Intron-containing β-tubulin transcripts in Cryptosporidium parvum cultured in vitro

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1191-1195 ◽  
Author(s):  
Xiaomin Cai ◽  
Cheryl A. Lancto ◽  
Mitchell S. Abrahamsen ◽  
Guan Zhu
Keyword(s):  
Life Cycle ◽  
Cryptosporidium Parvum ◽  
Late Life ◽  
Host Cells ◽  
Intron Splicing ◽  
Transcript Processing ◽  
Life Cycle Stages ◽  
Tubulin Mrna

The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the β-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the β-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced β-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12–72 h), at 48–72 h post-infection unprocessed β-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing β-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest of C. parvum development in vitro. This unusual observation prompts speculation that the abnormal intron-splicing of β-tubulin transcripts may be one of the factors preventing complete development of this parasite in vitro. Furthermore, the presence of both processed and unprocessed introns in β-tubulin transcripts in vitro may provide a venue for studying overall mechanisms for intron-splicing in this parasite.

Morphological characterization ofCryptosporidium parvumlife-cycle stages in anin vitromodel system

Parasitology ◽  
2009 ◽  
Vol 137 (1) ◽  
pp. 13-26 ◽  
Author(s):  
H. BOROWSKI ◽  
R. C. A. THOMPSON ◽  
T. ARMSTRONG ◽  
P. L. CLODE
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Protozoan Parasite ◽  
Morphological Characterization ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Life Cycle Stages ◽  
Complete Life Cycle ◽  
Behavioural Characteristics

SUMMARYCryptosporidium parvumis a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the potential to cause severe enteric disease. We describe the complete life cycle ofC. parvumin anin vitrosystem. Infected cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well as determining their time of occurrence after inoculation. Numerous stages ofC. parvumand their behaviour have been visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host interactions and the effect ofC. parvumon host cells were also visualized. An improved understanding of the parasite's biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.

In situ observations of Akashiwo sanguinea (Dinophyceae) displaying life cycle stages during blooms in a subtropical estuary

2017 ◽  
Vol 60 (6) ◽  
Author(s):  
Susan Badylak ◽  
Edward J. Phlips ◽  
Ashley Loren Mathews ◽  
Karen Kelley
Keyword(s):  
Life Cycle ◽  
Algal Bloom ◽  
Harmful Algal Bloom ◽  
Life Cycle Stages ◽  
Subtropical Estuary ◽  
In Situ Observations ◽  
Akashiwo Sanguinea

AbstractThis study reports on the harmful algal bloom (HAB) dinoflagellate

scholarly journals Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma

2020 ◽  
Vol 8 (5) ◽  
pp. 715 ◽  
Author(s):  
Samantha Gunasekera ◽  
Alireza Zahedi ◽  
Mark O’Dea ◽  
Brendon King ◽  
Paul Monis ◽  
...  
Keyword(s):  
Life Cycle ◽  
Host Cells ◽  
Protozoan Parasites ◽  
Future Directions ◽  
In Vivo Models ◽  
Severe Diarrhea ◽  
Transformed Cell Lines ◽  
Gnotobiotic Piglets

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine

1998 ◽  
Vol 44 (12) ◽  
pp. 1154-1160 ◽  
Author(s):  
Christian Chauret ◽  
Kerry Nolan ◽  
Ping Chen ◽  
Susan Springthorpe ◽  
Syed Sattar
Keyword(s):  
Cryptosporidium Parvum ◽  
Environmental Fate ◽  
Water Environment ◽  
Water Disinfection ◽  
Ottawa River ◽  
In Situ Conditions ◽  
Cryptosporidium Parvum Oocysts ◽  
St Lawrence River

Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2-µm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3; pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log10·day-1) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters.Key words: Cryptosporidium, survival, disinfection, biological antagonism.

Nippostrongylus brasiliensis: the effect of mitochondnal inhibitors on life-cycle stages

Parasitology ◽  
1984 ◽  
Vol 88 (1) ◽  
pp. 163-177 ◽  
Author(s):  
M. Fry ◽  
D. C. Jenkins
Keyword(s):  
Life Cycle ◽  
Electron Transport ◽  
Oxidative Phosphorylation ◽  
Adult Worm ◽  
Nippostrongylus Brasiliensis ◽  
Free Living ◽  
Mitochondrial Inhibitors ◽  
Life Cycle Stages ◽  
Transport Inhibitors

