Proteogenomic analysis of the total and surface-exposed proteomes of Plasmodium vivax salivary gland sporozoites

Abstract

Plasmodium falciparum and Plasmodium vivax cause the majority of human malaria cases. Research efforts predominantly focus on P. falciparum because of the clinical severity of infection and associated mortality rates. However, P. vivax malaria affects more people in a wider global range. Furthermore, unlike P. falciparum, P. vivax can persist in the liver as dormant hypnozoites that can be activated weeks to years after primary infection, causing relapse of symptomatic blood stages. This feature makes P. vivax unique and difficult to eliminate with the standard tools of vector control and treatment of symptomatic blood stage infection with antimalarial drugs. Infection by Plasmodium is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver. The most advanced malaria vaccine for P. falciparum (RTS,S, a subunit vaccine containing of a portion of the major sporozoite surface protein) conferred limited protection in Phase III trials, falling short of WHO-established vaccine efficacy goals. However, blocking the sporozoite stage of infection in P. vivax, before the establishment of the chronic liver infection, might be an effective malaria vaccine strategy to reduce the occurrence of relapsing blood stages. It is also thought that a multivalent vaccine comprising multiple sporozoite surface antigens will provide better protection, but a comprehensive analysis of proteins in P. vivax sporozoites is not available. To inform sporozoite-based vaccine development, we employed mass spectrometry-based proteomics to identify nearly 2,000 proteins present in P. vivax salivary gland sporozoites. Analysis of protein post-translational modifications revealed extensive phosphorylation of glideosome proteins as well as regulators of transcription and translation. Additionally, the sporozoite surface proteins CSP and TRAP, which were recently discovered to be glycosylated in P. falciparum salivary gland sporozoites, were also observed to be similarly modified in P. vivax sporozoites. Quantitative comparison of the P. vivax and P. falciparum salivary gland sporozoite proteomes revealed a high degree of similarity in protein expression levels, including among invasion-related proteins. Nevertheless, orthologs with significantly different expression levels between the two species could be identified, as well as highly abundant, species-specific proteins with no known orthologs. Finally, we employed chemical labeling of live sporozoites to isolate and identify 36 proteins that are putatively surface-exposed on P. vivax salivary gland sporozoites. In addition to identifying conserved sporozoite surface proteins identified by similar analyses of other Plasmodium species, our analysis identified several as-yet uncharacterized proteins, including a putative 6-Cys protein with no known ortholog in P. falciparum.

Malaria in India: The Need for New Targets for Diagnosis and Detection of Plasmodium vivax

“…[30] A recent report used clinical samples to identify 605 P. vivax proteins from blood stages of the parasite, [31] while another used clinical samples and blood feeding of mosquitoes to identify over a thousand sporozoite stage proteins. [32] More recently, papers have increased the coverage of the P. vivax proteome to over a thousand proteins; however, this was possible only by injecting non-human primates with the parasite. [33,34] …”

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

“…Protein O-fucosyltransferase 2 (POFUT2) initiates metazoan TSR Oglycosylation and plays an important role in the folding and stabilization of these proteins (12)(13)(14)(15)(16). Recently, TSR O-glycosylation has also been observed in Plasmodium falciparum and Plasmodium vivax (17,18): this O-glycan is initiated by a homologue of POFUT2 and is important for stabilizing proteins with TSR domains and efficient host infection (19). O-Glycosylation of TSR domains may also be an important consideration in vaccine design (20).…”

Implications of Plasmodium Glycosylation on Vaccine Efficacy and Design

“…Even so, homologs of several genes required for protein glycosylation are present and conserved in the genomes of Plasmodium spp. and metabolomic analyses of parasite material has demonstrated the presence of the nucleotide sugars required for protein glycosylation, which alluded to the existence of as yet undiscovered parasite glycans.With the aid of modern protein mass spectrometry methods, several research groups have recently tackled the issues of glycan lability and relatively small sample sizes to begin characterizing the true diversity of Plasmodium protein glycosylation [6][7][8][9][10][11][12]. Evidence for the synthesis of short N-linked glycans has been reported in asexual blood stages of Plasmodium falciparum [6], and there is every reason to expect that these modifications will be found in other life cycle stages as well.…”

“…Evidence for the synthesis of short N-linked glycans has been reported in asexual blood stages of Plasmodium falciparum [6], and there is every reason to expect that these modifications will be found in other life cycle stages as well. Cell surface proteomic studies of P. falciparum and P. vivax sporozoites, the two species responsible for the majority of malaria cases and deaths, have demonstrated that O-and C-linked glycans exist on the essential adhesive proteins, TRAP and CSP [8,10]. These abundant surface proteins are required for parasite infectivity and are prime vaccine candidates because sporozoites represent a population bottleneck in the life cycle as parasites pass from mosquito to human and establish an infection in the liver.…”

“…A good starting point would be to determine if patient antibody titres are greater for CSP or TRAP antigens in their glycosylated versus nonglycosylated states, and whether such antibodies are more 10.2217/fmb-2017-0284…”