Comparative molecular cell biology of phototrophic euglenids and parasitic trypanosomatids sheds light on the ancestor of Euglenozoa

Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual ‘J’ base (β-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear

Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals

Giardia lamblia mitosomes are believed to be vestigial mitochondria which lack a genome. Similar to higher eukaryotes, mitosomal proteins possess either N-terminal or internal mitosomal targeting sequences. To date, some components of the higher eukaryote archetypal mitochondrial protein import apparatus have been identified and characterized in Giardia mitosomes; therefore, it is expected that mitochondrial signals will be recognized by the mitosomal protein import system. To further determine the level of conservation of the Giardia mitosome protein import apparatus, we expressed mitochondrial proteins from higher eukaryotes in Giardia. These recombinant proteins include Tom20 and Tom22; two components of the mitochondrial protein import machinery. Our results indicate that N-terminal mitochondrial targeting sequence is recognized by the mitosomal protein import machinery; however, interestingly the internal mitochondrial targeting sequences of higher eukaryotes are not recognized by the mitosome. Our results indicate that Giardia mitosome protein transport machinery shows differential recognition of higher eukaryotic mitochondria transfer signals, suggesting a divergence of the transport system in G. lamblia. Therefore, our data support the hypothesis that the protein import machinery in Giardia lamblia mitosome is an incomplete vestigial derivative of mitochondria components

Localizing proteins in fixed Giardia lamblia and live cultured mammalian cells by confocal fluorescence microscopy

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes

Modeling long-term host cell-Giardia lamblia interactions in an in vitro co-culture system.

Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions

Pre-embedding double-label immunoelectron microscopy of chemically fixed tissue culture cells

Localization of specific proteins within cells at the nanometer level of resolution is central to understanding how these proteins function in cell processes such as motility and intracellular trafficking. Such localization can be achieved by combining transmission electron microscopy (TEM) with immunogold labeling. Here we describe a pre-embedding, indirect gold immunolabeling approach to localize two different proteins of interest with secondary antibodies labeled with gold particles of different sizes in cells grown on cover slips. In this protocol, the cells are immunolabeled prior to being embedded in an epoxy resin for ultrathin sectioning. The protocol also includes strategies for optimizing the balance between ultrastructure and antigen preservation, steps to minimize nonspecific antibody binding, and steps to optimize antibody penetration

Morphology and viability of Caco-2 cells in media mixes.

<p><b>A</b>) Images of Caco-2 cells grown to confluence in 8-chamber slides and cultured for 1, 2, and 5 days in 100% DMEM, 90% DMEM/10% <i>Giardia</i> media, or 75% DMEM/25% <i>Giardia</i> media. <b>B</b>) Caco-2 cells grown to confluence in 24-well plates and cultured with the media mixes, 100% DMEM, 90/10%, or 75/25%, for 1, 2, 5, and 7 days. Cell viability was determined using trypan blue exclusion with the number of blue cells compared to the total cell number to obtain percent viable. The data are represented as the percent change of experimental values when compared to the time-matched 100% DMEM culture conditions ± SD. (n = 1).</p

Effect of media mixes on MAPK activation in Caco-2 cells.

<p>Caco-2 cells were grown to confluence in 96-well plates, and cultured for 24 and 48 hours in the presence of the media mixes. Activation of stress-related kinases was measured using PACE assays. Activation of ERK (<b>A</b>), JNK (<b>B</b>), and p38 (<b>C</b>) was measured and compared to positive controls (EGF or sorbitol). Values are expressed as the mean fold over basal of 100% DMEM ± SEM. (n = 4).</p

Caspase-3 activity in Caco-2 cells incubated with <i>Giardia</i>.

<p><b>A</b>) Caco-2 cells grown in 6-well inserts or plates to confluence were inoculated with 350,000 parasites/cm²<i>Giardia</i> trophozoites. Caspase-3 activation as a marker for apoptosis was measured at 1 and 5 days using the Abcam Caspase-3 assay kit. Camptothecin (5 µM) was used as an inducer of caspase-3 activation. The data represent the percent change over control ± SEM assayed three times in duplicate. (n = 3) (p = 0.0071) <b>B</b>) Parasite density at 5 days in the plate and insert culture conditions.</p

<i>Giardia</i> trophozoites in the 21-day co-culture.

<p>The co-culture model was assembled as described. <b>A</b>) Live images of the model with parasites attached were obtained at 1 and 21 days. A trophozoite is identified with an arrow. Full size images of the model at day 1, 5, 13, and 21 are provided in the supplemental data (Figures S3–S6). <b>B</b>) <i>Giardia</i> growth curve over 21 days in the co-culture model. Trophozoites removed from the inserts with formononetin treatment were collected and counted with a hemocytometer at 1, 5, 13, and 21 days. Data represents the mean of four individual insert counts ± SD.</p

Effect of formononetin on stress activated kinases in Caco-2 cells.

<p>Caco-2 cells plated at 60,000 cells/ml were grown to confluence in 96-well plates. The cells were treated with control (DMSO) or 10 µM and 40 µM formononetin for 5 minutes. Activation of ERK (<b>A</b>), JNK (<b>B</b>), and p38 (<b>C</b>) were measured by PACE assays. EGF (100 ng/ml) treatment for 15 minutes was used as a positive control for ERK activation. Sorbitol (300 mM) treatment for 15 minutes was used to activate p38 and JNK. Values are expressed as the percent change over control of 100% DMEM ± SEM. (n = 4).</p