Free-living Amoebae Research Articles

"Yaraviridae": a proposed new family of viruses infecting Acanthamoeba castellanii.

Here, we propose the creation of the family "Yaraviridae", a new taxon to classify a virus infecting Acanthamoeba castellanii cells. Recently, we described the discovery of a new virus infecting free-living amoebae, yaravirus, which has features that strongly differ from those of all other viruses of amoebae described to date. Yaravirus particles are about 80 nm in diameter and have a dsDNA genome of ~45 kbp containing 74 ORFs, most of which (>90%) have no homologs in current databases. Together, these data support the creation of a new species ("Yaravirus brasiliense"), a new viral genus (here proposed as "Yaravirus"), and a new viral family (here proposed as "Yaraviridae") to classify yaravirus and other related viruses that may be described in the future. All of them are to be included into the existing realm Varidnaviria and the kingdom Bamfordvirae, due to the presence of a major capsid protein containing a double jelly-roll fold.

The Legionella Lqs-LvbR Regulatory Network Controls Temperature-Dependent Growth Onset and Bacterial Cell Density.

Legionella species are facultative intracellular pathogens that cause a life-threatening pneumonia termed Legionnaires' disease. Legionella pneumophila employs the Lqs-LvbR (Legionella quorum sensing-Legionella virulence and biofilm regulator) network to regulate virulence and motility, but its role for growth in media is ill-defined. Here, we report that compared to the L. pneumophila reference strain JR32, a ΔlqsR mutant showed a reduced lag phase at 30°C and reached a higher cell density at 45°C, while the ΔlqsA, ΔlqsS, and ΔlqsT mutants showed a longer lag phase and reached a lower cell density. A ΔlvbR mutant resumed growth like the parental strain at 30°C but exhibited a substantially reduced cell density at 45°C. Thus, LvbR is an important cell density regulator at elevated temperatures. Environmental and clinical L. pneumophila strains grew in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) medium after distinct lag phases with similar rates at 30°C, reached different cell densities at the optimal growth temperature of 40°C, and no longer grew at 50°C. Legionella longbeachae reached a rather low cell density at 40°C and did not grow at and beyond 45°C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and upon growth at 30°C or 45°C, the PlqsR, PlqsA, PlqsS, and PlvbR promoters from strain JR32 were expressed in these strains with distinct patterns. Taken together, our results indicate that the Lqs-LvbR network governs the temperature-dependent growth onset and cell density of the L. pneumophila reference strain JR32 and possibly also of environmental and clinical L. pneumophila isolates. IMPORTANCE Environmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires' disease, the incidence of which is on the rise worldwide. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a mechanism similar to that in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical, or biological means will reduce the incidence of Legionnaires' disease. Here, we show that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is determined not only by the temperature and nutrient availability but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation.

A history of over 40 years of potentially pathogenic free-living amoeba studies in Brazil - a systematic review.

Free-living amoeba (FLA) group includes the potentially pathogenic genera Acanthamoeba, Naegleria, Balamuthia, Sappinia, and Vermamoeba, causative agents of human infections (encephalitis, keratitis, and disseminated diseases). In Brazil, the first report on pathogenic FLA was published in the 70s and showed meningoencephalitis caused by Naegleria spp. FLA studies are emerging, but no literature review is available to investigate this trend in Brazil critically. Thus, the present work aims to integrate and discuss these data. Scopus, PubMed, and Web of Science were searched, retrieving studies from 1974 to 2020. The screening process resulted in 178 papers, which were clustered into core and auxiliary classes and sorted into five categories: wet-bench studies, dry-bench studies, clinical reports, environmental identifications, and literature reviews. The papers dating from the last ten years account for 75% (134/178) of the total publications, indicating the FLA topic has gained Brazilian interest. Moreover, 81% (144/178) address Acanthamoeba-related matter, revealing this genus as the most prevalent in all categories. Brazil’s Southeast, South, and Midwest geographic regions accounted for 96% (171/178) of the publications studied in the present work. To the best of our knowledge, this review is the pioneer in summarising the FLA research history in Brazil.

