PFE0565w, a Plasmodium falciparum Protein Expressed in Salivary Gland Sporozoites

Abstract

Abstract. Because malaria is still a significant problem worldwide, additional control methods need to be developed. The Plasmodium sporozoite is a good target for control measures because it displays dual infectivity for both mosquito and vertebrate host tissues. The Plasmodium falciparum gene, PFE0565w, was chosen as a candidate for study based on data from PlasmoDB, the Plasmodium database, indicating that it is expressed both at the transcriptional and protein levels in sporozoites, likely encodes a putative surface protein, and may have a potential role in the invasion of host tissues. Additional sequence analysis shows that the PFE0565w protein has orthologs in other Plasmodium species, but none outside of the genus Plasmodium. PFE0565w expresses transcript during both the sporozoite and erythrocytic stages of the parasite life cycle, where an alternative transcript was discovered during the erythrocytic stages. Data show that transcript is not present during axenic exoerythrocytic stages. Despite transcript being present in several life cycle stages, the PFE0565w protein is present only during the salivary gland sporozoite stage. Because the PFE0565w protein is present in salivary gland sporozoites, it could be a novel candidate for a pre-erythrocytic stage vaccine.

Expression profile of the Plasmodium falciparum intra-erythrocytic stage protein, PF3D7_1363700

“…Anopheles stephensi were reared as previously described [7,8] and used for maintaining the P. falciparum life cycle and parasite transmission. The P. falciparum NF54 strain and 3D7HT-GFP parasites (a constitutively green fluorescent protein (GFP)-expressing parasite line) were maintained according to a standard protocol [8,9] and used for mosquito-parasite infections.…”

“…The P. falciparum NF54 strain and 3D7HT-GFP parasites (a constitutively green fluorescent protein (GFP)-expressing parasite line) were maintained according to a standard protocol [8,9] and used for mosquito-parasite infections. Mosquito parasite transmission studies also were performed according to standard protocol [8].…”

“…The P. falciparum NF54 strain and 3D7HT-GFP parasites (a constitutively green fluorescent protein (GFP)-expressing parasite line) were maintained according to a standard protocol [8,9] and used for mosquito-parasite infections. Mosquito parasite transmission studies also were performed according to standard protocol [8]. Gametocyte cultures used for mosquito parasite transmission and for transcript and protein analysis were established by maintaining a 0.5% parasitaemia NF54 culture supplemented with 10% human serum (A+ male, Interstate Blood Bank) at a 3% haematocrit and changing medium every other day for 16 days [10,11].…”

“…Plasmodium falciparum axenic liver stage parasites were obtained in the absence of host hepatocytes as previously described [8,12]. …”

“…To further purify antibodies to reduce cross-reaction against tissues and pET-32a bacterial protein lysate, two purification methods were performed for either Western blot analysis or immunofluorescent assays (IFA). For antibodies used in Western blot analyses, anti-PF3D7_1363700 antibodies were purified as previously described by Schlarman and colleagues [8]. Antibodies for IFA were purified using a pre-absorption method as previously described [7], which consisted of pre-absorbing the antibodies against protein lysates of mosquito midguts, mosquito salivary glands, human RBCs and pET-32a bacterial protein lysates.…”