Cellular Response of Adapted and Non-Adapted Tetrahymena thermophila Strains to Europium Eu(III) Compounds.

Europium is one of the most reactive lanthanides and humans use it in many different applications, but we still know little about its potential toxicity and cellular response to its exposure. Two strains of the eukaryotic microorganism model Tetrahymena thermophila were adapted to high concentrations of two Eu(III) compounds (EuCl3 or Eu2O3) and compared to a control strain and cultures treated with both compounds. In this ciliate, EuCl3 is more toxic than Eu2O3. LC50 values show that this microorganism is more resistant to these Eu(III) compounds than other microorganisms. Oxidative stress originated mainly by Eu2O3 is minimized by overexpression of genes encoding important antioxidant enzymes. The overexpression of metallothionein genes under treatment with Eu(III) compounds supports the possibility that this lanthanide may interact with the -SH groups of the cysteine residues from metallothioneins and/or displace essential cations of these proteins during their homeostatic function. Both lipid metabolism (lipid droplets fusing with europium-containing vacuoles) and autophagy are involved in the cellular response to europium stress. Bioaccumulation, together with a possible biomineralization to europium phosphate, seems to be the main mechanism of Eu(III) detoxification in these cells.

Protozoan predation enhances stress resistance and antibiotic tolerance in Burkholderia cenocepacia by triggering the SOS response.

Bacterivorous protists are thought to serve as training grounds for bacterial pathogens by subjecting them to the same hostile conditions that they will encounter in the human host. Bacteria that survive intracellular digestion exhibit enhanced virulence and stress resistance after successful passage through protozoa but the underlying mechanisms are unknown. Here we show that the opportunistic pathogen Burkholderia cenocepacia survives phagocytosis by ciliates found in domestic and hospital sink drains, and viable bacteria are expelled packaged in respirable membrane vesicles with enhanced resistance to oxidative stress, desiccation, and antibiotics, thereby contributing to pathogen dissemination in the environment. Reactive oxygen species generated within the protozoan phagosome promote the formation of persisters tolerant to ciprofloxacin by activating the bacterial SOS response. In addition, we show that genes encoding antioxidant enzymes are upregulated during passage through ciliates increasing bacterial resistance to oxidative radicals. We prove that suppression of the SOS response impairs bacterial intracellular survival and persister formation within protists. This study highlights the significance of protozoan food vacuoles as niches that foster bacterial adaptation in natural and built environments and suggests that persister switch within phagosomes may be a widespread phenomenon in bacteria surviving intracellular digestion.

Quantitative proteomic analyses of a Pb-adapted Tetrahymena thermophila strain reveal the cellular strategy to Pb(II) stress including lead biomineralization to chloropyromorphite.

A strain of the protozoan ciliate Tetrahymena thermophila adapted to increasing Pb(II) concentrations over two years has shown that one of the resistance mechanisms to this extreme metal stress is the lead biomineralization to chloropyromorphite, one of the most stable minerals in the earth's crust. Several techniques such as microanalysis coupled to transmission and scanning electron microscopy (X-Ray Energy Disperse Spectroscopy), fluorescence microscopy and X-ray power diffraction analysis have revealed the presence of chloropyromorphite as crystalline aggregates of nano-globular structure, together with the presence of other secondary lead minerals. This is the first time that the existence of this type of biomineralization in a ciliate protozoan is described. The Pb(II) bioremediation capacity of this strain has shown that it can remove >90 % of the toxic soluble lead from the medium. A quantitative proteomic analysis of this strain has revealed the main molecular-physiological elements involved in adaptation to Pb(II) stress: increased activity of proteolytic systems against lead proteotoxicity, occurrence of metallothioneins to immobilize Pb(II) ions, antioxidant enzymes to mitigate oxidative stress, and an intense vesicular trafficking presumably involved in the formation of vacuoles where pyromorphite accumulates and is subsequently excreted, together with an enhanced energy metabolism. As a conclusion, all these results have been compiled into an integrated model that could explain the eukaryotic cellular response to extreme lead stress.

Heterologous Expression of the Phytochelatin Synthase CaPCS2 from Chlamydomonas acidophila and Its Effect on Different Stress Factors in Escherichia coli.

