Thioester-containing protein TEP15 promotes malaria parasite development in mosquitoes through negative regulation of melanization

Abstract

Background: Thioester-containing proteins (TEPs) serve as crucial effectors and regulatory components within the innate immune system of mosquitoes. Despite their significance, the mechanisms by which TEPs exert negative regulation on the immune response in mosquitoes remain inadequately understood. This study aims to elucidate the role of TEPs in the negative regulation of melanization, thereby advancing our comprehension of their regulatory function in the immune response.

Methods: We infected female Anopheles stephensi mosquitoes with Plasmodium yoelii by allowing them to feed on pre-infected female Kunming mice. Western blot, quantitative polymerase chain reaction, differential gene expression analyses, and gene silencing were then conducted. Student's t-test was used to analyze continuous variables, with statistical significance defined as p < 0.05.

Results: A. stephensi TEP15 (AsTEP15) negatively regulated mosquitos' innate immunity and promoted Plasmodium development. AsTEP15 knockdown induced mosquito resistance to malaria parasite melanization during the oocyst stage and significantly reduced sporozoite numbers. Further analysis showed that AsTEP15 mainly negatively affects the TEP1 and immune deficiency (IMD) pathway, thereby inhibiting melanization.

Conclusions: We describe a mosquito TEP that negatively regulates immunity, further enriching the functional diversity of TEP family members. In addition, our results suggest that oocysts may exploit TEPs to escape or inhibit mosquito immunity, highlighting potential targets for blocking malaria transmission.

Keywords: Anopheles stephensi; Plasmodium; AsTEP15; Melanization; Thioester-containing protein.

Fig. 1

AsTEP15 sequence analysis and its transcriptional changes following Plasmodium infection (a) sequence comparison. Schematic illustration of proteins including thioester structures: AsTEP15 (VectorBase accession number ASTE008693), human (Hu) CD109, and A2M. Green, complement (C) 3 isoform X1; blue, CD109 isoform; purple, α₂-macroglobulin-like TED domain; and red-brown, α₂-macroglobulin. The black vertical line shows the α₂-macroglobulin-like thioester bond-forming region. The black horizontal line shows the α₂-macroglobulin/complement system. b Unrooted phylogenetic tree of thioester-containing proteins. The evolutionary history was inferred using the neighbor-joining method. The optimal tree is shown. Evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. This analysis involved 56 amino acid sequences, with 5404 positions in the final dataset. Evolutionary analyses were conducted in MEGA11. The invertebrate TEP clades are blue shades, and CD109 clades are purple shades; C3, C4, and C5 clades are green shades; and A2M and CPAMD8 clades are red-brown shades. c AsTEP15 immunoblotting analysis. Total protein extracts from hemolymph and carcasses of non-fed female mosquitoes (n = 40). The 180 kd band represents putative full-length AsTEP15, and the 45 kd band represents the activation and subsequent hydrolysis of AsTEP15. Hemolymph was collected directly into the loading buffer, immunoblotted with anti-AsTEP15 rat antiserum, and revealed by anti-rat horseradish peroxidase-conjugated antibody. The blot was stripped and reprobed with a polyclonal antibody raised against the S7 protein, serving as a loading control. The molecular weight scale is shown on the right. d The mRNA expression level of AsTEP15 in mosquitoes (n = 15) fed with P. yoelii-infected blood and uninfected blood at the indicated time points post-blood-feeding was determined using real-time PCR. TEP15 transcript levels were normalized to the internal control transcript for ribosomal protein S7. For most of the time points, experiments were performed three times (error bars indicate standard errors)

Fig. 2

AsTEP15’s role in promoting Plasmodium development in mosquitoes. a Detection of RNA interference efficiency of AsTEP15. AsTEP15 mRNA levels in P. yoelii-parasite-infected mosquitoes (n = 15) determined using real-time PCR after AsTEP15 knockdown. b–c Number of salivary gland and hemolymph sporozoites in mosquitoes after AsTEP15 knockdown. The average number of salivary gland sporozoites (n = 15) b and hemolymph sporozoites (n = 20) (c) in mosquitoes infected with P. yoelii were compared after AsTEP15 knockdown. d Percentage of melanized oocysts in P. yoelii-parasite-infected mosquitoes (n = 22) at the indicated time points were compared after AsTEP15 knockdown. e Representative image of melanized oocysts in mosquitoes under a light microscope on day 12 post-blood feeding after AsTEP15 knockdown; scale bar 50 μm. Three individual experiments were performed

Fig. 3

AsTEP15 in regulating TEP1-mediated oocyst melanization. a Differential gene expression of immune-related genes in P. yoelii-infected mosquitoes after AsTEP15 knockdown. Heatmap comparing the differential expression of immune-related genes between double-stranded green fluorescent protein (dsGFP) and double-stranded TEP15 (dsTEP15) P. yoelii-infected mosquitoes (n = 30) on day 7 PI. b Detection of AsTEP1 expression change after AsTEP15 knockdown. TEP1 mRNA levels in P. yoelii-parasite-infected mosquitoes (n = 15) determined using real-time PCR following AsTEP15 knockdown at day 7 PI. c–d Number of salivary gland and hemolymph sporozoites in mosquitoes after AsTEP1 and AsTEP15 double knockdown. Average number of salivary glands (n = 20) (c) and hemolymph (n = 22) sporozoites (d) in P. yoelii-parasite-infected mosquitoes were compared with AsTEP1 and AsTEP15 knockdown. e Percentage of melanized oocysts after AsTEP1 and AsTEP15 double knockdown. Melanized oocyst percentage in P. yoelii-parasite-infected mosquitoes (n = 22) at the indicated time points were compared after AsTEP1 and AsTEP15 double knockdown

Fig. 4

AsTEP15 promotes malaria parasite development by negatively regulating the IMD pathway. a–b Number of salivary gland and hemolymph sporozoites in mosquitoes after AsTEP15 and AsRel2 double knockdown. The average number of salivary gland (n = 15) (a) and hemolymph (n = 20) sporozoites (b) in P. yoelii-infected mosquitoes were compared with AsTEP15 and AsRel2 double knockdown. c Melanized oocyst percentage after AsTEP15 and AsRel2 double knockdown. Melanized oocyst percentage in P. yoelii-infected mosquitoes (n = 22) at the indicated time points were compared after AsTEP15 and AsRel2 double knockdown. d Detection of Rel2 expression change after AsTEP15 and TEP1 double knockdown