If you have ever been bitten by a mosquito, you might already know that the red bump on your skin is your body’s immune response to the injected mosquito saliva. It turns out that when mosquitoes land on you, they salivate while searching for a blood vessel and then penetrate the skin with their needles. Unfortunately, when a mosquito has been infected with Plasmodium parasites, these parasites are transported along with mosquito saliva into the animal host, ultimately leading to malarial infection.
Mosquitoes have become an increasingly dangerous global threat—particularly in tropical or humid climates, where they not only thrive best but also transmit numerous viral, bacterial, and parasitic diseases including malaria and Zika. According to the World Health Organization, malaria remains a major global epidemic, with more than 200 million cases across 91 countries in 2016, but most occurring within sub-Saharan Africa. Although malaria had been eliminated in the United States in the 1950s, the CDC estimates that there are roughly 1,700 annual diagnoses of malaria across the country, a high incidence rate considering the severity of the disease. Since Plasmodium parasites enter an animal host during mosquito blood feeding, targeting the mosquito’s saliva appears to be one of the most plausible methods of preventing malaria and developing new antimalarial therapies.
However, the relationship between mosquito saliva and Plasmodium infection is disputed. While mosquito saliva has previously been believed to enhance pathogen transmission, recent studies suggest that saliva could provide the host protection from Plasmodium infection. Tyler Schleicher and Jing Yang recently co-authored a paper in Nature Communications with Erol Fikrig’s lab at the Yale School of Medicine that analyzed the ability of a specific mosquito salivary gland protein to decrease Plasmodium speed and movement. Because Plasmodium parasites are some of the fastest microbes known, proteins that can impede their speed have tremendous potential for novel antimalarial therapies.
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 blood-stage,” 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 the 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 salivary gland, but 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 mosGILT 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 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.