A Genetically Tractable Natural Mouse Model of Cryptosporidiosis Offers Insights into Host Protective Immunity

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCryptosporidiosis, initially recognized as an opportunistic infection associated with advanced HIV-AIDS, is now known as a leading cause of diarrheal disease in immunocompetent individuals around the world (, ). Young children are particularly vulnerable to infection with the apicomplexan parasite Cryptosporidium. In the Global Enteric Multicenter Study, the largest study of its kind to assess the etiology of early childhood diarrheal disease in sub-Saharan Africa and southeast Asia, Cryptosporidium was strongly associated with moderate-to-severe diarrhea and death across all study sites (). Children that survive these life-threatening Cryptosporidium infections remain prone to malnutrition and stunted growth, with measurable decreases in height-for-age scores (, , , ). A recent meta-analysis that accounted for growth faltering estimated over 12 million disability adjusted life years attributable to cryptosporidiosis in 2016 alone (). This massive impact on public health is exacerbated by the lack of tools to manage the disease; the current treatment for Cryptosporidium is of limited efficacy and new more potent medicines are still under development (, ) and no vaccine is available to prevent the infection.Cryptosporidiosis in humans is predominantly caused by Cryptosporidium hominis and Cryptosporidium parvum. Epidemiology in children, as well as studies in human volunteers and in different animal species, have shown the development of immunity and suggest that vaccination may prevent the disease (, ), but the lack of animal models has limited vaccine development. Gnotobiotic piglets are susceptible to C. hominis and calves to C. parvum, but large animal models require specialized facilities (, ). Experiments in immunocompetent mice have been performed with Cryptosporidium muris; however, this species differs significantly from those infecting humans: phylogenetically, biochemically, and in the anatomical site of infection (stomach versus the small intestine). Mice are naturally resistant to C. hominis and C. parvum, but can be rendered susceptible to the latter by chemical immune suppression () or genetic immune insufficiency (href="#bib24" rid="bib24" class=" bibr popnode">Griffiths et al., 1998, href="#bib54" rid="bib54" class=" bibr popnode">Mead et al., 1991). These models have been critical to our understanding of innate mechanisms of resistance to Cryptosporidium and affirmed the important role of T cells in parasite control (href="#bib11" rid="bib11" class=" bibr popnode">Borad and Ward, 2010, href="#bib53" rid="bib53" class=" bibr popnode">Mead, 2014). However, because these studies are performed in neonates, immunocompromised or naturally resistant mice, we know little about the mechanisms required for long-lived protection.Robust rodent models with natural progression that replicate human disease have proven transformative for many fields of infection biology (href="#bib50" rid="bib50" class=" bibr popnode">Martina et al., 2003, href="#bib64" rid="bib64" class=" bibr popnode">Ploss et al., 2009). In order to develop such a model for cryptosporidiosis, we isolated a species of Cryptosporidium that naturally colonizes the mouse small intestine. This parasite, Cryptosporidium tyzzeri, genetically resembles those that cause human cryptosporidiosis and causes similar disease pathology. We then use CRISPR-driven genome engineering to create stable transgenic C. tyzzeri that express luminescent and fluorescent reporters. With these transgenic parasites we demonstrate acquired resistance to infection in mice and importantly such resistance can be elicited through vaccination with an attenuated parasite. Moving forward this fully tractable mouse model of cryptosporidiosis will permit rigorous investigation of potential vaccination strategies and allow the dissection of the complex host-pathogen interactions of Cryptosporidium infection.