YSM Issue 91.3

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FOCUS medicine PHOTOGRAPHY BY KATE KELLY The basics of malaria transmission The biological carrier responsible for transmitting malaria is the infected female Anopheles mosquito, which delivers Plasmodium parasites into the skin of a host while also injecting its saliva. Sporozoites, a term for the infectious stage of the Plasmodium parasite life cycle, must then locate a nearby blood vessel in order to travel to the host liver and invade hepatocytes, or liver cells, thus establishing malaria infection. While navigating through the body, these sporozoites possess the ability to enter and maneuver within host cells as well as bypass the host immune system. This dynamic process of cell traversal therefore makes it difficult to catch the sporozoites once they have entered the dermis. “We are interested in the [Plasmodium] transmission stage…because blocking establishment of malaria at this earliest point represents a target that might present a better opportunity than treating the disease at the point when it progresses to millions of parasites in the bloodstage,” Schleicher said. The secreted saliva serves to prevent blood clotting and inflammation and thus better facilitate the blood meal for the mosquito. However, the array of proteins present in the saliva may also affect pathogen transmission by promoting pathogen survival or inhibiting their motility in the body. “Analyzing individual components of mosquito saliva allows for better characterization of novel protein-pathogen interactions,” Yang said. The researchers purified Plasmodium sporozoites from Anopheles mosquito saliva and identified a specific salivary gland protein that is directly involved with sporozoite transmission: a mosquito-equivalent of human gamma interferon inducible thiol reductase (GILT) protein, called mosGILT, that binds to the sporozoites and partially inhibits their movement. In humans, GILT is involved in protein unfolding as well as antigen processing. Because the interaction between the mosGILT protein and sporozoites is similar for different species of Plasmodium, the researchers used both human pathogen Plasmodium falciparum and the rodent parasite Plasmodium berghei to study the inhibitory properties of the mosGILT protein on parasite transmission. “It is important to know from our work that we provide evidence that the interaction between mosGILT and sporozoites occurs in both species of the parasite, suggesting this is a conserved interaction,” Schleicher emphasized. The inhibitory nature of mosGILT protein MosGILT is roughly twenty percent identical to both the human and mouse GILT, which is a useful measure for comparing protein function between the species. Conservation of key amino acid sequences suggests some similarity in the catalytic regions among the GILT proteins. However, the overall percent identity is low, and identical protein residues do not necessarily indicate the same protein activity. “In fact, the C-terminus is extended in mosGILT compared to the human and mouse equivalents, and when this portion is deleted, the mosGILT does not inhibit the motility of Plasmodium parasite as before, indicating that the C-terminus sequence is very important for the inhibitory ability of the protein,” Schleicher added. Although the exact interaction between mosGILT and sporozoites remains unclear, the researchers also discovered that mosGILT levels are high in the mosquito sal- “We hope to develop more effective treatments against mosquito-borne diseases.”
medicine FOCUS ivary gland, but it was not discernible in the saliva itself. “We don’t know how the mosGILT protein binds to the Plasmodium sporozoite, but our experiments with immunofluorescence staining and microscopy studies have shown us that mos- GILT binds to the surface of the parasite,” Yang said. With these pieces of data, the researchers concluded that the mosGILT and sporozoite interaction begins in the salivary gland of the mosquito at the sporozoite surface, and then mosGILT is carried from the gland and released with the saliva and sporozoites into the host. The sporozoites are still alive After sporozoite invasion of the hepatocytes, exoerythrocytic forms (EEF) develop and each release merozoites, the next stage of the Plasmodium life cycle. “Merozoites can invade red blood cells, leading to the blood-stage of the malaria infection and the clinical symptoms associated with malaria, such as chills and fever,” Yang