Phytochelatins (PCs) are cysteine-rich small peptides, enzymatically synthesized from reduced glutathione (GSH) by cytosolic enzyme phytochelatin synthase (PCS). The open reading frame (ORF) of the phytochelatin synthase CaPCS2 gene from the microalgae Chlamydomonas acidophila was heterologously expressed in Escherichia coli strain DH5α, to analyze its role in protection against various abiotic agents that cause cellular stress. The transformed E. coli strain showed increased tolerance to exposure to different heavy metals (HMs) and arsenic (As), as well as to acidic pH and exposure to UVB, salt, or perchlorate. In addition to metal detoxification activity, new functions have also been reported for PCS and PCs. According to the results obtained in this work, the heterologous expression of CaPCS2 in E. coli provides protection against oxidative stress produced by metals and exposure to different ROS-inducing agents. However, the function of this PCS is not related to HM bioaccumulation.

Arsenate and arsenite differential toxicity in Tetrahymena thermophila.

A comparative analysis of toxicities of both arsenic forms (arsenite and arsenate) in the model eukaryotic microorganism Tetrahymena thermophila (ciliate protozoa) has shown the presence of various detoxification mechanisms and cellular effects comparable to those of animal cells under arsenic stress. In the wild type strain SB1969 arsenate is almost 2.5 times more toxic than arsenite. According to the concentration addition model used in binary metallic mixtures their toxicities show an additive effect. Using fluorescent assays and flow cytometry, it has been detected that As(V) generates elevated levels of ROS/RNS compared to As(III). Both produce the same levels of superoxide anion, but As(V) also causes greater increases in hydrogen peroxide and peroxynitrite. The mitochondrial membrane potential is affected by both As(V) and As(III), and electron microscopy has also revealed that mitochondria are the main target of both arsenic ionic forms. Fusion/fission and swelling mitochondrial and mitophagy, together with macroautophagy, vacuolization and mucocyst extruction are mainly associated to As(V) toxicity, while As(III) induces an extensive lipid metabolism dysfunction (adipotropic effect). Quantitative RT-PCR analysis of some genes encoding antioxidant proteins or enzymes has shown that glutathione and thioredoxin metabolisms are involved in the response to arsenic stress. Likewise, the function of metallothioneins seems to be crucial in arsenic detoxification processes, after using both metallothionein knockout and knockdown strains and cells overexpressing metallothionein genes from this ciliate. The analysis of the differential toxicity of As(III) and As(V) shown in this study provides cytological and molecular tools to be used as biomarkers for each of the two arsenic ionic forms.

Interactions with Arsenic: Mechanisms of Toxicity and Cellular Resistance in Eukaryotic Microorganisms.

Arsenic (As) is quite an abundant metalloid, with ancient origin and ubiquitous distribution, which represents a severe environmental risk and a global problem for public health. Microbial exposure to As compounds in the environment has happened since the beginning of time. Selective pressure has induced the evolution of various genetic systems conferring useful capacities in many microorganisms to detoxify and even use arsenic, as an energy source. This review summarizes the microbial impact of the As biogeochemical cycle. Moreover, the poorly known adverse effects of this element on eukaryotic microbes, as well as the As uptake and detoxification mechanisms developed by yeast and protists, are discussed. Finally, an outlook of As microbial remediation makes evident the knowledge gaps and the necessity of new approaches to mitigate this environmental challenge.

Tetrahymena Glutathione Peroxidase Family: A Comparative Analysis of These Antioxidant Enzymes and Differential Gene Expression to Metals and Oxidizing Agents.

In the present work, an extensive analysis of the putative glutathione peroxidases (GPx) of the eukaryotic microorganism model Tetrahymena thermophila is carried out. A comparative analysis with GPx present in other Tetrahymena species and other very taxonomically diverse ciliates is also performed. A majority of ciliate GPx have replaced the selenocysteine (Sec) by Cys in its catalytic center, so they can be considered as phospholipid hydroperoxide glutathione peroxidases (PHGPx). Selenocysteine insertion sequence (SECIS) elements have been detected in several ciliate GPx that do not incorporate Sec in their amino acid sequences, and conversely, in other ciliate GPx with Sec, no SECIS elements are detected. These anomalies are analyzed and discussed. From the phylogenetic analysis using the ciliate GPx amino acid sequences, the existence of extensive intra- and interspecific gene duplications that produced multiple GPx isoforms in each species is inferred. The ancestral character of the selenoproteins is also corroborated. The analysis by qRT-PCR of six selected T. thermophila GPx genes has shown a quantitative differential expression between them, depending on the stressor (oxidizing agents, apoptotic inducer or metals) and the time of exposure.

Toxicity, Physiological, and Ultrastructural Effects of Arsenic and Cadmium on the Extremophilic Microalga Chlamydomonas acidophila.

