Dormant Cysts Research Articles

Enhanced astaxanthin production in Haematococcus lacustris by electrochemical stimulation of cyst germination

This study investigated the technical feasibility of using electrogermination to activate dormant cysts as an inoculum for subsequent 14-d photosynthetic astaxanthin production in Haematococcus lacustris. Electrotreatment affected the cell viability, surface charge, and morphology of H. lacustris cysts. At an optimal voltage of 2 V for 60 min, the cyst germination rate peaked at 44.6 % after 1 d, representing a 2.2-fold increase compared with that of the untreated control. Notably, electrogermination significantly enhanced both the astaxanthin content (44.9 mg/g cell) and productivity (13.2 mg/L/d) after 14 d of photobioreactor cultivation, corresponding to 1.7- and 1.5-fold increases compared with those in control, respectively. However, excessive electrotreatment, particularly at voltages exceeding 2 V or for durations beyond 60 min, did not enhance the astaxanthin production capability of H. lacustris. Proper optimization of renewable electrogermination can enable sustainable algal biorefinery to produce multiple bioactive products without compromising cell viability and astaxanthin productivity.

Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

A Review of Immunological and Neuropsychobehavioral Effects of Latent Toxoplasmosis on Humans.

Toxoplasmosis as a zoonotic disease has a worldwide distribution and can infect a wide range of animal hosts, as well as at least one third of the world's human population. The disease is usually mild or asymptomatic in immunocompetent individuals, but dormant tissue cysts survive especially in the brain for the host lifespan, known as latent toxoplasmosis (LT). Recent studies suggest that LT can have certain neurological, immunological psychological and behavioural effects on human including schizophrenia, bipolar disorder, Alzheimer's disease, depression, suicide anxiety and sleeping disorders. LT effects are controversial, and their exact mechanisms of action is not yet fully understood. This review aims to provide an overview of the potential effects, their basic mechanisms including alteration of neurotransmitter levels, immune activation in the central nervous system and induction of oxidative stress. Additionally, beneficial effects of LT, and an explanation of the effects within the framework of manipulation hypothesis, and finally, the challenges and limitations of the current research are discussed.

Dynamics, diversity, and roles of bacterial transmission modes during the first asexual life stages of the freshwater sponge Spongilla lacustris

BackgroundSponge-associated bacteria play important roles in the physiology of their host, whose recruitment processes are crucial to maintain symbiotic associations. However, the acquisition of bacterial communities within freshwater sponges is still under explored. Spongilla lacustris is a model sponge widely distributed in European rivers and lakes, producing dormant cysts (named gemmules) for their asexual reproduction, before winter. Through an in vitro experiment, this study aims to describe the dynamics of bacterial communities and their transmission modes following the hatching of these gemmules.ResultsAn overall change of bacterial β-diversity was observed through the ontology of the juvenile sponges. These temporal differences were potentially linked, first to the osculum acquisition and the development of a canal system, and then, the increasing colonization of the Chlorella-like photosymbionts. Gemmules hatching with a sterilized surface were found to have a more dispersed and less diverse microbiome, revealing the importance of gemmule epibacteria for the whole holobiont stability. These epibacteria were suggested to be vertically transmitted from the maternal tissues to the gemmule surface. Vertical transmission through the incorporation of bacterial communities inside of the gemmule, was also found as a dominant transmission mode, especially with the nitrogen fixers Terasakiellaceae. Finally, we showed that almost no ASVs were shared between the free-living community and the juveniles, suggesting that horizontal recruitment is unlikely to happen during the first stages of development. However, the free-living bacteria filtered are probably used as a source of nutrients, allowing an enrichment of copiotrophic bacteria already present within its microbiome.ConclusionsThis study brings new insight for a better understanding of the microbiome acquisition during the first stages of freshwater sponge development. We showed the importance of epibacterial communities on gemmules for the whole holobiont stability, and demonstrated the near absence of recruitment of free-living bacteria during the first stages.

Tolerance of dormant cysts of colpodid ciliates to gamma irradiation and cell repair after damage

Tolerance of dormant cysts of colpodid ciliates to gamma irradiation and cell repair after damage

Lactase can target cellular differentiation of Acanthamoeba castellanii belonging to the T4 genotype.