SUMMARYThe effects of mitochondrial inhibitors on the in vitro development of Nippostrongylus brasiliensis have been studied in free-living and parasitic life-cycle stages. Mitochondrial inhibitors were chosen as being representative of established electron transport inhibitors and oxidative phosphorylation inhibitors and uncouplers of the classical mammalian respiratory chain. All mitochondrial inhibitors tested were highly effective in killing or retarding development of free-living stages of N. brasiliensis. Free-living stages were particularly susceptible to such inhibitors upon hatching of embryonated eggs to 1st-stage larvae. Concentrations of inhibitors effective against free-living stages were consistent with their level of inhibition against isolated mitochondria from embryonated eggs and 3rd-stage infective larvae. Results suggest an absolute requirement in the development of free-living stages for the mammalian-like respiratory chain and associated oxidative phosphorylation. Electron transport inhibitors were effective in retarding at least the initial development of 4th-stage larvae to adults, but only antimycin A and azide produced a lasting effect leading to worm death. Oxidative phosphorylation inhibitors and uncouplers were ineffective against developing parasitic stages of N. brasiliensis. Experiments on whole-worm respiration indicated that most electron transport inhibitors were able to penetrate the adult worm, but oxidative phosphorylation inhibitors were without effect on whole-worm respiration. Results suggest that the mammalian-like electron transport chain is a necessary requirement to adult N. brasiliensis, but oxidative phosphorylation in the adult worm may not be required for development and survival in vitro although it could be necessary to support the parasite in vivo.

scholarly journals Role of CpSUB1, a Subtilisin-Like Protease, in Cryptosporidium parvum Infection In Vitro

2009 ◽  
Vol 8 (4) ◽  
pp. 470-477 ◽  
Author(s):  
Jane W. Wanyiri ◽  
Patsharaporn Techasintana ◽  
Roberta M. O'Connor ◽  
Michael J. Blackman ◽  
Kami Kim ◽  
...  
Keyword(s):  
Serine Protease ◽  
Cryptosporidium Parvum ◽  
Protease Activity ◽  
Diarrheal Disease ◽  
Genomic Sequence ◽  
Host Cells ◽  
Pcr Analysis ◽  
Apical Region ◽  
Gene Encoding

ABSTRACTThe apicomplexan parasiteCryptosporidiumis a significant cause of diarrheal disease worldwide. Previously, we reported that aCryptosporidium parvumsubtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity inC. parvumlysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in theC. parvumgenome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA fromC. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, forC. parvumlysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 byC. parvumlysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity inC. parvumand for processing of gp40/15. Importantly, the recombinant prodomain inhibitedC. parvuminfection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

scholarly journals Cryptosporidium parvum Pyruvate Kinase Inhibitors With in vivo Anti-cryptosporidial Efficacy

2022 ◽  
Vol 12 ◽  
Author(s):  
Shahbaz M. Khan ◽  
Xuejin Zhang ◽  
William H. Witola
Keyword(s):  
Pyruvate Kinase ◽  
Cryptosporidium Parvum ◽  
Diarrheal Disease ◽  
Severe Disease ◽  
The United States ◽  
Metabolic Energy ◽  
Host Cells ◽  
Infection Model

Cryptosporidium parvum is a highly prevalent protozoan parasite that causes a diarrheal disease in humans and animals worldwide. Thus far, the moderately effective nitazoxanide is the only drug approved by the United States Food and Drug Administration for treating cryptosporidiosis in immunocompetent humans. However, no effective drug exists for the severe disease seen in young children, immunocompromised individuals and neonatal livestock. C. parvum lacks the Krebs cycle and the oxidative phosphorylation steps, making it dependent solely on glycolysis for metabolic energy production. Within its glycolytic pathway, C. parvum possesses two unique enzymes, the bacterial-type lactate dehydrogenase (CpLDH) and the plant-like pyruvate kinase (CpPyK), that catalyze two sequential steps for generation of essential metabolic energy. We have previously reported that inhibitors of CpLDH are effective against C. parvum, both in vitro and in vivo. Herein, we developed an in vitro assay for the enzymatic activity of recombinant CpPyK protein and used it to screen a chemical compound library for inhibitors of CpPyK’s activity. The identified inhibitors were tested (at non-toxic concentrations) for efficacy against C. parvum using in vitro assays, and an in vivo mouse infection model. We identified six CpPyK inhibitors that blocked in vitro growth and proliferation of C. parvum at low micromolar concentrations (EC50 values ranging from 10.29 to 86.01 μM) that were non-toxic to host cells. Among those six compounds, two (NSC252172 and NSC234945) were found to be highly efficacious against cryptosporidiosis in immunocompromised mice at a dose of 10 mg/kg body weight, with very significant reduction in parasite load and amelioration of intestinal pathologies. Together, these findings have unveiled inhibitors for an essential molecular target in C. parvum and demonstrated their efficacy against the parasite in vitro and in vivo. These inhibitors are, therefore, potential lead-compounds for developing efficacious treatments for cryptosporidiosis.

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