explained. Since cell traversal and consequent evasion from the host immune system allow the parasites to survive in the body, the researchers also wanted to demonstrate that mosGILT did, in fact, negatively impact the sporozoite cell traversal activity. They incubated rGILT, or purified recombinant mosGILT, together with sporozoites and observed significantly reduced cell traversal activity. Additionally, pre-incubating hepatic cells and skin fibroblasts with rGILT did not lead to any indirect inhibition of cell traversal, further confirming that rGILT directly interacts with the sporozoites to reduce cell traversal and motility in the host. However, sporozoite viability was not directly affected when treated with rGILT in vitro. Instead, the percentage of EEF-positive liver cells increased, suggesting that the sporozoites had continued to infect the hepatocytes. “The viability assay in our paper was direct evidence that rGILT does not directly kill sporozoites or is immediately toxic to them…because [otherwise] we would expect the EEFs to decrease, as the sporozoites would be dying and not healthy enough to infect hepatic cells in culture,” Schleicher stated. Future directions The study’s findings that salivary gland protein mosGILT partially inhibits Plasmodium sporozoite motility and cell traversal has great consequences for the understanding of malaria transmission and development of new treatments. Next steps for the researchers include a similar attempt to identify the molecule on the surface of the sporozoites to which mosGILT binds. “If we are able to find the binding partner of mosGILT, we can better understand the mechanism of how mosGILT inhibits sporozoite motility through cell traversal and the overall reduction of speed,” Schleicher said. The researchers have already begun work to create mosGILT knockout mosquitoes using the CRISPR/Cas9 technique in order to study the mosGILT-sporozoite pathway. Furthermore, this research on mosGILT will aid the development of novel antimalarial therapies—perhaps through experimental manipulation of mosGILT activity or structure, or by synthesizing a new protein with a similar but more efficient inhibitory function. The researchers also understand the significance of their research to global health. “Our research not only applies to the Anopheles mosquito and malaria, but also to other mosquitoes that transmit notable viruses including Zika, and we hope to develop more effective treatments against mosquito-borne diseases,” Schleicher said. With these results, Schleicher and Yang are making progress toward uncovering the secrets of mosquito spit. ABOUT THE AUTHOR ANNA SUN ANNA SUN is a Molecular, Cellular and Developmental Biology major in Jonathan Edwards College ‘21. She hopes to study the interaction between technology and medicine in order to improve healthcare. Besides writing for the Yale Scientific Magazine, she is also involved in the MCDB Student Advisory Committee at Yale and dances with Danceworks. In her free time, she loves to spend time with her friends discovering the food scene in New Haven. THE AUTHOR WOULD LIKE TO THANK Jing Yang and Tyler Schleicher for sharing their time and enthusiasm about their research. FURTHER READING Schleicher, T. R, et. al.. “A Mosquito Salivary Gland Protein Partially Inhibits Plasmodium Sporozoite Cell Traversal and Tramission.” Nature Communications 9, no. 1 (July 25, 2018). www.yalescientific.org October 2018 Yale Scientific Magazine 17
Page 1 and 2: Yale Scientific Established in 1894
Page 3 and 4: TABLE OF CONTENTS VOL. 91 ISSUE NO.
Page 5 and 6: SCIENCE SURROUNDS US While the Yale
Page 7 and 8: Imagine if we could turn all the wo
Page 9 and 10: psychology NEWS WIRING OF PSYCHOPAT
Page 11 and 12: ecology NEWS HIPPOPOTAMI AS ENGINEE
Page 13 and 14: FOCUS cell biology The fundamental
Page 15: medicine FOCUS If you have ever bee
Page 19 and 20: FOCUS neuroscience Alzheimer’s is
Page 21 and 22: FOCUS applied physics L U M On June
Page 23 and 24: FOCUS materials science On June 10,
Page 25 and 26: SCIENCE IN THE LIBERAL ARTS: a refl
Page 27 and 28: applied physics FEATURE SEAS OF ELE
Page 29 and 30: physical chemistry FEATURE van der
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Page 33 and 34: SEIZURES biomedical engineering FEA
Page 35 and 36: epurposed By: Katie Schlick Rags to
Page 37 and 38: ALUMNI PROFILE ERIC FOSSUM (PhD ’
Page 39 and 40: As the string ensemble emerges, we

YSM Issue 91.3

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