The genome of Naegleria gruberi illuminates early eukaryotic versatility.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

Architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

First evidence of free-living Naegleria species in recreational lakes of Alberta, Canada.

Rising temperatures are increasing environmental habitats for thermotolerant pathogens, such as the so-called 'brain-eating amoeba', Naegleria fowleri. To the best of our knowledge, however, Naegleria species have not been reported in environmental water sources in Canada. We surveyed popular recreational lakes in Alberta, Canada during the summer bathing period to determine the presence or absence of Naegleria species. While N. fowleri was not isolated in this study, we identified other thermotolerant species, including Naegleria pagei, Naegleria gruberi, Naegleria jejuensis and Naegleria fultoni using culture-based methods, hence indicating the potential conditions to support N. fowleri. Ongoing monitoring and examination of water for pathogenic amoebae is recommended in order to assist in the public health management of water sources.

First report of free-living amoebae in watercourses in southern Brazil: molecular diagnosis and phylogenetic analysis of Vermamoeba vermiformis, Naegleria gruberi, and Acanthamoeba spp.

Free-living amoebae (FLA) are protozoa dispersed in different environments and are responsible for different infections caused to humans and other animals. Microorganisms such as Acanthamoeba spp., Vermamoeba sp., and Naegleria sp. are associated with diseases that affect the central nervous system, in addition to skin infections and keratitis, as occurs in the genus Acanthamoeba and with Vermamoeba vermiformis. Due to the concerns of these FLA in anthropogenic aquatic environments, this work aimed to identify these microorganisms present in waters of Porto Alegre, Brazil. One litre sample was collected in two watercourses during the summer of 2022 and inoculated onto non-nutrient agar plates containing heat-inactivated Escherichia coli. Polymerase chain reaction results indicated the presence of FLA of the genera Acanthamoeba, Vermamoeba, and Naegleria in the study areas. Genetic sequencing indicated the presence of V. vermiformis and Naegleria gruberi. These aquatic and anthropogenic environments can serve as a means of spread and contamination by FLA, which gives valuable information on public health in the city.

Naegleria australiensis isolated from a wastewater treatment station in Santiago Island, Cape Verde.

Despite the Naegleria genus being isolated from different natural environments such as water, soil, and air, not all Naegleria species are capable of causing infections in humans, and they are capable of completing their life cycle in environmental niches. However, the presence of this genus may suggest the existence of one of the highly pathogenic free-living amoeba (FLA) species: Naegleria fowleri or the brain-eating amoeba. This facultative parasitic protozoon represents a risk to public health, mainly related to domestic and agricultural waters. In this research, our main objective was to determine the existence of pathogenic protozoa in the Santa Cruz wastewater treatment plant, Santiago Island. Using 5 L of water we confirmed the presence of potentially pathogenic Naegleria australiensis, being the first report on Naegleria species in Cape Verde. This fact demonstrates the low efficiency in the treatment of wastewater and, consequently, a potential threat to public health. Nevertheless, more studies will be needed for the prevention and control of possible infections in this Macaronesian country.

Vestiges of the Bacterial Signal Recognition Particle-Based Protein Targeting in Mitochondria.

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.

Localization and functional characterization of the alternative oxidase in Naegleria.

The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome-mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so-called "brain-eating amoeba." Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free-living relatives.

Isolation and identification of potentially pathogenic free-living amoeba in dental-unit water samples.

Although the presence of free-living amoebae (FLAs) in various water sources has been reported, few studies have been surveyed on their abundance in medical-unit and dental-unit water samples. The current study aimed to identify morphological and molecular characteristics of FLA isolates in the water samples of the dental unit in Iran. A total of 232 water samples were collected from 17 dental units. Then, filtration and cultivation were conducted on a non-nutrient agar (NNA) medium. Also, polymerase chain reaction (PCR) assay and sequencing were performed by using the genus/species-specific primers plus a common primer set on positive samples. One hundred and sixty-six samples were positive for FLA by the microscopic method, whereas 114 samples were positive by the molecular method with a common primer set. Considering the PCR assay with genus/species-specific primers, 23.27% (54/232) samples were identified as Acanthamoeba spp. (belonging to T4 genotype), 36.63% (85/232) as Vermamoeba vermiformis, and 1.72% (4/232) as Vahlkampfiidae family (Naegleria lovaniensis). These results highlight a need to improve filtration systems in dental units and periodic screenings for FLA in dental-unit water.

Molecular identification and phylogenetic analysis of free-living amoeba in the water resources of Arak, Iran.

