Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Abstract

Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum. Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of hostparasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites. KEYWORDS 3D model, Cryptosporidium, in vitro culture, intestinal epithelial cells C ryptosporidium spp. are apicomplexan parasites of global importance that cause diarrheal disease in humans and animals worldwide (1, 2). Though immunocompetent individuals experience self-limiting or asymptomatic infection, immunocompromised hosts, such as untreated patients with HIV infection or AIDS (3) and malnourished children (1, 2) in resource-limited settings, may experience severe diarrhea, wasting, and death. In a recent case-control study, Cryptosporidium was one of four pathogens responsible for moderate to severe diarrhea in children under the age of 5 years and was associated with an increased risk of death in children from 1 to 2 years of age in seven countries in sub-Saharan Africa and South Asia (4). The only FDA-approved drug for the treatment of cryptosporidiosis is nitazoxanide, a drug with broad antiparasitic properties (5). However, this drug is not effective in immunocompromised patients (6) and has not been widely tested in malnourished children in resource-limited countries. Though significant improvements in water purification methods have occurred (7) since the outbreak of waterborne cryptosporidiosis in Milwaukee, WI, in 1993 (8), industrialized countries are seeing increased numbers of cases, largely due to recreational water outbreaks (9).Unlike many other apicomplexan parasites which often require more than one host to complete their life cycles (10), Cryptosporidium completes its entire life cycle in a

A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni

“…In line with our results for C. jejuni and the tissue-engineered 3D model, various other 3D models including those cultured in the rotating wall vessel bioreactor (RWV) or organs-on-achip are promising new tools for infection research and have been applied to study a variety of pathogens, such as S. Typhimurium, Pseudomonas aeruginosa, or Mycobacterium tuberculosis [84][85][86][87][88][89]. For example, 3D models grown in the RWV successfully recapitulated infection outcomes, such as SPI-1 independent invasion of host cells by S. Typhimurium [84], as previously observed in vivo [90].…”

Recent Breakthroughs and Ongoing Limitations in Cryptosporidium Research

“…Enteroids are stem cell-derived 3D structures that can be generated from crypts derived from human intestinal biopsies, can be passaged indefinitely, and are a more physiological alternative to transformed cell lines 28, 29 . This 3D ex vivo model supports robust C. parvum infection and results in the production of oocysts 30 . The use of murine-derived enteroids for C. parvum infection has also been reported 31 .…”

Advanced bioengineering technologies for preclinical research

“…These models mimicked the oxygen gradients existing in vivo in the gut lumen and allowed for the selective survival of bacteria in aerobic, microaerobic and anaerobic conditions [55]. In a follow-up study, this model was used to study the effects of long-term intestinal infection of Cryptosporidium parvum parasite [56]. Three-dimensional microtissues of lung mucosa bearing epithelial and stromal cellular components (fibroblasts) were produced on collagen gels and employed to investigate the role of Staphylococcus aureus exotoxins in tissue pathology associated with pneumonia ( Figure 3(c)) [57,58].…”