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Acanthamoeba castellanii is a widespread Free-Living Amoeba (FLA) that can cause severe ocular or cerebral infections in immunocompetent and immunocompromised patients, respectively, besides its capacity to transport diverse pathogens. During their life cycle, FLA can alternate between a vegetative form, called a trophozoite, and a latent and resistant form, called a cyst. This resistant form is characterized by the presence of a cell wall containing two layers, namely the ectocyst and the endocyst, mainly composed of cellulose and proteins. In the present work, we aimed to stimulate Acanthamoeba castellanii excystment by treating their cysts with a cellulolytic enzyme, i.e., cellulase, or two proteolytic enzymes, i.e., collagenase and pepsin. While 11 days were necessary to obtain total excystment in the control at 27°C, only 48 h were sufficient at the same temperature to obtain 100% trophozoites in the presence of 25 U/mL cellulase, 50 U/mL collagenase or 100 U/mL pepsin. Additionally, more than 96% amoebae have excysted after only 24 h with 7.5 U/mL cellulase at 30°C. Nevertheless, no effect of the three enzymes was observed on the excystment of Balamuthia mandrillaris and Vermamoeba vermiformis. Surprisingly, A. castellanii trophozoites excysted in the presence of cellulase displayed a markedly shorter doubling time at 7 h, in comparison to the control at 23 h. Likewise, trophozoites doubled their population in 9 h when both cellulose and cellulase were added to the medium, indicating that Acanthamoeba cyst wall degradation products promote their trophozoite proliferation. The analysis of cysts in epifluorescent microscopy using FITC-lectins and in electron microscopy revealed a disorganized endocyst and a reduction of the intercystic space area after cellulase treatment, implying that these cellular events are preliminary to trophozoite release during excystment. Further studies would be necessary to determine the signaling pathways involved during this amoebal differentiation process to identify new therapeutic targets for the development of anti-acanthamoebal drugs.

International audience; Clotrimazole (CTZ) and cyclodextrin (CD) inclusion complexes having improved apparent water solubility were obtained from phase solubility diagrams. β-CD (1.5% w/w) and hydroxypropyl-β-CD (40% w/w) offered poor CTZ solubility enhancements (12 and 384 times, respectively). Unexpectedly, the apparent solubility of CTZ was 9980 times increased from 0.4 µg.mL(-1) (1.42 μM) without CD to 4.89 mg.mL(-1) (14.9 mM) using randomly-methylated β-CD (Me-β-CD) (40% w/w). This is the highest apparent CTZ solubility improvement ever reported in the literature using conventional CDs. Quantitative nuclear magnetic resonance ((1) H-NMR) coupled with two-dimensional nuclear Overhauser effect (NOESY) experiments and molecular docking calculations showed that the highest interactions with Me-β-CD were reported for CTZ two phenyl groups. A lower interaction was reported for chlorophenyl, while imidazole had the weakest interaction with Me-β-CD. Copyright © 2015 John Wiley & Sons, Ltd.

The 24-alkylated sterols have been shown previously to be absent in membranes of amphotericin B (AmB)-resistant Leishmania donovani promastigotes, suggesting that the S- adenosyl-l-methionine:C-24-Delta-sterol-methyltransferase (SCMT or ERG6) was not functional or not expressed in AmB-resistant (AmB-R) parasites. From an L. donovani wild-type clone, we cloned two cDNAs with an identical open reading frame encoding a putative SCMT, the enzyme responsible for a first sterol methylation at the C-24 position. The two cDNAs differed by their 3'-untranslated region (3'-UTR) and 5'-UTR sequences. One transcript (A) had a normal structure with a spliced leader and was highly expressed in normal cells but absent in AmB-R cells. The other (B), which did not possess the spliced leader sequence, was weakly expressed in normal cells but strongly expressed in AmB-R cells. As a functional test, ERG6 null mutant Saccharomyces cerevisiae yeasts were transformed using the pYES2.1 TOPO TA expression vector containing the candidate SCMT1/ERG6 coding sequence cloned from L. donovani. The transformed yeasts exhibited C-24 alkylated sterol expression, mainly ergosterol, within their membranes, proving that the isolated cDNA encodes on a SCMT responsible for sterol methylation. In AmB-R L. donovani promastigotes, the absence of the normal transcript (A) and the expression of an abnormal species (B) devoid of a spliced leader could explain the absence of sterol methylation in these cells. Further studies using a homologous system will allow us to draw conclusions about the relationship between SCMT expression and AmB resistance in Leishmania.

18 p.-6 fig.-1tab., This work aims to evaluate whether nanoassemblies (NanoSb) made from antimony(V) complexes with octanoyl-N-methylglucamide (SbL8) or decanoyl-N-methylglucamide (SbL10) would effectively target the infection sites in visceral and cutaneous leishmaniases (VL and CL). NanoSb were investigated regarding stability at different pHs, accumulation of Sb in the macrophage host cell and liver, and in vitro and in vivo activities in models of leishmaniasis. The kinetic stability assay showed that NanoSb are stable at neutral pH, but release incorporated lipophilic substance after conformational change in media that mimic the gastric fluid and the parasitophorous vacuole. NanoSb promoted greater accumulation of Sb in macrophages and in the liver of mice after parenteral administration, when compared to conventional antimonial Glucantime®. SbL10 was much more active than Glucantime® against intramacrophage Leishmania amastigotes and less cytotoxic than SbL8 against macrophages. The in vitro SbL10 activity was further enhanced with co-incorporated miltefosine. NanoSb showed high antileishmanial activity in the L. donovani murine VL after parenteral administration and moderate activity in the L. amazonensis murine CL after topical treatment. This study supports the ability of NanoSb to effectively deliver a combination of Sb and co-incorporated drug to host cell and infected tissues, in a better way than Glucantime® does., This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), grant numbers 425332/2018-7, 306198/2021-5 and 401390/2020-9, Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil), grant number PPM-00372-17 and RED-00202-22, Brazilian Synchrotron Light Laboratory/MCT, grant number SAXS1-14258, Agence Nationale de la Recherche (France), grant number ANR-11-IDEX-0003-02, Région Ile-de-France (DIM1Health) and COST Action number CA21111. L.R. is supported by Ministerio de Ciencia e Innovación, Subdirección General de Redes y Centros del Investigación Cooperativa-FEDER, grant number RD16/0027/0010/AEI/10.13039/501100011033. F.F. was recipient of fellowships from CNPq, “Chaire Jean d’Alembert” (IdEx Université Paris-Saclay) and “Chaire d’Excellence DIM1Health” (Région Ile-de-France). A.C.B.O., G.S.R. and V.M.C.V. were recipient of a studentship from CNPq.