurring inside the promastigote stage, different assays were performed in two different species of Leishmania. Methods We focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure, mitochondrial membrane potential, and chromatin condensation among others. Results The results showed that four molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing. Conclusions The present results highlight the potential use of phenalenone derivatives against Leishmania species and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents.

Leishmaniasis and Chagas disease are neglected tropical diseases that cause problems in developing countries. The causative agents, Leishmania spp. and Trypanosoma cruzi, produce a clinical picture that can be fatal for the patient, such as Chagas heart disease, visceral leishmaniasis and megacolon, among others. Current treatments for these diseases are not very effective and highly toxic, since they require very prolonged treatments. The development of innovative, effective and safe drugs to fight infections caused by these parasites remains a challenge. For this reason, in recent years, there has been an increase in the search for new therapies. In this study, the antikinetoplastid activity of 13 sesquiterpene lactones obtained from Palythoa aff. clavata was screened against L. amazonensis, L. donovani and T. cruzi. The results revealed that the sesquiterpene lactones anhydroartemorin (2), cis,trans-costunolide-14-acetate (3) and 4-hydroxyarbusculin A (11) were the most selective against the kinetoplastid species studied. These molecules seem to induce the mechanisms involved in an apoptotic-like death or programmed cell death (PCD) in the kinetoplastids, and since they do not cause necrosis, the inflammatory events associated with this type of cell death will not be triggered.

The neglected infection known as Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, results in more than 7000 deaths per year, with an increasing number of cases in non-endemic areas such as Europe or the United States. Moreover, with the current available therapy, only two compounds which are active against the acute phase of the disease are readily available. In addition, these therapeutic agents display multiple undesired side effects such as high toxicity, they are expensive, the treatment is lengthy and the resistant strain has emerged. Therefore, there is a need to find new compounds against Chagas disease which should be active against the parasite but also cause low toxicity to the patients. In the present work, the activity of novel acrylonitriles against Trypanosoma cruzi was evaluated as well as the analysis of the physiological events induced in the treated parasites related to the cell death process. Hence, the characteristic features of an apoptosis-like process such as chromatin condensation and mitochondrial membrane potential, among others, were studied. From the 32 compounds tested against the epimastigote stage of T. cruzi, 11 were selected based on their selectivity index to determine if these compounds were able to induce programmed cell death (PCD) in the treated parasites. Furthermore, acrylonitriles Q5, Q7, Q19, Q27 and Q29 were shown to trigger physiological events related in the PCD. Therefore, this study highlights the therapeutic potential of acrylonitriles as novel trypanocidal agents., Projects PI18/01380 FIS, Spanish Ministry of Science, Innovation and Universities, RD16/0027/0001 of the programme of Redes Temáticas de Investigación Cooperativa (RICET), FIS, Spanish Ministry of Science, Innovation and Universities; C.J.B.-E. and D.S.-H. by ACIISI, I.S by RICET, all cofounded by FEDER. Project “Iniciación a la actividad investigadora, 2019” from Universidad de La Laguna (Ministerio de Ciencia e Innovación y Universidades). This research was funded by the Spanish Ministry of Science, Innovation and Universities (MICINN), State Research Agency (AEI) and the European Regional Development Funds (ERDF) (PGC2018-094503-B-C21). S.D.-H. thanks La Laguna University and Cajasiete for a pre-doctoral contract.

Recently, the search for novel therapeutic agents against Acanthamoeba species has been focused on the evaluation of natural resources. Among them, marine microorganisms have risen as a source of bioactive compounds with the advantage of the ability to obtain unlimited and constant amounts of the compounds in contrast to other natural sources such as plants. Furthermore, marine actinomycetes have recently been reported as highly rich in bioactive agents including salinosporamides, xiamycines, indolocarbazoles, naphtyridines, phenols, dilactones such as antimycines and macrolides among others. In this study, staurosporine (STS) was isolated from a strain of Streptomyces sanyensis and tested against Acanthamoeba to characterize the therapeutic potential of STS against this protozoan parasite. We have established that STS is active against both stages of the Acanthamoeba life cycle, by the activation of Programmed Cell Death via the mitochondrial pathway of the trophozoite. We have also established that STS has relatively low toxicity towards a macrophage cell line. However, previous studies have highlighted higher toxicity levels induced on other vertebrate cell lines and future research to lower these toxicity issues should be developed.

