Genome assembly impacts annotation quality, gene family member estimates, and genetic variation analyses.
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.
Fig 5. TgFBXL2 localizes to a perinucleolar compartment.
Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages require substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct perinucleolar sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNAseq data show that TgFBXL2 conditional depletion induces the expression of stage-specific genes including a a large cohort of genes necessary for sexual commitment. Together, these data suggest that TgFBXL2 is a latent guardian of stage specific gene expression in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of development.
Carlos G Baptista, Sarah Hosking, Elisabet Gas-Pascual, Loic Ciampossine, Steven Abel, Mohamed-Ali Hakimi, Victoria Jeffers, Karine Le Roch, Christopher M West, Ira J Blader. PLoS Pathog. 2024 May 30;20(5):e1012269. doi: 10.1371/journal.ppat.1012269.
Radical cure of Plasmodium vivax malaria must include elimination of quiescent ‘hypnozoite’ forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.
S. P. Maher, M. A. Bakowski, A. Vantaux, E. L. Flannery, C. Andolina, M. Gupta, Y. Antonova-Koch, M. Argomaniz, M. Cabrera-Mora, B. Campo, A. T. Chao, A. K. Chatterjee, W. T. Cheng, E. Chuenchob, C. A. Cooper, K. Cottier, M. R. Galinski, A. Harupa-Chung, H. Ji, S. B. Joseph, T. Lenz, S. Lonardi, J. Matheson, S. A. Mikolajczak, T. Moeller, A. Orban, V. Padín-Irizarry, K. Pan, J. Péneau, J. Prudhomme, C. Roesch, A. A. Ruberto, S. S. Sabnis, C. L. Saney, J. Sattabongkot, S. Sereshki, S. Suriyakan, R. Ubalee, Y. Wang, P. Wasisakun, J. Yin, J. Popovici, C. W. McNamara, C. J. Joyner, F. Nosten, B. Witkowski, K. G. Le Roch, D. E. Kyle. 2024. eLife13:RP98221, https://doi.org/10.7554/eLife.98221.1
Fig 1 Progression of the schizont stages during the intracellular development of Sarcocystis neurona expressing yellow fluorescent protein. Post-invasion, the merozoites convert into schizonts that progressively develop into early-, mid-, and late-schizonts while undergoing a form of asexual reproduction called endopolygeny. In the final step in endopolygeny, the mature schizont forms 64 haploid merozoites fully equipped to egress and invade new host cells. S. neurona, Sn; DAPI-stained host cell nuclei, HCN.
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.
The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.
Rudo Kieft, Yang Zhang, Haidong Yan, Robert J Schmitz, Robert Sabatini. Nucleic Acids Res. 2024 May 23:gkae392. doi: 10.1093/nar/gkae392.
Background: Like other oviparous organisms, the gonotrophic cycle of mosquitoes is not complete until they have selected a suitable habitat to oviposit. In addition to the evolutionary constraints associated with selective oviposition behavior, the physiological demands relative to an organism’s oviposition status also influence their nutrient requirement from the environment. Yet, studies that measure transmission potential (vectorial capacity or competence) of mosquito-borne parasites rarely consider whether the rates of parasite replication and development could be influenced by these constraints resulting from whether mosquitoes have completed their gonotrophic cycle.
Methods: Anopheles stephensi mosquitoes were infected with Plasmodium berghei, the rodent analog of human malaria, and maintained on 1% or 10% dextrose and either provided oviposition sites (‘oviposited’ herein) to complete their gonotrophic cycle or forced to retain eggs (‘non-oviposited’). Transmission potential in the four groups was measured up to 27 days post-infection as the rates of (i) sporozoite appearance in the salivary glands (‘extrinsic incubation period’ or EIP), (ii) vector survival and (iii) sporozoite densities.
Results: In the two groups of oviposited mosquitoes, rates of sporozoite appearance and densities in the salivary glands were clearly dependent on sugar availability, with shorter EIP and higher sporozoite densities in mosquitoes fed 10% dextrose. In contrast, rates of appearance and densities in the salivary glands were independent of sugar concentrations in non-oviposited mosquitoes, although both measures were slightly lower than in oviposited mosquitoes fed 10% dextrose. Vector survival was higher in non-oviposited mosquitoes.
Conclusions: Costs to parasite fitness and vector survival were buffered against changes in nutritional availability from the environment in non-oviposited but not oviposited mosquitoes. Taken together, these results suggest vectorial capacity for malaria parasites may be dependent on nutrient availability and oviposition/gonotrophic status and, as such, argue for more careful consideration of this interaction when estimating transmission potential. More broadly, the complex patterns resulting from physiological (nutrition) and evolutionary (egg-retention) trade-offs described here, combined with the ubiquity of selective oviposition behavior, implies the fitness of vector-borne pathogens could be shaped by selection for these traits, with implications for disease transmission and management. For instance, while reducing availability of oviposition sites and environmental sources of nutrition are key components of integrated vector management strategies, their abundance and distribution are under strong selection pressure from the patterns associated with climate change.