The cytotoxicity of cadmium (Cd), arsenate (As(V)), and arsenite (As(III)) on a strain of Chlamydomonas acidophila, isolated from the Rio Tinto, an acidic environment containing high metal(l)oid concentrations, was analyzed. We used a broad array of methods to produce complementary information: cell viability and reactive oxygen species (ROS) generation measures, ultrastructural observations, transmission electron microscopy energy dispersive x-ray microanalysis (TEM-XEDS), and gene expression. This acidophilic microorganism was affected differently by the tested metal/metalloid: It showed high resistance to arsenic while Cd was the most toxic heavy metal, showing an LC50 = 1.94 µM. Arsenite was almost four-fold more toxic (LC50= 10.91 mM) than arsenate (LC50 = 41.63 mM). Assessment of ROS generation indicated that both arsenic oxidation states generate superoxide anions. Ultrastructural analysis of exposed cells revealed that stigma, chloroplast, nucleus, and mitochondria were the main toxicity targets. Intense vacuolization and accumulation of energy reserves (starch deposits and lipid droplets) were observed after treatments. Electron-dense intracellular nanoparticle-like formation appeared in two cellular locations: inside cytoplasmic vacuoles and entrapped into the capsule, around each cell. The chemical nature (Cd or As) of these intracellular deposits was confirmed by TEM-XEDS. Additionally, they also contained an unexpected high content in phosphorous, which might support an essential role of poly-phosphates in metal resistance.

Selenium cytotoxicity in Tetrahymena thermophila: New clues about its biological effects and cellular resistance mechanisms.

Selenium is an essential micronutrient but at high concentrations can produce severe cytotoxicity and genomic damage. We have evaluated the cytotoxicity, ultrastructural and mitochondrial alterations of the two main selenium inorganic species; selenite and selenate, in the eukaryotic microorganism Tetrahymena thermophila. In this ciliate, selenite is more toxic than selenate. Their LC50 values were calculated as 27.65 µM for Se(IV) and 56.88 mM for Se(VI). Significant levels of peroxides/hydroperoxides are induced under low-moderate selenite or selenate concentrations. Se(VI) exposures induce an immediate mitochondrial membrane depolarization. Selenium treated cells show an intense vacuolization and some of them present numerous discrete and small electrondense particles, probably selenium deposits. Mitochondrial fusion, an intense swelling in peripheral mitochondria and mitophagy are detected in selenium treated cells, especially in those exposed to Se (IV). qRT-PCR analysis of diverse genes, encoding relevant antioxidant enzymes or other proteins, like metallothioneins, involved in an environmental general stress response, have shown that they may be crucial against Se(IV) and/or Se (VI) cytotoxicity.

AP-1 (bZIP) Transcription Factors as Potential Regulators of Metallothionein Gene Expression in Tetrahymena thermophila.

Metallothioneins (MT) are multi-stress proteins mainly involved in metal detoxification. MT gene expression is normally induced by a broad variety of stimulus and its gene expression regulation mainly occurs at a transcriptional level. Conserved motifs in the Tetrahymena thermophila MT promoters have been described. These motifs show a consensus sequence very similar to AP-1 sites, and bZIP type transcription factors might participate in the MT gene expression regulation. In this research work, we characterize four AP-1 transcription factors in each of four different analyzed Tetrahymena species, detecting a high conservation among them. Each AP-1 molecule has its counterpart in the other three Tetrahymena species. A comparative qRT-PCR analysis of these AP-1 genes have been carried out in different T. thermophila strains (including metal-adapted, knockout and/or knockdown strains among others), and under different metal-stress conditions (1 or 24 h Cd2+, Cu2+, or Pb2+ treatments). The possible interaction of these transcription factors with the conserved AP-1 motifs present in MT promoters has been corroborated by protein-DNA interaction experiments. Certain connection between the expression patterns of the bZIP and MT genes seems to exist. For the first time, and based on our findings, a possible gene expression regulation model including both AP-1 transcription factors and MT genes from the ciliate T. thermophila has been elaborated.

Population genetic structure, introgression, and hybridization in the genus Rhizophora along the Brazilian coast.

Mangrove plants comprise plants with similar ecological features that have enabled them to adapt to life between the sea and the land. Within a geographic region, different mangrove species share not only similar adaptations but also similar genetic structure patterns. Along the eastern coast of South America, there is a subdivision between the populations north and south of the continent's northeastern extremity. Here, we aimed to test for this north-south genetic structure in Rhizophora mangle, a dominant mangrove plant in the Western Hemisphere. Additionally, we aimed to study the relationships between R. mangle, R. racemosa, and R. × harrisonii and to test for evidence of hybridization and introgression. Our results confirmed the north-south genetic structure pattern in R. mangle and revealed a less abrupt genetic break in the northern population than those observed in Avicennia species, another dominant and widespread mangrove genus in the Western Hemisphere. These results are consistent with the role of oceanic currents influencing sea-dispersed plants and differences between Avicennia and Rhizophora propagules in longevity and establishment time. We also observed that introgression and hybridization are relevant biological processes in the northeastern coast of South America and that they are likely asymmetric toward R. mangle, suggesting that adaptation might be a process maintaining this hybrid zone.