Can Free Living Acanthamoeba Act as a Trojan Horse for SARS-Cov-2 on Viral Survival and Transmission in the Environment? A Narrative Review.

Acanthamoeba is a free-living amoeba that has been found on surfaces, air, water and various environmental sources around the world. It enters the human body through the respiratory tract via the nose. Of note, amoebae are well known to act as a reservoir for various pathogenic microorganisms including bacteria and viruses such as Adenoviruses and Mimivirus. Given that SARS-CoV-2 of the Coronaviridae family is transmitted through the respiratory tract, and the Trojan horse nature of Acanthamoeba, it has been suggested that amoebae act as a vector in the transmission of SARS-CoV-2. The aim of this study was to provide an opinion of the possibility of the coexistence of Acanthamoeba with SARS-CoV-2.

Isolation and Identification of Free Living Amoeba from Patients and Contact Lens Users in Iran.

Background:Free-living amoebae (FLA) such as Acanthamoeba spp., are considered as opportunistic and pathogenic protozoans. Acanthamoeba granulomatous encephalitis (AGE) is a serious threat for immunodeficient patients and Acanthamoeba keratitis (AK) for contact lens users. We aimed to identify the presence of free living amoebae in nasal swabs of patients and contact lens users in Qazvin, Iran.Methods:During 2019, 251 nasal and oral swabs (including the pharynx and mouth) were collected from patients with diabetes, AIDS and those under periodic dialysis in Qazvin, Iran. In addition, 27 soft contact lenses were collected from the participants. Following DNA extraction, PCR and sequencing were conducted to identify the genotypes of the amoeba. Phylogenetic analysis of the identified sequences was performed using MEGA 7 software.Results:A strain of Acanthamoeba belonging to the T3 genotype was isolated from hemodialysis patients. Two specimens of Acanthamoeba with T3 genotype were isolated from keratitis patients.Conclusion:The clinicians should pay attention to the possible complication of this organism because this amoeba is potentially pathogenic for immunocompromised patients. Since the amoeba is present in environmental resources, the use of contact lenses should be accompanied by considering proper hygiene.

Assessment of the Impact of Treatment and Distribution on the Occurrence of Nontuberculous Mycobacteria and Free-Living Amoebae in Full-Scale Drinking Water Systems

Assessment of the Impact of Treatment and Distribution on the Occurrence of Nontuberculous Mycobacteria and Free-Living Amoebae in Full-Scale Drinking Water Systems

Polypyrrole-conjugated zinc oxide nanoparticle as antiamoebic drugs against Acanthamoeba castellanii

The presence of free-living amoebae that cause infections to the central nervous system, and cornea is one of the concerning health issues. Acanthamoeba spp. a genus of free-living amoebae can cause rare but deadly granulomatous amoebic encephalitis and blinding Acanthamoeba keratitis. Due to the limited and mostly ineffective treatment options against Acanthamoeba infections, new treatment modalities must be developed, such as use of nanotechnology for targeted therapeutics. Herein, this study was devised to test the amoebicidal effects of polymer conjugated with metal oxide nanoparticles against a clinical isolate of A. castellanii (T4). These nanoconjugates were synthesized and characterized by Field Emission Scanning Electron Microscope and Fourier Transform Infrared Spectroscopy. The amoebicidal assay was conducted to study the results of antiacanthamoebic action of Polypyrrole (PPy)-Zinc Oxide (ZnO). The results of antiacanthamoebic evaluation demonstrated that PPy-ZnO inhibited the viability of A. castellanii. Therefore, PPy-ZnO can be a potential drug candidate for the treatment of Acanthamoeba induced infections.

Isolation and identification of free-living amoeba from contact lenses: Thermal and osmotic tolerance in relation to their pathogenicity.

Isolation and identification of free-living amoeba from contact lenses: Thermal and osmotic tolerance in relation to their pathogenicity.