In this work, the presence of free-living amoebae (FLA) in dishcloths collected from human activity related places was evaluated. Once in the laboratory, 6 cm2 pieces of each dishcloth were cut and washed with Page’s Amoeba Solution (PAS) in sterile tubes. After washing, the dishcloth pieces were removed, and the tubes were centrifuged (1500 rpm for 10 min). The obtained pellets were seeded onto 2% non-nutrient agar (NNA) plates, incubated at room temperature and were monitored daily an inverted microscope. Once clonal cultures were obtained (only one type of FLA observed), molecular analyses were carried out in order to characterize the isolated FLA strains at the genus/genotype level. From the 31 dishcloths which were processed, FLA strains were isolated in NNA plates in 13 the samples (13/31, 42%). However, and due to bacterial overgrowth, only six strains were characterized at the molecular level (PCR and sequencing). Among the PCR positive strains, 83.33% (5/6) of the PCR positive samples belonged to Acanthamoeba genus (80% (4/5) to genotype T4 and 20% (1/5) to genotype T11). Furthermore, one strain was identified as a member of Allovahlkampfia genus using both morphological and molecular approaches. To the best of our knowledge, this is the first report on the isolation of Allovahlkampfia genus from dishcloths and in the Spanish territory. The presence of FLA in dishcloths should raise awareness to improve hygienic strategies in food- and domestic-related environments, in order to prevent contamination with these protozoa, which are able to be pathogenic and even to act as vehicles of other pathogenic agents.

Objective: To evaluate the combination of several statins (atorvastatin, fluvastatin and simvastatin) and azoles (voriconazole, posaconazole and itraconazole) against Acanthamoeba spp. Methods: The efficiency of the different drug combinations against the trophozoite stage of different Acanthamoeba strains were evaluated by Alamar Blue assay. Effect on the cyst stage was observed by inverted microscope. Cytotoxicity of combinations of azoles and statins was evaluated by measuring the release of lactate dehydrogenase from a murine macrophage cell line. Results: Combinations of any of the tested statins and voriconazole or posaconazole were more efficient in inhibiting Acanthamoeba compared to statins or azoles individually. The drug combinations at the combined inhibitory concentrations 50% showed lower toxicity compared to that of the compounds alone. Conclusions: The combinations of statins together with voriconazole and posaconazole are more efficient than these drugs alone, and these combinations have lower cytotoxicity in mammalian cell lines.

Leishmaniasis produces approximately-one million of new cases annually, making it one of the most important tropical diseases. As current treatments are not fully effective and are toxic, it is necessary to develop new therapies that are more effective and less toxic, and cause a controlled cell death, with which we can avoid the immunological problems caused by necrosis. In this work 32 acrylonitriles were studied in vitro against Leishmania amazonensis. Three compounds Q20 (12.41), Q29 (11.2) and Q31 (11.56) had better selectivity than the reference compound, miltefosine (11.14) against promastigotes of these parasites, for this reason they were selected to determine their mechanism of action to know the cell death type of they produce. The results of the mechanisms of action show that these three acrylonitriles tested produce chromatin condensation, decreased mitochondrial membrane potential, altered plasma permeability and production of reactive oxygen species. All these characteristic events seem to indicate programmed cell death. Therefore, this study demonstrates the activity of acrylonitriles derivatives as possible leishmanicidal agents., This work was funded by PI18/01380 from the Instituto de Salud Carlos III, Spain; RICET (project RD16/0027/0001) from the Programa Redes Temáticas de Investigación Cooperativa, FIS (Ministerio Español de Salud, Madrid, Spain); CB21/13/00100 Consorcio Centro De Investigación Biomédica En Red M.P. (CIBER) de Enfermedades Infecciosas, Inst. de Salud Carlos III, Madrid, Spain and Cabildo de Tenerife, MEDI and FDCAN and Spanish Ministry of Science, Innovation and Universities (MICINN), State Research Agency (AEI) and the European Regional Development Funds (ERDF) (21/0587). Spanish Ministry of Science, Innovation and Universities (MICINN), State Research Agency (AEI), the European Regional Development Funds (ERDF) (PGC2018–094503-B-C21), C.J.B.-E. (TESIS2020010057) and D.S.N.-H. (TESIS2019010065) was funded by a grant from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información cofunded by FEDER. S.D.-H thanks La Laguna University and Ministry of Science and Innovation for a Junior Posdoctoral contract.

