The first biennial Cryptosporidium meeting was held on 10–12 March 2024 in Philadelphia, PA, USA. The organizers, Dr Boris Striepen and Dr Christopher Hunter, welcomed more than 130 attendees to the University of Pennsylvania School of Veterinary Medicine. The meeting opened with a panel discussion featuring a diverse group of researchers, clinicians, non-profit and industry partners who offered unique insights into the problems of cryptosporidiosis. Seven research themed sessions (‘Impact of cryptosporidiosis’, ‘Population genetics’, ‘Genomics and new tools for research and translation’, ‘Parasite cell and developmental biology’, ‘Host–parasite interaction and immunity’, ‘Cryptosporidium metabolism and emerging targets’, and ‘Immunity to Cryptosporidium and vaccines’), as well as two poster sessions completed the meeting. A farewell dinner in the domed Asia gallery of the Penn Museum was organized for all the attendees. The meeting was graciously supported by the Bill and Melinda Gates Foundation, the Burroughs Wellcome Fund, Novartis, Zoetis, and several centers and departments of the University of Pennsylvania. In this TrendsTalk, we invited the session chairs to highlight the innovative research and discoveries presented during the inaugural Cryptosporidium meeting.
Wes van Voorhis, Joyce Siwila, Jessica C Kissinger, Natalia Bayona Vásquez, Guy Robinson, Rodrigo Baptista, Asis Khan, Amandine Guérin, Yi-Wei Chang, Zannatun Noor, N Bishara Marzook, Sumiti Vinayak, Sam Arnold, Chelsea Marie, Robert K M Choy, Mattie C Pawlowic, Rajiv S Jumani. Trends Parasitol. 2024 Jun;40(6):431-438. doi: 10.1016/j.pt.2024.04.005
Purpose of review: Here we highlight the significant contribution that genomics-based approaches have had on the field of Cryptosporidium research and the insights these approaches have generated into Cryptosporidium biology and transmission.
Recent findings: There are advances in genomics, genetic manipulation, gene expression, and single-cell technologies. New and better genome sequences have revealed variable sub-telomeric gene families and genes under selection. RNA expression data now include single-cell and post-infection time points. These data have provided insights into the Cryptosporidium life cycle and host-pathogen interactions. Antisense and ncRNA transcripts are abundant. The critical role of the dsRNA virus is becoming apparent.
Summary: The community’s ability to identify genomic targets in the abundant, yet still lacking, collection of genomic data, combined with their increased ability to assess function via gene knock-out, is revolutionizing the field. Advances in the detection of virulence genes, surveillance, population genomics, recombination studies, and epigenetics are upon us.
Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes.
Importance: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.
Sriveny Dangoudoubiyam, Jamie K Norris, Sivaranjani Namasivayam, Rodrigo de Paula Baptista, Naila Cannes do Nascimento, Joseph Camp, Christopher L Schardl, Jessica C Kissinger, Daniel K Howe. mSphere. 2024 May 29:e0011124. doi: 10.1128/msphere.00111-24.
Jessica Kissinger and Dennis Kyle received the Lamar Dodd Creative Research Award during UGA’s Honors Week. The award recognizes established investigators whose overall scholarly body of work has had a major impact on the field of study and has established the investigator’s international reputation as a leader in the field.
Jessica Kissinger, Distinguished Research Professor in the Franklin College of Arts and Sciences’ genetics department and former director of the UGA Institute of Bioinformatics, has focused her interdisciplinary career on the question of how parasites evolve. She has been a driving force behind the groundbreaking effort to create and maintain novel bioinformatics databases covering omics data for hundreds of dangerous pathogens. The Eukaryotic Pathogen, Vector, and Host Informatics Resources knowledgebase (VEuPathDB.org) is an integrated, centralized resource for data mining on more than 500 organisms. Databases searches are free, permitting researchers to gain insights into and test hypotheses that may pave the way for new approaches to treating or preventing diseases such as malaria and Cryptosporidium (a waterborne parasite). Kissinger has used the databases and other bioinformatics tools to make remarkable discoveries.
Dennis E. Kyle, professor of cellular biology and infectious diseases in the Franklin College of Arts and Sciences and the College of Veterinary Medicine, is one of the top parasitologists in the world. Kyle serves as director of the Center for Tropical and Emerging Global Diseases, and some of his most recent work focuses on discovery of new drugs that eliminate dormant vivax malaria that can linger in the liver. His group has discovered new drug series that target the dormant liver stages and is moving these novel therapeutics through preclinical studies. He also works on Naegleria fowleri, a rare but deadly parasite known as “brain-eating amoebae.” More than 97% of people infected with these amoebae die within two weeks. Kyle has conducted research into that pathogen, leading to effective repurposed drugs and the first rapid, sensitive diagnostic method.
Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.