The free living Acanthamoeba spp. are ubiquitous amoebae associated with potentially blinding disease known as Acanthamoeba keratitis (AK) and a fatal central nervous system infection granulomatous amoebic encephalitis (GAE). With the inherent ability of cellular differentiation, it can phenotypically transform to a dormant cyst form from an active trophozoite form. Acanthamoeba cysts are highly resistant to therapeutic agents as well as contact lens cleaning solutions. One way to tackle drug resistance against Acanthamoeba is by inhibiting the formation of cysts from trophozoites. The biochemical analysis showed that the major component of Acanthamoeba cyst wall is composed of carbohydrate moieties such as galactose and glucose. The disaccharide of galactose and glucose is lactose. In this study, we analyzed the potential of lactase enzyme to target carbohydrate moieties of cyst walls. Amoebicidal assessment showed that lactase was ineffective against trophozoite of A. castellanii but enhanced amoebicidal effects of chlorhexidine. The lactase enzyme did not show any toxicity against normal human keratinocyte cells (HaCaT) at the tested range. Hence, lactase can be used for further assessment for development of potential therapeutic agents in the management of Acanthamoeba infection as well as formulation of effective contact lens disinfectants.

Marine microfossils: Tiny archives of ocean changes through deep time.

Microorganisms have inhabited the oceans since the dawn of Earth. Some of them have organic walls and some produce mineral tests that are usually composed of carbonate minerals or silica. They can therefore be preserved with original parts during sedimentary deposition or fossilized through permineralization or carbonization processes. The most common marine fossil groups studied by micropaleontologists are cyanobacteria, coccolithophores, dinoflagellates, diatoms, silicoflagellates, radiolarians, foraminifers, red and green algae, ostracods, and pteropods. Dormant or reproductive cysts can also be used for determinations of the fossil microbiota. Microfossils can be studied in petrographic slides prepared from rocks or separated from loosely consolidated rocks by disaggregation or dissolution and wet sieving. Their presence is sometimes recognized by biomarkers. Transmitted light microscopy and reflected light stereomicroscopy are necessary for micropaleontological studies whereas scanning electronic microscopy (SEM) aids research on the tiniest fossils and reveals fine skeletal details. Microorganisms have influenced the oxygenation of water and the atmosphere, as well as Earth's carbon cycle and have contributed to the formation of sedimentary rocks. By studying microfossils, paleontologists depict the age of the rock and identify depositional environments. Such studies help us recognize periods of stress in Earth's history and understand their influence on living organisms. Biogenic rocks, made of microfossils, can be used as raw materials, such as fossil fuels, building stone, or additives for the food industry, agricultural, or cosmetic purposes.

Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30°C, 37°C, and 40°C) and osmotolerance (0.5M, 1M, and 1.5M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

The impact of selected abiotic factors on Artemia hatching process through real-time observation of oxygen changes in a microfluidic platform

Current studies on abiotic impacts on Artemia, a crustacean which is widely used in aquaculture, and ecotoxicology, often focus on endpoint analysis (e.g., hatching rates, survival). Here, we demonstrate that a mechanistic understanding can be obtained through measurement of oxygen consumption in real-time over an extended time period in a microfluidic platform. The platform enables high level control of the microenvironment and direct observation of morphological changes. As a demonstration, temperature and salinity are chosen to represent critical abiotic parameters that are also threatened by climate change. The hatching process of Artemia consists of four different stages: hydration, differentiation, emergence, and hatching. Different temperatures (20, 35, and 30 °C) and salinities (0, 25, 50, and 75 ppt) are shown to significantly alter the duration of hatching stages, metabolic rates, and hatchability. Specifically, the metabolic resumption of dormant Artemia cysts was significantly enhanced at higher temperatures and moderate salinity, however, the time needed for this resumption was only dependent on higher temperatures. Hatchability was inversely related to the duration of the differentiation stage of hatching, which persisted longer at lower temperatures and salinities. The current approach of investigation of metabolism and corresponding physical changes can be employed to study hatching processes of other aquatic species, even those with low metabolic rate.