The aim of the present study was to detect free-living amoeba (FLA) in the water resources of Arak, Iran using molecular tools. A total of 154 samples were collected from different water supplies. Molecular analyses, sequencing, and phylogenetic study were conducted to confirm the species and genotypes of FLA. Fisher exact test was used to determine the significance. Of 154 water samples, 19 (12.3%) samples were tested positive for FLA. Three genotypes of Acanthamoeba including T4, subtype D, and T5 were identified among the isolates. The pathogenicity assay showed that the isolate of Acanthamoeba in drinking water was highly pathogenic. Three species of Naegleria, including N. australiensis, N. pagei, and N. gruberi were found among the samples. Six isolates of Vermamoeba were identified as V. vermiformis. Meanwhile, three other species including Vannella sp., Vahlkampfia avara, and Stenamoeba polymorpha were also recovered from the water samples. Statistical analysis showed a significant difference between the various water resources contaminated with FLA. This is the first study to reveal the presence of S. polymorpha in water sources in Iran. According to the findings of the present study, health officials should be beware of potential public health impacts of FLA in water resources.

Molecular characterization and phylogenetic analysis of FLA from different water sources in Egypt.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. In addition to their natural distribution, some species have been documented as pathogenic to humans. The main aim of the current study was the molecular identification, sequencing and phylogenetic analysis of morphologically detected FLA in water sources in El-Qalyubia, Egypt. A total of 96 water samples were collected from different water sources. Each water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive FLA were subjected to PCR, PCR products were sequenced and the obtained sequences were phylogenetically analysed. FLA were found in 41 water samples examined (42.7%). Nile water and groundwater were the sources with the highest prevalence rates (83.3 and 62.5%, respectively). Naegleria italica was first identified in Egypt from the waters of the Nile. In addition, Vahlkampfia spp. and Hartmannella spp. were also detected. However, other FLA species, including Acanthamoeba spp. and the pathogenic Naegleria fowleri, previously reported in Egypt, were not included in this study. The recent identification of these FLA in the Egyptian waters related to human populations indicates the need for more phylogenetic studies using larger sample sizes to investigate their potential threat to human health.

Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation.

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl-AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

Virtual Screening of Alkaloid and Terpenoid Inhibitors of SMT Expressed in Naegleria sp.

The pathogenic form of thermophilic Naegleria sp. i.e., Naegleria fowleri, also known as brain eating amoeba, causes primary amoebic encephalitis (PAM) with a >97% fatality rate. To date, there are no specific drugs identified to treat this disease specifically. The present antimicrobial combinatorial chemotherapy is hard on many patients, especially children. Interestingly, Naegleria fowleri has complex lipid biosynthesis pathways like other protists and also has a strong preference to utilize absorbed host lipids for generating energy. The ergosterol biosynthesis pathway provides a unique drug target opportunity, as some of the key enzymes involved in this pathway are absent in humans. Sterol 24-C Methyltransferase (SMT) is one such enzyme that is not found in humans. To select novel inhibitors for this enzyme, alkaloids and terpenoids inhibitors were screened and tested against two isozymes of SMT identified in N. gruberi (non-pathogenic) as well as its homolog found in yeast, i.e., ERG6. Five natural product derived inhibitors i.e., Cyclopamine, Chelerythrine, Berberine, Tanshinone 2A, and Catharanthine have been identified as potential drug candidates based on multiple criteria including binding affinity, ADME scores, absorption, and, most importantly, its ability to cross the blood brain barrier. This study provides multiple leads for future drug exploration against Naegleria fowleri.

Centriole assembly at a glance.

The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle - from assembly to disappearance - focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.

The extrachromosomal elements of the Naegleria genus: How little we know.

There are currently 47 characterized species in the Naegleria genus of free-living amoebae. Each amoeba has thousands of extrachromosomal elements that are closed circular structures comprised of a single ribosomal DNA (rDNA) copy and a large non-rDNA sequence. Despite the presence of putative open reading frames and introns, ribosomal RNA is the only established transcript. A single origin of DNA replication (ori) has been mapped within the non-rDNA sequence for one species (N. gruberi), a finding that strongly indicates that these episomes replicate independently of the cell's chromosomal DNA component. This article reviews that which has been published about these interesting DNA elements and by analyzing available sequence data, discusses the possibility that different phylogenetically related clusters of Naegleria species individually conserve ori structures and suggests where the rRNA promoter and termination sites may be located.

Current report on the prevalence of free-living amoebae (FLA) in natural hot springs: a systematic review.

