Free-living Protozoa Research Articles

Extracellular Vesicles from Naegleria fowleri Induce IL-8 Response in THP-1 Macrophage.

Extracellular vesicles (EVs) released from pathogenic protozoans play crucial roles in host–parasite communication and disease pathogenesis. Naegleria fowleri is a free-living protozoan causing primary amoebic meningoencephalitis, a fatal disease in the central nervous system. This study aims to explore the roles of N. fowleri-derived EVs (Nf-EVs) in host–pathogen interactions using the THP-1 cell line as a model. The Nf-EVs were isolated from the N. fowleri trophozoite culture supernatant using sequential centrifugation and characterized by nanoparticle tracking analysis and transmission electron microscopy. The functional roles of Nf-EVs in the apoptosis and immune response induction of THP-1 monocytes and macrophages were examined by flow cytometry, quantitative PCR, and ELISA. Results showed that Nf-EVs displayed vesicles with bilayer membrane structure approximately 130–170 nm in diameter. The Nf-EVs can be internalized by macrophages and induce macrophage responses by induction of the expression of costimulatory molecules CD80, CD86, HLA-DR, and CD169 and the production of cytokine IL-8. However, Nf-EVs did not affect the apoptosis of macrophages. These findings illustrate the potential role of Nf-EVs in mediating the host immune cell activation and disease pathogenesis.

Free-living protozoa (ciliophora, excavata and amoebozoa) in two water sources for human supply in the municipality of Blumenau, SC

The knowledge of the diversity of free-living protozoa in aquatic ecosystems represents an important tool in environmental quality management. It has been widely used in studies evaluating the water quality of fresh-water sources with environmental variables. Therefore, this study aimed to verify the diversity of free-living protozoa in two water sources used as a public supply for human consumption in the municipality of Blumenau, SC, for 12 months. Water samples were collected monthly for analysis of environmental variables and for identification of organisms. In total, 39 taxa were recorded in P1 and P2, both in the readings performed on the day of collection and in the cultivation. In P1, 11 taxa were identified in the readings of the day of collection, and after reading the cultivation, 20 new taxa were recorded. P2 presented 12 taxa in the readings performed on the day of collection and 10 new taxa in the cultivations' readings. Ciliates were the most expressive group in the period, with 69.5% frequency in P1 and 66.1% in P2. The results of the environmental variables in conjunction with the survey of the free-living protozoa of P1 and P2 did not show marked differences between the points. Still, they contributed to the knowledge of the diversity of protozoa in aquatic environments.

An Extremely Streamlined Macronuclear Genome in the Free-Living Protozoan Fabrea salina.

Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there are two distinct nuclei in ciliates with separate germline and somatic cell functions. Here, we assembled a near-complete macronuclear genome of Fabrea salina, which belongs to one of the oldest clades of ciliates. Its extremely minimized genome (18.35 Mb) is the smallest among all free-living heterotrophic eukaryotes and exhibits typical streamlined genomic features, including high gene density, tiny introns, and shrinkage of gene paralogs. Gene families involved in hypersaline stress resistance, DNA replication proteins, and mitochondrial biogenesis are expanded, and the accumulation of phosphatidic acid may play an important role in resistance to high osmotic pressure. We further investigated the morphological and transcriptomic changes in the macronucleus during sexual reproduction and highlighted the potential contribution of macronuclear residuals to this process. We believe that the minimized genome generated in this study provides novel insights into the genome streamlining theory and will be an ideal model to study the evolution of eukaryotic heterotrophs.

Antiproliferation and Antiencystation Effect of Class II Histone Deacetylase Inhibitors on Acanthamoeba castellanii.