My name is Grace Vick and I am a 4th year infectious diseases PhD candidate in Vasant Muralidharan’s lab. I’m originally from North Carolina and received my Bachelor’s of Science in Biology from Western Carolina University. After graduating undergraduate, I completed an internship at the Defense Forensic Science Center doing forensic biology research. After that, I spent 2 years as an ORISE Fellow at the Centers for Disease Control and Prevention, studying and identifying genetic markers of multi-drug resistant strains of Neisseria gonorrhoeae. I came straight to UGA through the ILS program after my fellowship at CDC.
What made you want to study science?
Ever since I was little, I’ve always spent a lot of time being outside in nature and enjoyed figuring out the intricacies of how things work. During my undergraduate, I was able to explore the different areas of science and found the molecular biology of genetics to be an interesting field that is highly translatable and still vastly unknown. After I spent a few years gaining lab experience and an appreciation for the public health concerns of infectious diseases at the CDC, I knew I wanted to pursue a PhD in that field which brought me to UGA.
Why did you choose UGA?
My experience at the CDC offered the opportunity to learn about diseases and public health issues across all sectors and countries, which led me to learn more about parasitic diseases. Previously, I knew nothing about these diseases but as I learned more about their complex and fascinating life cycles and how these diseases of poverty impact people around the world, I was captivated by this research. Because I was really interested in spending my PhD studying infectious and parasitic diseases, I found out about the CTEGD at UGA and that is what brought me here. The CTEGD is a really wonderful environment for trainees to be exposed to exciting and diverse parasitology research, and I’ve really enjoyed my experience here.
What is your research focus and why did you choose it?
Our lab works on the deadliest form of malaria, Plasmodium falciparum. P. falciparum kills over half a million people each year, with the majority of those deaths being children under the age of 5. Our lab is interested in understanding the molecular mechanisms that are essential to asexual blood stage of this parasite. My work specifically focuses on determining the role of previously unknown proteins that we have discovered are essential for asexual stage invasion of merozoites into host red blood cells. Using a combination of genetic engineering, molecular, and cellular biology techniques, I aim to determine the molecular function of these proteins in the human asexual stage invasion of red blood cells.
Have you received any awards or honors?
In addition to receiving the NIH T32 Predoctoral Fellowship, I have been invited to present at multiple national and international conferences such as Molecular Parasitology Meeting in Massachusetts and Molecular Approaches in Malaria in Lorne, Australia where I won a poster award.
What are your career goals?
When I graduate with my doctoral degree, I hope to either join governmental research or the industry sector. If I decided to head into governmental work, I would choose a career at the CDC where I could continue working in the parasitology research field and apply current public health policies to the international parasitology field. If I decide to join the biomedical industry sector, I would want to work in Research and Design at a company that designs therapeutics and diagnostics for disease prevention and treatment.
What do you hope to do for your capstone experience?
I would really love to experience fieldwork in a malaria-endemic region. I think having the experience of meeting people and learning firsthand how this disease affects millions of people every day would be very eye-opening for me since I have only seen the lab side of malaria. The ability to experience fieldwork would give me a broader experience with how malaria is researched and treated outside of the lab environment and in rural lab environments. I would love to visit Africa or South East Asia to conduct fieldwork in a malaria-endemic environment.
What is your favorite thing about Athens?
Obviously, I love the food in Athens! I love going downtown to grab food and drinks on the weekend. Otherwise, I enjoy getting out and exploring the green spaces and parks that Athens has to offer such as Sandy Creek and the North Oconee Greenway with my husband and dog.
Any advice for a student interested in this field?
I would say the best advice is to read and soak up as much as you can about parasitology both before you get into the field and after. A lot of research has overlap between different parasites and it’s helpful to know about other parasitic diseases that might not be your main focus. Plus, parasites are fun! 🙂 My other advice in general for starting graduate school is to always reach out to students in labs you’re interested in joining. Students are pretty much always willing to help give clear insight into lab dynamics, mentorship of the PI, and generally how life working in that lab is. That information is all really helpful to know when choosing which lab to join!
Support trainees like Grace by giving today to the Center for Tropical & Emerging Global Diseases.
Figure 1. The gap in our understanding of how hepatocytes eliminate Plasmodium.
The Plasmodium parasites that cause malaria undergo asymptomatic development in the parenchymal cells of the liver, the hepatocytes, prior to infecting erythrocytes and causing clinical disease. Traditionally, hepatocytes have been perceived as passive bystanders that allow hepatotropic pathogens such as Plasmodium to develop relatively unchallenged. However, now there is emerging evidence suggesting that hepatocytes can mount robust cell-autonomous immune responses that target Plasmodium, limiting its progression to the blood and reducing the incidence and severity of clinical malaria. Here we discuss our current understanding of hepatocyte cell-intrinsic immune responses that target Plasmodium and how these pathways impact malaria.
Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor “domesticated endogenous viruses” (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs). Large quantities of DNA-containing IV virions are produced in ovary calyx cells during the pupal and adult stages of female wasps. Females parasitize host insects by injecting eggs and virions into the body cavity. After injection, virions rapidly infect host cells which is followed by expression of IV genes that promote the successful development of wasp offspring. IV genomes consist of two components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus that produce virions. In this study, we generated a chromosome-scale genome assembly for Hyposotor didymator that harbors H. didymator ichnovirus (HdIV). We identified a total of 67 HdIV loci that are amplified in calyx cells during the wasp pupal stage. We then focused on an HdIV gene, U16, which is transcribed in calyx cells during the initial stages of replication. Sequence analysis indicated that U16 contains a conserved domain in primases from select other viruses. Knockdown of U16 by RNA interference inhibited virion morphogenesis in calyx cells. Genome-wide analysis indicated U16 knockdown also inhibited amplification of HdIV loci in calyx cells. Altogether, our results identified several previously unknown HdIV loci, demonstrated that all HdIV loci are amplified in calyx cells during the pupal stage, and showed that U16 is required for amplification and virion morphogenesis.
Ange Lorenzi, Fabrice Legeai, Véronique Jouan, Pierre-Alain Girard, Michael R Strand, Marc Ravallec, Magali Eychenne, Anthony Bretaudeau, Stéphanie Robin, Jeanne Rochefort, Mathilde Villegas, Gaelen R Burke, Rita Rebollo, Nicolas Nègre, Anne-Nathalie Volkoff. PLoS Pathog. 2024 Apr 25;20(4):e1011980. doi: 10.1371/journal.ppat.1011980.
Assistant Professor Tania Rozario has received a seed grant from the Hypothesis Fund to develop a new approach to advance tapeworm research, particularly on the little understood topic of regeneration.
As a child in Malaysia Rozario was fascinated with the world around her. Her interest was fostered by her grandfather who was an amateur botanist and science teacher. After reading about NASA in a kid’s science magazine, she wrote a letter to them. Their willingness to engage with her inspired her to see science as a real career choice.
“I was exposed to science at an early age,” said Rozario. “But what had the biggest impact on my decision to become a scientist was doing undergraduate research.”
By the time she enrolled in graduate school at the University of Virginia, she knew she wanted to study regeneration. She focused on developmental biology and embryology as she needed a strong foundation in these disciplines to pursue her future research. She returned to regeneration during her post-doctoral training in the Newmark laboratory at the Morgridge Institute for Research. It was then that she started her work in tapeworm regeneration.
“I was drawn to the untapped potential in tapeworms to understand basic biological functions,” she said. “Tapeworms have a complex lifecycle and are difficult to study in the lab – so there’s a challenge there too.”
The mechanisms of regeneration are poorly understood in tapeworms. Stem cells are responsible for regeneration. The Rozario lab wants to know what is special about the stem cells and signals in the “neck” as this tissue is the only tissue capable of regenerating new segments, despite the fact that there are stem cells everywhere in the tapeworm body.
Rozario’s work is shedding light on tapeworm biology and the broader understanding of stem cells in other organisms. Here, a distribution of cycling stem cells (multicolored) is shown within the regeneration-competent tapeworm neck. (Photo courtesy of Tania Rozario)
“Tapeworms can grow very large, but regeneration only happens from a tiny part,” explained Rozario. “We want to know what genes are controlling it but right now we don’t have sufficient tools.”
With the gene-editing tool CRISPR/Cas, researchers have been making remarkable strides in understanding genes in many organisms. However, there is no evidence that transgenesis, the process in which genes are inserted into an organism, works in tapeworms.
This is where the seed grant from the Hypothesis Fund comes in.
“They have scouts who are looking for unconventional science – research where although there may be risk or uncertainty that it will work, it could have a transformational effect if it does,” said Rozario.
The Hypothesis Fund provides seed grants for bold ideas at the earliest stage of research, often before any preliminary data have been generated.
“There are a number of barriers to getting CRISPR/Cas to work in an organism,” said Rozario in response to the risk of this project.
She lists three things that are needed to successfully use CRISPR/Cas: the right type of organism, access to an early development stage, and the expertise.
“We are in a good position to make this work,” further explained Rozario.
The Rozario lab has successfully developed a number of tools to better study the tapeworm in the lab. Since tapeworms produce both male and female gametes in every segment there is plenty of early development stage material to work with.
“Thanks to this gift, we are able to bring in post-doctoral researcher Olufemi Akinkuotu from the University of Pennsylvania School of Veterinary Medicine,” said Rozario. “He has specific training in developing gene-editing tools in parasitic nematodes, which are distantly related to tapeworms but share many parallel challenges.”
While there is still a risk that CRISPR/Cas won’t work in tapeworms, if it does the payoff could be huge – not only for understanding the basic biology of tapeworms, but to further our understanding of stem cells in other organisms.