Exploring the protist microbiome: The diversity of bacterial communities associated with Arcella spp. (Tubulina: Amoebozoa)

Research on protist-bacteria interactions is increasingly relevant as these associations are now known to play important roles in ecosystem and human health. Free-living amoebae are abundant in all environments and are frequent hosts for bacterial endosymbionts including pathogenic bacteria. However, to date, only a small fraction of these symbionts have been identified, while the structure and composition of the total symbiotic bacterial communities still remains largely unknown. Here, we use the testate amoeba Arcella spp. as model organisms to investigate the specificity and diversity of Arcella-associated microbial communities. High-throughputamplicon sequencing from the V4 region of the 16S rRNA gene revealed high diversity in the bacterial communities associated with the wild Arcella spp. To investigate the specificity of the associated bacterial community with greater precision, we investigated the bacterial communities of two lab-cultured Arcella species, A. hemispherica and A. intermedia, grown in two different media types. Our results suggest that Arcella-bacteria associations are species-specific, and that the associated bacterial community of lab-cultured Arcella spp. remains distinct from that of the surrounding media. Further, each host Arcella species could be distinguished based on its bacterial composition. Our findings provide insight into the understanding of eukaryotic-bacterial symbiosis.

Histological Changes in the Kidneys and Heart in Experimental Acanthamoebiasis in Immunocompetent and Immunosuppressed Hosts

The course of Acanthamoeba spp. infection depends on the age and immune status of the host, and the virulence of the Acanthamoeba spp. strain. Some strains of free-living amoebae exhibit organ specificity, during the course of infection, while others may cause changes in many organs or completely lose pathogenicity. The aim of the current study was to investigate the pathological properties of Acanthamoeba spp. isolated from a patient with acute myeloid leukemia and atypical pneumonia (AM22). Moreover, the objective was to investigate the histopathological changes in the kidneys and heart of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. Amoebae were re-isolated from both the kidneys and hearts of the inoculated mice, although no cysts or trophozoites of the amoebae were detected in microscopic slides of the fragments of these organs. Acanthamoeba spp. induced changes in the kidney and heart weight of infected mice. In immunocompetent and immunosuppressed Acanthamoeba spp. infected mice, we found some histopathological changes, including areas with less acidic cytoplasm and a relaxation of muscle fibers. In further studies, it is important to analyze changes in gene and protein expressions in the heart and kidneys of hosts with disseminated acanthamoebiasis to better understand the course of infection in these organs, because the results of histological analysis varied depending on the immune status and duration of infection.

Genotyping and phylogenetic analysis of free-living amoeba (Acanthamoeba and Naegleria) in treated and untreated water in the northeastern provinces of Iran

Abstract Free-living amoebae (FLA) are widely distributed protozoa in natural or man-made aquatic environments without the need for a host organism for survival. Several strains of FLA are known to be pathogenic. As of date, there is inadequate data on the geographical distribution of FLA in northeastern and northern Iran. This study aimed to investigate the prevalence and genotype distribution of Acanthamoeba and Naegleria in drinking water and surface water samples in northern and northeastern Iran. A total of 60 water samples were collected and filtered from various sources for the presence of amoebae. DNA extraction was performed, and PCR confirmed the presence of FLA. PCR products were sequenced to identify the species/genotype. Phylogenetic relationships and taxonomic status constructed using MEGA X software. The findings on growth media showed 35% (21/60) and 26% (16/60) were positive for Acanthamoeba and Naegleria, respectively, while PCR analysis also obtained similar results. All isolates of Acanthamoeba were identified as T4 genotype. Poor water quality, as well as insufficient preservation and treatment, might indicate that chlorine disinfection is ineffective in removing contamination of amoebas in treated water samples. Therefore, regular water quality monitoring is essential to control amoeba's growth, reducing the risk of human infections with FLA.

Survey and first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea, Turkey.