Acanthamoeba is a ubiquitous and free-living protozoan pathogen responsible for causing Acanthamoeba keratitis (AK), a severe corneal infection inflicting immense pain that can result in permanent blindness. A drug-based treatment of AK has remained arduous because Acanthamoeba trophozoites undergo encystment to become highly drug-resistant cysts upon exposure to harsh environmental conditions such as amoebicidal agents (e.g., polyhexanide, chloroquine, and chlorohexidine). As such, drugs that block the Acanthamoeba encystation process could result in a successful AK treatment. Histone deacetylase inhibitors (HDACi) have recently emerged as novel therapeutic options for treating various protozoan and parasitic diseases. Here, we investigated whether novel HDACi suppress the proliferation and encystation of Acanthamoeba. Synthetic class II HDACi FFK29 (IIa selective) and MPK576 (IIb selective) dose-dependently decreased the viability of Acanthamoeba trophozoites. While these HDACi demonstrated a negligible effect on the viability of mature cysts, Acanthamoeba encystation was significantly inhibited by these HDACi. Apoptosis was slightly increased in trophozoites after a treatment with these HDACi, whereas cysts were unaffected by the HDACi exposure. The viability of human corneal cells was not affected by HDACi concentrations up to 10 μmol/L. In conclusion, these synthetic HDACi demonstrated potent amoebicidal effects and inhibited the growth and encystation of Acanthamoeba, thus highlighting their enormous potential for further development.

Scuticociliatosis caused by Philasterides dicentrarchi.

The ciliate Philasterides dicentrarchi has been previously identified as a new agent of scuticociliatosis in marine fish. The parasite can cause high mortalities in fish reared on farms or kept in aquariums. P. dicentrarchi is usually a free-living protozoan but can become an opportunistic histophagous parasite causing rapid lethal systemic infections in cultured fish. This review provides information about the morphology and biology of the scuticociliate P. dicentrarchi, as well as information about the pathological and immunological reactions of the host in response to the infection with the parasite. The epidemiology and the control strategies of the disease are also reviewed.

Production of Oral Vaccines Based on Virus-Like Particles Pseudotyped with Protozoan-Surface Proteins.

Giardia lamblia is the only known parasite that can inhabit the harsh upper gastrointestinal tract, where most of the digestive proteases are secreted. Intestinal and free-living protozoa express surface proteins containing an extraordinarily high percentage of cysteine. These cysteine-rich variant-specific surface proteins (VSPs) form a dense coat on the entire surface of Giardia trophozoites, that coat protects the parasite inside the host intestine. VSPs not only are resistant to proteolytic digestion, extreme pH and temperatures, but also stimulate host immune responses. These properties can be used to protect as well as to increase the immunogenicity of vaccine antigens for oral administration. The incorporation of VSPs onto virus-like particles bearing viral antigens allows oral administration of these vaccines, protecting the antigens from degradation and generating robust and protective immune responses. In this chapter we describe the development of this versatile vaccine platform for the generation of safe, stable, and efficient oral vaccines, including their production and validation, as well as the characterization of immune response to oral immunization.

Investigating Bacterial and Free-Living Protozoa Diversity in Biofilms of Hot Water Pipes of Apartment Buildings in the City of Riga (Latvia)

Background: Biofilms, when formed on the surfaces of water pipes, can be responsible for a wide range of water quality and operational problems. We sought to assess the bacterial and free-living protozoa (FLP) diversity, in relation to the presence of Legionnaire's disease-causing bacteria Legionella pneumophila (L. pneumophila) in 45 biofilms of hot water distribution system pipes of apartment buildings in Riga, the capital city of Latvia.Results: 16S rRNA amplicon sequencing (metataxonomics) revealed that each biofilm contained 224 rather evenly distributed bacterial genera and that most common and most abundant were two genera, completely opposites in terms of their oxygen requirements: the obligately anaerobic Thermodesulfovibrio and the strictly aerobic Phenylobacterium. Water temperature and north-south axis (i.e., different primary water sources) displayed the most significant effect on the inter-sample variations, allowing us to re-construct three sub-networks (modules) of co-occurring genera, one involving (potentially FLP-derived) Legionella spp. Pangenome-based functional profile predictions suggested that all three may be dominated by pathways related to the development and maintenance of biofilms, including quorum sensing and nutrient transport, as well as the utilization of various energy sources, such as carbon and nitrogen. In our 18S rRNA amplicon sequencing data, potential hosts of L. pneumophila were detected in 11 out of 12 biofilm samples analyzed, however, in many cases, their relative abundance was very low (<1%). By validating our findings using culture-based methods, we detected L. pneumophila (serogroups 2, 3, 6 and 9) in nine (20%) biofilms, whereas FLP (mostly Acanthamoeba, Vahlkampfidae and Vermamoeba spp.) were present in six (~13%) biofilms. In two biofilms, L. pneumophila and its potential hosts were detected simultaneously, using culture-based methods.Conclusions: Overall, our study sheds light on the community diversity of hot water biofilms and predicts how several environmental factors, such as water temperature and source might shape it.