Nigel Yarlett, Mary Morada, Deborah A Schaefer, Kevin Ackman, Elizabeth Carranza, Rodrigo de Paula Baptista, Michael W Riggs, Jessica Kissinger. PLoS Pathog. 2024 Feb 28;20(2):e1011992. doi: 10.1371/journal.ppat.1011992.
Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.
Sebastian Shaw, Ian S Cohn, Rodrigo P Baptista, Guoqin Xia, Bruno Melillo, Fiifi Agyabeng-Dadzie, Jessica C Kissinger, Boris Striepen. Proc Natl Acad Sci USA. 2024 Jan 2;121(1):e2313210120. doi: 10.1073/pnas.2313210120.
The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) is a Bioinformatics Resource Center funded by the National Institutes of Health with additional funding from the Wellcome Trust. VEuPathDB supports >600 organisms that comprise invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Since 2004, VEuPathDB has analyzed omics data from the public domain using contemporary bioinformatic workflows, including orthology predictions via OrthoMCL, and integrated the analysis results with analysis tools, visualizations, and advanced search capabilities. The unique data mining platform coupled with >3000 pre-analyzed data sets facilitates the exploration of pertinent omics data in support of hypothesis driven research. Comparisons are easily made across data sets, data types and organisms. A Galaxy workspace offers the opportunity for the analysis of private large-scale datasets and for porting to VEuPathDB for comparisons with integrated data. The MapVEu tool provides a platform for exploration of spatially resolved data such as vector surveillance and insecticide resistance monitoring. To address the growing body of omics data and advances in laboratory techniques, VEuPathDB has added several new data types, searches and features, improved the Galaxy workspace environment, redesigned the MapVEu interface and updated the infrastructure to accommodate these changes.
Jorge Alvarez-Jarreta, et al. Nucleic Acids Res. 2023 Nov 11:gkad1003. doi: 10.1093/nar/gkad1003
Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.
Sivaranjani Namasivayam, Cheng Sun, Assiatu B Bah, Jenna Oberstaller, Edwin Pierre-Louis, Ronald Drew Etheridge, Cedric Feschotte, Ellen J Pritham, Jessica C Kissinger. Proc Natl Acad Sci U S A. 2023 Nov 7;120(45):e2308569120. doi: 10.1073/pnas.2308569120.
They say what doesn’t kill you makes you stronger. Whoever coined that adage had probably never heard of Plasmodium.
It’s a microscopic parasite, invisible to the naked eye but common in tropical and subtropical regions throughout the world. Each year, millions of people are infected by Plasmodium and exposed to an even more debilitating—and often deadly—disease: malaria.
Malaria is one of the deadliest diseases known to man. It can lead to extreme illness, marked by fever, chills, headaches and fatigue. More than half the world’s population is at risk of contracting the disease, and those who develop relapsing infections suffer a host of associated costs.
Limited educational opportunities and wage loss lead to an often unbreakable cycle of poverty. Vulnerable populations are most at risk.
“When I’m teaching in an endemic area like Africa, it isn’t unusual to find a student who needs to sleep during part of the workshop because they have malaria,” researcher Jessica Kissinger said.
It’s a challenge she and her collaborators in the University of Georgia’s Center for Tropical and Emerging Global Diseases (CTEGD) are trying to combat.
When the Center was established in 1998, there were only a couple of faculty members studying Plasmodium. Now, 25 years later, it has become a world-class powerhouse of multidisciplinary malaria research. Scientists examine various species of the dangerous parasite, studying its life cycle and the mosquito that transmits it.
While Plasmodium seems to have superpowers that allow it to evade detection and resist treatment, CTEGD researchers are working together to innovate and transfer science from the lab to interventions on the ground.
A 50,000-piece puzzle with no edges
Plasmodium is a complex organism, and studying it is like putting together a jigsaw puzzle. Some researchers contribute pieces related to the blood or liver stages of the parasite’s lifecycle, while others provide insights about hosts interactions. One way UGA’s research connects with the global effort to eradicate malaria is PlasmoDb—a resource derived in part from Kissinger’s research that is now part of a host of databases under the umbrella of The Eukaryotic Pathogen, Vector and Host information Resource (VEuPathDB).
“Our group has been able to help many others when their research question crosses into an –omic,” Kissinger said, referring to in-house shorthand for domains like genomics, proteomics and metabolomics.
Kissinger, Distinguished Research Professor of genetics in the Franklin College of Arts & Sciences, became interested in malaria and Plasmodium during her postdoctoral training at the National Institutes of Health (NIH). Working from an evolutionary biology perspective, she’s interested in how the parasite has changed over time.
“I see it as an arms race,” Kissinger said. “I want to understand what moves they have and can make.”
To understand the parasite, you must dive deep into its genetic code.
