'Targeting the feast of a sleeping beast': Nutrient and mineral dependencies of encysted Acanthamoeba castellanii.

Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta-oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully.

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba.

BACKGROUND: The encystation of Acanthamoeba leads to the development of resilient cysts from vegetative trophozoites. This process is essential for the survival of parasites under unfavorable conditions. Previous studies have reported that, during the encystation of A. castellanii, the expression levels of encystation-related factors are upregulated. However, the regulatory mechanisms for their expression during the encystation process remains unknown. Proteins in the sirtuin family, which consists of nicotinamide adenine dinucleotide-dependent deacetylases, are known to play an important role in various cellular functions. In the present study, we identified the Acanthamoeba silent-information regulator 2-like protein (AcSir2) and examined its role in the growth and encystation of Acanthamoeba. METHODS: We obtained the full-length sequence for AcSir2 using reverse-transcription polymerase chain reaction. In Acanthamoeba transfectants that constitutively overexpress AcSir2 protein, SIRT deacetylase activity was measured, and the intracellular localization of AcSir2 and the effects on the growth and encystation of trophozoites were examined. In addition, the sirtuin inhibitor salermide was used to determine whether these effects were caused by AcSir2 overexpression RESULTS: AcSir2 was classified as a class-IV sirtuin. AcSir2 exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation. In trophozoites, AcSir2 overexpression led to greater cell growth, and this growth was inhibited by treatment with salermide, a sirtuin inhibitor. When AcSir2 was overexpressed in the cysts, the encystation rate was significantly higher; this was also reversed with salermide treatment. In AcSir2-overexpressing encysting cells, the transcription of cellulose synthase was highly upregulated compared with that of control cells, and this upregulation was abolished with salermide treatment. Transmission electron microscope-based ultrastructural analysis of salermide-treated encysting cells showed that the structure of the exocyst wall and intercyst space was impaired and that the endocyst wall had not formed. CONCLUSIONS: These results indicate that AcSir2 is a SIRT deacetylase that plays an essential role as a regulator of a variety of cellular processes and that the regulation of AcSir2 expression is important for the growth and encystation of A. castellanii.

The most abundant cyst wall proteins of Acanthamoeba castellanii are lectins that bind cellulose and localize to distinct structures in developing and mature cyst walls.

BACKGROUND: Acanthamoeba castellanii, which causes keratitis and blindness in under-resourced countries, is an emerging pathogen worldwide, because of its association with contact lens use. The wall makes cysts resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. METHODOLOGY/PRINCIPAL FINDINGS: Transmission electron microscopy and structured illumination microscopy (SIM) showed purified cyst walls of A. castellanii retained an outer ectocyst layer, an inner endocyst layer, and conical ostioles that connect them. Mass spectrometry showed candidate cyst wall proteins were dominated by three families of lectins (named here Jonah, Luke, and Leo), which bound well to cellulose and less well to chitin. An abundant Jonah lectin, which has one choice-of-anchor A (CAA) domain, was made early during encystation and localized to the ectocyst layer of cyst walls. An abundant Luke lectin, which has two carbohydrate-binding modules (CBM49), outlined small, flat ostioles in a single-layered primordial wall and localized to the endocyst layer and ostioles of mature walls. An abundant Leo lectin, which has two unique domains with eight Cys residues each (8-Cys), localized to the endocyst layer and ostioles. The Jonah lectin and glycopolymers, to which it binds, were accessible in the ectocyst layer. In contrast, Luke and Leo lectins and the glycopolymers, to which they bind, were mostly inaccessible in the endocyst layer and ostioles. CONCLUSIONS/SIGNIFICANCE: The most abundant A. castellanii cyst wall proteins are three sets of lectins, which have carbohydrate-binding modules that are conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique to Acanthamoebae (8-Cys of Leo). Cyst wall formation is a tightly choreographed event, in which lectins and glycopolymers combine to form a mature wall with a protected endocyst layer. Because of its accessibility in the ectocyst layer, an abundant Jonah lectin is an excellent diagnostic target.

Neuroleptic Drug Targets a Brain-Eating Amoeba: Effects of Promethazine on Neurotropic Acanthamoeba castellanii.

