Cryptosporidium is a leading cause of severe diarrheal disease around the world. Recent field research points to parasite sex as the source of hypertransmissibility fueling continental sweeps, while new laboratory studies took advantage of Cryptosporidium sex to build powerful experimental tools to understand the parasite and its interaction with the host.
In the United States, Cryptosporidium was responsible for one of the largest recorded disease outbreaks, the 1993 Milwaukee incident that infected >400 000 residents through contaminated drinking water. To this day, half of all US outbreaks associated with recreational water are due to this parasite. Globally, infants and toddlers carry the most severe burden of disease and death, and cryptosporidiosis is a key consequence and cause of chronic malnutrition [1]. There are neither efficient drugs nor vaccines, and cryptosporidiosis is an important yet neglected disease in those most vulnerable.
Multiple Cryptosporidium species and strains infect humans, and this can occur through water and food contamination, human-to-human contact, or zoonotically. Cryptosporidium hominis predominates in urban settings, while rural life and exposure to animals increases the incidence of Cryptosporidium parvum, Cryptosporidium meleagridis, and Cryptosporidium cuniculus, which are transmitted from cattle, poultry, or rabbits, respectively. The pressure to adapt results in rapid evolution and drives host specificity and speciation. However, what defines a species in Cryptosporidium has been the topic of vigorous discussion, as is the extent to which genetic exchange occurs within and across species. New data from the field and the laboratory show that parasite sex enables extensive exchange and promotes adaptation and spread.
The United States is currently experiencing a rise in cryptosporidiosis cases linked to the emergence of a hypertransmissible C. hominis subtype. Using comparative genomics of field isolates, Huang and colleagues [2] tracked the evolution of this parasite and show its origin to be the recombination of strains independently imported from Europe and Africa. Similar studies in Africa and Asia support local anthroponotic transmission of C. hominis and again demonstrate significant recombination fueling admixture and adaptation of potential pathogenesis factors [3, 4]. Recombination was similarly implicated in the evolution of C. parvum. Here the authors point to zoonotic spill over and spillback between humans and animals as a consequence and cause of recombination [5].
Recent laboratory studies investigating the molecular detail of the parasite's life cycle provide mechanistic context for this high recombination frequency. Cryptosporidium has a single host life cycle with all stages residing within the epithelial cells of the intestinal (or respiratory) tract. Live-cell microscopy of infected cultures revealed that the parasite follows an intrinsic developmental program, resulting in sex every 2 days [6]. Deviating from previous models, these studies along with single-cell transcriptional studies demonstrated a stripped-down life cycle of only 3 intracellular stages: asexual, male, and female. Importantly, progression to sex is obligatory, and occurs numerous times over the course of a single infection. The high frequency of mating explains the high level of recombination suggested by the population genetics surveys. Frequent sex thus provides a remarkably efficient avenue for the rapid (and potentially worrisome) spread of genes that confer parasite benefit, including transmissibility, virulence, or drug resistance.
What are the boundaries of sex? Host specificity governs the probability of encounter among different parental parasites and thus is a likely isolating factor. However, sexual isolation through gamete or meiotic incompatibility may generate additional boundaries. To rigorously test this, we established an experimental model to conduct genetic crosses in Cryptosporidium [7]. We developed phenylalanyl transfer RNA synthetase as a new selectable marker gene in addition to neo. This allowed us to engineer C. parvum parasites with differential drug resistance, which were used to coinfect mice, which were then treated with 2 drugs at once. This straightforward procedure was highly efficient and selected for large numbers of double-drug–resistant recombinant progeny, to the exclusion of parental parasites. Using genetic crosses also enabled more sophisticated genetic engineering, including the isolation of complex conditional gene deletions, and another marker gene, published in 2023 by Hanna et al [8], further extends these possibilities.
Interestingly, recombinant progeny was also obtained when 2 different species, C. parvum, derived from cattle, and Cryptosporidium tyzzeri, derived from mice, were crossed. This demonstrates the feasibility of interspecies crosses, should they meet in a broadly susceptible host (in this case an immunodeficient mouse); however, we note that this was less efficient than crosses within species, and, at least in initial analysis, appeared to result in fewer chromosomal crossovers.
We believe the most significant impact of this technology to lie in the promise of forward genetics. The fundamental mechanisms of Cryptosporidium host-parasite interaction remain largely unknown. Parasite strains that differ in virulence, persistence, and host specificity have been identified, and genetic crosses offer an unbiased road to the ab initio discovery of the genes and mechanisms that underlie this biology. Previous work on the related parasites that cause malaria and toxoplasmosis has demonstrated the power of this approach to make discoveries [9]. This work relied on cloning of individual progeny, followed by painstaking parallel phenotypic and genotypic analyses. Currently, Cryptosporidium cloning by limiting dilution is not feasible. Fortunately, this can be overcome using population-based strategies. Bulk segregant analysis measures the frequency of alleles in populations under selection to identify and map those loci associated with the trait under selection [10]. Thus, any phenomenon of parasite biology for which there is natural variation and that can be experimentally framed as a selection, is now open to mechanistic study.
Understanding parasite sex is at the very heart of understanding Cryptosporidium and how it continuously adapts to its changing hosts. Parasite sex also offers a powerful experimental tool for discovery that we believe to likely be transformational.
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Author notes
Potential conflicts of interest. All authors: No reported conflicts. Both authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
