Characterization of the SUF FeS cluster synthesis machinery in the amitochondriate eukaryote Monocercomonoides exilis.

Monocercomonoides exilis is the first known amitochondriate eukaryote. Loss of mitochondria in M. exilis ocurred after the replacement of the essential mitochondrial iron-sulfur cluster (ISC) assembly machinery by a unique, bacteria-derived, cytosolic SUF system. It has been hypothesized that the MeSuf pathway, in cooperation with proteins of the cytosolic iron-sulfur protein assembly (CIA) system, is responsible for the biogenesis of FeS clusters in M. exilis, yet biochemical evidence is pending. Here, we address the M. exilis MeSuf system and show that SUF genes, individually or in tandem, support the loading of iron-sulfur (FeS) clusters into the reporter protein IscR in Escherichia coli. The Suf proteins MeSufB, MeSufC, and MeSufDSU interact in vivo with one another and with Suf proteins of E. coli. In vitro, the M. exilis Suf proteins form large complexes of varying composition and hence may function as a dynamic biosynthetic system in the protist. The putative FeS cluster scaffold MeSufB-MeSufC (MeSufBC) forms multiple oligomeric complexes, some of which bind FeS clusters and form selectively only in the presence of adenosine nucleotides. The multi-domain fusion protein MeSufDSU binds a PLP cofactor and can form higher-order complexes with MeSufB and MeSufC. Our work demonstrates the biochemical property of M. exilis Suf proteins to act as a functional FeS cluster assembly system and provides insights into the molecular mechanism of this unique eukaryotic SUF system.

On the host specificity and genetic diversity of Iodamoeba bütschlii: Observations from short amplicon-based next-generation sequencing.

Iodamoeba is a single-celled intestinal parasite, which is common in humans in certain parts of the world, and also in pigs. For the first time, we provide DNA-based evidence of goat, dromedary, fallow deer, and donkey as hosts of Iodamoeba and show that Iodamoeba-specific nucleotide sequences from these four hosts do not appear to overlap with those of humans, unlike those from pigs. We moreover show that similar strains of Iodamoeba can be found in Madagascar, Western Sahara, and Ecuador and that intra-sample diversity is typically extensive across even small fragments of DNA in both human and non-human hosts.

New geographical records and molecular investigation of the ciliate Spirostomum.

Spirostomum is a genus of large ciliates, and its species are distributed worldwide. However, there has been limited research conducted on their geographical distribution and genomics. We obtained nine samples of ciliates from eight regions in Liaoning Province, China, and conducted a study on their geographical distribution and characteristics. Morphological and second-generation high-throughput sequencing methods were applied to identify the species, and a phylogenetic tree was established to gain a deeper understanding of the geographical distribution and evolutionary relationships of Spirostomum in Northeast China. The results identified Spirostomum yagiui and Spirostomum subtilis as a newly recorded species in Northeast China region. There are now five species of Spirostomum that have been recorded in China, and new details on the genomic characteristics of Spirostomum yagiui were provided. In addition, this study also identified the main branches of Spirostomum teres and Spirostomum minus in northern China, and provided a theoretical basis for the existence of hidden species. Spirostomum yagiui is the first species in the family Spirostomidae to have undergone mitochondrial genome sequencing.

A gene-rich mitochondrion with a unique ancestral protein transport system.