Genome plasticity in response to stress in Tetrahymena thermophila: selective and reversible chromosome amplification and paralogous expansion of metallothionein genes.

Extreme stress situations can induce genetic variations including genome reorganization. In ciliates like Tetrahymena thermophila, the approximately 45-fold ploidy of the somatic macronucleus may enable adaptive responses that depend on genome plasticity. To identify potential genome-level adaptations related to metal toxicity, we isolated three Tetrahymena thermophila strains after an extended adaptation period to extreme metal concentrations (Cd2+ , Cu2+ or Pb2+ ). In the Cd-adapted strain, we found a approximately five-fold copy number increase of three genes located in the same macronuclear chromosome, including two metallothionein genes, MTT1 and MTT3. The apparent amplification of this macronuclear chromosome was reversible and reproducible, depending on the presence of environmental metal. We also analysed three knockout (KO) and/or knockdown (KD) strains for MTT1 and/or MTT5. In the MTT5KD strain, we found at least two new genes arising from paralogous expansion of MTT1, which encode truncated variants of MTT1. The expansion can be explained by a model based on somatic recombination between MTT1 genes on pairs of macronuclear chromosomes. At least two of the new paralogs are transcribed and upregulated in response to Cd2+ . Altogether, we have thus identified two distinct mechanisms, both involving genomic plasticity in the polyploid macronucleus that may represent adaptive responses to metal-related stress.

Extreme metal adapted, knockout and knockdown strains reveal a coordinated gene expression among different Tetrahymena thermophila metallothionein isoforms.

Metallothioneins (MT) constitute a superfamily of small cytosolic proteins that are able to bind metal cations through numerous cysteine (Cys) residues. Like other organisms the ciliate Tetrahymena thermophila presents several MT isoforms, which have been classified into two subfamilies (Cd- and Cu-metallothioneins). The main aim of this study was to examine the specific functions and transcriptional regulation of the five MT isoforms present in T. thermophila, by using several strains of this ciliate. After a laboratory evolution experiment over more than two years, three different T. thermophila strains adapted to extreme metal stress (Cd2+, Cu2+ or Pb2+) were obtained. In addition, three knockout and/or knockdown strains for different metallothionein (MT) genes were generated. These strains were then analyzed for expression of the individual MT isoforms. Our results provide a strong basis for assigning differential roles to the set of MT isoforms. MTT1 appears to have a key role in adaptation to Cd. In contrast, MTT2/4 are crucial for Cu-adaptation and MTT5 appears to be important for Pb-adaptation and might be considered as an "alarm" MT gene for responding to metal stress. Moreover, results indicate that likely a coordinated transcriptional regulation exists between the MT genes, particularly among MTT1, MTT5 and MTT2/4. MTT5 appears to be an essential gene, a first such report in any organism of an essential MT gene.

The Tetrahymena metallothionein gene family: twenty-one new cDNAs, molecular characterization, phylogenetic study and comparative analysis of the gene expression under different abiotic stressors.

BACKGROUND: Ciliate metallothioneins (MTs) are included in family 7 of the MT superfamily. This family has been divided into two main subfamilies: 7a or CdMTs and 7b or CuMTs. All ciliate MTs reported have been isolated from different Tetrahymena species and present unique features with regard to standard MTs. Likewise, an expression analysis has been carried out on some of MT genes under metal stress, corroborating their classification into two subfamilies. RESULTS: We isolated 21 new cDNAs from different Tetrahymena species to obtain a wider view of the biodiversity of these conserved genes. Structural analysis (cysteine patterns) and an updated phylogenetic study both corroborated the previous classification into two subfamilies. A new CuMT from a Tetrahymena-related species Ichthyophthirius multifiliis was also included in this general analysis. We detected a certain tendency towards the presentation of a CdMT tri-modular structure in Borealis group species with respect to Australis group. We report for the first time a semi-complete paralog duplication of a CdMT gene originating a new CdMT gene isoform in T. malaccensis. An asymmetry of the codon usage for glutamine residues was detected between Cd- and CuMTs, and the phylogenetic implications are discussed. A comparative gene expression analysis of several MT genes by qRT-PCR revealed differential behavior among them under different abiotic stressors in the same Tetrahymena species. CONCLUSIONS: The Tetrahymena metallothionein family represents a quite conserved protein structure group with unique features with respect to standard MTs. Both Cd- and CuMT subfamilies present very defined and differentiated characteristics at several levels: cysteine patterns, modular structure, glutamine codon usage and gene expression under metal stress, among others. Gene duplication through evolution seems to be the major genetic mechanism for creating new MT gene isoforms and increasing their functional diversity.

