Periodic Reporting for period 1 - SugarBlock (Unraveling the protein glycosylation of Plasmodium falciparum is crucial for development of novel therapeutics against malaria)
Periodo di rendicontazione: 2016-08-29 al 2018-08-28
The project aims to increase our limited understanding of protein glycosylation patterns in the Plasmodium spp. parasites, the causative agent of malaria. Glycosylation modifies protein by enzymatically adding glycans during protein synthesis. It plays crucial biological roles such as cellular recognition, signal transduction, modulation of immune response and host-pathogen interactions. The common types include N-linked, O-linked and GPI-anchor protein glycosylation and the less studied C-mannosylation, O-fucosylation and O-GlcNAcylation. Currently our knowledge of their expression patterns in the parasites remains incomplete. O-fucosylation was recently identified on 2 major Plasmodium proteins, CSP and TRAP, which are common targets in malaria vaccine development. The design of the only approved malaria vaccine, RTS,S, is partially based on the specific domains of CSP, which are amenable to protein glycosylation. Its implication in vaccine design remains to be determined. Another key recent advance showed that unknown α-galactose (α-gal)-containing proteins in Plasmodium sporozoites induced the production of antibodies which were able to provide complete protection in malaria-infected mice. Such level of protection is not achieved with the current vaccine, but much remains unclear about the molecular characteristics of these glycosylated proteins in various parasite forms found in the different stages of the complex life cycle of the parasites. These include sporozoites and merozoites and the various blood-stage parasitic forms (rings, trophozoites, schizonts) and gametocytes. In this project, we studied protein glycosylation of sporozoites, merozoites and blood-stage parasites, with a focus on α-gal containing proteins in Plasmodium falciparum (Pf), the most virulent form of human malaria, and Plasmodium berghei (Pb), the rodent form which serves as a good model to study human malaria. We also investigated the proteins found in the salivary glands of Pf-infected and non-infected mosquitoes.
In order to study the glycans in the extracellular sporozoites and merozoites, it is crucial that they are of high purity, free from mosquito and human rbc debris respectively. The collection and purification of Pb sporozoites from infected mosquitoes were performed at the partner organization during the 6-month period. After dissecting hundreds of infected mosquitoes, we successfully obtained over 10 million Pb sporozoites with purity greater than 97% using state-of-the-art fluorescence-activated cell sorting (FACS) flow cytometry. However, due to the minute size of sporozoites, only less than 100 μg protein could be extracted and were insufficient for protein glycosylation study. Obtaining larger quantities proved to be challenging due to variations in mosquito infection rates, which were difficult to control and took months to stabilize. The alternate source from commercially available sporozoites was isolated using methods that did not produce sporozoites of high purity for glycan analysis. Instead we acquired Pf-infected and non-Pf-infected mosquito salivary glands for further proteomics analysis. Nevertheless, we have established the parameters using FACS to sort fluorescently-labelled sporozoites as previously known method (density gradient) used to separate sporozoites from mosquito debris did not result in such high purity. As for merozoites, using existing optimized methods in our laboratory, we successfully obtained highly purified and large quantities of tightly synchronized Pf merozoites. We also isolated sufficient quantities of other blood-stage parasites including rbc-infected rings, trophozoites, schizonts and gametocytes. As these samples inevitably contained rbc debris, uninfected rbcs were also collected simultaneously as controls. Using lectins, anti-α-gal (m86) and blood stage-specific antibodies, we investigated the protein glycosylation in the extracted protein samples. The results showed specific α-gal expression patterns, which appeared to relate to their growth cycle during the blood-stage development. They were not detected in the mature parasites (merozoites, schizonts and gametocytes) but was repeatedly observed in the young parasites that exist as rings and trophozoites in the rbcs. As it was not detected in the uninfected rbcs, we concluded that the α-gal epitopes were from the parasites and may be under specific developmental regulation. We further validated the results using α-galactosidase enzyme capable of removing α-gal structure from the proteins. The type of protein glycosylation was then determined using enzymatic or chemical reactions to study the glycan-protein linkage. The results indicated that these glycans were not likely to be O-linked or GPI-anchor protein glycosylation, while signals seemed to be slightly reduced with N-linked deglycosylation. In the late phase of the project, isolation of the glycan-bound protein(s) from the extracted protein mixture was performed using immunoprecipitation and m86 antibody and analysed using LC-MS/MS analysis. In addition to blood-stage parasites, comparative analysis of proteins obtained from Pf-infected and non-infected salivary glands of Anopheles mosquito was performed. Preliminary analysis identified a total of 1053 non-redundant proteins with 193 and 3 of those uniquely present in the infected and non-infected samples, respectively. Given the reported significance of α-gal in malaria, we are currently working with collaborators to gain further insights into the data obtained.
Every year, malaria affects over 200 million people worldwide causing nearly half a million deaths, mostly in children aged below 5. Despite research supporting that the disease is preventable and treatable with effective drugs, malaria remains a major public health problem. It is estimated that 50% of the global population is at risk of malaria. The only vaccine available, RTS,S, offers only short-term protection in young children and has to be implemented along with control measures such as the use of insecticidal nets and sprays to achieve meaningful public health benefits. Additionally, the wide use of both single and a combination of anti-malaria drugs leads to resistance. There is an urgent need to develop more effective vaccines and drugs to control and ultimately eliminate the disease. To our knowledge, the presence of α-gal in blood stage parasites has not been reported. Interestingly our results showed that their expression appeared to be regulated. The α-gal epitope is of great importance in xenotransplantation as the use of non-primates as sources of organ transplantation has led to organ rejection. This is because evolutionary forces have eliminated the ability of humans to express the α-gal epitope but they are found abundantly on non-primates and some pathogens. Humans have as much as 1% of circulating natural anti-α-gal antibodies. Therefore, the viability of exploiting such natural mechanism of protection against malaria makes these α-gal containing proteins an attractive therapeutic candidate for immunization. We hope our findings will contribute to increased knowledge of Plasmodium biology and help in the development of more effective malaria vaccines.