Fig 1.
SDS-PAGE analysis of neat SGE, human receptor proteins and human receptor interacting SGPs.
(A) A. aegypti SGE analysis showing 9 prominent bands and many smaller bands. The red font intense band labels with molecular weights of 56/65 kDa and 31 kDa, were previously analysed to contain seven and thirteen different proteins dominated by apyrase and D7(AEEL006424 and AEEL006417) proteins, respectively [42]. (B) CD14, CD86, DC-SIGN, and CD4 only control. (C) CD4-interacting SGPs. (D) CD14- interacting SGPs. (E) CD86-interacting SGPs. (F) DC-SIGN interacting SGPs. SGS1 is a 380 kDa protein. The identified lower molecular weight bands might be due to cleaved products as this protein is known to have three predicted cleavage sites [40]. (G) TLR4-interacting proteins. Due to post-translational modifications and similar molecular weight of SGPs, a single band could contain more than one protein merged into that band.
Table 1.
Human receptor protein binding SGPs identified by pull-down and MS analysis.
Fig 2.
Human receptor proteins-SGPs interacting network.
CD4, CD14, CD86, DC-SIGN and TLR4 are the human receptor proteins. All the SGPs identified here were previously reported to be present in the mosquito saliva [35]. Malate dehydrogenase (AAEL001593) has not been reported in the mosquito saliva.
Table 2.
Roles of the receptor binding SGPs in virus transmission/infection.
Fig 3.
Interaction of recombinant ectodomain CD4 and DC-SIGN proteins with individual recombinant SGPs.
ELISA results of SGPs interaction with CD4 protein and DC-SIGN protein. The mosquito SGPs include: AaVA-1, D7 (AAEL006417), Neutrophil stimulating protein 1 (NeST1), Lymphotoxin-β receptor inhibitor (LTRIN), and Ae. aegypti bacteria-responsive protein 1 (AgBR1). Bovine serum albumin (BSA) was used as a negative control. LTRIN and AgBR1 were not previously identified in our pull-down experiment. Statistical significance was calculated using the two-way ANOVA on GraphPad Prism version 7.0 and represented as p values: *p < 0.05, ***p < 0.001, ****p< 0.0001. The cut-off value of negative control was calculated as the mean + three times the standard deviation at the value of 0.066 which is shown the dashed line before applying the statistical analysis.
Fig 4.
AaVA-1 and Nest1 enhance the activation of T cells.
Human peripheral blood mononuclear cells (PBMC) with mosquito proteins at different concentration, 24 hours later activation marker CD69 and CD25 were measured. (A-C). Frequency of CD69 expression on CD4+ T cells was compared after different concentration of AaVA-1 (A), D7 (AAEL006417) (B), or Nest1 (C) treated PBMC cells. (D-F). Frequency of CD25 expression on CD4+ T cells was compared after different concentration of AVAa-1 (D), D7 (AAEL006417) (E), or Nest1 (F) treated PBMC cells. (G-I). Mean fluorescence of intensity of CD25 expression on CD4+ T cells was compared after different concentration of AaVA-1 (G), D7 (AAEL006417) (H), or NeST1 (I) treated PBMC cells.
Fig 5.
Schematics of the potential role of SGPs interactions with human host receptor proteins on pathogen transmission and or infection.
(A) SGP binds to human immune receptor proteins resulting in host immunosuppression, thereby, promoting viral infection and replication. (B) SGP binds to human receptor protein and blocks the binding site of invading pathogens on the receptor resulting in the abrogation of pathogen cellular entry and reduced overall infection. (C) Anticoagulant SGP or proteins with anti-hemostatic activities hijacked by human receptor making it possible for blood to clot easily at the bite site resulting in the difficult acquisition of blood by feeding mosquitoes. This may cause the mosquito to inject more pathogen-infected saliva which could lead to enhanced pathogen transmission. Conversely, excessive blood clot could deter mosquito from further feeding resulting in a lower deposition of pathogens and lower transmission or infection.