Kissinger paired her work in Plasmodium genomics with her interest in computing by helping create the database with information from the Plasmodium genome project completed in 2002. The Malaria Host-Pathogen Interaction Center, one of her projects at UGA, was a seven-year, multi-institutional effort funded, in part, by NIH to create data sets that could be used in systems biology of the host-pathogen interaction during the development of disease.
“Wouldn’t it be neat if, from the beginning of infection all the way to cure, you knew everything that was going on in the organism all the time?” Kissinger said, noting the project’s goal.
They generated terabytes of data that, along with data from the global research community, are publicly accessible and reusable through PlasmoDB and other resources.
Being part of a group that is studying so many different aspects of malaria helps put Kissinger’s research into perspective. Now, in addition to understanding the parasite, she also thinks about tools needed to facilitate research from peers.
High-tech solutions rely on basic research
David Peterson, professor of infectious diseases in the College of Veterinary Medicine, noted that low-tech solutions have mitigated malaria’s human costs. He acknowledged, however, that their long-term goals required more.
“We have to acknowledge that low-tech solutions, such as mosquito nets, have saved lives,” Peterson said. “But to develop the high-tech solutions that will one day end malaria, we need basic research.”
Pregnant women are particularly vulnerable to malaria because their existing immunity to malaria fails to protect them during pregnancy. Placental malaria often results in premature birth and low birth weight.
Peterson is interested in a binding protein that allows the parasite to adhere to the placenta. While many P. falciparum parasites have only one gene copy that encodes the placental binding protein, Peterson is investigating Plasmodiumisolates with two or more slightly different copies.
But why isn’t one copy enough?
That is the primary question Peterson is focused on. He wants to understand how Plasmodium uses extra copies to evade the immune system, distinguishing the role of each requires tools that Vasant Muralidharan, associate professor of cellular biology, has.
Muralidharan’s interest began when he contracted malaria himself. Through access to good health care, he made a full recovery, but the pain he endured remained. He wanted to understand this parasite. Even more, he wanted to make an impact with research.
His graduate training focused on biophysics, but soon his interest in Plasmodium resurfaced. He discovered there was a lack of tools to study the parasite on a genetic level.
“It’s like a house of cards, and each card is a gene,” Muralidharan said. “You can remove one and see what happens—does the house fall or remain standing?”
In the days before CRISPR/Cas9, there wasn’t a precise way to remove genes. Muralidharan is among the pioneers of gene-editing techniques in Plasmodium.
Like Peterson, Muralidharan focuses on proteins secreted by the parasite. He studies the largely unknown process that allows the parasite to invade a red blood cell (RBC), replicate and escape. The lack of tools was a major hindrance, so Muralidharan created new ones.
These tools have been used by Muralidharan’s CTEGD and CDC colleagues to see how drugs might fail. Muralidharan’s laboratory can create mutant Plasmodium parasites that become resistant to a particular drug, and genome sequence databases allow researchers to check if that mutant is already circulating in malaria endemic regions.
Building a research bridge to endemic regions
Plasmodium vivax is the predominant malaria parasite in Southeast Asia. It causes “relapsing malaria” during which some parasites go “dormant” after entering the liver instead of reproducing. This phase is a major obstacle for current treatments.
CTEGD Director Dennis Kyle, GRA Eminent Scholar Chair in Antiparasitic Drug Discovery and head of the Department of Cellular Biology, became fascinated with the Plasmodium parasite early in his career, spending time living in Thailand and working in refugee camps where malaria is prevalent.
“When I first got to the refugee camp and saw the situation people were living in, I questioned my decision to become a scientist in the lab instead of becoming a physician,” Kyle said, recalling a camp he worked in that housed about 1,300 kids between the ages of 2 and 15. “There was a guy who was a leader in the group who probably had no more than an early high school education. He said, ‘Look at what you can do—you might generate something that would benefit all of us. The physicians we have in the camp can only work on a few people at a time.’”
Kyle’s laboratory is looking to repurpose medications that have antimalarial properties, a safe way to reduce the development time from lab to clinical use. He’s optimistic we will see a drug treatment that eliminates vivax malaria.
“That’s where UGA is playing a major role,” he said. “The Gates Foundation funded us to develop tools to study the dormant parasite in the liver. And we’ve been successful.”
“We use mouse models to delve into the fundamental host-parasite interactions, which you cannot do practicallyin humans,” Kurup said. “Our understanding of these fundamental processes gives rise to newer and better vaccination approaches and drugs.”
Like other Plasmodium researchers, Joyner became interested in parasites at an early age. During an undergraduate parasitology class, he discovered how little was known about P. vivax. He was already interested in how diseases develop, so for graduate school he focused on the liver stage of vivax malaria. However, it was a difficult task.
“At the time, the technologies weren’t there,” Joyner said. “Dennis was working on his system, but it wasn’t on the scene yet. I changed from studying the parasite to studying the animal model to understand pathogenesis and immunology in humans.”