Leishmaniasis and Chagas are among the most significant neglected tropical diseases. Due to several drawbacks with the current chemotherapy, developing new antikinetoplastid drugs has become an urgent issue. In the present work, a bioassay-guided investigation of the root bark of Maytenus chiapensis on Leishmania amazonensis and Trypanosoma cruzi led to the identification of two D:A-friedo-nor-oleanane triterpenoids (celastroloids), 20β-hydroxy-tingenone (celastroloid 5) and 3-O-methyl-6-oxo-tingenol (celastroloid 8), as promising antikinetoplastid leads. They displayed higher potency on L. amazonensis promastigotes (50% inhibitory concentrations [IC(50)s], 0.44 and 1.12 μM, respectively), intracellular amastigotes (IC(50)s, 0.83 and 1.91 μM, respectively), and T. cruzi epimastigote stage (IC(50)s, 2.61 and 3.41 μM, respectively) than reference drugs miltefosine and benznidazole. This potency was coupled with an excellent selectivity index on murine macrophages. Mechanism of action studies, including mitochondrial membrane potential (Δψm) and ATP-level analysis, revealed that celastroloids could induce apoptotic cell death in L. amazonensis triggered by the mitochondria. In addition, the structure-activity relationship is discussed. These findings strongly underline the potential of celastroloids as lead compounds to develop novel antikinetoplastid drugs.

36 p.-6 fig.-4 tab., Pathogenic and opportunistic free-living amoebae such as Acanthamoeba spp. can cause keratitis (Acanthamoeba keratitis [AK]), which may ultimately lead to permanent visual impairment or blindness. Acanthamoeba can also cause rare but usually fatal granulomatous amoebic encephalitis (GAE). Current therapeutic options for AK require a lengthy treatment with nonspecific drugs that are often associated with adverse effects. Recent developments in the field led us to target cAMP pathways, specifically phosphodiesterase. Guided by computational tools, we targeted the Acanthamoeba phosphodiesterase RegA. Computational studies led to the construction and validation of a homology model followed by a virtual screening protocol guided by induced-fit docking and chemical scaffold analysis using our medicinal and biological chemistry (MBC) chemical library. Subsequently, 18 virtual screening hits were prioritized for further testing in vitro against Acanthamoeba castellanii, identifying amoebicidal hits containing piperidine and urea imidazole cores. Promising activities were confirmed in the resistant cyst form of the amoeba and in additional clinical Acanthamoeba strains, increasing their therapeutic potential. Mechanism-of-action studies revealed that these compounds produce apoptosis through reactive oxygen species (ROS)-mediated mitochondrial damage. These chemical families show promise for further optimization to produce effective antiacanthamoebal drugs., Funding from RICET (RD16/0027/0010) and (RD16/0027/0001), FEDER funds (PI18/01380), MECD (Grant FPU15/1465 to V. S.-P.) is acknowledged, M. R. B. was also funded by RICET and I. S. was funded by Agustin de Betancourt Programme.

The in vitro activity against Leishmania spp. of five novel designed compounds, phenalenone derivatives, is described in this study. Previous works have shown that some phenalenones present leishmanicidal activity, some of which could induce programmed cell death events in L. amazonensis parasites. In this research, we focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure and mitochondrial membrane potential, among others. The results showed that the new derivatives have comparable or better activity and selectivity than the commonly prescribed anti-leishmanial drug. This result was obtained by inducing stronger mitochondrial depolarization or more intense phosphatidylserine exposure than miltefosine, highlighting compound 8 with moreover 9-times better selectivity index. In addition, the new five molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing.

