Advanced imaging of microorganisms, including protists, is challenging due to their small size. Specimen expansion prior to imaging is thus beneficial to increase resolution and cellular details. Here, we present a sample preparation workflow for improved observations of the single-celled eukaryotic pathogen Giardia intestinalis (Excavata, Metamonada). The binucleated trophozoites colonize the small intestine of humans and animals and cause a diarrhoeal disease. Their remarkable morphology includes two nuclei and a pronounced microtubular cytoskeleton enabling cell motility, attachment and proliferation. By use of expansion and confocal microscopy, we resolved in a great detail subcellular structures and organelles of the parasite cell. The acquired spatial resolution enabled novel observations of centrin localization at Giardia basal bodies. Interestingly, non-luminal centrin localization between the Giardia basal bodies was observed, which is an atypical eukaryotic arrangement. Our protocol includes antibody staining and can be used for the localization of epitope-tagged proteins, as well as for differential organelle labelling by amino reactive esters. This fast and simple technique is suitable for routine use without a superresolution microscopy equipment.
Giardia intestinalis is a globally important microbial pathogen with considerable public health, agricultural, and economic burden. Genome sequencing and comparative analyses have elucidated G. intestinalis to be a taxonomically diverse species consisting of at least eight different sub-types (assemblages A-H) that can infect a great variety of animal hosts, including humans. The best studied of these are assemblages A and B which have a broad host range and have zoonotic transmissibility towards humans where clinical Giardiasis can range from asymptomatic to diarrheal disease. Epidemiological surveys as well as previous molecular investigations have pointed towards critical genomic level differences within numerous molecular pathways and families of parasite virulence factors within assemblage A and B isolates. In this study, we explored the necessary machinery for the formation of vesicles and cargo transport in 89 Canadian isolates of assemblage A and B G. intestinalis. Considerable variability within the molecular complement of the endolysosomal ESCRT protein machinery, adaptor coat protein complexes, and ARF regulatory system have previously been reported. Here, we confirm inter-assemblage, but find no intra-assemblage variation within the trafficking systems examined. This variation includes losses of subunits belonging to the ESCRTIII as well as novel lineage specific duplications in components of the COPII machinery, ARF1, and ARFGEF families (BIG and CYTH). Since differences in disease manifestation between assemblages A and B have been controversially reported, our findings may well have clinical implications and even taxonomic, as the membrane trafficking system underpin parasite survival, pathogenesis, and propagation.
- MeSH
- feces parazitologie MeSH
- genomika MeSH
- genotyp MeSH
- Giardia lamblia * MeSH
- giardiáza * parazitologie MeSH
- lidé MeSH
- veřejné zdravotnictví MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Kanada MeSH
Giardia intestinalis, a cosmopolitan gastrointestinal protist, is detected mainly in patients with clinical giardiasis in high-income countries. In contrast, there is very little information on the presence of Giardia in asymptomatic individuals. Therefore, the aim of this study was to determine the presence and prevalence of Giardia in gut-healthy volunteers in the Czech Republic and to perform a comparative evaluation of different diagnostic methods, since Giardia diagnostics is complicated. Our results confirmed that the qPCR method is the most sensitive method for detecting Giardia and revealed a prevalence of 7% (22/296) in asymptomatic individuals. In most cases, the colonization intensity ranged from 10-1-101. A conventional PCR protocol targeting the TPI gene was used to identify the assemblages. However, this protocol had limited sensitivity for Giardia amplification, effectively detecting colonization above an intensity of 104. In addition, Giardia was detected in 19% of the animals, which were closely associated with the study participants. However, due to methodological limitations, zoonotic transmission could not be clearly confirmed. Notably, contact with animals proved to be the only factor that had a significant impact on the incidence of Giardia in gut-healthy humans.
- MeSH
- feces MeSH
- genotyp MeSH
- Giardia lamblia * genetika MeSH
- giardiáza * epidemiologie diagnóza MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- prevalence MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes.
- MeSH
- anaerobióza MeSH
- Giardia lamblia * genetika metabolismus MeSH
- lidé MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- proteiny obsahující železo a síru * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.