Mitochondria originated from an ancient endosymbiosis involving an alphaproteobacterium.1,2,3 Over time, these organelles reduced their gene content massively, with most genes being transferred to the host nucleus before the last eukaryotic common ancestor (LECA).4 This process has yielded varying gene compositions in modern mitogenomes, including the complete loss of this organellar genome in some extreme cases.5,6,7,8,9,10,11,12,13,14 At the other end of the spectrum, jakobids harbor the most gene-rich mitogenomes, encoding 60-66 proteins.8 Here, we introduce the mitogenome of Mantamonas sphyraenae, a protist from the deep-branching CRuMs supergroup.15,16 Remarkably, it boasts the most gene-rich mitogenome outside of jakobids, by housing 91 genes, including 62 protein-coding ones. These include rare homologs of the four subunits of the bacterial-type cytochrome c maturation system I (CcmA, CcmB, CcmC, and CcmF) alongside a unique ribosomal protein S6. During the early evolution of mitochondria, gene transfer from the proto-mitochondrial endosymbiont to the nucleus became possible thanks to systems facilitating the transport of proteins synthesized in the host cytoplasm back to the mitochondrion. In addition to the universally found eukaryotic protein import systems, jakobid mitogenomes were reported to uniquely encode the SecY transmembrane protein of the Sec general secretory pathway, whose evolutionary origin was however unclear. The Mantamonas mitogenome not only encodes SecY but also SecA, SecE, and SecG, making it the sole eukaryote known to house a complete mitochondrial Sec translocation system. Furthermore, our phylogenetic and comparative genomic analyses provide compelling evidence for the alphaproteobacterial origin of this system, establishing its presence in LECA.

Molecular basis of phenotypic plasticity in a marine ciliate.

Phenotypic plasticity, which involves phenotypic transformation in the absence of genetic change, may serve as a strategy for organisms to survive in complex and highly-fluctuating environments. However, its reaction norm, molecular basis, and evolution remain unclear in most organisms, especially microbial eukaryotes. In this study, we explored these questions by investigating the reaction norm, regulation, and evolution of phenotypic plasticity in the cosmopolitan marine free-living ciliates Glauconema spp., which undergo significant phenotypic changes in response to food shortages. This study led to the de novo assembly of macronuclear genomes using long-read sequencing, identified hundreds of differentially expressed genes associated with phenotypic plasticity in different life stages, validated the function of two of these genes, and revealed that the reaction norm of body shape in response to food density follows a power-law distribution. Purifying selection may be the dominant evolutionary force acting on the genes associated with phenotypic plasticity, and the overall data support the hypothesis that phenotypic plasticity is a trait maintained by natural selection. This study provides novel insight into the developmental genetics of phenotypic plasticity in non-model unicellular eukaryotes, and sheds light on the complexity and long evolutionary history of this important survival strategy.

Sodium Metabisulfite Inhibits Acanthamoeba Trophozoite Growth through Thiamine Depletion.

Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine.

Telomere-to-telomere Genome Assembly of the Clubroot Pathogen Plasmodiophora Brassicae.

Plasmodiophora brassicae (Woronin, 1877), a biotrophic, obligate parasite, is the causal agent of clubroot disease in brassicas. The clubroot pathogen has been reported in more than 80 countries worldwide, causing economic losses of hundreds of millions every year. Despite its widespread impact, very little is known about the molecular strategies it employs to induce the characteristic clubs in the roots of susceptible hosts during infection, nor about the mechanisms it uses to overcome genetic resistance. Here, we provide the first telomere-to-telomere complete genome of P. brassicae. We generated ∼27 Gb of Illumina, Oxford Nanopore, and PacBio HiFi data from resting spores of strain Pb3A and produced a 25.3 Mb assembly comprising 20 chromosomes, with an N50 of 1.37 Mb. The BUSCO score, the highest reported for any member of the group Rhizaria (Eukaryota: 88.2%), highlights the limitations within the Eukaryota database for members of this lineage. Using available transcriptomic data and protein evidence, we annotated the Pb3A genome, identifying 10,521 protein-coding gene models. This high-quality, complete genome of P. brassicae will serve as a crucial resource for the plant pathology community to advance the much-needed understanding of the evolution of the clubroot pathogen.

Measurements of soil protist richness and community composition are influenced by primer pair, annealing temperature, and bioinformatics choices.

Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic options that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and protist taxon richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition using metabarcoding.IMPORTANCEEcosystem functioning is driven by the activity and interactions of the microbial community, in both aquatic and terrestrial environments. Protists are a group of highly diverse, mostly unicellular microbes whose identity and roles in terrestrial ecosystem ecology have been largely ignored until recently. This study highlights the importance of choices researchers make, such as primer pair, on the results and conclusions about protist diversity and community composition in soils. In order to better understand the roles protist taxa play in terrestrial ecosystems, biases in methodological and analytical choices should be understood and acknowledged.

