FBN30 in wild Anopheles gambiae functions as a pathogen recognition molecule against clinically circulating Plasmodium falciparum in malaria endemic areas in Kenya

Abstract

Malaria is a worldwide health problem that affects two-thirds of the world population. Plasmodium invasion of anopheline mosquitoes is an obligatory step for malaria transmission. However, mosquito-malaria molecular interactions in nature are not clear. A genetic variation within mosquito fibrinogen related-protein 30 (FBN30) was previously identified to be associated with Plasmodium falciparum infection in natural Anopheles gambiae populations at malaria endemic areas in Kenya, and reducing FBN30 expression by RNAi makes mosquitoes more susceptible to P. berghei. New results show that FBN30 is a secreted octamer that binds to both P. berghei and clinically circulating P. falciparum from malaria endemic areas in Kenya, but not laboratory P. falciparum strain NF54. Moreover, the natural genetic mutation (T to C) within FBN30 signal peptide, which changes the position 10 amino acid from phenylalanine to leucine, reduces protein expression by approximately half. This change is consistent to more susceptible An. gambiae to P. falciparum infection in the field. FBN30 in natural An. gambiae is proposed to work as a pathogen recognition molecule in inhibiting P. falciparum transmission in malaria endemic areas.

Figure 1

FBN30 is secreted from insect Hi5 cells and forms octamers. (a) The non-reducing (NON-RD) (without β-ME) and the reducing (RD) conditions of 12% SDS-PAGE analysis were performed. FBN30 was detected with anti-FBN30 antibody. A specific band with a molecular mass of ~ 33 kDa (bottom arrow labeled, corresponding to insect cell expressed recombinant FBN30 protein) under reducing condition was detected from the medium (right panel), indicating that the recombinant FBN30 is a secreted protein. Under non-reducing conditions, a specific band of ~66 kDa (top arrow labeled) was detected with anti-FBN30, suggesting that FBN30 forms a homodimer through disulfide bond. (b) The FBN30 protein from the concentrated medium was applied onto a size exclusion chromatography using a Superdex-200 increase column and the FBN30 in each eluted fraction was quantified with ELISA assays. The molecular mass of FBN30 ranged from 212–274 kDa, suggesting that the native FBN30 exists as an octamer, which is formed by four disulfide-bond linked homodimers.

Figure 2

FBN30 binds to P. berghei infected red blood cells and ookinetes. (a) Interaction between the recombinant FBN30 protein and P. berghei iRBC by ELISA. The lysates of P. berghei-infected red blood cells (iRBC) and uninfected mouse red blood cells (RBC) were used to determine the interaction between FBN30 and P. berghei parasites. OD₄₀₅ values and standard deviations were obtained from 3 replicates. The OD₄₀₅ value in P. berghei iRBC was 1.86 folds of the uninfected mouse RBC, and the difference is significant (p < 0.01). (b) Interaction between FBN30 protein and P. berghei parasites determined by indirect immunofluorescence assays (IFA). Images in the first row represent non-infected mouse RBCs. Images in the second row represent mouse P. berghei iRBCs and ookinetes. Images in the third row obtained by merging first and second rows to show the co-localization of FBN30 and parasites. Arrows show the location of P. berghei gametocytes. (c) FBN30 does not bind to P. berghei sporozoites as determined by IFA. Images in the first row are the control group by replacing FBN30 with equal amount of BSA in IFA assays. The first and second columns show P. berghei parasites (green color) and FBN30 (red color) respectively. Merging column one and two generated the third column.

Figure 3

FBN30 interacts with clinically circulating Plasmodium falciparum isolates. (a) FBN30 does not bind to laboratory P. falciparum strain NF54. There is no significant difference between uninfected RBC and NF54 infected RBC in ELISA assays. (b) The ELISA data show that significantly more FBN30 bound to blood lysates from the malaria infected patients (15 individuals) than that from the uninfected human subjects (6 individuals) (p = 0.018). (c) FBN30 interacts with the cultured wild type P. falciparum ookinetes determined by IFA. Images in the first row are the control group in which the anti-FBN30 was replaced with BSA. Arrows show the location of ookinetes.

Figure 4

Expression efficiency of two variants of FBN30 in mosquito cell lines. Moss55 cells(left) and Sua5B cells(right) were the mosquito cells transfected with vector. After transfection assays with the same amount of recombinant plasmids containing either FBN30 (T) or (C), the western blot assays detected one specific band corresponding to the recombinant FBN30 protein. The band intensity was measured with Image J software. The expression ratios of FBN30 (T) to (C) were 1.9 and 2.5 folds in Moss 55 and Sua5B respectively.