- MeSH
- genomika MeSH
- Giardia lamblia * genetika MeSH
- Giardia genetika MeSH
- giardiáza * parazitologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Buffaloes represent an important economic resource for several regions of the world including Romania. In the present study, we examined 104 faecal samples collected from 38 buffalo calves (2-11 weeks old) from household rearing systems in Romania for gastrointestinal parasites. All samples were tested using the saturated salt flotation, McMaster and modified Ziehl-Nielsen staining methods. PCR coupled with sequencing isolates were used to identify assemblages of Giardia lamblia (Kunstler, 1882) and species of Cryptosporidium Tyzzer, 1907. Overall, 33 out of 38 examined buffalo calves were infected with different gastrointestinal parasites: 16 had single infections and 17 had mixed infections with two or three parasites. Species of Eimeria Schneider, 1875 (32/38; 84%) were the most prevalent parasites; eight species were identified according to the oocyst morphology, including the pathogenic E. bareillyi (Gill, Chhabra et Lall, 1963) which was detected for the first time in buffaloes from Romania. The nematodes Toxocara vitulorum (Goeze, 1782) (11/38; 37%) and Strongyloides papillosus (Wedl, 1856) (6/38; 16%) were also detected. Cryptosporidium spp. were found in four (11%) buffalo calves; two of them were molecularly identified as C. ryanae Fayer, Santin et Trout, 2008, and another one clustered in the same clade with C. ryanae, C. bovis Fayer, Santin et Xiao, 2005, and C. xiaoi Fayer et Santin, 2009. Giardia duodenalis assemblage E was also molecularly detected in a single (2.6%) buffalo calf. The presence of other buffaloes in the same barn was identified as a risk factor for infection with T. vitulorum. Our results indicate extensive parasitic infections in buffalo calves from northwestern Romania and underline the necessity of prophylactic treatments for T. vitulorum and E. bareillyi.
- MeSH
- buvoli parazitologie MeSH
- Cryptosporidium * MeSH
- Eimeria * MeSH
- feces parazitologie MeSH
- Giardia lamblia * genetika MeSH
- kryptosporidióza * epidemiologie parazitologie MeSH
- paraziti * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Rumunsko MeSH
Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges' role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia's unconventional actin cytoskeleton has an important role in supporting parasite attachment.
- MeSH
- aktiny metabolismus MeSH
- Giardia lamblia * genetika metabolismus MeSH
- Giardia metabolismus MeSH
- giardiáza * parazitologie MeSH
- paraziti * metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.
- MeSH
- CRISPR-Cas systémy * MeSH
- editace genu metody MeSH
- Giardia lamblia * genetika MeSH
- guide RNA, Kinetoplastida MeSH
- lidé MeSH
- tetraploidie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.
BACKGROUND: The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study. Eukaryotes control mitochondrial inheritance mainly due to the presence of the genetic component; still only little is known about the segregation of mitochondria to daughter cells during cell division. Additionally, anaerobic eukaryotic microbes evolved a variety of genomeless mitochondria-related organelles (MROs), which could be theoretically assembled de novo, providing a distinct mechanistic basis for maintenance of stable mitochondrial numbers. Here, we approach this problem by studying the structure and inheritance of the protist Giardia intestinalis MROs known as mitosomes. RESULTS: We combined 2D stimulated emission depletion (STED) microscopy and focused ion beam scanning electron microscopy (FIB/SEM) to show that mitosomes exhibit internal segmentation and conserved asymmetric structure. From a total of about forty mitosomes, a small, privileged population is harnessed to the flagellar apparatus, and their life cycle is coordinated with the maturation cycle of G. intestinalis flagella. The orchestration of mitosomal inheritance with the flagellar maturation cycle is mediated by a microtubular connecting fiber, which physically links the privileged mitosomes to both axonemes of the oldest flagella pair and guarantees faithful segregation of the mitosomes into the daughter cells. CONCLUSION: Inheritance of privileged Giardia mitosomes is coupled to the flagellar maturation cycle. We propose that the flagellar system controls segregation of mitochondrial organelles also in other members of this supergroup (Metamonada) of eukaryotes and perhaps reflects the original strategy of early eukaryotic cells to maintain this key organelle before mitochondrial fusion-fission dynamics cycle as observed in Metazoa was established.