Giardia intestinalis reshapes mucosal immunity toward a Type 2 response that attenuates inflammatory bowel-like diseases.

Diarrheal diseases are the second leading cause of death in children worldwide. Epidemiological studies show that co-infection with Giardia intestinalis decreases the severity of diarrhea. Here, we show that Giardia is highly prevalent in the stools of asymptomatic school-aged children. It orchestrates a Th2 mucosal immune response, characterized by increased antigen-specific Th2 cells, IL-25, Type 2-associated cytokines, and goblet cell hyperplasia. Giardia infection expanded IL-10-producing Th2 and GATA3+ Treg cells that promoted chronic carriage, parasite transmission, and conferred protection against Toxoplasma gondii-induced lethal ileitis and DSS-driven colitis by downregulating proinflammatory cytokines, decreasing Th1/Th17 cell frequency, and preventing collateral tissue damage. Protection was dependent on STAT6 signaling, as Giardia-infected STAT6-/- mice no longer regulated intestinal bystander inflammation. Our findings demonstrate that Giardia infection reshapes mucosal immunity toward a Type 2 response, which confers a mutualistic protection against inflammatory disease processes and identifies a critical role for protists in regulating mucosal defenses.

Phenotypic plasticity and the effects of thermal fluctuations on specialists and generalists.

Classical theories predict that relatively constant environments should generally favour specialists, while fluctuating environments should be selected for generalists. However, theoretical and empirical results have pointed out that generalist organisms might, on the contrary, perform poorly under fluctuations. In particular, if generalism is underlaid by phenotypic plasticity, performance of generalists should be modulated by the temporal characteristics of environmental fluctuations. Here, we used experiments in microcosms of Tetrahymena thermophila ciliates and a mathematical model to test whether the period or autocorrelation of thermal fluctuations mediate links between the level of generalism and the performance of organisms under fluctuations. In the experiment, thermal fluctuations consistently impeded performance compared with constant conditions. However, the intensity of this effect depended on the level of generalism: while the more specialist strains performed better under fast or negatively autocorrelated fluctuations, plastic generalists performed better under slow or positively autocorrelated fluctuations. Our model suggests that these effects of fluctuations on organisms' performance may result from a time delay in the expression of plasticity, restricting its benefits to slow enough fluctuations. This study points out the need to further investigate the temporal dynamics of phenotypic plasticity to better predict its fitness consequences under environmental fluctuations.

Eukaryotic genomic data uncover an extensive host range of mirusviruses.

A recent marine metagenomic study has revealed the existence of a novel group of viruses designated mirusviruses, which are proposed to form an evolutionary link between two realms of double-stranded DNA viruses, Varidnaviria and Duplodnaviria. Metagenomic data suggest that mirusviruses infect microeukaryotes in the photic layer of the ocean, but their host range remains largely unknown. In this study, we investigated the presence of mirusvirus marker genes in 1,901 publicly available eukaryotic genome assemblies, mainly derived from unicellular eukaryotes, to identify potential hosts of mirusviruses. Mirusvirus marker sequences were identified in 915 assemblies spanning 227 genera across eight supergroups of eukaryotes. The habitats of the putative mirusvirus hosts included not only marine but also other diverse environments. Among the major capsid protein (MCP) signals in the genome assemblies, we identified 85 sequences that showed high sequence and structural similarities to reference mirusvirus MCPs. A phylogenetic analysis of these sequences revealed their distant evolutionary relationships with the seven previously reported mirusvirus clades. Most of the scaffolds with these MCP sequences encoded multiple mirusvirus homologs, suggesting that mirusviral infection contributes to the alteration of the host genome. We also identified three circular mirusviral genomes within the genomic data of the oil-producing thraustochytrid Schizochytrium sp. and the endolithic green alga Ostreobium quekettii. Overall, mirusviruses probably infect a wide spectrum of eukaryotes and are more diverse than previously reported.

Facultative symbiont virulence determines horizontal transmission rate without host specificity in Dictyostelium discoideum social amoebas.