Evolutive History of Leishmania Genus and Differential Diagnosis of Clinical Important Species Based on a Unique Kinetoplastida Chitinase

Introduction: Leishmaniasis, a neglected infectious disease affecting humans, domestic, and wild animals, is caused by 20 from 53 Leishmania genus species and transmitted by sandflies. Leishmania genus, belonging to the Trypanosomatidae family and Kinetoplastida order, are grouped into five subgroups according to the biogeographic and evolutive history of parasites and hosts, leading to incongruences and paraphyly. The GH18 Leishmania chitinase, which is encoded by a species-specific single-copy gene, conserved in the basal groups of trypanosomatids, and absent in the genus Trypanosoma, was evaluated as a phylogenetic marker and a diagnostic target. Methods: Primers were designed to detect Leishmania in its host biological samples and obtain the chitinase sequence of species that are unavailable in public databanks. The GH18 chitinase gene and its genomic context were evaluated phylogenetically. A protocol was developed to discriminate Leishmania subgenera by adopting polymerase chain reaction (PCR) and restriction fragment length polymorphism and using in silico tools. The adopted PCR method for detecting a partial 953 bp GH18 chitinase-encoding gene represented high sensibility and specificity on DNA of isolated parasites and was used as negative controls, Trypanosoma cruzi, and DNA from Leishmania hosts. Results: Preservation of the chitinase locus in the aquatic free-living protozoan Bodo saltans disclosed a primitive common origin. Based on the comparative analysis, the amino acid sequence of GH18 trypanosomatidae chitinase demonstrated its high similarity to that of chitinase from marine prokaryotes and protozoans. Phylogenetic reconstruction based on chitinase corroborated the Supercontinent Origins Theory for Leishmania. Conclusion: The chitinase-encoding gene was effectively detected in biological samples and thus could be considered for differential molecular diagnosis among Leishmania clinical important species worldwide.

Infection of Slugs with Theronts of the Ciliate Protozoan, Tetrahymena rostrata.

Tetrahymena rostrata is a free-living ciliated protozoan and is a facultative parasite of some species of terrestrial mollusks. It is a potential biopesticide of pest slugs, such as the grey field slug, which cause considerable damage to crops. T. rostrata has several developmental forms. Homogeneous preparations of the feeding stage cells (trophonts) and excysted stage cells (theronts) were compared for their ability to infect and kill Deroceras reticulatum slugs. Theronts were more effective and remained viable and infective, even after prolonged starvation.

Kinetic Analysis of Acanthamoeba castellanii Infected with Giant Viruses Quantitatively Revealed Process of Morphological and Behavioral Changes in Host Cells.