Twenty‐five years of acanthamoeba diagnostics in Austria

AbstractAcanthamoeba spp. are ubiquitous free‐living amoebae and do not need a host. They can, however, cause disease upon accidental contact with a potential host. The most common Acanthamoeba infection in humans is the so‐called Acanthamoeba keratitis (AK), occurring mainly in contact lens wearers.The first case of AK was reported in the early seventies and in the mid‐eighties the association between AK and contact lens wear was discovered. Today, acanthamoebae are among the most important causative agents of keratitis in contact lens wearers. AK often shows a severe progression, which is due to a lack of awareness but also to the lack of specific treatment. The dormant cysts pose a particular problem, often residing within the tissue and leading to reinfection after termination of treatment. In industrialized countries the annual incidence of AK lies between 0.1–1 cases per 100 000 inhabitants, with a marked regional variation depending on contact lens wear habits and mode of water supply.Our institution is the Austrian reference laboratory for Acanthamoeba diagnostics. All samples from patients with suspected AK are screened for Acanthamoeba spp. by culture and PCR in parallel, and all detected amoebae are genotyped. We currently have an annual incidence of AK of around 0.2 cases per 100 000 inhabitants, with >90% of cases occurring in contact lens wearers. In contrast to other countries, we do not see any seasonality and in contrast to the early years, today, males and females are equally represented, the most affected group being the 21–30‐year‐olds. The vast majority of cases are unilateral and many cases have a prolonged progression, in some cases infections remain viable for >1 yr. The predominant genotype is Acanthamoeba T4, other common genotypes are T3, T5, T6, T10 and T11.

Pleiotropic Roles of Cholesteryl Sulfate during Entamoeba Encystation: Involvement in Cell Rounding and Development of Membrane Impermeability.

ABSTRACTEntamoeba histolytica, a protozoan parasite, causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of cysts, the only form that is able to transmit to a new host. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed “encystation.” During encystation, cell morphology is markedly changed; motile amoeboid cells become rounded, nonmotile cells. Concomitantly, cell components change and significant fluctuations of metabolites occur. Cholesteryl sulfate (CS) is a crucial metabolite for encystation. However, its precise role remains uncertain. To address this issue, we used in vitro culture of Entamoeba invadens as the model system for the E. histolytica encystation study and identified serum-free culture conditions with CS supplementation at concentrations similar to intracellular CS concentrations during natural encystation. Using this culture system, we show that CS exerts pleiotropic effects during Entamoeba encystation, affecting cell rounding and development of membrane impermeability. CS dose dependently induced and maintained encysting cells as spherical maturing cysts with almost no phagocytosis activity. Consequently, the percentage of mature cysts was increased. CS treatment also caused time- and dose-dependent development of membrane impermeability in encysting cells via induction of de novo synthesis of dihydroceramides containing very long N-acyl chains (≥26 carbons). These results indicate that CS-mediated morphological and physiological changes are necessary for the formation of mature cysts and the maintenance of the Entamoeba life cycle. Our findings also reveal important morphological aspects of the process of dormancy and the control of membrane structure.IMPORTANCE Entamoeba histolytica causes a parasitic infectious disease, amoebiasis. Amoebiasis is a global public health problem with a high occurrence of infection and inadequate clinical options. The parasite alternates its form between a proliferative trophozoite and a dormant cyst that enables the parasite to adapt to new environments. The transition stage in which trophozoites differentiate into cysts is termed “encystation.” Cholesteryl sulfate is essential for encystation; however, its precise role remains to be determined. Here, we show that cholesteryl sulfate is a multifunctional metabolite exerting pleiotropic roles during Entamoeba encystation, including the rounding of cells and the development of membrane impermeability. Such morphological and physiological changes are required for Entamoeba to produce cysts that are transmissible to a new host, which is essential for maintenance of the Entamoeba life cycle. Our findings are therefore relevant not only to Entamoeba biology but also to general cell and lipid biology.

Sirtinol Supresses Trophozoites Proliferation and Encystation of Acanthamoeba via Inhibition of Sirtuin Family Protein.