In facultative symbioses, only a fraction of hosts are associated with symbionts. Specific host and symbiont pairings may be the result of host-symbiont coevolution driven by reciprocal selection or priority effects pertaining to which potential symbiont is associated with a host first. Distinguishing between these possibilities is important for understanding the evolutionary forces that affect facultative symbioses. We used the social amoeba, Dictyostelium discoideum, and its symbiont, Paraburkholderia bonniea, to determine whether ongoing coevolution affects which host-symbiont strain pairs naturally cooccur within a facultative symbiosis. Relative to other Paraburkholderia, including another symbiont of D. discoideum, P. bonniea features a reduced genome size that indicates a significant history of coevolution with its host. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness for naturally cooccurring (native) host and symbiont pairings compared to novel pairings. We show for the first time that P. bonniea symbionts can horizontally transmit to new amoeba hosts when hosts aggregate together during the social stage of their life cycle. Here we find evidence for a virulence-transmission trade-off without host specificity. Although symbiont strains were significantly variable in virulence and horizontal transmission rate, hosts and symbionts responded similarly to associations in native and novel pairings. We go on to identify candidate virulence factors in the genomes of P. bonniea strains that may contribute to variation in virulence. We conclude that ongoing coevolution is unlikely for D. discoideum and P. bonniea. The system instead appears to represent a stable facultative symbiosis in which naturally cooccurring P. bonniea host and symbiont pairings are the result of priority effects.

Surface currents shape protist community structure across the Indo-Pacific.

Biogeographic structure in marine protist communities is shaped by a combination of dispersal potential and environmental selection. High-throughput sequencing and global sampling efforts have helped better resolve the composition and functions of these communities in the world's oceans using both molecular and visual methods. However, molecular barcoding data are critically lacking across the Indo-Pacific, a region widely considered the epicenter of marine biodiversity. To fill this gap, we characterized protist communities in four sampling regions across Indonesia that represent the latitudinal, longitudinal, and human population gradients of the region: Lombok, Wakatobi, Misool, and Waigeo. We show high spatial structuring in marine protist communities across Indonesia, and biotic factors appear to play little role in driving this observed structure. Our results appear to be driven by abiotic factors linked to surface current patterns across the Indo-Pacific as a result of: (1) a choke point in circulation at the Indonesian Throughflow leading to low diatom diversity in Lombok, Wakatobi, and Misool; (2) an increase in nutrient availability at the edge of the Halmahera Eddy in Waigeo, leading to an increase in diatom diversity; and/or (3) seasonal variations in protist communities in line with shifts in velocity of the Indonesian Throughflow. Overall, our results highlight the importance of abiotic factors in shaping protist communities on broad geographic scales over biotic, top-down pressures, such as grazing from higher trophic levels.

Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila.

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Species-specific predation determines the feeding impacts of six soil protist species on bacterial and eukaryotic prey.

Predatory protists play a central role in nutrient cycling and are involved in other ecosystem functions by predating the microbiome. While most soil predatory protist species arguably are bacterivorous, some protist species can prey on eukaryotes. However, studies about soil protist feeding mainly focused on bacteria as prey and rarely tested both bacteria and eukaryotes as potential prey. In this study, we aimed to decipher soil predator-prey interactions of three amoebozoan and three heterolobosean soil protists and potential bacterial (Escherichia coli; 0.5-1.5 µm), fungal (Saccharomyces cerevisiae; 5-7 µm) and protist (Plasmodiophora brassicae; 3-5 µm) prey, either as individual prey or in all their combinations. We related protist performance (relative abundance) and prey consumption (qPCR) to the protist phylogenetic group and volume. We showed that for the six soil protist predators, the most suitable prey was E. coli, but some species also grew on P. brassicae or S. cerevisiae. While protist relative abundances and growth rates depended on prey type in a protist species-specific manner, phylogenetic groups and volume affected prey consumption. Yet we conclude that protist feeding patterns are mainly species-specific and that some known bacterivores might be more generalist than expected, even preying on eukaryotic plant pathogens such as P. brassicae.

Microbial Community Response to Granular Peroxide-Based Algaecide Treatment of a Cyanobacterial Harmful Algal Bloom in Lake Okeechobee, Florida (USA).