Acanthamoeba spp. has recently been reported to express diverse group of ion channels and receptors that are expressed by human cells which bind drugs that are used in noninfectious diseases. Bioinformatics computational tools, growth assays, and 3D structural modeling have enabled the discovery of primitive muscarinic receptors, voltage-gated calcium channels, and ion transport pumps such as Na-K ATPase in this protist pathogen. The significance of the reported receptors and ion channels in the biology of Acanthamoeba is yet to be determined. We selected promethazine, which is a known antagonist of proteins like dopaminergic, histaminergic, muscarinic receptors, and calmodulin, to determine its effects on the growth and proliferation of trophozoites and cysts of Acanthamoeba spp. In order to elucidate the receptors involved in the effects produced by promethazine, we also performed individual experiments on Acanthamoeba trophozoites and cysts in the presence of the agonist of the above-mentioned receptors. Our results show that promethazine in the range of 60-100 µg/mL proved to be amoebicidal for Acanthamoeba trophozoites and at slightly higher doses ranging around 125-250 µg/mL also showed partial cysticidal effects. We also show the evidence of homology between the human targets of promethazine and similar targets in Acanthamoeba by the use of bioinformatic computational tools and 3D modeling. Promethazine and its structural analogs, because of being FDA-approved, have a wider margin of safety that can be tested as potential anti- Acanthamoeba agents in diseases like keratitis and encephalitis caused by this protist pathogen.

Evidence of human-like Ca2+ channels and effects of Ca2+ channel blockers in Acanthamoeba castellanii.

The evolution of voltage-gated calcium channel (Cav) in eukaryotes is an area of interest for biologists worldwide. The CLAN CL0030 and its family Ion_Trans 2 PF 07885 have been known to be present in prokaryotes, but the origin of these ion channels in Acanthamoeba spp. is yet to be determined. We inferred the origin of primitive forms of two-pore channels like proteins, human-like Cav 1.1 of L-type, and Cav subunit alpha-2/delta-1 in Acanthamoeba spp. early during evolution. By in-depth investigation into genomics, transcriptomics, use of bioinformatics tools and experimentations done with drugs like amlodipine and gabapentin on Acanthamoeba spp., we show the evidence of primitive forms of these channels in this protist pathogen. Genomics and transcriptomics of proteins ACA1_167020, 092610, and 270170 reflected their cellular expression in Acanthamoeba spp. We performed amino acid sequence homology, 3D structural modeling, ligand binding predictions, and dockings. Bioinformatics and 3D structural models show similarities between ACA1_167020, 092610, 270170, and different types of known human Cav. We show amoebicidal effects of amlodipine and gabapentin on Acanthamoeba spp., which can help design their structural analogs to target pathogenic genotypes of Acanthamoeba in diseases like Acanthamoeba keratitis and granulomatous amoebic encephalitis.

Molecular and Biochemical Properties of a Cysteine Protease of Acanthamoeba castellanii.

Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.

Molecular and biochemical characterization of key enzymes in the cysteine and serine metabolic pathways of Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. METHODS: The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. RESULTS: The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. CONCLUSIONS: This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.

Effect of oxidative stress on vital indicators of Acanthamoeba castellanii (T4 genotype).

Acanthamoeba has 22 genotypes with the T4 genotype being the main causative agent of amoebic granulomatous encephalitis and keratitis. Because the molecular mechanisms of the immune defenses of neutrophils and macrophages against histoparasites are based on oxidative stress, parasites may rely on their antioxidant systems to preclude immune defenses. Therefore, understanding of the effect of oxidative stress on vital characteristics of Acanthamoeba castellanii (T4 genotype) and the antioxidant defense responses of Acanthamoeba to oxidative status will cast light on immune cell-parasite interactions. Acanthamoeba T4 cells were cultured in RPMI-1640 medium containing different concentrations of hydrogen peroxide (H2O2). The survival of Acanthamoeba was evaluated by MTT assay and the IC50 concentration was calculated. The total antioxidant capacity (TAC) of the parasite was determined by the cupric reducing antioxidant capacity (CUPRAC) method. Malondialdehyde (MDA) as a marker of lipid peroxidation, protein carbonyl content as a measure of oxidized protein, total thiol (-SH) groups present on proteins as a major source of cellular antioxidants, and total oxidant status (TOS) were evaluated by colorimetric methods. The reactive oxygen species level increased markedly after induction of oxidative stress by the treatment of Acanthamoeba T4 with H2O2. Exposure to H2O2 also significantly increased the MDA and protein carbonyl content. The TOS level and total thiol groups also increased in the treated group compared to those in untreated parasites, although the results were not statistically significant. The TAC level was found to be significantly higher in H2O2-treated parasites, confirming that the parasite fosters its total antioxidant capacity to overcome oxidative conditions. This study showed that under oxidative stress, the defense reactions of the parasite are in part mediated by increasing its antioxidant activity, which is important for the survival of the parasite.

Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii.

