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In vitro culture of human-infecting Encephalitozoon spp. for genome sequencing with minimal host contaminant
Anne Caroline Mascarenhas Dos Santos1, Pingdong Liang1, Oscar X Juárez1
1Biology Department, Illinois Institute of Technology, Chicago, Illinois, United States of America.
Abstract:
Microsporidia are obligate intracellular parasites infecting a wide range of hosts, including humans. They can cause severe infectious diseases if left untreated, particularly in immunocompromised individuals. The propagation of the human-infecting microsporidian species in vitro is essential for generating sufficient material for genomics studies, yet existing protocols often lack detail, accessibility, or strategies to minimize host DNA contamination. Here, we present an optimized, reproducible protocol for culturing Encephalitozoon spp. in human foreskin fibroblasts (HFF-1), designed to produce high-yield and high-quality genomic DNA with minimal host DNA for downstream sequencing experiments. In one month, our method yielded approximately one billion spores, which were purified using mechanical disruption, filtration, detergent treatment, and DNase I treatment to remove free host DNA. The reduction of host DNA was validated through PCR, and next-generation sequencing - with Illumina, PacBio and Oxford Nanopore - revealing that 83-97% of the reads mapped to microsporidian genomes. This protocol enabled the generation of the first telomere-to-telomere assemblies for E. cuniculi, E. hellem, and E. intestinalis. Our workflow provides a robust framework for producing microsporidian genomic material suitable for advanced genomics applications, with potential adaptability to other intracellular pathogens.
Insights
We developed a new protocol to culture microsporidia in human cells, yielding abundant, pure spores for genomic DNA extraction. This method significantly reduces host DNA contamination, enabling high-quality sequencing and assembly of microsporidian genomes.
Area of Science:
- Microbiology
- Genomics
- Parasitology
Background:
- Microsporidia are obligate intracellular parasites that infect diverse hosts, including humans.
- Infections can cause severe disease, especially in immunocompromised individuals.
- Existing in vitro propagation methods for human-infecting species are often inadequate for genomics studies due to low yield and host DNA contamination.
Purpose of the Study:
- To develop an optimized and reproducible protocol for culturing Encephalitozoon spp. in human foreskin fibroblasts (HFF-1).
- To generate high-yield, high-quality genomic DNA with minimal host DNA contamination for downstream sequencing.
- To enable the generation of complete microsporidian genome assemblies.
Main Methods:
- Culturing Encephalitozoon spp. in HFF-1 cells.
- Purification of microsporidian spores using mechanical disruption, filtration, detergent treatment, and DNase I treatment.
- Validation of host DNA reduction using PCR and next-generation sequencing (Illumina, PacBio, Oxford Nanopore).
Main Results:
- The protocol yielded approximately one billion spores in one month.
- 83-97% of sequencing reads mapped to microsporidian genomes after purification.
- Enabled the first telomere-to-telomere genome assemblies for E. cuniculi, E. hellem, and E. intestinalis.
Conclusions:
- This optimized protocol provides a robust framework for producing microsporidian genomic material suitable for advanced genomics.
- The method significantly minimizes host DNA contamination, facilitating high-quality genome sequencing.
- The workflow is potentially adaptable for culturing and studying other intracellular pathogens.