The occurrence of potentially pathogenic free-living amoebae (FLA) in natural hot springs is considered a public health concern. FLAs are known to cause serious health outcomes to a wide spectrum of mammalian hosts. The present study aimed to provide the distribution of isolated cases of FLAs in hot springs through a systematic review process of available published articles online. Relevant studies are published between January 2010 and January 2020 involving the isolation of Naegleria spp., Acanthamoeba spp., Balamuthia spp., Sappinia spp., and Vermamoeba spp. in natural hot springs in the United States, South America, North America, Europe, Asia, and Africa. Articles were identified through a search of PubMed and Google Scholar databases. Out of 94 articles screened, a total of 20 articles are included in the study with consideration of established inclusion and exclusion criteria. The most common FLAs isolated in hot springs are Acanthamoeba spp. (134; 48.5%) and Naegleria spp. (127; 46.0%). Other FLAs isolated in hot springs include Balamuthia spp. (2; 0.7%) and Vermamoeba spp. (13; 4.7%). FLA in hot springs used for recreational and medical purposes is a potential source of infection. It is recommended that strict surveillance and maintenance of hot springs be implemented to prevent potential future infection.

Inhibition of Fatty Acid Oxidation as a New Target To Treat Primary Amoebic Meningoencephalitis.

Primary amoebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoeba Naegleria fowleri The amoeba migrates along the olfactory nerve to the brain, resulting in seizures, coma, and, eventually, death. Previous research has shown that Naegleria gruberi, a close relative of N. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several already-approved inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine, and valproic acid inhibited growth of N. gruberi We then tested these compounds on N. fowleri and found etomoxir, perhexiline, and thioridazine to be effective growth inhibitors. Hence, not only are lipids the preferred food source for N. gruberi, but also oxidation of fatty acids seems to be essential for growth of N. fowleri Inhibition of fatty acid oxidation could result in new treatment options, as thioridazine inhibits N. fowleri growth in concentrations that can be reached at the site of infection. It could also potentiate currently used therapy, as checkerboard assays revealed synergy between miltefosine and etomoxir. Animal testing should be performed to confirm the added value of these inhibitors. Although the development of new drugs and randomized controlled trials for this rare disease are nearly impossible, inhibition of fatty acid oxidation seems a promising strategy as we showed effectivity of several drugs that are or have been in use and that thus could be repurposed to treat PAM in the future.

Identification and characterisation of a cryptic Golgi complex in Naegleria gruberi.

Although the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here, we identify the Golgi complex in Naegleria, one of the first descriptions of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species of Naegleria and show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker (Ng)CopB by fluorescence in Naegleria gruberi, identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immunoelectron microscopy reveals that NgCOPB localises to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.This article has an associated First Person interview with the first author of the paper.

Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.

Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

Isolation and identification of free-living amoebae isolated from well water in Istanbul.

Free-living amoebae (FLA) are ubiquitous protozoa commonly found in water and soil environments. FLA belonging to various genera, including Acanthamoeba, Balamuthia, Naegleria, and Vermamoeba, can cause opportunistic and non-opportunistic infections in humans and animals such as keratitis or meningoencephalitis. In addition, some of them serve as hosts for a large number of pathogenic bacteria, yeasts, and viruses. The purpose of the present study was to assess the prevalence and molecular characterization of FLA in well water samples in Istanbul. Ten well water samples were collected from the taps and the presence of FLA was monitored both by the culture and polymerase chain reaction methods. FLA were isolated in 8 out of the 10 samples (80%) included in this study. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotypes T3 and T4, and Vermamoeba vermiformis in the investigated well water samples. This study reports for the first time the detection of Acanthamoeba genotype T3 in well water samples in Istanbul. The presence of potentially pathogenic amoebae in habitats related to human activities supports the relevance of FLA as a potential public health concern.

Isolation and molecular identification of free-living amoebae (Naegleria spp., Acanthamoeba spp. and Vermamoeba spp.) from mineral springs in Guilan Province, northern Iran.

Free-living amoebae (FLA) include many genera which cause serious diseases such as sight-threatening keratitis, cutaneous ulcers and fatal encephalitis. This study was conducted due to the lack of research regarding genotypes Acanthamoeba, Naegleria and Vermamoeba in mineral springs of Guilan Province in northern Iran. Twenty-five water samples were collected from mineral springs in Guilan Province. After filtration through nitrocellulose membrane, samples were cultured on non-nutrient agar plates. The morphological key of Page was used to identify free-living amoebae (FLA) using an inverted microscope. Positive cultures were analyzed by polymerase chain reaction (PCR) and genotypes based on the NCBI database. Eleven (44%) samples were positive for Acanthamoeba, Naegleria and Vermamoeba. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba castellanii (three case isolates belonged to T4 genotype), three cases of Vermamoeba vermiformis, and two cases of N. australiensis, two cases of N. pagei and one case of N. gruberi. According to our research the occurrence of Acanthamoeba, Naegleria spp. and Vermamoeba spp. in mineral springs could be hazardous for high risk persons. Regular monitoring and posting warning signs of these waters by health planners could prevent free-living amoebae mediated diseases.