The encystation of Acanthamoeba leads to the development of metabolically inactive and dormant cysts from vegetative trophozoites under unfavorable conditions. These cysts are highly resistant to anti-Acanthamoeba drugs and biocides. Therefore, the inhibition of encystation would be more effective in treating Acanthamoeba infection. In our previous study, a sirtuin family protein—Acanthamoeba silent-information regulator 2-like protein (AcSir2)—was identified, and its expression was discovered to be critical for Acanthamoeba castellanii proliferation and encystation. In this study, to develop Acanthamoeba sirtuin inhibitors, we examine the effects of sirtinol, a sirtuin inhibitor, on trophozoite growth and encystation. Sirtinol inhibited A. castellanii trophozoites proliferation (IC50=61.24 μM). The encystation rate of cells treated with sirtinol significantly decreased to 39.8% (200 μM sirtinol) after 24 hr of incubation compared to controls. In AcSir2-overexpressing cells, the transcriptional level of cyst-specific cysteine protease (CSCP), an Acanthamoeba cysteine protease involved in the encysting process, was 11.6- and 88.6-fold higher at 48 and 72 hr after induction of encystation compared to control. However, sirtinol suppresses CSCP transcription, resulting that the undegraded organelles and large molecules remained in sirtinol-treated cells during encystation. These results indicated that sirtinol sufficiently inhibited trophozoite proliferation and encystation, and can be used to treat Acanthamoeba infections.

Probing the Peculiarity of EhRabX10, a pseudoRab GTPase, from the Enteric Parasite Entamoeba histolytica through In Silico Modeling and Docking Studies.

Entamoeba histolytica (Eh) is a pathogenic eukaryote that often resides silently in humans under asymptomatic stages. Upon indeterminate stimulus, it develops into fulminant amoebiasis that causes severe hepatic abscesses with 50% mortality. This neglected tropical pathogen relies massively on membrane modulation to flourish and cause disease; these modulations range from the phagocytic mode for food acquisition to a complex trogocytosis mechanism for tissue invasion. Rab GTPases form the largest branch of the Ras-like small GTPases, with a diverse set of roles across the eukaryotic kingdom. Rab GTPases are vital for the orchestration of membrane transport and the secretory pathway responsible for transporting the pathogenic effectors, such as cysteine proteases (EhCPs) which help in tissue invasion. Rab GTPases thus play a crucial role in executing the cytolytic effect of E. histolytica. First, they interact with Gal/Nac lectins required for adhering to the host cells, and then, they assist in the secretion of EhCPs. Additionally, amoebic Rab GTPases are vital for encystation because substantial vesicular trafficking is required to create dormant amoebic cysts. These cysts are the infective agent and help to spread the disease. The absence of a “bonafide” vesicular transport machinery in Eh and the existence of a diverse repertoire of amoebic Rab GTPases (EhRab) hint at their contribution in supporting this atypical machinery. Here, we provide insights into a pseudoRab GTPase, EhRabX10, by performing physicochemical analysis, predictive 3D structure modeling, protein-protein interaction studies, and in silico molecular docking. Our group is the first one to classify EhRabX10 as a pseudoRab GTPase with four nonconserved G-motifs. It possesses the basic fold of the P-loop containing nucleotide hydrolases. Through this in silico study, we provide an introduction to the characterization of the atypical EhRabX10 and set the stage for future explorations into the mechanisms of nucleotide recognition, binding, and hydrolysis employed by the pseudoEhRab GTPase family.

A gastric acid condition enhances the microbial killing effect of ethanol

Tolerance to extremely low pH (< pH 3) is a crucial strategy that enables microorganisms to survive in the human stomach cavity. We found that the killing effect of ethanol on dormant cysts of soil protist Colpoda cucullus, and pathogenic bacteria such as Klebsiella pneumoniae and enterohemorrhagic Escherichia coli O157 was enhanced under stomach acid's low pH condition. This result suggests that gastric-acid-tolerant microorganisms can be partly killed by drinking alcoholic beverages during meals. Keywords: Acid-tolerant microorganisms, ethanol, bactericidal effect.