ABSTRACTMost virus-infected cells show morphological and behavioral changes, which are called cytopathic effects. Acanthamoeba castellanii, an abundant, free-living protozoan, serves as a laboratory host for some viruses of the phylum Nucleocytoviricota—the giant viruses. Many of these viruses cause cell rounding in the later stages of infection in the host cells. Here, we show the changes that lead to cell rounding in the host cells through time-lapse microscopy and image analysis. Time-lapse movies of A. castellanii cells infected with Mimivirus shirakomae, kyotovirus, medusavirus, or Pandoravirus japonicus were generated using a phase-contrast microscope. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) and used it to analyze these time-lapse movies. Image analysis revealed that the process leading to cell rounding varies among the giant viruses; for example, M. shirakomae infection did not cause changes for some time after the infection, kyotovirus infection caused an early decrease in the number of cells with typical morphologies, and medusavirus and P. japonicus infection frequently led to the formation of intercellular bridges and rotational behavior of host cells. These results suggest that in the case of giant viruses, the putative reactions of host cells against infection and the putative strategies of virus spread are diverse.IMPORTANCE Quantitative analysis of the infection process is important for a better understanding of viral infection strategies and virus-host interactions. Here, an image analysis of the phase-contrast time-lapse movies displayed quantitative differences in the process of cytopathic effects due to the four giant viruses in Acanthamoeba castellanii, which were previously unclear. It was revealed that medusavirus and Pandoravirus japonicus infection led to the formation of a significant number of elongated particles related to intercellular bridges, emphasizing the importance of research on the interaction of viruses with host cell nuclear function. Mimivirus shirakomae infection did not cause any changes in the host cells initially, so it is thought that the infected cells can actively move and spread over a wider area, emphasizing the importance of observation in a wider area and analysis of infection efficiency. These results suggest that a kinetic analysis using the phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) reveals the infection strategies of each giant virus.

Serological Study of Infection with the Chlamydia - Like Microorganism 'Simkania Negevensis' in Patients Undergoing Hemodialysis

Background: Infection is a common complication of hemodialytic treatment. Many of these infections are due to sepsis, primarily arising from the vascular access site. Simkania negevensis (S. negevensis) is an obligate intracellular bacterium belonging to the family Simkaniaceae in the Chlamydial order, which is able to survive and grow as an amoeba resisting microorganism in trophozoites and cysts of Acanthamoeba and other free-living protozoa, which probably represent its natural reservoirs. Objective: The study aimed to early diagnose and minimize the risks of Simkania negevensis infection in hemodialysis patients. Patients and Methods: This cross-sectional study was performed on 78 hemodialysis patients, and they were treated with regular hemodialysis. Patients were subjected to the following: complete history taking, full clinical assessment, laboratory investigation (complete blood count, measurement of serum IgG antibody against S. negevensisin hemodialysis patients and water samples were collected from hemodialysis circuits for detection of the occurrence of S. negevensis infection). Results: There was no significant difference in the age and gender between the patients undergoing hemodialysis with negative water samples for Simkania compared to hemodialysis with positive water for S. negevensis. Regarding hypertension (HTN) and diabetes mellitus (DM), there was no significant difference between the patients undergoing hemodialysis with negative water samples for Simkania compared to hemodialysis with positive water for S. negevensis. IgG levels are significantly higher in patients during hemodialysis when the water samples were positive for Simkania. Conclusion: Our study detected the occurrence of S. negevensis in hemodialysis patients. When the patients were undergoing hemodialysis with positive water for Simkania, we detected significant rise of IgG antibodies against S. negevensis. Significant area under curve with significant cutoff >6.1 for IgG with sensitivity 73.3% and specificity 80.0%.

Microfluidic guillotine reveals multiple timescales and mechanical modes of wound response in Stentor coeruleus

BackgroundWound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus, a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism.ResultsHere we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales of wound repair and of mechanical modes of wound response in Stentor. We found that Stentor requires ~ 100–1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ~ 8–80 μm2/s, faster than most other single cells reported in the literature. Further, we characterized three distinct mechanical modes of wound response in Stentor: contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor, was surprisingly not important for the contraction mode and was of low importance for the others.ConclusionsWhile events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved.

Inhibitory effect of host ocular microenvironmental factors on chlorhexidine digluconate activity.