Neglected tropical diseases such as leishmaniasis and American trypanosomiasis represent an increasing health problem. Current treatments are not satisfactory which remains an urgent need for novel, cheap and safe chemotherapies. In the course of our ongoing search for new potential anti-protozoal agents, this study aimed to perform a bio-guided fractionation of Inula viscosa (Asteraceae) using in vitro assays against three strains of Leishmania and Trypanosma genus. Eight known compounds were identified from the ethanolic extract of leaves, sesquiterpenoids (3 and 4) and flavonoids (5 and 6) were characterized as the main bioactive constituents. Sesquiterpene lactones 3 and 4 (IC50 values between 4.99 and 14.26 μM) showed promising antiparasitic activity against promastigotes of L. donovani, L. amazonensis and epimastigotes of T. cruzi. Their structures were successfully characterized by spectroscopic techniques including 1D and 2D NMR experiments. Furthermore, the main bioactive compounds 4, 5 and 6 displayed higher potency (IC50 values between 0.64 and 2.13 μM) against amastigotes of L. amazonensis than miltefosine (IC50 3.11 μM), and a low toxicity on macrophages cell line (SI > 45). The analysis of structure-activity relationship (SAR) of the anti-protozoal activity revealed that lactonization or oxidation enhanced the biological profile, suggesting that the hydrophobic moiety was presumably involved in the activity by increasing the affinity and/or cell membrane permeability. In order to get an insight into the mechanism of action of these compounds, programmed cell death (PCD) experiments were performed, and the obtained results suggest that the reported compounds induced PCD in the treated parasites. These results highlight that sesquiterpenoids and flavonoids from I. viscosa could constitute an interesting scaffold for the development of novel antikinetoplastid agents.

Free living, cosmopolitan amoebae from Acanthamoeba genus present a serious risk to human health. As facultative human parasites, these amoebae may cause Acanthamoeba keratitis (AK). Acanthamoeba keratitis is a severe, vision-threatening corneal infection with non-specific symptoms. The number of reported AK cases worldwide has been increasing every year. Moreover, 90% of Acanthamoeba keratitis cases are related to contact lens use. Wearing and storage contact lenses not in accordance with the physicians and manufacturers recommendations are the primary key risk factors of this disease. Amoebae can easily adhere to the contact lens surface and transmit to the corneal epithelium. Preventing amoebae adhesion to the contact lens surface could significantly decrease the number of AK infections. Until now, the effective therapy against AK is still under development. Currently proposed therapies are mainly limited to the chlorhexidine digluconate combined with propamidine isethionate or hexamidine applications, which are insufficient and very toxic to the eye. Due to lack of effective treatment, looking for new potential preventive agents is crucial to decrease the number of Acanthamoeba keratitis infections, especially among contact lens users. Nanoparticles have been already included in several novel therapies against bacteria, viruses, fungi, and protist. However, their anti-amoebic potential has not been fully tested yet. The aim of this study was to assess silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) anti-amoebic activity and influence on the amoebae adhesion to the surface of four different groups of contact lenses&mdash, classified according to the Food and Drugs Administration (FDA) guidelines. The obtained results show that both tested nanoparticles were effective against Acanthamoeba trophozoites and decreased the amoebae adhesion to the contact lens surface. AgNPs showed better anti-amoebic activity to cytotoxicity dependence and reduced amoebae adhesion in a wider spectrum of the tested contact lenses. Our studies also confirmed that ionization next to hydration of the contact lens material is a crucial parameter influencing the Acanthamoeba adhesion to the contact lens surface. In conclusion, silver nanoparticles might be considered as a novel preventive agent against Acanthamoeba keratitis infection.

According to the World Health Organization, leishmaniasis is considered as a major neglected tropical disease causing an enormous impact on global public health. Available treatments were complicated due to the high resistance, toxicity, and high cost. Therefore, the search for novel sources of anti-leishmania agents is an urgent need. In the present study, an in vitro evaluation of the leishmanicidal activity of the essential oil of Tunisian chamomile (Matricaria recutita L.) was carried out. Chamomile essential oil exhibits a good activity on promastigotes forms of L. amazonensis and L. infantum with a low inhibitory concentration at 50% (IC50) (10.8 ± 1.4 and 10.4 ± 0.6 μg/mL, respectively). Bio-guided fractionation was developed and led to the identification of (−)-α-bisabolol as the most active molecule with low IC50 (16.0 ± 1.2 and 9.5 ± 0.1 μg/mL for L. amazonensis and L. infantum, respectively). This isolated sesquiterpene alcohol was studied for its activity on amastigotes forms (IC50 = 5.9 ± 1.2 and 4.8 ± 1.3 μg/mL, respectively) and its cytotoxicity (selectivity indexes (SI) were 5.4 and 6.6, respectively). The obtained results showed that (−)-α-bisabolol was able to activate a programmed cell death process in the promastigote stage of the parasite. It causes phosphatidylserine externalization and membrane damage. Moreover, it decreases the mitochondrial membrane potential and total ATP levels. These results highlight the potential use of (−)-α-bisabolol against both L. amazonensis and L. infantum, and further studies should be undertaken to establish it as novel leishmanicidal therapeutic agents.

Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK) which mainly affects contact lens wearers and it is commonly related to poor hygiene of contact lenses and their cases. Moreover, treatment of AK is complex due to the existence of a highly resistant cyst stage and if not diagnosed early has poor prognosis, leading to blindness and/or keratoplasty. Even though AK is increasing worldwide as well as awareness among patients and clinicians, it is still a poorly studied pathogen. Additionally, a remaining question to be answered is whether these opportunistic pathogens are present in the ocular surface of healthy contact lens wearers since they are the main group at risk.In order to carry out this study, sterile Schirmer strip tests were collected from a group of individuals all of them contact lens wearers who were attending a local ophthalmology clinic in Tenerife, Canary Islands, Spain. The collected samples (100 eyes of 50 patients) were cultured in 2% Non-Nutrient Agar (NNA) plates and positive plates (16) were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains belonged to Acanthamoeba genotype T4 and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. In conclusion, the ocular surface of contact lens wearers included in this study was colonized by potentially pathogenic strains of Acanthamoeba and should be considered as a risk for AK infection in this region and worldwide.

Screening of a designed collection of mono-substituted amino-1H-phenalen-1-ones against promastigote forms of L. donovani and L. amazonensis, identified seven compounds with anti-leishmanial activities comparable or better than the commonly prescribed anti-leishmanial drug, miltefosine. Structure-activity analysis revealed that appendages containing a basic tertiary nitrogen were favored, and that the position of the appendage also affected their potency. Like miltefosine, several of these active compounds significantly reduced the mitochondrial membrane potential in promastigotes. Further studies in amastigotes of L. amazonensis revealed that compounds 14, 15 and 33 were more active and more selective than miltefosine, with sub-micromolar potencies and selectivity indices >100.

Free-living amoebae of genus Acanthamoeba are opportunistic pathogens widely distributed in the environment, and are the causative agents of several humans’ infections, such as Acanthamoeba keratitis, Granulomatous Amoebic Encephalitis and also disseminated infections. The existence of the cyst stage complicates Acanthamoeba therapy as it is highly resistant to antibiotics and physical agents. All these facts reinforced the necessity to find and develop an effective therapy against Acanthamoeba infections. In the present study, we are interested to several seaweeds species collected from the Tunisian coasts and belonging to the 3 phyla (brown, green and red algae). The aim was to quantify the Total Phenolic Compounds in different organic extract, to evaluate antioxidant capacity (DPPH and ABTS) and to study the antiprotozoal activity against A. castellanii Neff. The parasites have been inhibited by all extracts with an IC50 ranged from 52,3±1.8 μg/mL for ethyl acetate extract, to 134,6±0.7 μg/mL for the hexanic one for the various species studied.

Free-living amoebae (FLA) are protozoa that are widely distributed in the environment mainly in water and soil related habitats. These amoebae have also been reported to be associated with some bacterial pathogens for humans such as Campylobacter spp. The species C. jejuni is the causative agent of about 90% of human campylobacteriosis cases worldwide and this disease may even end up in severe autoimmune sequelae as Guillain-Barré syndrome (GBS). In this study, the interactions between the strain Acanthamoeba castellanii Neff and Campylobacter jejuni was investigated. Campylobacter jejuni was coincubated with Acanthamoeba castellanii Neff trophozoites at different temperatures, in order to evaluate the C. jejuni ability to grow in presence A. castellanii culture and Acanthamoeba Conditioned Medium (ACM). C. jejuni was coincubated with A. castellanii axenic culture at different temperatures in aerobic conditions. Our results revealed that bacteria were still cultivable (Blood Agar medium, at 37 °C, in microaerophilic atmosphere) after a 14 days C. jejuni - A. castellanii coculture, comparing with C. jejuni alone, which was only cultivable for 24 h.

Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been reported to be associated with bacterial human pathogens such as Legionella spp. Campylobacter spp or Vibrio cholerae among others. Including within Vibrio spp. V. harveyi (Johnson and Shunk, 1936) is a bioluminescent marine bacteria which has been found swimming freely in tropical marine waters, being part of the stomach and intestine microflora of marine animals, and as both a primary and opportunistic pathogen of marine animals. Our aim was to study the interactions between Vibrio harveyi and Acanthamoeba castellanii Neff. Firstly, in order to analyze changes in it cultivability, V. harveyi was coincubated with A. castellanii Neff axenic culture and with Acanthamoeba Conditioned Medium (ACM) at different temperatures in aerobic conditions. Interestingly, at 4 °C and 18-20 °C bacteria were still cultivable in marine agar, at 28 °C, in aerobic conditions, but there weren't significant differences comparing with the controls. We also noted an enhanced migration of Acanthamoeba toward V. harveyi on non-nutrient agar plates compared to controls with no bacteria.

Acanthamoeba infections cause serious humans diseases, such as amoebic keratitis and granulomatous amoebic encephalitis. Melaleuca essential oil has been reported to be effective in treating bacterial and fungal infections. However, the anti-parasitic effects of this essential oil are not well studied. In the present study, we first characterized the composition of the essential oil, extracted from the Tunisian Melaleuca styphelioides leaves, and then tested its effect on the Acanthamoeba castellanii Neff. Gas chromatography–mass spectrometry (GC-MS) analysis revealed that the major common compounds were Caryophyllene oxide (23.42%), Spathulenol (20.5%), Isoaromadendrene epoxide (7.45%), Ledol (5.98%), α-Pinene (3.82%), Isopinocarveol (2.18%). Our data also showed that M. styphelioides essential oil inhibited the growth of Acanthamoeba with an IC50 value of 69.03 ± 9.17 μg/ml. This work suggests M. styphelioides essential oil as a potential anti amoeba drug. Nevertheless, further studies are still needed to further verify the cytotoxicity of the studied oil on human macrophages and check its applicability to treat Acanthamoeba infections in vivo.

The protozoan parasite Leishmania causes a spectrum of diseases and there are over 1 million infections each year. Current treatments are toxic, expensive, and difficult to administer, and resistance to them is emerging. In this study, we screened the antileishmanial activity of the Pathogen Box compounds from the Medicine for Malaria Venture against Leishmania amazonensis, and compared their structures and cytotoxicity. The compounds MMV676388 (3), MMV690103 (5), MMV022029 (7), MMV022478 (9) and MMV021013 (10) exerted a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and intracellular amastigotes. Moreover, studies on the mechanism of cell death showed that compounds 3 and 5 induced an apoptotic process while the compounds 7, 9 and 10 seem to induce an autophagic mechanism. The present findings underline the potential of these five molecules as novel therapeutic leishmanicidal agents.

Leishmaniasis and American trypanosomiasis are parasitic diseases that cause significant clinical, social and economic impact on the population of tropical and subtropical countries. Their current treatment is limited and presents multiple drawbacks, including high toxicity, high cost, lengthy treatment plans, as well as the emergence of resistant species. Therefore, there is a need to find new lead compounds with high potency against parasites and low toxicity in patients. In the present work, the bioguided fractionation of an endemic plant from the Canary Islands, Withania aristata, led to the identification of withanolide-type metabolites (1–3) with leishmanicidal and trypanocidal activities. Compounds 1 and 3 showed a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and T. cruzi epimastigotes, higher than the reference drugs, miltefosine and benznidazole, respectively. Moreover, compounds 1–3 were more potent (IC50 0.055–0.663 µM) than the reference drug against the intracellular amastigote stage of L. amazonensis, with a high selectivity index on murine macrophage cells (SI 58.66–216.73). Studies on the mechanism of death showed that the compounds induced programmed cell death or that which was apoptosis-like. The present findings underline the potential of withanolides as novel therapeutic antikinetoplastid agents.