CCA-Addition Gone Wild: Unusual Occurrence and Phylogeny of Four Different tRNA Nucleotidyltransferases in Acanthamoeba castellanii.

tRNAs are important players in the protein synthesis machinery, where they act as adapter molecules for translating the mRNA codons into the corresponding amino acid sequence. In a series of highly conserved maturation steps, the primary transcripts are converted into mature tRNAs. In the amoebozoan Acanthamoeba castellanii, a highly unusual evolution of some of these processing steps was identified that are based on unconventional RNA polymerase activities. In this context, we investigated the synthesis of the 3'-terminal CCA-end that is added posttranscriptionally by a specialized polymerase, the tRNA nucleotidyltransferase (CCA-adding enzyme). The majority of eukaryotic organisms carry only a single gene for a CCA-adding enzyme that acts on both the cytosolic and the mitochondrial tRNA pool. In a bioinformatic analysis of the genome of this organism, we identified a surprising multitude of genes for enzymes that contain the active site signature of eukaryotic/eubacterial tRNA nucleotidyltransferases. In vitro activity analyses of these enzymes revealed that two proteins represent bona fide CCA-adding enzymes, one of them carrying an N-terminal sequence corresponding to a putative mitochondrial target signal. The other enzymes have restricted activities and represent CC- and A-adding enzymes, respectively. The A-adding enzyme is of particular interest, as its sequence is closely related to corresponding enzymes from Proteobacteria, indicating a horizontal gene transfer. Interestingly, this unusual diversity of nucleotidyltransferase genes is not restricted to Acanthamoeba castellanii but is also present in other members of the Acanthamoeba genus, indicating an ancient evolutionary trait.

Differentially Expressed Gene Profile of Acanthamoeba castellanii Induced by an Endosymbiont Legionella pneumophila.

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term 'integral component of membrane' were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

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.

Life without post-transcriptional addition of G-1: two alternatives for tRNAHis identity in Eukarya.

The identity of tRNA(His) is strongly associated with the presence of an additional 5'-guanosine residue (G-1) in all three domains of life. The critical nature of the G-1 residue is underscored by the fact that two entirely distinct mechanisms for its acquisition are observed, with cotranscriptional incorporation observed in Bacteria, while post-transcriptional addition of G-1 occurs in Eukarya. Here, through our investigation of eukaryotes that lack obvious homologs of the post-transcriptional G-1-addition enzyme Thg1, we identify alternative pathways to tRNA(His) identity that controvert these well-established rules. We demonstrate that Trypanosoma brucei, like Acanthamoeba castellanii, lacks the G-1 identity element on tRNA(His) and utilizes a noncanonical G-1-independent histidyl-tRNA synthetase (HisRS). Purified HisRS enzymes from A. castellanii and T. brucei exhibit a mechanism of tRNA(His) recognition that is distinct from canonical G-1-dependent synthetases. Moreover, noncanonical HisRS enzymes genetically complement the loss of THG1 in Saccharomyces cerevisiae, demonstrating the biological relevance of the G-1-independent aminoacylation activity. In contrast, in Caenorhabditis elegans, which is another Thg1-independent eukaryote, the G-1 residue is maintained, but here its acquisition is noncanonical. In this case, the G-1 is encoded and apparently retained after 5' end processing, which has so far only been observed in Bacteria and organelles. Collectively, these observations unearth a widespread and previously unappreciated diversity in eukaryotic tRNA(His) identity mechanisms.

Pathogenic Acanthamoeba castellanii Secretes the Extracellular Aminopeptidase M20/M25/M40 Family Protein to Target Cells for Phagocytosis by Disruption.

Acanthamoeba is free-living protist pathogen capable of causing a blinding keratitis and granulomatous encephalitis. However, the mechanisms of Acanthamoeba pathogenesis are still not clear. Here, our results show that cells co-cultured with pathogenic Acanthamoeba would be spherical and floated, even without contacting the protists. Then, the Acanthamoeba protists would contact and engulf these cells. In order to clarify the contact-independent pathogenesis mechanism in Acanthamoeba, we collected the Acanthamoeba-secreted proteins (Asp) to incubate with cells for identifying the extracellular virulent factors and investigating the cytotoxicity process. The Asps of pathogenic Acanthamoeba express protease activity to reactive Leu amino acid in ECM and induce cell-losing adhesion ability. The M20/M25/M40 superfamily aminopeptidase protein (ACA1_264610), an aminopeptidase be found in Asp, is upregulated after Acanthamoeba and C6 cell co-culturing for 6 h. Pre-treating the Asp with leucine aminopeptidase inhibitor and the specific antibodies of Acanthamoeba M20/M25/M40 superfamily aminopeptidase could reduce the cell damage during Asp and cell co-incubation. These results suggest an important functional role of the Acanthamoeba secreted extracellular aminopeptidases in the Acanthamoeba pathogenesis process. This study provides information regarding clinically pathogenic isolates to target specific molecules and design combined drugs.

DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation.

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

Identification and Characterization of Protein Arginine Methyltransferase 1 in Acanthamoeba castellanii.

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.

Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba.

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.

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.

Cytotoxic activity and degradation patterns of structural proteins by corneal isolates of Acanthamoeba spp.

BACKGROUND: Proteolytic enzymes secreted by trophozoites (amoebic secretome) are suggested as the main virulence factor involved in the severity of Acanthamoeba keratitis. The degradation profile of the main glycoprotein components of anterior and posterior portions of the cornea and the cytopathic effect of secretomes on endothelial cells by contact-independent mechanism were evaluated. METHODS: Trophozoites were isolated primarily from corneal tissue samples (n = 11) and extracellular proteins were collected from axenic cell culture supernatants. The molecular weights of proteolytic enzymes were estimated by zymography. Enzymatic cleavage of laminin and fibronectin substrates by amoebic secretome was investigated and cluster analysis was applied to the proteolysis profiles. Primary cultures of endothelial cells were used in both qualitative and quantitative assays of cytophatogenicity. RESULTS: Differential patterns of proteolysis were observed among the Acanthamoeba secretomes that were analysed. The uniformity of laminin degradation contrasted with the diversity of the proteolysis profiles observed in the fibronectin substrate. Acanthamoeba secretome extracted from four clinical isolates was shown to be toxic when in contact with the endothelial cell monolayer (p < 0.01). Induction of apoptosis and membrane permeability, at different percentual values, were suggested as the main mechanisms that could induce endothelial cell death when in contact with amoebic secretome. CONCLUSIONS: Our results provide evidence that virulence factors secreted by Acanthamoeba trophozoites can be related to an increased pathogenicity pattern by an independent contact-trophozoite mechanism, through induction of endothelial cell death by apoptosis at a higher percentage than providing the lack of cell viability by the membrane-associated pore-forming toxin activity.

Juglone induces cell death of Acanthamoeba through increased production of reactive oxygen species.

Juglone (5-hydroxy-1,4-naphthoquinone) is a major chemical constituent of Juglans mandshruica Maxim. Recent studies have demonstrated that juglone exhibits anti-cancer, anti-bacterial, anti-viral, and anti-parasitic properties. However, its effect against Acanthamoeba has not been defined yet. The aim of this study was to investigate the effect of juglone on Acanthamoeba. We demonstrate that juglone significantly inhibits the growth of Acanthamoeba castellanii at 3-5 µM concentrations. Juglone increased the production of reactive oxygen species (ROS) and caused cell death of A. castellanii. Inhibition of ROS by antioxidant N-acetyl-l-cysteine (NAC) restored the cell viability. Furthermore, our results show that juglone increased the uptake of mitochondrial specific dye. Collectively, these results indicate that ROS played a significant role in the juglone-induced cell death of Acanthamoeba.

Absence of a universal element for tRNAHis identity in Acanthamoeba castellanii.

The additional G(-1) nucleotide on tRNA(His) is a nearly universal feature that specifies tRNA(His) identity in all three domains of life. In eukaryotes, the G(-1) identity element is obtained by a post-transcriptional pathway, through the unusual 3'-5' polymerase activity of the highly conserved tRNA(His) guanylyltransferase (Thg1) enzyme, and no examples of eukaryotic histidyl-tRNAs that lack this essential element have been identified. Here we report that the eukaryote Acanthamoeba castellanii lacks the G(-1) identity element on its tRNA(His), consistent with the lack of a gene encoding a bona fide Thg1 ortholog in the A. castellanii genome. Moreover, the cytosolic histidyl-tRNA synthetase in A. castellanii exhibits an unusual tRNA substrate specificity, efficiently aminoacylating tRNA(His) regardless of the presence of G(-1). A. castellanii does contain two Thg1-related genes (encoding Thg1-like proteins, TLPs), but the biochemical properties we associate here with these proteins are consistent with a function for these TLPs in separate pathways unrelated to tRNA(His) metabolism, such as mitochondrial tRNA repair during 5'-editing.