Acanthamoeba spp. are free-living protozoan that cause a serious human eye disease called Acanthamoeba keratitis (AK). Several new and effective medical therapy for AK patients remains highly debated and therefore, CHG is still considered one of the first lines of treatment for AK patients. We hypothesized that ocular microenvironmental factors are responsible for Acanthamoeba drug resistance and clinical AK treatment failure. To investigate the influence of the ocular surface on CHG treatment, we tested the effect of several ocular elements on the anti-amoeba activity of CHG. The suspected inhibitory elements, including mucin, albumin, human and amoeba cell lysates, live and heat-killed bacteria, and cornea, were added to the amoebicidal activity platform, where amoeba was incubated with CHG at varying concentrations. Mucin showed a significant inhibitory effect on CHG activity against Acanthamoeba castellanii In contrast, albumin did not affect CHG treatment. Furthermore, human and amoeba cell lysates as well as live and heat-killed bacterial suspensions also significantly inhibited CHG activity. Additionally, we found that pig corneas also reduced CHG activity. In contrast, dry eye drops and their major component, propylene glycol, which is commonly used as eyewash material, did not have an impact on CHG activity. Our results demonstrate the effect of ocular microenvironmental factors on CHG activity and suggest that these factors may play a role in the development of amoeba resistance to CHG and treatment failure.

Unravelling the removal mechanisms of bacterial and viral surrogates in aerobic granular sludge systems

The aerobic granular sludge (AGS) process is an effective wastewater treatment technology for organic matter and nutrient removal that has been introduced in the market rapidly. Until now, limited information is available on AGS regarding the removal of bacterial and viral pathogenic organisms present in sewage. This study focussed on determining the relation between reactor operational conditions (plug flow feeding, turbulent aeration and settling) and physical and biological mechanisms on removing two faecal surrogates, Escherichia coli and MS2 bacteriophages. Two AGS laboratory-scale systems were separately fed with influent spiked with 1.0 × 106 CFU/100 mL of E. coli and 1.3 × 108 PFU/100 mL of MS2 bacteriophages and followed during the different operational phases. The reactors contained only granular sludge and no flocculent sludge. Both systems showed reductions in the liquid phase of 0.3 Log10 during anaerobic feeding caused by a dilution factor and attachment of the organisms on the granules. Higher removal efficiencies were achieved during aeration, approximately 1 Log10 for E. coli and 0.6 Log10 for the MS2 bacteriophages caused mainly by predation. The 18S sequencing analysis revealed high operational taxonomic units (OTUs) of free-living protozoa genera Rhogostoma and Telotrochidium concerning the whole eukaryotic community. Attached ciliates propagated after the addition of the E. coli, an active contribution of the genera Epistylis, Vorticella, and Pseudovorticella was found when the reactor reached stability. In contrast, no significant growth of predators occurred when spiking the system with MS2 bacteriophages, indicating a low contribution of protozoa on the phage removal. Settling did not contribute to the removal of the studied bacterial and viral surrogates.

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay.

Background:The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene.Methods:Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique. Sensitivity, specificity, accuracy and likelihood ratio were evaluated.Results:Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype.Conclusion:Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.

Differential expression of Acanthamoeba castellanii proteins during amoebic keratitis in rats

Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.

Environmental abiotic and biotic factors affecting the distribution and abundance of Naegleria fowleri.

ABSTRACT Naegleria fowleri is a free-living protozoan that resides in soil and freshwater. Human intranasal amoebae exposure through water or potentially dust particles can culminate in primary amoebic meningoencephalitis, which generally causes death. While many questions remain regarding pathogenesis, the microbial ecology of N. fowleri is even less understood. This review outlines current knowledge of the environmental abiotic and biotic factors that affect the distribution and abundance of N. fowleri. Although the impacts of some abiotic factors remain poorly investigated or inconclusive, N. fowleri appears to have a wide pH range, low salinity tolerance and thermophilic preference. From what is known about biotic factors, the amoebae preferentially feed upon bacteria and are preyed upon by other free-living amoebae. Additional laboratory and environmental studies are needed to fill in knowledge gaps, which are crucial for surveillance and management of N. fowleri in freshwaters. As surface water temperatures increase with climate change, it is likely that this amoeba will pose a greater threat to human health, suggesting that identifying its abiotic and biotic preferences is critical to mitigating this risk.

Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms

The water-borne bacterium Legionella pneumophila is the causative agent of Legionnaires' disease. In the environment, the opportunistic pathogen colonizes different niches, including free-living protozoa and biofilms. The physiological state(s) of sessile Legionella in biofilms and their functional consequences are not well understood. Using single-cell techniques and fluorescent growth rate probes as well as promoter reporters, we show here that sessile L. pneumophila exhibits phenotypic heterogeneity and adopts growing and nongrowing ("dormant") states in biofilms and microcolonies. Phenotypic heterogeneity is controlled by the Legionella quorum sensing (Lqs) system, the transcription factor LvbR, and the temperature. The Lqs system and LvbR determine the ratio between growing and nongrowing sessile subpopulations, as well as the frequency of growth resumption ("resuscitation") and microcolony formation of individual bacteria. Nongrowing L. pneumophila cells are metabolically active, express virulence genes and show tolerance toward antibiotics. Therefore, these sessile nongrowers are persisters. Taken together, the Lqs system, LvbR and the temperature control the phenotypic heterogeneity of sessile L. pneumophila, and these factors regulate the formation of a distinct subpopulation of nongrowing, antibiotic tolerant, virulent persisters. Hence, the biofilm niche of L. pneumophila has a profound impact on the ecology and virulence of this opportunistic pathogen.

Role of amoebae for survival and recovery of ‘non-culturable’ Helicobacter pylori cells in aquatic environments

ABSTRACT Helicobacter pylori is a fastidious Gram-negative bacterium that infects over half of the world's population, causing chronic gastritis and is a risk factor for stomach cancer. In developing and rural regions where prevalence rate exceeds 60%, persistence and waterborne transmission are often linked to poor sanitation conditions. Here we demonstrate that H. pylori not only survives but also replicates within acidified free-living amoebal phagosomes. Bacterial counts of the clinical isolate H. pylori G27 increased over 50-fold after three days in co-culture with amoebae. In contrast, a H. pylori mutant deficient in a cagPAI gene (cagE) showed little growth within amoebae, demonstrating the likely importance of a type IV secretion system in H. pylori for amoebal infection. We also demonstrate that H. pylori can be packaged by amoebae and released in extracellular vesicles. Furthermore, and for the first time, we successfully demonstrate the ability of two free-living amoebae to revert and recover viable but non-cultivable coccoid (VBNC)-H. pylori to a culturable state. Our studies provide evidence to support the hypothesis that amoebae and perhaps other free-living protozoa contribute to the replication and persistence of human-pathogenic H. pylori by providing a protected intracellular microenvironment for this pathogen to persist in natural aquatic environments and engineered water systems, thereby H. pylori potentially uses amoeba as a carrier and a vector of transmission.

Exploring the Histone Acetylation Cycle in the Protozoan Model Tetrahymena thermophila.

The eukaryotic histone acetylation cycle is composed of three classes of proteins, histone acetyltransferases (HATs) that add acetyl groups to lysine amino acids, bromodomain (BRD) containing proteins that are one of the most characterized of several protein domains that recognize acetyl-lysine (Kac) and effect downstream function, and histone deacetylases (HDACs) that catalyze the reverse reaction. Dysfunction of selected proteins of these three classes is associated with human disease such as cancer. Additionally, the HATs, BRDs, and HDACs of fungi and parasitic protozoa present potential drug targets. Despite their importance, the function and mechanisms of HATs, BRDs, and HDACs and how they relate to chromatin remodeling (CR) remain incompletely understood. Tetrahymena thermophila (Tt) provides a highly tractable single-celled free-living protozoan model for studying histone acetylation, featuring a massively acetylated somatic genome, a property that was exploited in the identification of the first nuclear/type A HAT Gcn5 in the 1990s. Since then, Tetrahymena remains an under-explored model for the molecular analysis of HATs, BRDs, and HDACs. Studies of HATs, BRDs, and HDACs in Tetrahymena have the potential to reveal the function of HATs and BRDs relevant to both fundamental eukaryotic biology and to the study of disease mechanisms in parasitic protozoa.