Microsatellite markers for studies with the carnivorous plant Philcoxia minensis (Plantaginaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the critically endangered carnivorous species Philcoxia minensis (Plantaginaceae) for further population genetic studies aiming at its conservation. METHODS AND RESULTS: We identified 29 clones containing 40 microsatellites from a genomic enriched library. A total of 27 primer pairs were developed and evaluated in 30 individuals of a natural P. minensis population. Seventeen markers successfully presented amplification products within the expected size range, of which 12 were polymorphic. The expected and observed heterozygosities ranged from 0.03 to 0.65 and from 0.00 to 0.77, respectively. Positive transferability with the related species P. bahiensis was observed for the same 17 markers. CONCLUSIONS: The 12 polymorphic microsatellite markers are suitable for studies in genetic diversity and structure, mating system, and gene flow in P. minensis and also may be useful for similar issues regarding the related species P. bahiensis.

Breastfeeding duration rates and factors affecting continued breastfeeding among infants born at an inner-city US Baby-Friendly hospital.

The effects of Baby-Friendly status on breastfeeding duration in the United States have not been published. The objectives of this study were to obtain breastfeeding rates at 6 months among babies born in a US Baby-Friendly hospital and to assess factors associated with continued breastfeeding at 6 months. The authors randomly selected 350 medical records of infants born in 2003 at Baby-Friendly Boston Medical Center. Of 336 eligible infants, 248 (74%) attended the 6-month well-child visit and 37.1% (92/248) were breastfeeding at 6 months. In multivariate logistic regression, the likelihood of breastfeeding at 6 months was decreased by presence of a feeding problem in the hospital (AOR 0.27; 95% CI 0.07-0.99), whereas the likelihood of breastfeeding at 6 months increased with maternal age (AOR 1.05; 95% CI 1.00-1.10) and for mothers born in Africa (AOR 4.29; 95% CI 1.36-13.5) or of unrecorded birthplace (AOR 3.29; 95% CI 1.38-7.85). Breastfeeding duration is traditionally poor in low-income, black populations in the United States. Among a predominantly low-income and black population giving birth at a US Baby-Friendly hospital, breastfeeding rates at 6 months were comparable to the overall US population.

A randomized, controlled trial of the effectiveness of community-based case management in insuring uninsured Latino children.

BACKGROUND: Lack of health insurance adversely affects children's health. Eight million US children are uninsured, with Latinos being the racial/ethnic group at greatest risk for being uninsured. A randomized, controlled trial comparing the effectiveness of various public insurance strategies for insuring uninsured children has never been conducted. OBJECTIVE: To evaluate whether case managers are more effective than traditional methods in insuring uninsured Latino children. DESIGN: Randomized, controlled trial conducted from May 2002 to August 2004. SETTING AND PARTICIPANTS: A total of 275 uninsured Latino children and their parents were recruited from urban community sites in Boston. INTERVENTION: Uninsured children were assigned randomly to an intervention group with trained case managers or a control group that received traditional Medicaid and State Children's Health Insurance Program (SCHIP) outreach and enrollment. Case managers provided information on program eligibility, helped families complete insurance applications, acted as a family liaison with Medicaid/SCHIP, and assisted in maintaining coverage. MAIN OUTCOME MEASURES: Obtaining health insurance, coverage continuity, the time to obtain coverage, and parental satisfaction with the process of obtaining insurance for children were assessed. Subjects were contacted monthly for 1 year to monitor outcomes by a researcher blinded with respect to group assignment. RESULTS: One hundred thirty-nine subjects were assigned randomly to the intervention group and 136 to the control group. Intervention group children were significantly more likely to obtain health insurance (96% vs 57%) and had approximately 8 times the adjusted odds (odds ratio: 7.78; 95% confidence interval: 5.20-11.64) of obtaining insurance. Seventy-eight percent of intervention group children were insured continuously, compared with 30% of control group children. Intervention group children obtained insurance significantly faster (mean: 87.5 vs 134.8 days), and their parents were significantly more satisfied with the process of obtaining insurance. CONCLUSIONS: Community-based case managers are more effective than traditional Medicaid/SCHIP outreach and enrollment in insuring uninsured Latino children. Case management may be a useful mechanism to reduce the number of uninsured children, especially among high-risk populations.