Seventeen new cephalotane-type diterpenoids, fortalides A-Q (1-17), along with five known analogues, were isolated from the seeds of Cephalotaxus fortunei var. alpina. Their structures were determined by extensive spectroscopic methods, as well as electronic circular dichroism (ECD) and X-ray crystallographic data analyses. Some isolates exhibited unusual structural features that were first found in cephalotane-type diterpenoids, such as the occurrence of the 7-oxabicyclo[4.1.1]octane moiety in 14 and 15 and the cis-arrangement of 3-OH and Me-19 in 9. Besides, the antiplasmodial activity of these compounds was evaluated in this study.
Zhan-Peng Ge, Bin Zhou, Flavia M Zimbres, Reagan S Haney, Qun-Fang Liu, Yan Wu, Maria B Cassera, Jin-Xin Zhao, Jian-Min Yue. Org Biomol Chem. 2022 Nov 4. doi: 10.1039/d2ob01748b
Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite’s lifecycle.
Amélie Vantaux, Julie Péneau, Caitlin A Cooper, Dennis E Kyle, Benoit Witkowski, Steven P Maher. Front Microbiol. 2022 Sep 23;13:976606. doi: 10.3389/fmicb.2022.976606.
UGA’s Samarchith “Sam” Kurup, assistant professor of cellular biology, has been awarded a five-year National Institutes of Health grant to study the natural immune response to the Plasmodium parasite—which causes malaria—in liver cells. (photo credit: Lauren Corcino)
Samarchith “Sam” Kurup grew up in India, and he’s always been aware of the impact of malaria.
In 2020 there were an estimated 241 million cases of malaria worldwide and an estimated 627,000 deaths, according to a recently released World Health Organization Fact Sheet. Eighty percent of the malaria-related deaths in Africa are children under the age of 5. The relapsing nature of the disease leads to educational and employment loss that has long-term economic impacts for both the individual as well as society.
Kurup began his training in veterinary medicine in India, where he became hooked on parasitology, then continued his studies at UGA. While pursuing his Ph.D. he worked in Rick Tarleton’s lab, studying a parasitic disease that affects both animals and humans—his first introduction to human immunology. He continued his training in immunology as a postdoctoral researcher in John Harty’s lab at the University of Iowa.
Combining parasitology with immunology prepared him to tackle malaria.
Malaria is one of the most studied parasitic diseases, yet the Plasmodium parasite that causes it keeps evading attempts to treat the infection in humans. This is largely due to its complex life cycle and the ability of the parasite to evolve drug resistance. In addition to life stages that occur in the mosquito, which transmits the Plasmodium parasite to humans, there are two life stages in humans—a short phase of initial development in the liver, followed by an infection of the blood cells that causes clinical disease.
“A lot of research has been focused on the blood stage in humans, as this is when a person is symptomatic,” said Kurup, assistant professor of cellular biology in the Franklin College of Arts and Sciences. “But we now recognize that if we want to stop malaria, we need to stop it in its tracks in the liver before accessing the blood, and for that we need to understand the liver stage.”
Kurup, a member of UGA’s Center for Tropical and Emerging Global Diseases, trained in parasitology and immunology. He hopes that uncovering how the human immune system naturally fights malaria in the liver stage will lead to an effective malaria vaccine. (photo credit: Lauren Corcino)
Kurup has been awarded a five-year National Institutes of Health grant to study the natural immune response to the Plasmodium parasite in liver cells.
“The liver stage is short and can be difficult to study in the laboratory,” he said. “There are also practical and ethical limitations to studying the liver stage of malaria in humans. We are hoping to tease apart the basic principles of immune responses during this stage using the mouse model.”
Kurup’s preliminary studies have shown that a group of signaling proteins called type 1 interferons play a role in the destruction of Plasmodium parasites in the liver. His newly funded project will fill a gap in the malaria knowledge base by using a combination of in vitro study and in vivo experiments to determine the molecular processes that eliminate Plasmodium parasites in liver cells. His group recently developed a transgenic parasite line that can be used to genetically alter its host cell.
“This strain is a game changer for our line of research because we can now determine how our liver cells would naturally eliminate the parasite, and maybe why it sometimes fails,” he said.
In a study recently published in Cell Reports, Kurup and colleagues used the genetically altered parasite to inhibit signaling by type 1 interferons and showed that this protein has a direct role in the control of malaria. Their study also revealed that other natural immune mechanisms may be active in controlling malaria in liver cells. The project funded by the new grant will delve further into these mechanisms.
“In addition to taking us a step closer to the control and possible eradication of malaria, this project will expand our knowledge so that we can better reduce the burdens of this illness in our society,” he said.
Kurup is hopeful that uncovering how the human immune system naturally fights malaria in the liver stage will lead to an effective malaria vaccine.
“I really believe that bringing together our knowledge in parasitology and approaches in immunology is key to uncovering new information on this elusive life stage in malaria,” he said. “There is no better place to do this, considering the intellectual and material resources we have at our disposal at UGA and the CTEGD.”
Background: Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands.
Methods: This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11-15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17-29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields.
Results: The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17-29 days post-blood meal) for either parasite strain.
Conclusions: Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.
The resilience of Plasmodium vivax, the most widely-distributed malaria-causing parasite in humans, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasite’s influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax-infected hepatocytes at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response are upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform therapeutic targets against P. vivax liver-stage infection.