Transcriptome analysis reveals the encystment-related lncRNA expression profile and coexpressed mRNAs in Pseudourostyla cristata

Ciliated protozoans form dormant cysts for survival under adverse conditions. The molecular mechanisms regulating this process are critical for understanding how single-celled eukaryotes adapt to the environment. Despite the accumulated data on morphology and gene coding sequences, the molecular mechanism by which lncRNAs regulate ciliate encystment remains unknown. Here, we first detected and analyzed the lncRNA expression profile and coexpressed mRNAs in dormant cysts versus vegetative cells in the hypotrich ciliate Pseudourostyla cristata by high-throughput sequencing and qRT-PCR. A total of 853 differentially expressed lncRNAs were identified. Compared to vegetative cells, 439 and 414 lncRNAs were upregulated and downregulated, respectively, while 47 lncRNAs were specifically expressed in dormant cysts. A lncRNA-mRNA coexpression network was constructed, and the possible roles of lncRNAs were screened. Three of the identified lncRNAs, DN12058, DN20924 and DN30855, were found to play roles in fostering encystment via their coexpressed mRNAs. These lncRNAs can regulate a variety of physiological activities that are essential for encystment, including autophagy, protein degradation, the intracellular calcium concentration, microtubule-associated dynein and microtubule interactions, and cell proliferation inhibition. These findings provide the first insight into the potentially functional lncRNAs and their coexpressed mRNAs involved in the dormancy of ciliated protozoa and contribute new evidence for understanding the molecular mechanisms regulating encystment.

Stage-Specific De Novo Synthesis of Very-Long-Chain Dihydroceramides Confers Dormancy to Entamoeba Parasites

ABSTRACTAmoebiasis is a parasitic disease caused by Entamoeba histolytica infection and is a serious public health problem worldwide due to ill-prepared preventive measures as well as its high morbidity and mortality rates. Amoebiasis transmission is solely mediated by cysts. Cysts are produced by the differentiation of proliferative trophozoites in a process termed “encystation.” Entamoeba encystation is a fundamental cell differentiation process and proceeds with substantial changes in cell metabolites, components, and morphology, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites that function as key factors for encystation. However, a comprehensive lipid analysis has not been reported, and the involved lipid metabolic pathways remain largely unknown. Here, we exploited the state-of-the-art untargeted lipidomics and characterized 339 molecules of 17 lipid subclasses. Of these, dihydroceramide (Cer-NDS) was found to be among the most induced lipid species during encystation. Notably, in encysting cells, amounts of Cer-NDS containing very long N-acyl chains (≥26 carbon) were more than 30-fold induced as the terminal product of a de novo metabolic pathway. We also identified three ceramide synthase genes responsible for producing the very-long-chain Cer-NDS molecules. These genes were upregulated during encystation. Furthermore, these ceramide species were shown to be indispensable for generating membrane impermeability, a prerequisite for becoming dormant cyst that shows resistance to environmental assault inside and outside the host for transmission. Hence, the lipid subclass of Cer-NDS plays a crucial role for Entamoeba cell differentiation and morphogenesis by alternating the membrane properties.IMPORTANCE Entamoeba is a protozoan parasite that thrives in its niche by alternating its two forms between a proliferative trophozoite and dormant cyst. Cysts are the only form able to transmit to a new host and are differentiated from trophozoites in a process termed “encystation.” During Entamoeba encystation, cell metabolites, components, and morphology drastically change, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites. However, the involved lipid species and their metabolic pathways remain largely unknown. Here, we identified dihydroceramides (Cer-NDSs) containing very long N-acyl chains (C26 to C30) as a key metabolite for Entamoeba encystation by our state-of-the-art untargeted lipidomics. We also showed that these Cer-NDSs are critical to generate the membrane impermeability, a prerequisite for this parasite to show dormancy as a cyst that repels substances and prevents water loss. Hence, ceramide metabolism is essential for Entamoeba to maintain the parasitic lifestyle.

No Light, No Germination: Excitation of the Rhodospirillum centenum Photosynthetic Apparatus Is Necessary and Sufficient for Cyst Germination.

