New Tools to Speed Research on Parasite-Caused Diarrheal Disease

NIAID Now | June 25, 2019

Segment of mouse small intestine showing infection (red dots) with Cryptosporidium tyzzeri parasites.

Credit: Muthugapatti Kandasamy, Adam Sateriale, and Boris Striepen

The waterborne intestinal parasite Cryptosporidium is the second leading cause of moderate-to-severe diarrheal disease worldwide. The parasite has been long recognized as an opportunistic infection in people whose immune systems are compromised by chronic infections, such as HIV. More recently, awareness has grown about Cryptosporidium’s profound impact on the health and well-being of very young children with intact immune systems. However, efforts to address this public health challenge are hampered by the lack of a robust animal model of the infection and by the difficulty of growing the parasite in the lab, making it hard for researchers to test candidate drugs or vaccines.

That may now be changing, thanks to research by two independent groups of NIAID-supported scientists. One team, led by Boris Striepen, Ph.D., of the University of Pennsylvania School of Veterinary Medicine, developed a mouse model of Cryptosporidium infection that mimics human disease and used it to study both the course of a natural infection and the animals’ response to an experimental vaccine.

The research began with a rather unglamorous task—collecting mouse droppings from farms around Athens, Georgia. Thirty percent of the collected fecal pellets yielded a previously unknown Cryptosporidium species (C. tyzzeri), the full genome of which the team sequenced. They determined that the mouse version of Cryptosporidium was genetically very similar to species that infect people. The investigators added a firefly gene to the C. tyzzeri parasites, causing them to produce light, which allowed the team to track how the parasites moved into mouse small intestines and established infection.

Dr. Striepen and his colleagues introduced the modified C. tyzzeri into groups of lab mice with fully functioning immune systems as well as into mice genetically engineered to lack certain immune system cells or components. By comparing the course of infection in the two kinds of mice, the team determined that two immune components—T cells and the infection-fighting interferon-gamma protein—are critical to the animals’ ability to control the parasite. The findings in mice mirror what is known about the human response to Cryptosporidium infection. Knowledge about which aspects of immunity are essential to parasite control opens new avenues to developing vaccines, which could provide protection against infection by stimulating similar immune responses without exposure to the parasite itself.

In another series of experiments, the team investigated whether a vaccine containing live, but weakened, C. tyzzeri oocysts—the spore-like infective form of the parasite—would protect mice from subsequent infection with whole parasites. Mice with intact immune systems were protected by the vaccine, while mice lacking T cells or the ability to produce interferon-gamma were not, underscoring the importance of those two immune factors in controlling infection.

“In future work we will use this new model to understand the mechanistic basis of immunity to Cryptosporidium and to test candidate drugs and vaccines,” said Dr. Striepen. 

A second key advance in Cryptosporidium research was announced by NIAID-supported investigators from Washington University School of Medicine in St. Louis. The group, led by L. David Sibley, Ph.D., achieved a long-sought goal of growing all life-stages of the parasite in living cells. Previously, researchers had to collect parasite oocysts from infected calves and grow them in mouse cells or in human cell lines derived from cancerous tissue. Invariably, the parasites would die after a few days and would not progress through their complete life-cycle.

With Washington University School of Medicine colleague Thaddeus S. Stappenbeck, M.D., Ph.D., Dr. Sibley and his team used mouse intestinal stem cells to grow “mini-guts” in lab dishes. These clusters contain all the cell types and structural complexity of a full-sized intestine and yielded an environment where parasite oocysts thrived. For the first time, every stage of the parasites’ full life-cycle can be studied in the lab, noted the researchers.

So far, the technique has been used to grow one species of Cryptosporidium (C. parvum), which infects children and the young of other mammals. A closely related species (C. hominis) infects only humans and has proved challenging to culture in the lab. Developing a system to grow all the life-stages of C. hominis is an important goal of future work in Dr. Sibley’s lab, he said.

“There’s a lot of basic research that still needs to be done,” said Dr. Sibley. “But this system provides an important path forward. We can now use genetic approaches to study the role of individual genes and thereby identify important targets for improved therapies.”

Both papers on Cryptosporidium were published on June 20, 2019, in Cell Host & Microbe.

Learn more about NIAID research support for other neglected tropical diseases.

References: A Sateriale et al. A genetically tractable, natural mouse model of cryptosporidiosis offers insights into host protective immunity. Cell Host & Microbe DOI: 10.1016/j.chom.2019.05.006 (2019).

G Wilke et al. A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability. Cell Host & Microbe DOI:10.106/j.chom.2019.05.007 (2019).

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