Life cycle progression and sexual development of the apicomplexan parasite Cryptosporidium parvum

Abstract

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early childhood mortality. Currently there are no fully effective treatments or vaccines available. Parasite transmission occurs through ingestion of oocysts, through either direct contact or contaminated water or food. Oocysts are meiotic spores and the product of parasite sex. Cryptosporidium has a single host lifecycle where both asexual and sexual processes unfold in the intestine of infected hosts. Here we use the ability to genetically engineer Cryptosporidium to make life cycle progression and parasite sex tractable. We derive reporter strains to follow parasite development in culture and infected mice and define the genes that orchestrate sex and oocyst formation through mRNA sequencing of sorted cells. After two days, parasites in cell culture show pronounced sexualization, but productive fertilization does not occur and infection falters. In contrast, in infected mice male gametes successfully fertilize female parasites, leading to meiotic division and sporulation. To rigorously test for fertilization, we devised a two-component genetic crossing assay employing a Cre recombinase activated reporter. Our findings suggest obligate developmental progression towards sex in Cryptosporidium, which has important implications for the treatment and prevention of the infection.

Defining Stage-Specific Activity of Potent New Inhibitors of Cryptosporidium parvum Growth In Vitro

“…Collectively, these assays provide a set of guidelines for evaluating future compounds to define the stages of the life cycle that they affect and for defining time-and concentration-dependent killing. One of the limitations of using transformed cell lines such as HCT-8 cells for growing C. parvum is that the parasites do not proceed past gamont development (20), and parasite numbers decline over time after reaching a peak between 48 and 72 hpi (31,32). Although previous studies have tried to estimate "time-to-kill" rates in response to treatment in HCT-8 cells (8,17), it is not possible to perform classical washout and recovery experiments using such transformed lines due to the fact that replication ceases after a few days.…”

“…2A). In both HCT-8 and ALI cultures, C. parvum undergoes asexual development and formation of gamonts; however, fertilization is blocked in HCT-8 cultures (20), while it proceeds to oocyst development in ALI cultures (22). To determine the stage against which each compound is most active, we developed methods to define the proportion of each stage present at defined time points during infection.…”

“…C. parvum can be propagated for several rounds of asexual growth in a variety of tumor cell lines in vitro, and although it develops into gametocytes, it does not complete the sexual cycle to form oocysts. Thus, only short-term propagation is possible in these systems (20). Recent efforts have developed new platforms to alleviate this restriction and have led to organoid-based systems that use human stem cell-derived cultures to propagate C. parvum and to produce oocysts that are infectious to mice (21).…”

Stage-Specific Long Non-coding RNAs inCryptosporidium parvumas Revealed by Stranded RNA-Seq

“…Type I meronts are thought to be capable of reinvading adjacent cells generating an asexual cycle (Fayer 2008), while Type II meronts contribute to the formation of microgamonts (male form) or macrogamonts (female form) to complete the sexual stages (Bouzid, Hunter et al 2013). Conventional monolayer cell culture does not permit completion of the life cycle much beyond 48 hours post-infection (hpi) ( Figure 1A), for as of yet poorly understood reasons but gametogenesis does occur (Tandel, English et al 2019). The lack of in vitro culture has historically impeded the development of new drugs and vaccines for this medically important parasite.…”

“…Under UV irradiation, C. parvum oocysts have shown a vital stress-induced gene expression response according to microarray data (Zhang, Guo et al 2012). mRNA expression related to gametocyte and oocyst formation were studied using RNA sequencing of sorted cells (Tandel, English et al 2019). Yet, little is known about the regulation of key developmental transitions.…”