Rhodospirillum centenum is a Gram-negative alphaproteobacterium that is capable of differentiating into dormant cysts that are metabolically inactive and desiccation resistant. Like spores synthesized by many Gram-positive species, dormant R. centenum cysts germinate in response to an environmental signal, indicating that conditions favor survival and proliferation. Factors that induce germination are called germinants and are often both niche and species specific. In this study, we have identified photosynthesis as a niche-specific germinant for R. centenum cyst germination. Specifically, excitation of wild-type cysts suspended in a nutrient-free buffer with far-red light at >750 nm results in rapid germination. This is in stark contrast to mutant strains deficient in photosynthesis that fail to germinate upon exposure to far-red light under all assayed conditions. We also show that photosynthesis-induced germination occurs in a carbon- and nitrogen-free buffer even in strains that are deficient in carbon or nitrogen fixation. These results demonstrate that photosynthesis not only is necessary for germination but is itself sufficient for the germination of R. centenum cysts.IMPORTANCE Environmental cues that signal Gram-positive spores to germinate (termed germinants) have been identified for several Bacillus and Clostridium species. These studies showed that germinants are niche and species specific. For example, Clostridium difficile spores sense bile salts as a germinant as their presence informs these cells of an intestinal environment. Bacillus fastidiosus spores use uric acid as a germinant that is present in soil and poultry litter as this species inhabits poultry litter. It is evident from these studies that dormant cells sample their environment to assess whether conditions are advantageous for the propagation and survival of vegetative cells. To date, a limited number of germinants have been defined for only a few Gram-positive spore-forming species. Beyond that group, there is scant information on what cues signal dormant cells to exit dormancy. In our study, we show that the versatile Gram-negative photosynthetic bacterium Rhodospirillum centenum uses light-driven photosynthesis, and not the availability of nutrients, to trigger the germination of dormant cysts. This use of light-driven photosynthesis as a germinant is surprising as this species is also capable of growing under dark conditions using exogenous carbon sources for energy. Consequently, photosynthetic growth appears to be the preferred growth mechanism by this species.

TgIF2K-B Is an eIF2α Kinase in Toxoplasma gondii That Responds to Oxidative Stress and Optimizes Pathogenicity.

Toxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not yet clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eukaryotic initiation factor 2 (eIF2). Here, we show that the Toxoplasma eIF2 kinase TgIF2K-B is activated in response to oxidative stress and affords protection. Knockout of the TgIF2K-B gene, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasite's ability to establish persistent infection in its host.IMPORTANCEToxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite's oxidative stress response and its ability to persist in the host as a latent infection.

Epidemiology, Pathophysiology, Diagnosis, and Management of Cerebral Toxoplasmosis.

Toxoplasma gondii is known to infect a considerable number of mammalian and avian species and a substantial proportion of the world's human population. The parasite has an impressive ability to disseminate within the host's body and employs various tactics to overcome the highly regulatory blood-brain barrier and reside in the brain. In healthy individuals, T. gondii infection is largely tolerated without any obvious ill effects. However, primary infection in immunosuppressed patients can result in acute cerebral or systemic disease, and reactivation of latent tissue cysts can lead to a deadly outcome. It is imperative that treatment of life-threatening toxoplasmic encephalitis is timely and effective. Several therapeutic and prophylactic regimens have been used in clinical practice. Current approaches can control infection caused by the invasive and highly proliferative tachyzoites but cannot eliminate the dormant tissue cysts. Adverse events and other limitations are associated with the standard pyrimethamine-based therapy, and effective vaccines are unavailable. In this review, the epidemiology, economic impact, pathophysiology, diagnosis, and management of cerebral toxoplasmosis are discussed, and critical areas for future research are highlighted.

DNA content in Acanthamoeba during two stress defense reactions: Encystation, pseudocyst formation and cell cycle

During environmental stress, the vegetative cells of the facultative pathogenic amoeba Acanthamoeba castellanii reversibly differentiate into resistant dormant stages, namely, cysts or pseudocysts. The type of resistant stage depends on the nature and duration of the stressor. Cell differentiation is accompanied by changes in morphology and cellular metabolism. Moreover, cell differentiation is also expected to be closely linked to the regulation of the cell cycle and, thus, to cellular DNA content. While the existence of the resistant stages in A. castellanii is well known, there is no consensus regarding the relationship between differentiation and cell cycle progression. In the present work, we used flow cytometry analysis to explore the changes in the DNA content during Acanthamoeba encystation and pseudocyst formation. Our results strongly indicate that A. castellanii enters encystation from the G2 phase of the cell cycle. In contrast, differentiation into pseudocysts can begin in the G1 and G2 phases. In addition, we present a phylogenetic analysis and classification of the main cell cycle regulators, namely, cyclin-dependent kinases and cyclins that are found in the genome of A. castellanii.