Infection with Acanthamoeba spp. may result in granulomatous amoebic encephalitis and Acanthamoeba keratitis. Water is an important habitat where Acanthamoeba species thrive. Therefore, studying the occurrence of this free-living amoeba in water sources will help understand the infection dynamics. The aim of the study was to survey and report on the presence of Acanthamoeba spp. in water resources from the Ordu and Giresun provinces in Black Sea. Acanthamoeba spp. was found in 1/17 natural spring water samples from Ordu and in 2/18 from Giresun. Acanthamoeba species were not detected in any of the investigated tap water samples. Sequencing of the (SSU) rDNA gene resulted in the identification of haplotype I (Acanthamoeba genotype: KJ094684). T4 (8.6%) was the only isolated genotype in both Ordu and Giresun provinces. This is the first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea. The occurrence of Acanthamoeba species in natural spring water sources should be considered as a potential risk for human infection, especially to high-risk populations.

Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing

Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires’ disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing.

What Is the Weight of a Single Amoeba and Why Does It Matter?

Cell size is an important variable in the study of cellular growth, metabolism, and the cell cycle. The large size of Amoeba proteus and the ease with which it can be collected and cultured have made it a star in biology education—and it was a model for research on cell biology before the introduction of molecular genetic methods. Measuring the cytoplasmic density of a single amoeba without modern instrumentation seems like a difficult task, but this was done with supreme accuracy in the 1940s. The solution was based on the familiar Cartesian diver that is used to demonstrate Archimedes’s principle. It required the fabrication of a tiny diver that would respond to the additional mass of a cell. Experiments using this method allowed investigators to study changes in size and density associated with feeding, starvation, and cell division. This research is an illustration of the ingenuity of cell biologists in the pre-molecular genetic era of their field, which is often overlooked by contemporary scientists. The consideration of the mass, density, and buoyancy of free-living amoebas encourages a new hypothesis about the evolution of testate amoebas.

Co-Existence of Free-Living Amoebae and Potential Human Pathogenic Bacteria Isolated from Rural Household Water Storage Containers.

Simple SummaryIn many households in rural communities, water needed for drinking and cooking is fetched from rivers, fountains, or boreholes shared by the community. The water is then stored in various storage containers for several days without treatment and exposed to several conditions that could potentially contaminate the water and cause diseases. If the storage containers are not regularly and properly cleaned, biofilms can form inside the containers. Several microorganisms can be found inside the biofilm that can potentially cause diseases in humans. One such group of organisms is called free-living amoebae, which graze on the bacteria found inside the biofilm. Several of these potentially harmful bacteria have adapted and can survive inside these free-living amoebae and potentially cause diseases when ingested by humans.This study investigated the co-existence of potential human pathogenic bacteria and free-living amoebae in samples collected from stored water in rural households in South Africa using borehole water as a primary water source. Over a period of 5 months, a total of 398 stored water and 392 biofilm samples were collected and assessed. Free-living amoebae were identified microscopically in 92.0% of the water samples and 89.8% of the biofilm samples. A further molecular identification using 18S rRNA sequencing identified Vermamoeba vermiformis, Entamoeba spp., Stenamoeba spp., Flamella spp., and Acanthamoeba spp. including Acanthamoeba genotype T4, which is known to be potentially harmful to humans. Targeted potential pathogenic bacteria were isolated from the water samples using standard culture methods and identified using 16S rRNA sequencing. Mycobacterium spp., Pseudomonas spp., Enterobacter spp., and other emerging opportunistic pathogens such as Stenotrophomonas maltophilia were identified. The results showed the importance of further studies to assess the health risk of free-living amoebae and potential human pathogenic bacteria to people living in rural communities who have no other option than to store water in their homes due to water shortages.

Contact lenses contamination by Acanthamoeba spp. in Upper Egypt.