Partial characterization of Acanthamoeba castellanii (T4 genotype) DNase activity.

The deoxyribonuclease (DNase) activities of Acanthamoeba castellanii belonging to the T4 genotype were investigated. Using zymographic assays, the DNase activities had approximate molecular masses of 25 and 35 kDa. A. castellanii DNases exhibited activity at wide-ranging temperature of up to 60 °C and at pH ranging from 4 to 9. The DNases activities were unaffected by proteinase-K treatment, divalent cations such as Ca(++), Cu(++), Mg(++), and Zn(++), or divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) or sodium dodecyl sulfate (SDS). The non-reliance on divalent cations and homology data suggests that A. castellanii DNases belong to the class of eukaryotic lysosomal DNase II but exhibit robust properties. The DNases activity in A. castellanii interfered with the genomic DNA extraction. Extraction methods involving EDTA, SDS, and proteinase-K resulted in low yield of genomic DNA. On the other hand, these methods resulted in high yield of genomic DNA from human cells suggesting the robust nature of A. castellanii DNases that are unaffected by reagents normally used in blocking eukaryotic DNases. In contrast, the use of chaotropic agent such as guanidine thiocyanate improved the yield of genomic DNA from A. castellanii cells significantly. Further purification and characterization of Acanthamoeba DNases is needed to study their non-classic distinct properties and to determine their role in the biology, cellular differentiation, cell cycle progression, and arrest of Acanthamoeba.

Molecular cloning and expression of phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthetic pathway from Acanthamoeba castellanii.

Free-living amoebae of the genus Acanthamoeba are widespread protozoans that can cause serious infectious diseases. This study characterised phosphoglycerate dehydrogenase (PGDH) and phosphoserine aminotransferase (PSAT) in the phosphorylated serine biosynthetic pathway of Acanthamoeba castellanii. The PGDH gene encodes a protein of 442 amino acids with a calculated molecular weight of 47.7 kDa and an isoelectric point (pI) of 7.64. Meanwhile, the PSAT gene encodes a protein of 394 amino acids with a calculated molecular weight of 43.8 kDa and a pI of 5.80. Confocal microscopy suggests that PGDH is mainly diffused in the cytoplasm, whereas PSAT is located in the inner part of the cell membrane. The messenger RNA (mRNA) expression levels of PGDH and PSAT vary depending on growth state under consecutive culture conditions. No significant changes in the mRNA expression levels of both PGDH and PSAT occur after the incubation of L-serine with Acanthamoeba. This result indicates that exogenous serine exerts no influence on the expression of these genes and that the so-called feedback inhibition of both PGDH and PSAT in Acanthamoeba differs from that in bacteria or other organisms. We propose that the enzymes in the phosphorylated serine biosynthetic pathway function in amoeba growth and proliferation.

Kinetic characterization of the ATPase and actin-activated ATPase activities of Acanthamoeba castellanii myosin-2.

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Functional expression of the Acanthamoeba castellanii alternative oxidase in Escherichia coli; regulation of the activity and evidence for Acaox gene function.

To evidence Acanthamoeba castellanii alternative oxidase (AcAOX) gene product function, we studied alterations in the levels of mRNA and protein and AcAOX activity during growth in amoeba batch culture. Moreover, heterologous expression of AcAOX in AOX-deficient Escherichia coli confirmed by the protein immunodetection and functional studies was performed. Despite the presence of native bo and bd quinol oxidases in E. coli membrane, from which the latter is known to be cyanide-resistant, functional expression of AcAOX in E. coli conferred cyanide-resistant benzohydroxamate-sensitive respiration on the bacteria. Moreover, AcAOX activity in transformed bacteria was stimulated by GMP and inhibited by ATP, indicating that AcAOX is regulated by mutual exclusion of purine nucleotides, which was previously demonstrated in the mitochondria of A. castellanii.

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.

The ecto-3'-nucleotidase activity of Acanthamoeba castellanii trophozoites increases their adhesion to host cells through the generation of extracellular adenosine.

Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3'-nucleotidase/nuclease (3'-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3'-AMP, and this activity is similar to that observed in other protists. The addition of 3'-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3'-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3'-AMP. We believe that ecto-3'-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3'-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.

Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.