Cyanobacterial harmful algal blooms (cyanoHABs) occur in fresh water globally. These can degrade water quality and produce toxins, resulting in ecological and economic damages. Thus, short-term management methods (i.e., algaecides) are necessary to rapidly mitigate the negative impacts of cyanoHABs. In this study, we assess the efficacy of a hydrogen peroxide-based algaecide (PAK® 27) on a Microcystis dominated bloom which occurred within the Pahokee Marina on Lake Okeechobee, Florida, USA. We observed a significant reduction in chlorophyll a (96.81%), phycocyanin (93.17%), and Microcystis cell counts (99.92%), and a substantial reduction in microcystins (86.7%) 48 h after treatment (HAT). Additionally, there was a significant shift in bacterial community structure 48 HAT, which coincided with an increase in the relative abundance of photosynthetic protists. These results indicate that hydrogen peroxide-based algaecides are an effective treatment method for cyanoHAB control and highlight their effects on non-target microorganisms (i.e., bacteria and protists).

Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania.

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

The Plasmodiophora brassicae Golgi-localized UPF0016 protein PbGDT1 mediates calcium but not manganese transport in yeast and Nicotiana benthamiana.

Manganese and calcium homeostasis and signalling, in eukaryotic organisms, are regulated through membrane located pumps, channels and exchangers, including the Mn2+/Ca2+ uncharacterized protein family 0016 (UPF0016). Here we show that Plasmodiophora brassicae PbGDT1 is a member of the UPF0016 and an ortholog of Saccharomyces cerevisiae Gdt1p (GCR Dependent Translation Factor 1) protein involved in manganese homeostasis as well as the calcium mediated stress response in yeast. PbGDT1 complemented the ScGdt1p and ScPMR1 (Ca2+ ATPase) double null mutant under elevated calcium stress but not under elevated manganese conditions. In both yeast and Nicotiana benthamiana, PbGDT1 localizes to the Golgi apparatus, with additional ER association in N. benthamiana. Expression of PbGDT1 in N. benthamiana, suppresses BAX-triggered cell death, further highlighting the importance of calcium homeostasis in maintaining cell physiology and integrity in a stress environment.

Molecular phylogenetic analyses support the validity of Ceratiomyxa porioides (Amoebozoa, Eumycetozoa) at species level.

The frequently encountered macroscopic slime molds of the genus Ceratiomyxa have long been recognized by mycologists and protistologists for hundreds of years. These organisms are amoebozoan amoebae that live and grow inside and on the surface of decaying wood. When conditions are favorable, they form subaerial sporulating structures called fruiting bodies which take on a variety of forms. These forms are typically some arrangement of column and/or branches, but one is uniquely poroid, forming folds instead. Originally, this poroid morphology was designated as its own species. However, it was not always clear what significance fruiting body morphology held in determining species. Currently, Ceratiomyxa fruticulosa var. porioides, the poroid form, is considered a taxonomic variety of Ceratiomyxa fruticulosa based on morphological designation alone. Despite its long history of observation and study, the genus Ceratiomyxa has been paid little molecular attention to alleviate these morphological issues. We have obtained the first transcriptomes of the taxon C. fruticulosa var. porioides and found single gene phylogenetic and multigene phylogenomic support to separate it from C. fruticulosa. This provides molecular evidence that fruiting body morphology does correspond to species level diversity. Therefore, we formally raise Ceratiomyxa porioides to species level.

Metabolic responses of Euglena gracilis under photoheterotrophic and heterotrophic conditions.

The protist Euglena gracilis has various trophic modes including heterotrophy and photoheterotrophy. To investigate how cultivation mode influences metabolic regulation, the chemical composition of cellular metabolites of Euglena gracilis grown under heterotrophic and photoheterotrophic conditions was monitored from the early exponential phase to the mid-stationary phase using two different techniques, i.e, nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). The combined metabolomics approach allowed an in-depth understanding of the mechanism of photoheterotrophic and heterotrophic growth for biomolecule production. Heterotrophic conditions promoted the production of polar amino and oxygenated compounds such as proteins and polyphenol compounds, especially at the end of the exponential phase while photoheterotrophic cells enhanced the production of organoheterocyclic compounds, carbohydrates, and alkaloids.