Acanthamoeba spp. are one of the free-living amoeba that spread worldwide causing keratitis. Owing to the increase in the use of lenses, whether for medical or cosmetic purposes, the incidence of disease increases every year. Contamination of the lenses with the Acanthamoeba trophozoites or cysts may lead to eye infection and cause sight-threatening keratitis in human. We isolated Acanthamoeba spp. from new lenses, used lenses, and contact lens disinfecting solutions and identified them based on morphological characteristics and molecular test. New and used lenses and contact lens disinfecting solutions were cultured on monogenic media. Light and scanning electron microscope was used to identify Acanthamoeba spp. morphological features. Genotype identification was also evaluated using PCR sequencing of 18S rRNA gene specific primer pair JDP1 and JDP2. A hundred samples were examined, 29 (29%) were infected with Acanthamoeba spp. That belonged to two strains of Acanthamoeba (Acanthamoeba 41 and Acanthamoeba 68). 18S rRNA of the Acanthamoeba 41 had 99.69% sequence identity to Acanthamoeba castellanii clone HDU-JUMS-2, whereas Acanthamoeba 68 had 99.74% similar pattern to that of Acanthamoeba sp. isolate T4 clone ac2t4 that are morphologically identified as Acanthamoeba polyphaga. The obtained data revealed that the isolated strains belong to T4 genotype that was evolutionarily similar to strains isolated in Iran. Cosmetic lenses and disinfectant solutions are a major transmissible mode for infection. This genotype is common as the cause of Acanthamoeba keratitis. To avoid infection, care must be taken to clean the lenses and their preservative solutions and prevent contamination with the parasite.

Legionella effector LegA15/AnkH contains an unrecognized cysteine protease-like domain and displays structural similarity to LegA3/AnkD, but differs in host cell localization.

Legionella pneumophila is a human pathogen that causes Legionnaires' disease, a severe form of pneumonia. It can be found in various aquatic environments ranging from cooling towers to ponds. In addition to causing disease in humans, it can also infect free-living amoebae commonly found in various aquatic environments. Once inside a human lung macrophage, it creates a niche called the Legionella-containing vacuole where it can evade phagolysosomal degradation and replicate. During infection, normal cellular functions are hijacked by proteins that are secreted by the pathogen, called bacterial effectors. Here, the structural characterization of the effector LegA15/AnkD is reported. The protein contains an ankyrin-repeat domain followed by a cysteine protease-like (CPL) domain with a putative catalytic triad consisting of His268-Asn290-Cys361. The CPL domain shows similarity to the CE clan in the MEROPS database, which contains ubiquitin-like hydrolases. The C-terminal segment of LegA15, including the CPL domain, shows structural similarity to another effector, LegA3/AnkH, while they share only 12% sequence identity. When expressed in mammalian cells, LegA15 is localized within the cytoplasm, in contrast to LegA3, which localizes to the nucleus.

The transcriptome of Balamuthia mandrillaris trophozoites for structure-guided drug design

Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a ‘brain-eating’ disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.

Trogocytosis in Unicellular Eukaryotes.

Trogocytosis is a mode of internalization of a part of a live cell by nibbling and is mechanistically distinct from phagocytosis, which implies internalization of a whole cell or a particle. Trogocytosis has been demonstrated in a broad range of cell types in multicellular organisms and is also known to be involved in a plethora of functions. In immune cells, trogocytosis is involved in the “cross-dressing” between antigen presenting cells and T cells, and is thus considered to mediate intercellular communication. On the other hand, trogocytosis has also been reported in a variety of unicellular organisms including the protistan (protozoan) parasite Entamoeba histolytica. E. histolytica ingests human T cell line by trogocytosis and acquires complement resistance and cross-dresses major histocompatibility complex (MHC) class I on the cell surface. Furthermore, trogocytosis and trogocytosis-like phenomena (nibbling of a live cell, not previously described as trogocytosis) have also been reported in other parasitic protists such as Trichomonas, Plasmodium, Toxoplasma, and free-living amoebae. Thus, trogocytosis is conserved in diverse eukaryotic supergroups as a means of intercellular communication. It is depicting the universality of trogocytosis among eukaryotes. In this review, we summarize our current understanding of trogocytosis in unicellular organisms, including the history of its discovery, taxonomical distribution, roles, and molecular mechanisms.

A survey of Free-Living Amoebae (FLAs) in Nasal Mucosa of Healthy Individuals using Culture-based Method

A survey of Free-Living Amoebae (FLAs) in Nasal Mucosa of Healthy Individuals using Culture-based Method