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆8,14-sterol (<95%). The combination of [2H3-methyl]methionine incubation to intact cultures showing C28-ergosterol incorporates 2-2H atoms and C29-7-dehydroporiferasterol incorporates 5 2H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ∆24(28)- and ∆25(27)-olefin pathways to C28- and C29-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C28/C29-∆5,7-sterols, viable encysted cells (mature cyst) possess mostly C29-∆5-sterol and non-viable encysted cells possess C28/C29-∆5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ∆5,7-sterol biosynthesis (to ∆5,7) - metabolism (to ∆5 or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.

Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells.

Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.

Comparison of Proteins Secreted into Extracellular Space of Pathogenic and Non-pathogenic Acanthamoeba castellanii.

Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins , 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.

Trehalose During Two Stress Responses in Acanthamoeba: Differentiation Between Encystation and Pseudocyst Formation.

The non-reducing disaccharide trehalose can serve as a protectant against a range of environmental stressors, such as heat, cold, or dehydration, in both prokaryotes and eukaryotes, with the exception of vertebrates. Here, we analyzed trehalose metabolism in the facultatively parasitic organism Acanthamoeba castellanii, known to respond to unfavorable external conditions by forming two resistant stages: a cyst, produced in the case of chronic stress, and a pseudocyst, formed in reaction to acute stress. The possible role of trehalose in the resistant stages was investigated using a combination of bioinformatic, molecular biological and biochemical approaches. Genes for enzymes from a widespread trehalose-6-synthase-trehalose-6-phosphate phosphatase (TPS-TPP) pathway and a prokaryotic trehalose synthase (TreS) pathway were identified. The expression patterns of the genes during encystation and pseudocyst formation were analyzed and correlated with the time course of cellular trehalose content determined mass spectrometrically. The data clearly demonstrate fundamental differences between encystation and pseudocyst formation at the level of cellular metabolism.

Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis.

Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus.IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimivirus and provide new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating, and morphogenesis. Furthermore, we demonstrate that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, allow us to present an ultimate model for the mimivirus replication cycle.

An observational study of phagocytes and Klebsiella pneumoniae relationships: different behaviors.

Klebsiella pneumoniae is a bacterium that can be in relation with free living amoebae like Acanthamoeba castellanii in natural environments such as soil and water. This pathogen, which is responsible for community-acquired pneumonia and for nosocomial infections, also has interactions with host defense mechanisms like macrophages. As it has been shown that A. castellanii shares some traits with macrophages, in particular the ability to phagocyte bacteria, we have studied the uptake and the fate of the bacteria after contact with the two phagocytic cells. In our conditions, K. pneumoniae growth was increased in coculture in presence of A. castellanii or Thp-1 macrophagic cells and bacterial development was also increased by A. castellanii supernatant. In addition, we showed that the presence of the bacteria had a negative effect on the macrophages whereas it does not affect amoeba viability. Using gentamicin, which kills bacteria outside cells, we showed that only macrophages were able to internalize K. pneumoniae. This result was confirmed by electron microscopy. We have consequently reported some differences in bacterial uptake and internalization between a free living amoeba and macrophagic cells, highlighting the fact that results obtained with this amoebal model should not be extrapolated to the relationships between K. pneumoniae and macrophages.

Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway.

Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections.

Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51).

In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 µM), whereas inhibition by fluconazole was weak (IC50, 30 µM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.

Acanthamoeba castellanii Proteases are Capable of Degrading Iron-Binding Proteins as a Possible Mechanism of Pathogenicity.

Acanthamoeba castellanii, a free-living amoeba, is an amphizoic organism that can behave as an opportunistic pathogen, causing granulomatous amoebic encephalitis in immunocompromised patients or infecting immunocompetent individuals via cutaneous lesions, sinusoidal infections, or amoebic keratitis. Therefore, this amoeba could be in contact with different iron-binding proteins, such as lactoferrin in tears and mucosa and transferrin and hemoglobin in blood. Iron is a vital and necessary element for host metabolism but also for parasite survival. Accordingly, parasites have developed iron uptake mechanisms, one of which is the utilization of proteases to degrade host iron-binding proteins. In this work, we performed a partial biochemical characterization of A. castellanii proteases at different pHs and utilizing protease inhibitors with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copolymerized with different iron-binding proteins. We describe for the first time the presence of several cysteine proteases in a total A. castellanii crude extract and in conditioned culture medium precipitated with ethanol. These amoebic peptidases degraded human holo-lactoferrin, holo-transferrin, hemoglobin, and horse spleen ferritin; some of these proteases were substrate specific, and others degraded multiple substrates. These proteases could be considered virulence factors that promote iron acquisition from the host.

A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel ß-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.

Identification of unusual phospholipid fatty acyl compositions of Acanthamoeba castellanii.

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms.

Down-regulation of cellulose synthase inhibits the formation of endocysts in Acanthamoeba.

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.