Anthony A Ruberto, Steven P Maher, Amélie Vantaux, Chester J Joyner, Caitlin Bourke, Balu Balan, Aaron Jex, Ivo Mueller, Benoit Witkowski, Dennis E Kyle. Front Cell Infect Microbiol. 2022 Aug 25;12:986314. doi: 10.3389/fcimb.2022.986314. eCollection 2022.
Malaria is a devastating disease impacting over half of the world’s population. Plasmodium parasites that cause malaria undergo obligatory development and replication in hepatocytes before infecting red blood cells and initiating clinical disease. While type I interferons (IFNs) are known to facilitate innate immune control to Plasmodium in the liver, how they do so has remained unresolved, precluding the manipulation of such responses to combat malaria. Utilizing transcriptomics, infection studies, and a transgenic Plasmodium strain that exports and traffics Cre recombinase, we show that direct type I IFN signaling in Plasmodium-infected hepatocytes is necessary to control malaria. We also show that the majority of infected hepatocytes naturally eliminate Plasmodium infection, revealing the potential existence of anti-malarial cell-autonomous immune responses in such hepatocytes. These discoveries challenge the existing paradigms in Plasmodium immunobiology and are expected to inspire anti-malarial drugs and vaccine strategies.
UGA’s Lọla Fagbami, winner of a Burroughs Wellcome Fund 2022 Postdoctoral Diversity Enrichment Program fellowship, is a native of Lagos, Nigeria, who relocated to the United States with her family in the late 1990s. She is passionate about expanding scientific literacy through outreach and mentoring as well as refuting chemophobia—the fear of or aversion to chemicals and chemistry. (Photo by Lauren Corcino)
Fagbami, UGA’s first PDEP Fellow, conducts research on the human malaria parasite Plasmodium falciparum at the Center for Tropical and Emerging Global Diseases. She works with Vasant Muralidharan, associate professor of cellular biology in the Franklin College of Arts and Sciences, who nominated her for the award.
“Dr. Fagbami has excellent training in metabolomics, mass spectrometry and Plasmodium drug discovery. Her exceptional work as a graduate student has shown how human malaria-causing parasites use metabolic adaptation to induce antimalarial drug resistance. Dr. Fagbami is a fearless, highly intelligent, accomplished and outstanding scientist who will be a leader in our field,” Muralidharan wrote in his nomination letter.
“Her research project addresses a major gap in the field that has enormous implications for malaria elimination and eradication efforts,” he added.
The PDEP award provides $60,000 over three years to support career-development activities for historically excluded minority postdoctoral fellows pursuing academic careers in biomedical or medical research, according to the Burroughs Wellcome Fund.
“This award is an investment in me as a scientist and leader and will help advance my career to the next level,” Fagbami said. “I am excited to join the extraordinary community of PDEP scholars and also connect with program alumni who have successfully made the transition to research independence.”
Fagbami earned a B.S. in chemistry at Emory University, an M.B.S. and an M.P.H. in health policy at Rutgers University, and a Ph.D. in chemical biology at Harvard University.
The Plasmodium falciparum protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood. The deep sequencing of the var2csa DBL3X domain in placental blood from forty-nine primigravid and multigravid women living in malaria-endemic western Kenya revealed numerous unique sequences within individuals in association with chronic PM but not gravidity. Additional analysis unveiled four distinct sequence types that were variably present in mixed proportions amongst the study population. An analysis of the abundance of each of these sequence types revealed that one was inversely related to infant gestational age, another was inversely related to placental parasitemia, and a third was associated with chronic PM. The categorization of women according to the type to which their dominant sequence belonged resulted in the segregation of types as a function of gravidity: two types predominated in multigravidae whereas the other two predominated in primigravidae. The univariate logistic regression analysis of sequence type dominance further revealed that gravidity, maternal age, placental parasitemia, and hemozoin burden (within maternal leukocytes), reported a lack of antimalarial drug use, and infant gestational age and birth weight influenced the odds of membership in one or more of these sequence predominance groups. Cumulatively, these results show that unique var2csa sequences differentially appear in women with different PM exposure histories and segregate to types independently associated with maternal factors, infection parameters, and birth outcomes. The association of some var2csa sequence types with indicators of pathogenesis should motivate vaccine efforts to further identify and target VAR2CSA epitopes associated with maternal morbidity and poor birth outcomes.
Eldin Talundzic, Stephen Scott, Simon O Owino, David S Campo, Naomi W Lucchi, Venkatachalam Udhayakumar, Julie M Moore, David S Peterson. Pathogens. 2022 Apr 28;11(5):520. doi: 10.3390/pathogens11050520
The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.
Peter Christensen, Annie Racklyeft, Kurt E Ward, Jessica Matheson, Rossarin Suwanarusk, Adeline C Y Chua, Osamu Kaneko, Htin Lin Aung, Laurent Rénia, Nadia Amanzougaghene, Victor Magneron, Julien Lemaitre, Roger Le Grand, Dennis Kyle, Pablo Bifani, Gregory M Cook, Georges Snounou, Bruce Russell. Parasitol Int. 2022 Apr 22;89:102589. doi: 10.1016/j.parint.2022.102589. Online ahead of print.
Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite’s ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesized that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalyzed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.
Alona Botnar, Grant Lawrence, Steven P Maher, Amélie Vantaux, Benoît Witkowski, Justine C Shiau, Emilio F Merino, David De Vore, Christian Yang, Cameron Murray, Maria B Cassera, James W Leahy, Dennis E Kyle. Int J Parasitol. 2022 Apr 18;S0020-7519(22)00066-2. doi: 10.1016/j.ijpara.2022.03.003.