Joyner joined UGA after completing his postdoctoral training at Emory University, where he developed a non-mouse animal model to study vivax malaria.
“We have to go to [Thailand] where people are infected and collect blood samples and then feed mosquitoes these samples to do the necessary studies,” Kyle said. “That’s been very impactful. We’ve gotten a lot of data out of it, and now with Chet’s model it all can be done under one roof.”
Joyner wants to understand the human immune response with a focus on vaccine development. Building on Muralidharan’s and other researchers’ findings of how the parasite interacts with the RBCs, Joyner’s vaccine program targets a specific protein in the parasite that inhibits the development of immunity.
“My colleagues have shown that if you knock this protein out in the parasite, the immune response in mice is actually great, and we are now working together to evaluate this in non-mouse models.” Joyner said.
Joyner also has collaborated with Belen Cassera, professor of biochemistry, to screen drug compounds. Cassera’s training focused on metabolism to find drug targets. She is particularly interested in how a drug functions.
“If we understand how the drug works, it will help us predict potential side effects in humans,” Cassera said. “We can’t predict everything, but knowing how it works gives you some confidence in whether it will work in humans.”
Cassera is focused on finding drugs that will treat the more lethal Plasmodium falciparum, the predominant species in Africa, which is rapidly becoming resistant to current treatments. Her work is complementary to Kyle’s.
“They run certain assays for the liver-stage infection, and our lab benefits because we want to know if the drug we are developing is specific for the blood stage or can tackle all stages,” Cassera said.
Don’t forget the mosquito
“Malaria is a vector-borne disease transmitted by a mosquito. You need to tackle not only the parasite in the human but also stop its transmission,” Cassera said. “CTEGD is unique because we can study the whole life cycle, including the mosquito.”
Michael Strand, H.M. Pulliam Chair of Entomology in the College of Agricultural and Environmental Sciences and a National Academy of Sciences Fellow, is an expert on parasite-host interactions. Instead of the human host, he is interested in mosquitoes. Recent work indicates blood feeding behavior of mosquitoes strongly affects malaria parasite development while the gut microbiota of mosquitos could lead to new ways to control populations. Having the SporoCore insectory on campus aids his research.
Established in 2020, SporoCore, under the management of Ash Pathak, assistant research scientist in the Department of Infectious Diseases, provides both uninfected and Plasmodium-infected Anopheles stephensi mosquitoes to researchers at UGA and other institutions. Like Joyner’s animal model, the insectory allows for research to be done in the U.S. that would otherwise require field work in an endemic country.
Old-school interventions like mosquito nets, combined with new drug therapies, have reduced the number of malaria deaths, which declined over the last 30 years before rising slightly during the COVID-19 pandemic. Great strides have been made to control and treat malaria—but not enough. New tools, like the ones being developed at CTEGD, are needed to keep pushing malaria’s morbidity and mortality rates in the right direction.
“The hard part—what can’t be done easily with the tools we already have—is being done,” Kyle said. “We just need new tools, which is one of the things that our center is really a leader in.”
Parasitic diseases caused by kinetoplastid parasites are a burden to public health throughout tropical and subtropical regions of the world. TriTrypDB (https://tritrypdb.org) is a free online resource for data mining of genomic and functional data from these kinetoplastid parasites and is part of the VEuPathDB Bioinformatics Resource Center (https://veupathdb.org). As of release 59, TriTrypDB hosts 83 kinetoplastid genomes, nine of which, including Trypanosoma brucei brucei TREU927, Trypanosoma cruzi CL Brener and Leishmania major Friedlin, undergo manual curation by integrating information from scientific publications, high-throughput assays and user submitted comments. TriTrypDB also integrates transcriptomic, proteomic, epigenomic, population-level and isolate data, functional information from genome-wide RNAi knock-down and fluorescent tagging, and results from automated bioinformatics analysis pipelines. TriTrypDB offers a user-friendly web interface embedded with a genome browser, search strategy system and bioinformatics tools to support custom in silico experiments that leverage integrated data. A Galaxy workspace enables users to analyze their private data (e.g., RNA-sequencing, variant calling, etc.) and explore their results privately in the context of publicly available information in the database. The recent addition of an annotation platform based on Apollo enables users to provide both functional and structural changes that will appear as ‘community annotations’ immediately and, pending curatorial review, will be integrated into the official genome annotation.
Achchuthan Shanmugasundram, David Starns, Ulrike Böhme, Beatrice Amos, Paul A Wilkinson, Omar S Harb, Susanne Warrenfeltz, Jessica C Kissinger, Mary Ann McDowell, David S Roos, Kathryn Crouch, Andrew R Jones. PLoS Negl Trop Dis. 2023 Jan 19;17(1):e0011058. doi: 10.1371/journal.pntd.0011058.