Related Experiment Videos
Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal
Katherine Wasson1, Peter A Barry
1Center for Comparative Medicine, School of Veterinary Medicine, University of California, Davis, California 95616, USA. kwasson@ucdavis.edu
Abstract:
Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.
Insights
This study investigated microsporidial spore activation and invasion, finding that Encephalitozoon intestinalis DNA enters host cells regardless of spore viability, but replication is inhibited by heat treatment or albendazole.
Area of Science:
- Microbiology
- Parasitology
- Molecular Biology
Background:
- Microsporidia are obligate intracellular pathogens.
- Human infections often caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis lead to chronic diarrhea.
- Understanding microsporidial spore activation and invasion is crucial for developing treatments.
Purpose of the Study:
- To define the kinetics of in vitro Encephalitozoon intestinalis replication.
- To investigate signals involved in microsporidial spore activation and invasion.
- To evaluate the efficacy of heat treatment and albendazole on E. intestinalis infection.
Main Methods:
- Real-time quantitative PCR was used to quantify parasite gene copy number.
- Segments of small subunit ribosomal RNA and polar tube protein 2 genes were targeted.
- Murine colon carcinoma cells were infected with E. intestinalis spores.
Main Results:
- Parasite DNA was detected in host cells as early as 4 hours post-inoculation.
- Genome replication was completed by 36 hours, with progeny released at 72 hours.
- Heat treatment inhibited replication but not DNA transfer; albendazole suppressed replication.
Conclusions:
- E. intestinalis completes its life cycle within the host cell turnover time.
- Invasion into host cells is independent of spore viability.
- Real-time quantitative PCR is a sensitive method for monitoring microsporidial infections.